molecular characterisation of microorganisms within the

Molecular characterisation of microorganisms within the

stump-tailed lizard tick, Amblyomma albolimbatum,

in Western Australia

Samuel Elliot

Bachelor of Science

Submitted in fulfilment of the requirements for the degree of

Bachelor of Sciences with Honours.

School of Veterinary and Life Sciences

Murdoch University, 2021

II

Declaration

I declare this thesis is my own account of my research and contains as its main content, work

which has not been previously submitted for a degree at any tertiary education institution.

Samuel Elliot

III

Abstract

Ticks pose a major threat to the health and wellbeing of humans, domesticated animals and wildlife.

Recently, a bacterial community study (16S rRNA) on the stump-tailed lizard tick, Amblyomma

albolimbatum, removed from bobtails, Tiliqua rugosa, in Western Australia (WA), revealed the

presence of Coxiella, Francisella and Rickettsia DNA sequences. However, resolution to species

level could not be achieved due to the short sequences generated through next generation

sequencing. In addition, oocytes proposed to be Hemolivia sp. have been identified within the gut

epithelium of A. albolimbatum; however molecular data confirming the species is lacking. Here we

present for the first time, molecular data supporting the presence of Coxiella burnetii, Francisella sp.

nov., Rickettsia spp. and Hemolivia mariae within A. albolimbatum in WA. Three hundred and six

ticks, morphologically identified as A. albolimbatum, were removed from 88 T. rugosa lizards at the

Kanyana Wildlife Rehabilitation Centre, Perth, WA from 2013 to 2015. A subset of 142 ticks were

subjected to genomic DNA extraction and screened for Coxiella, Francisella, Rickettsia using genus-

specific assays and haemoparasites using a nested 18S rRNA assay followed by Sanger

sequencing. Coxiella burnetii was found in a single A. albolimbatum based on 16S and GroEL genes.

Coxiella burnetii is the causative agent of Q fever and coxiellosis in people and animals, respectively,

and therefore, its presence is of major concern. Secondly, a Francisella sp. nov. based on a

concatenated alignment of 4,230 bp (16S, tpiA, prfB, rpoA, dnaA) was generated with a 3% genetic

distance to F. persica, a known soft tick endosymbiont. Interestingly, there was evidence of three

Rickettsia spp. which warrants further investigation as two were closely related to the Spotted Fever

Rickettsia spp. group. Lastly, two samples had a 100% genetic identity to H. mariae at the 18S

gene, which is consistent with the previous report of Hemolivia within A. albolimbatum and that ticks

have an important role for the sylvatic lifecycle of this parasite. Further research is required to

determine the prevalence of these microbes within questing A. albolimbatum ticks and the role they

play in the health of T. rugosa and wildlife rehabilitation workers.

IV

Acknowledgments

Firstly, I would like to thank my supervisors. My primary supervisor Dr. Charlotte Oskam, I struggle

with words to express the depth of gratitude I have for you taking me on as an honours student. You

have been so supportive, approachable, and generous with your time. My Co-supervisors Dr. Jill

Austen and Dr. Amanda Barbosa, you have both been so helpful and approachable when I have

needed help with anything over the last year. All of you have been so engaging and inspiring. Thank

you to the Vector and Waterborne Pathogens Research Group, I am constantly blown away by how

intelligent and motivated you are. Siobhon Egan, thank you for your patience and willingness to help

towards the bombardment of questions I had when I was beginning my lab work. Professor Peter

Irwin, thank you for allowing us the space to use your desk for the duration of this project while you

were not using it. A special thanks to Xavier Barton, A special thanks to Xavier Barton, who I shared

a lot of troubleshooting and other moments with on this learning journey. Thank you to Ruby

Mckenna, for lending me your lab manual that gave insight into this project, a representation of the

collaborative nature of science. I would also like to thank Dr Shane Tobe, although not an official

supervisor, has been encouraging and willing to give thought provoking advice regarding this project.

I express thanks to Dr Paola Magni for the pictures of the bobtails and the team at the Australian

Rickettsial Reference Laboratory for the use of their Rickettsia culture for my positive control.

I would like to thank my father Derek, who provided me with technical computer related support

through my bachelor’s degree; my mother Susan; and to my brother Rory for lending me money to

purchase a laptop when I started university.

Thanks to Tim for letting me work one day week and supplying me with coffee. Trent and Zoe, thank

you for being amazing over the last year. Finally, A special thanks to Jack Elwin, for your patience

and allowing me to live in your home during the course of this project. You’re a bloody legend, thank

you!

V

Table of Contents

Declaration II

Abstract III

Acknowledgements IV

Table of Contents V

List of Figures VII

List of Tables VIII

List of Abbreviations IX

CHAPTER ONE – INTRODUCTION 1

1.1 Bobtails (Tiliqua rugosa) 1

1.2 Ticks 1

1.2.1 Tick life cycle 2

1.2.2 Ticks of Australia 3

1.2.3 Bobtail ticks 5

1.3 Tick associated microbes and diseases 6

1.3.1 Australian tick-borne diseases 7

1.3.1.1 Coxiella 8

1.3.1.2 Francisella 10

1.3.1.3 Rickettsia 11

1.3.1.4 Hemolivia 12

1.4 Molecular detection of tick associated microbes 13

1.4.1 Polymerase chain reaction (PCR) 13

1.4.1.1 16S rRNA gene 14

1.4.1.2 Additional genes 15

1.5 Aims and Hypotheses 15

CHAPTER TWO – MATERIALS AND METHODS 17

2.1 Sample acquisition 17

2.2 DNA extraction 17

2.3 PCR assays 18

2.3.1 Coxiella detection using conventional PCR 18

2.3.2 Francisella detection using conventional PCR 20

2.3.3 Rickettsia detection using conventional PCR 22

2.3.4 Piroplasm detection using nested PCR 24

2.4 Agarose gel electrophoresis and Sanger sequencing 24

2.5 Phylogenetic analysis 25

CHAPTER THREE – RESULTS 27

3.1 Molecular characterisation of Coxiella 27

3.2 Molecular characterisation of Francisella 35

3.3 Molecular characterisation of Rickettsia 47

3.4 Molecular characterisation of haemoprotozoa 69

VI

CHAPTER FOUR – DISCUSSION 72

4.1 Microbial species of interest 72

4.1.1 Coxiella burnetii confirmed within A. albolimbatum 72

4.1.2 Francisella sp. nov. within A. albolimbatum 75

4.1.3 Rickettsia spp. identified within A. albolimbatum 78

4.1.4 Hemolivia mariae confirmed within A. albolimbatum 81

4.2 Limitations and Future directions 83

4.3 Conclusions 84

References 85

Appendix 97

VII

List of Figures

Figure 1.1 Three host life cycle of a female ixodid tick 3

Figure 1.2 A. albolimbatum parasitising ear canal of a bobtail 5

Figure 1.3 Bobtail parasitised by A. albolimbatum 5

Figure 3.1 Agarose gel electrophoresis of Coxiella 16S (short) PCR products 27

Figure 3.2 Agarose gel electrophoresis of Coxiella 16S (short) PCR products 27

Figure 3.3 Agarose gel electrophoresis of Coxiella 16S (long) PCR products 28

Figure 3.4 Agarose gel electrophoresis of Coxiella GroEL PCR products 28

Figure 3.5 Phylogenetic analysis of 1,140 bp 16S rRNA of Coxiella 31

Figure 3.6 Phylogenetic analysis of 558 bp GroEL of Coxiella 32

Figure 3.7 Phylogenetic analysis of 1,694 bp concatenated 16S and GroEL of Coxiella 34

Figure 3.8 Agarose gel electrophoresis of Francisella 16S PCR products 36

Figure 3.9 Agarose gel electrophoresis of Francisella tpiA PCR products 36

Figure 3.10 Agarose gel electrophoresis of Francisella rpoA PCR products 37

Figure 3.11 Agarose gel electrophoresis of Francisella prfB PCR products 37

Figure 3.12 Agarose gel electrophoresis of Francisella dnaA PCR products 37

Figure 3.13 Agarose gel electrophoresis of Francisella dnaA PCR products 38

Figure 3.14 Phylogenetic analysis of 1,022 bp of 16S rRNA of Francisella 40

Figure 3.15 Phylogenetic analysis of 521 bp of tpiA of Francisella 41

Figure 3.16 Phylogenetic analysis of 900 bp of prfB of Francisella 42

Figure 3.17 Phylogenetic analysis of 723 bp of rpoA of Francisella 43

Figure 3.18 Phylogenetic analysis of 954 bp of dnaA of Francisella 45

Figure 3.19 Phylogenetic analysis of 4,230 bp of concatenated 16S, tpiA, prfB, rpoA, and dnaA of

Francisella 46

Figure 3.20 Agarose Gel Electrophoresis of Rickettsia gltA (long 650bp) PCR products 48

Figure 3.21 Agarose Gel Electrophoresis of Rickettsia gltA (short 381bp) PCR products 48

Figure 3.22 Agarose Gel Electrophoresis of Rickettsia ompA and ompB PCR products 49

Figure 3.23 Agarose Gel Electrophoresis of Rickettsia ompA PCR products 49

Figure 3.24 Agarose Gel Electrophoresis of Rickettsia ompB PCR products 50

Figure 3.25 Agarose Gel Electrophoresis of Rickettsia 17 kDa PCR products 50

Figure 3.26 Agarose Gel Electrophoresis of Rickettsia sca4 PCR products 50

Figure 3.27 Phylogenetic analysis of 344 bp of gltA of Rickettsia 55

Figure 3.28 Phylogenetic analysis of 634 bp of gltA of Rickettsia 56

Figure 3.29 Phylogenetic analysis of 517 bp of ompA of Rickettsia 58

Figure 3.30 Phylogenetic analysis of 251 bp of ompB of Rickettsia 59

Figure 3.31 Phylogenetic analysis of 394 bp of 17 kDa of Rickettsia 61

Figure 3.32 Phylogenetic analysis of 775 bp of sca4 of Rickettsia 63

Figure 3.33 Phylogenetic analysis of 644 bp of concatenated 17 kDa and gltA of Rickettsia 64

Figure 3.34 Phylogenetic analysis of 1,435 bp of concatenated ompA, ompB and gltA of

Rickettsia 65

Figure 3.35 Phylogenetic analysis of 1,639 bp of concatenated ompA, ompB, and sca4 of

Rickettsia 66

Figure 3.36 Phylogenetic analysis of 1,482 bp of concatenated ompB, 17 kDa, and sca4 of

Rickettsia 67

Figure 3.37 Phylogenetic analysis of 633 bp of concatenated ompB and, 17 kDa of Rickettsia 68

Figure 3.38 Agarose Gel Electrophoresis of Haemaprotozoa 18S 70

Figure 3.39 Phylogenetic analysis of 723 bp of 18S of Haemaprotozoa 71

VIII

List of Tables

Table 2.1 Primers and PCR cycling conditions for Coxiella detection 20

Table 2.2 Primers and PCR cycling conditions for Francisella detection 21

Table 2.3 Primers and PCR cycling conditions for Rickettsia detection 23

Table 2.4 Primers and PCR cycling conditions for haemoprotozoa detection 24

Table 2.5 Best-fit nucleotide substitution model for each gene locus 26

Table 3.1 Summary of positive samples from Coxiella assays 29

Table 3.2 BLAST results from 16S and GroEL Coxiella positive samples 29

Table 3.3 Summary of positive samples from Francisella assays 35

Table 3.4 BLAST results from 16S, tpiA, dnaA prfB and rpoA Francisella positive samples 39

Table 3.5 Summary of positive samples from Rickettsia assays 47

Table 3.6 BLAST results from gltA, ompA, ompB, 17 kDa, and sca4 Rickettsia sequences 53

Table 3.7 Summary of positive samples from haemoprotozoa assays 69

Table 3.8 BLAST results from 18S haemoprotozoa sequences 69

IX

List of Abbreviations

17 kDa 17 kDa lipoprotein precursor antigen gene AGRF Australian Genome Research Facility BLAST Basic Local Alignment Sequencing Tool bp base pairs DNA Deoxyribonucleic acid dnaA chromosomal replication initiator protein alpha subunit dNTP deoxyribonucleotide triphosphate EBC Extraction blank control EtOH Ethanol gltA citrate synthase gene GroEL GTR HKY K2

Chaperonin protein gene General Time Reversible Hasegawa-Kishino-Yano Kimura 2-parameter

MEGA Molecular Evolutionary Genetic Analysis software MgCl Magnesium chloride NCBI National Centre for Biotechnology Information nt Nucleotide NTC No-template control ompA Outer membrane protein A ompB Outer membrane protein B PBS Phosphate-buffered saline PCR Polymerase Chain Reaction pgm phosphoglucomutase prfB peptide chain release factor 2 beta subunit rpm Revolutions per minute rpoA DNA-directed RNA polymerase alpha subunit rRNA Ribosomal ribonucleic acid sca4 T92 TN93

120 kDa antigen gene Tamura 3-parameter Tamura-Nei

taq Thermus aquaticus DNA polymerase tpiA Triose-phosphate isomerase alpha subunit TBD Tick-borne disease TBP Tick-borne pathogen ZOTU Zero Operational Taxonomic Unit

1

CHAPTER ONE

INTRODUCTION

In 2019, bacterial community profiling of the 16S rRNA gene was conducted on the stump-tailed

lizard tick, Amblyomma albolimbatum, as part of an ongoing research to characterise and understand

the microbial diversity within Australian ticks (Mckenna, 2019). Several microbial taxa of interest,

those that were related to known tick borne pathogens including those of zoonotic potential, were

identified. However, due to relatively short sequences, taxonomic resolution could not be achieved

to determine the identity of the microbes present within this tick species. Herein this literature review,

a brief description of the vertebrate host Tiliqua rugosa (referred to throughout as ‘bobtail’), followed

by a background of ticks (with a focus on hard ticks), associated microbes (taxa of interest), and

technology used to characterise these microbes will be outlined to provide context for this thesis.

1.1 Bobtails (Tiliqua rugosa)

First described in 1825 by John Edward Gray as Trachydosaurus rugosus, Tiliqua rugosa is a

scincidae lizard; a short tailed, slow moving species with a heavily armoured body found in varying

colours from dark brown to cream (Norval et al, 2019; Pianka and Vitt, 2003). Tiliqua rugosa has four

recognised subspecies, three of which are only found in Western Australia, collectively known as

bobtail, although also known as the stump tailed lizard and the sleepy lizard. The fourth subspecies

of bobtail, Tiliqua rugosa asper, is the only species native to eastern Australia, is also known as the

shingleback (Norval and Gardner, 2019). Tiliqua rugosa rugosa, the subspecies relative to this

project, is distributed in south-west Australia; found throughout and surrounding bushland of Perth.

1.2 Ticks

Ticks are obligate haematophagous arthropods and the most important vector of infectious

microorganisms concerning companion animals and livestock (Parola and Raoult, 2001). Ticks are

members of the phylum Arthropoda. Within this phylum, ticks are assigned to a subphylum

2

Chelierata and then class Arachnida shared with scorpions, spiders, and mites (Guglielmone et al.,

2010). Ticks and mites share a subclass Acari though ticks are classified again into the order

Ixodidae. All ticks are categorised in three families: Ixodidae (hard ticks), Argasidae (soft ticks) and

Nuttalliellidae. Presently there are approximately 900 species of ticks classified globally; over 700

species of hard ticks are currently recognised in Ixodidae and around 200 species of soft ticks are

currently recognised in Argasidae (Guglielmone et al., 2010). Nuttalliellidae, is a third and the

smallest family consisting of a single species, Nuttalliella namaqua, specific and endemic to South

Africa (Jongejan and Uilenberg, 2004).

1.2.1 Tick life cycle

The life cycle of most ixodid ticks consist of four stages spanning one–three hosts: egg, larvae,

nymph, and either female or male adult (Bull and King, 1981; Barker and Walker, 2014). Transition

from one life stage to the next is dependent on a blood meal acquired from a host prior to moulting.

Each life stage requires only one blood meal and full engorgement can take between three days and

three weeks, though duration of feeding varies depending on species, instar, and host (Chilton and

Bull, 1991; Barker and Walker, 2014). This is contrasted by male ticks not completely engorging,

though they require smaller blood meals for sustenance (Barker and Walker, 2014).

Three-host ticks life cycle is as follows: When finished developing in eggs, larvae will hatch around

several weeks and then begin questing for a host, usually a host sharing the environment (Kerr and

Bull, 2006). Once a host is found, larvae will engorge from their blood meal and detach, usually when

host are in their refuge sites, before moulting to a nymph (Petney et al., 1983; Petney and Bull,

1981). This process is repeated by the nymph, moulting to an adult male or female (Smyth, 1973;

Barker and Walker, 2014). Male ticks remain on the host attempting to mate with multiple females.

The male will transfer the sperm sac to the female. Female ticks will only mate once prior to

engorgement. Once engorged to repletion, females will detach from their host and have enough

sperm to fertilize all their eggs before they die (Parola and Raoult, 2001). Eggs are always laid in the

3

environment and not on the host (Barker et al., 2014). Life cycle of a three-host tick can vary from

six months to several years depending on environmental conditions and host availability (Barker and

Walker, 2014).

Figure 1.1. Three host life cycle of a female ixodid tick (Apanaskevich and Oliver, 2014).

1.2.2 Ticks of Australia

Australia is home to a range of native and unique ticks. At least 74 valid species are currently known

to exist in Australia; 69 of those species are native to Australia, and five were introduced following

European arrival over 230 years (Barker et al., 2014; Ash et al., 2017; Kwak et al., 2018). The five

introduced tick species are comprised of three hard tick species: Haemaphysalis longicornis (bush

tick), Rhipicephalus australis (Australian cattle tick), and Rhipicephalus linnaei (brown dog tick; syn

Rh. sanguineus sensu lato, tropical lineage; Šlapeta et al. 2021); and two soft ticks: Argas persicus

(poultry tick) and Otobius megnini (spinose ear tick) (Graves and Stenos, 2017).

4

Within Australia, there are 57 native hard ticks belonging to the genera Amblyomma, Bothriocroton,

Haemaphysalis and Ixodes, and 12 native soft ticks belonging to two genera, Argas and

Ornithodoros (Barker and Walker, 2014). Since 2017, three new Ixodes species have been

described. Morphological and molecular characterisation by Ash et al. (2017) confirmed Ixodes

woyliei to be the first new species of Ixodes tick species described in over 50 years. Ixodes woyliei

has a high predilection for the endangered marsupial Bettongia penicillate, otherwise known as the

woylie (Ash et al., 2017). Ixodes heathi, an endangered tick found on a critically endangered

mountain pygmy possum (Burramys parvus), was discovered shortly afterwards in 2018 (Kwak et

al., 2018). More recently, Barker et al. (2019) described the species Ixodes barkeri removed from

the short-beaked echidna, Tachyglossus aculeatus (Barker, 2019). The description of these novel

species of tick reveals the importance of accurate species descriptions and the addition of molecular

data.

Companion or domesticated animals are frequently parasitised by 17 ticks in Australia,

approximately 54 species of tick are known to parasitise reptiles, birds and wild mammals, and only

a few species are known to bite humans (Ash et al., 2017; Barker et al., 2014; Graves and Stenos,

2017). The species known to bite humans are the following: 11 hard ticks, Amblyomma triguttatum

(ornate kangaroo tick), Bothriocroton auruginans (wombat tick), B. hydrosauri (southern reptile tick),

Haemaphysalis bancrofti (wallaby tick), Haemaphysalis longicornis (bush tick), Ixodes cornuatus

(southern paralysis tick), Ixodes hirsti (Hirst's marsupial tick), I. holocyclus (paralysis tick), I. tasmani

(common marsupial tick), Rhipicephalus (Boophilus) australis (Australian cattle tick) and R. linnaei

(brown dog tick); and five soft ticks: A. persicus (poultry tick), Argas robertsi (Robert’s bird tick),

Ornithodoros capensis (seabird soft tick), O. gurneyi (kangaroo soft tick), O. megnini (spinose ear

tick). Though these species of tick have been documented to bite humans, it is most likely due to

opportunity rather than the ticks specifically questing for humans (Barker and Walker, 2014).

Similarly ticks not recorded to bite humans may still parasitise people if given the opportunity, though

there are no specific records of this occurring.

5

1.2.3 Bobtail ticks

Australia has more reptile-specific ticks than any other region, which is most likely due to the

abundance and species richness of reptiles (Barker and Walker, 2014; Natusch, 2018). Among the

current 74 tick species documented in Australia, 13 are known to parasitise reptiles. All ticks

historically found on the bobtail are three reptile ticks: A. albolimbatum (the stump-tail lizard tick); A.

limbatum (the reptile tick); and B. hydrosauri (the southern reptile tick).

Ticks commonly parasitise the bobtail under scales and in the external auditory meatus (Figure 1.1

and 1.2) (Smyth, 1973; Barker et al., 2014; Barker and Walker, 2014). Although the bobtail is the

primary host for these three ticks, these ticks have been reported to parasitise, albeit less frequently,

other vertebrate species. For example, B. hydrosauri has been found on other reptile hosts including

lizards, snakes, a terrestrial turtle, and has been reported to parasitise mammals such as cattle,

horses and humans (Barker and Walker, 2014).

Figure 1.2. Amblyomma albolimbatum parasitising ear canal of a bobtail. Source: Dr Paola Magni.

Figure 1.3. Bobtail parasitised with A. albolimbatum. Source: Dr Paola Magni.

6

Smyth (1973) identified and detailed a geographic and host distribution of three species of reptile

tick widespread in Australia. Further research by Professor C. Michael Bull revealed valuable insights

into the biology and ecology of ticks from his long-term study into the parapatric boundaries of hard

ticks A. limbatum and B. hydrosauri (formerly Aponoma hydrosauri) associated with the bobtail,

showing that when any pair of ticks met there is at most 2kms of overlap and clear boundaries

between species (Belan and Bull, 1995). All three tick species occur in Western Australia; however,

Amblyomma limbatum has a northerly distribution, occurring in dryer, more tropical parts of the state;

contrasted by B. hydrosauri and A. albolimbatum having more southern and south-western

distributions, respectively (Sharrad and King, 1981; Godfrey and Gardner, 2017). Interest currently

surrounds ticks known to parasitise reptiles and the putative link to tick-borne disease in humans.

For example, B. hydrosauri has been attributed to causing Flinders Island spotted fever due to

transmission of the bacterium Rickettsia honei (Graves and Stenos, 2017; Whiley et al., 2016).

1.3 Tick associated microbes and diseases

Globally, ticks are established as an important arthropod vector for transmitting a range of microbes

detrimental to the health of its vertebrate host (Colwell et al., 2011). Every tick species has

environment and biotope requirements that determine their geographic distribution, and

subsequently, areas associated with tick-borne diseases (TBD) (Parola and Raoult, 2001). While

identifying the presence of a pathogenic organism within a tick warrants caution, it should be noted

that the presence does not necessarily lead to transmission or result in disease. For transmission of

a pathogenic species to a vertebrate host to occur, the microbe must be present within the salivary

glands of a tick during feeding (Hooper and Gordon, 2001; Parola and Raoult, 2001). Some of the

most important zoonotic TBDs include anaplasmosis (Anaplasma spp.), babesiosis (Babesia spp.),

Lyme borreliosis (Borrelia burgdorferi sensu lato complex), tick-borne encephalitis (Flavivirus),

tularaemia (Francisella tularensis), Crimean-Congo haemorrhagic fever (Nairovirus), and spotted

fever (Rickettsia spp.) (Springer et al. 2021).

7

In addition to pathogenic microbes, ticks also harbour a full suite of microorganisms, referred to as

the ‘microbiome’, including bacteria, fungi, protozoa, and viruses, that can be commensal, symbiotic

or pathogenic/parasitic (Narasimhan and Fikrig, 2015). Of note, tick symbionts share a close

phylogenetic relationship to many tick borne pathogens (TBP) (Bonnet et al., 2017). Ticks depend

on symbiotic relationships with endosymbionts of the genera Coxiella, Francisella, Rickettsia, and

Wolbachia, to provide nutrients missing from their diets, aid in reproduction, assist in moulting and

may also promote or inhibit the acquisition or transmission of some TBPs (Gerhart et al., 2016;

Bonnet et al., 2017).

Ticks can also cause non-infectious illness directly to their hosts, such as tick paralysis, mammalian

meat allergy, autoimmune disorders and post-infection fatigue are also reported to occur following a

tick bite in Australia (van Nunen, 2015; Graves and Stenos, 2017). However, non-infectious

diseases, along with fungi and viruses, will not be discussed further as they are beyond the scope

of this thesis.

1.3.1 Australian tick-borne diseases

Compared to overseas, very few Australian tick species are attributed to transmitting tick-borne

pathogens which cause disease (Stenos and Graves, 2017). At present, only three TBD are

recognised; two Rickettsia species, Rickettsia australis and Rickettsia honei which cause

Queensland Tick Typhus (QTT) and Flinders Island spotted fever (FISF), respectively; and Coxiella

burnetii, the causative agent of Q fever. Bothriocroton hydrosauri and I. holocyclus are both known

to harbour and transmit R. honei and R. australis, respectively (Panetta et al., 2017). Both I. tasmani

(common marsupial tick) and I. cornuatus (southern paralysis tick) also have been associated with

R. australis and known to bite humans (Barker and Walker, 2014; Graves and Stenos, 2017). First

discovered on Flinders Island, Tasmania, FISF is an Australian TBD that has been detected in

patients from other parts of Australia (Unsworth et al., 2005). The paralysis tick, I. holocyclus, is

endemic to east coast Australia and causes QTT and Q Fever due to R. australis and C. burnetii

8

respectively (Stenos and Graves, 2017). Withal, the Ornate kangaroo tick, A. triguttatum, a human

biting tick, can be located through central, northern and Western Australia (WA) and has been linked

with the transmission of C. burnetii and a proposed novel SFG pathogen, R. gravesii (Li et al., 2010;

Graves and Stenos, 2017).

1.3.1.1 Coxiella

Coxiella is a genus of gram-negative, obligate intracellular bacteria of the phlyum Proteobacteria;

order Legionellales; family Coxiellaceae (Maurin and Raoult, 1999). The genus Coxiella currently

comprised of three identified species: Coxiella cheraxi, a pathogen of the redclaw crayfish (Cherax

quadricarinatus); Coxiella massilliensis, an etiological agent to humans; and Coxiella burnetii, the

causative agent of Q fever (Angelakis et al., 2016; Duron et al., 2015; Tan and Owens, 2000).

Originally classified as a Rickettsia species ‘Rickettsia burnetii’, C. burnetii was reclassified from

alphaproetobacteria to gammaproteobacteria after phylogenetic analyses of the 16S rRNA gene

locus revealed a genetic relationship closer to Legionellales than Rickettsiales (Leclerque and

Kleespies, 2008; Marrie et al., 2015).

Coxiella burnetii is a zoonotic pathogen that causes the highly infectious disease Query fever (Q

fever) in humans, and coxiellosis in animals (Derrick, 1937; Duron et al., 2015). With the exception

of New Zealand, C. burnetii has been reported to occur worldwide and has a sylvatic life cycle

associated with wildlife, domestic animals, and ticks (Maurin and Raoult, 1999). Human acquisition

of C. burnetii is predominantly through inhalation of aerosols, though Q fever can occur through

contact with infectious tissues and excretions from domestic ruminants and companion animals

(Gregory et al., 2019; Maurin and Raoult, 1999). Most animals infected with C. burnetii are

asymptomatic, however bronchopneumonia, fever, and inflammation of the placenta post-abortion

have been known to occur (Babudieri , 1959; Palmer et al., 1983; Waldman, 1978). In addition,

female animals infected with C. burnetii are known to shed high amounts of the infectious bacteria

in urine, faeces, milk and parturient material (Tan and Owens, 2000). Q fever is considered a

9

notifiable disease across Australia by the Department of Health and is the only endemic tick

associated notifiable disease impacting human health in Australia.

Currently more than 40 tick species have been associated with C. burnetii and Coxiella species

(Parola and Raoult, 2001; Duron et al., 2015). Phylogenetic analyses of the Coxiella genus

emphasised a high genetic diversity and showed the genus can be split into four main clades (Duron

et al., 2015). The Coxiella endosymbionts have been identified within all clades, however C. burnetii

clusters into a unique subclade indicating the species is likely to have evolved from a Coxiella tick

endosymbiont; the closest species to C. burnetii are Coxiella strains associated with soft ticks from

Ornithodoros and Argas. Additionally, Coxiella species genetically related to C. burnetii identified as

endosymbionts in soft and hard ticks, lack virulent characteristics (Duron et al., 2015).

Although more commonly associated with mammals, evidence of C. burnetii has been reported in

water snakes (Natrix natrix); rat snakes (Ptyas korros); Indian python (python molurus); and tortoise

(Kachuga sp.) (Yadav and Sethi, 1979). In addition, overseas reptile ticks have previously been

associated with C. burnetii. The tortoise tick Hyalomma aegyptium (Blanc 1961; Široký, 2007), is

primarily found on Testudo genus tortoises in the Mediterranean region, and has a three host life

cycle alternating between tortoises, other reptiles, and mammals (Široký et al., 2007). The monitor

lizard tick, Amblyomma exornatum, harboured natural infection of C. burnetii in Guinea-Bissau

(Arthur, 1962). Additionally, Kim et al. (1978) has described outbreak of Q fever in New York among

humans removing the African tick, Amblyomma nutalli, from ball python (Python regius). Similarly, a

Coxiella endosymbiont has also been reported in A. nuttalli from Africa (Nardi et al., 2021). Most

recently, two separate studies on Australian reptile ticks, A. albolimbatum and B. undatum, removed

from bobtail and goanna respectively, observed Coxiella sp. reads (proposed C. burnetii) in next

generation sequence (NGS) data (Panetta et al., 2017; Mckenna, 2019). However, both studies were

unsuccessful in confirming the presence of C. burnetii via conventional PCR.

10

1.3.1.2 Francisella

The family Francisellaceae consists of a single genus Francisella which are small coccobacillary,

aerobic, gram-negative, obligate intracellular, non-sporulating bacteria and belong to the phylum

Proteobacteria; class Gammaproteobacteria; order Thiotrichales (Olsufiev et al., 1959; Gerhart et

al., 2016). First identified in 1912 as a plague-like disease of rodents, tularaemia is a highly infectious

zoonotic disease caused by F. tularensis, affecting humans and animals, and considered endemic

in the Northern Hemisphere (Eden et al., 2017). Francisella tularensis can be divided into several

subspecies ranging in pathogenicity: F. tularensis tularensis, which is life threatening; F. tularensis

holartica, causes less severe disease; F. tularensis mediasiatca shows similar virulence to F. t.

holartica; and F. t. novicida, regarded as least virulent (Larson et al., 2020). Acquisition of tularaemia

usually occur through direct contact with infected animals, infected water, inhalation of contaminated

aerosols and tick vectors (Eden et al., 2017). Importantly, while tularaemia is considered a notifiable

disease in Australia, there are currently no reports of ticks transmitting the disease to humans within

Australia. However, tularaemia was reported in Tasmania, Australia in 2011, when an adult human

was bitten by a ringtail possum. The causative agent was proposed to be F. t. holartica, based upon

16S rRNA sequence and serology (Eden et al., 2017; Jackson et al., 2012). Francisella tularensis

has a much broader host range than most other zoonotic pathogens, and has been reported in

species of mammals, birds, amphibians and invertebrates (Mörner and Addison, 2001). In addition

to Francisella causing human disease, closely related Francisella endosymbionts have previously

been described in ticks overseas, for example: Argas, Ornithodoros, Dermacentor, and Amblyomma

ticks.

In 2003, Francisella hispaniensis was isolated from a human in the Northern Territory following a

foot cut received in brackish water (Whipp et al., 2003). Francisella DNA has also been sequenced

from reptile ticks, A. helvoum, and A. varanese, removed from snakes in Thailand, and in a tick

commonly found on reptiles, A. fimbriatum, in the Northern Territory, Australia (Vilcins et al., 2009;

Sumrandee et al., 2014). Following this, Aravena- Román et al. (2015) reported a Francisella sp.

with a 99.8% genetic similarity to F. hispaniensis at the 16S rRNA locus, becoming the first

documented case of bacteraemia attributed to F. hispaniensis in WA; the route of transmission

11

however remains unknown. Furthermore, 16S sequences generated by NGS identified a Francisella

sp. 99% genetically similar to F. hispaniensis was observed in A. alblolimbatum parasitising the

sleepy lizard T. rugosa, was recently reported in Perth, WA (Mckenna, 2019). Polymerase chain

reaction (PCR) developed by Forsman et al, (1994) targeting the 16S rRNA gene of Francisella is

considered to be efficient and cost effective, however is limited by how confidently the assay can

differentiate between species of Francisella (Roux and Raoult, 2000); therefore, additonal genes

need to be considered to identify species more confidently.

While not associated with reptiles, a recent study reported a high prevalence and abundance of

Francisella sequences in the red kangaroo tick, A. triguttatum, a common human biting tick, from

Western Australia (Egan et al., 2020).

1.3.1.3 Rickettsia

The genus Rickettsia are small, gram-negative, pleomorphic coccobalilli bacteria from the phylum

Proteobacteria; class Alphaproteobacteria; order Rickettsiales; family Rickettsiaceae (Roux and

Raoult, 2000). Rickettsia are associated with causing spotted fever and tick-associated typhus

diseases, and are commonly transmitted via hard ticks (Parola et al., 2005). Based on genotypic and

phenotypic differences, the genus can be divided into three groups of disease causing species: The

spotted fever group (SFG), comprised of species mostly associated with ticks; the typhus group

(TG), includes R. typhi and R. prowazekii usually transmitted to humans by fleas and lice; and the

ancestral group, comprised of a single species, R. bellii, the earliest diverging species of Rickettsia

(Ogata et al., 2006; Vilcins et al., 2009; Ogrzewalska et al., 2016; Parola et al., 2016; Abdad et al.,

2017). Several Spotted fever Group (SFG) and Typhus Group (TG) Rickettsia have been recognised

as causing disease in humans (Ogata et al., 2006).

Despite its use for microbiome studies, the 16S rRNA gene is highly conserved within the genus

Rickettsia, and phylogenetic analysis of the 16S cannot be used to confidently differentiate between

12

species, warranting more suitable gene targets (Sekeyova et al., 2001). For example, a recent

microbiome investigation into A. albolimbatum ticks collected from T. rugosa, found several zero

operation taxonomic units (ZOTU) related to rickettsial DNA, suggesting the presence of multiple

Rickettsia spp. Independently, researchers at the Australia Rickettsial Reference Laboratory, have

recently sequenced a single Rickettsia sp. from the same tick species and concluded that sequences

clustered in a distinct subclade to R. gravesii (Tadepalli et al., 2021). However, because each group

utilised different gene assays, it is currently unknown how related the Rickettsia species from both

groups are.

1.3.1.4 Hemolivia

A variety of protistan parasites are present in the blood of vertebrates and are transmitted by

haematophagous vectors, such as ticks. The genus Hemolivia are haemogregarine parasites in the

family Karyolsidae from the sub-order Adeleiorina of the phylum Apicomplexans, and infect

exothermic vertebrates (Carreno and Barta, 1999). There are currently three known species of

Hemolivia: Hemolivia stellata; Hemolivia mariae; and Hemolivia mauratanica. Hemolivia stellata and

H. maratanica are known to infect the cane toad and tortoises respectively. The parasitic protist

Hemolivia mariae infects the erythrocytes of lizards, such as Tiliqua rugosa (Smallridge and

Paperna, 1997; Barker and Walker, 2014). Hemolivia mariae is transmitted by at least two ixodid tick

species known to be a definite host and vector, Amblyomma limbatum and Bothriocroton hydrosauri;

though A. limbatum has been reported to be more susceptible to H. mariae infection (O’Donoghue,

2017; C. Smallridge and Bull, 2001; Spratt and Beveridge, 2018). Amblyomma albolimbatum has

also been implicated as a host for H. mariae based on morphological characteristics and differences

between their host range and geographical distribution; thus, ruling out H. stellata (Moller, 2014).

However, molecular testing is still required to confirm the identity of the Hemolivia species present.

It has been proposed that ticks become infected with H. mariae when taking a blood meal from an

infected bobtail. Similarly, bobtails can become infected with H. mariae after ingesting infected ticks

13

(Barker and Walker, 2014; Smallridge and Bull, 2001). However, studies show that tick load on

infected and non-infected lizards are not significantly different; there is also no evidence of infection-

induced mortality in ticks (Smallridge and Bull, 2001). Furthermore, T. rugosa infected with H. mariae

do not have the energy expenditure of those that do not, resulting in a decrease in their physical

activity and therefore a decrease in their territory. Moreover, B. hydrosauri are also identified to

negatively impact the physical capability of their reptilian hosts (Bouma et al., 2007; Barker and

Walker, 2014). While the presence of H. mariae has been documented in hard ticks, the zoonotic

potential is unknown.

1.4 Molecular detection of tick associated microbes

While serology and culture are the gold standard for the detection of infectious diseases, serology is

limited by lag between initial infection and the development of antibodies (Stenos et al., 2005).

Moreover, culture-based methods can take up to 60 days to produce a positive result with some

pathogens unable to be detected by traditional culture base techniques (Stenos et al., 2005;

Unsworth et al., 2005). For the purpose of this study, the following section will discuss how

polymerase chain reaction (PCR) has been used for the molecular characterisation of microbial taxa

of interest within ticks.

1.4.1 Polymerase chain reaction (PCR)

Polymerase chain reaction is an effective molecular technique allowing DNA fragments to be

amplified within a few hours. This is particularly useful when there is limited quantity of the original

DNA; in some cases, a single molecule (Pierce, 2017). Substantially quicker than tradition methods

such as culture and serological methods, PCR can detect low quantity organisms from a variety of

sample types, due to higher sensitivity, making it a valuable molecular tool (Mullis and Faloona,

1987; Stewart et al., 2017).

14

PCR (coupled with Sanger sequencing) can also be designed to be highly sensitive and specific

making it a valuable diagnostic method. Sequences acquired via PCR and Sanger Sequencing can

be compared with databases composed of reference sequences; this provides a method to draw

inference from phylogenetic relationships of an amplified sequence with prior categorised taxa.

Furthermore, these molecular techniques have been used for detection of bacteria such as Coxiella

and Rickettsia in Australian ticks (Cooper et al., 2013; Izzard et al., 2009). This makes PCR a

particularly useful tool for differentiation of closely related species such as Spotted Fever group

(SFG) and typhus group Rickettsia which cannot be set apart when employing serology (Tadepalli

et al., 2021). Due to the overlap of geographical location known for B. hydrosauri, and confirmed

cases of R. honei isolated from patients, Whiley et al. (2016) conducted a study using real-time PCR

(qPCR) to perform assays on B. hydrosauri collected from T. rugosa from mainland South Australia,

discovering Rickettsia species in all tick samples. While R. honei was identified within ticks, the study

also suggested that B. hydrosauri could be a vector for multiple Rickettsia species.

1.4.1.1 16S rRNA gene

The ubiquitous 16S ribosomal RNA gene (16S) in prokaryotic organisms is fundamental, and

responsible for encoding the small subunit ribosomal RNA molecules of ribosomes (Woese and Fox,

1977; Větrovský and Baldrian, 2013). The gene is universally distributed among the bacterial

domain, making identification and characterisation among different taxa from a variety of sources,

including ticks possible to analyse using phylogenetics (Clarridge, 2004; Větrovský and Baldrian,

2013). Occurring between a single copy and up to 15 copies depending on the taxa, the 16S rRNA

is approximately 1,500 bp long and features both conserved regions and nine hypervariable regions

(V1 – V9) (Klappenbach et al., 2001; Dias et al., 2020) . The conserved regions allow for appropriate

PCR primers target for various taxa due to slow evolution rate of this region (Woese and Fox, 1977).

The nine hypervariable domains of 16S exhibit varying degrees of sequence diversity; among some

bacteria genera the hypervariable regions are highly conserved between species, limiting species-

level identification (Greay et al., 2018; Dias et al., 2020). Although, this limits the 16S rRNA

15

sequencing method due to potential sequence similarity between closely related species, such as

endosymbionts and pathogens (Větrovský and Baldrian, 2013). Thus, further characterisation of

alternative genes is sometimes necessary to more confidently determine a species.

1.4.1.2 Additional genes

Targeting the 16S rRNA has proved a valuable tool and is still widely used, particularly for

microbiome analyses, however it does not suffice for certain taxa, such as Rickettsia, as previously

outlined (Mckenna, 2019). This is due to the conserved nature of the gene, rendering phylogenetic

analysis based on the 16S gene more unreliable, especially concerning members of the SFG

(Fournier et al., 2003; Roux et al., 1997). Additionally, the 16S gene is not adequate for determining

virulence; as the region does not usually encode virulence factors (Clarridge, 2004). In 1997, a study

on enteric bacteria investigating the RNA polymerase b-subunit (rpoB) gene as an alternative to the

16S RNA, it was reported that the rpoB gene is more reliable between some genera (Mollet et al.,

1997). Similarly the citrate synthase gene (gltA) of Rickettsia provides more accuracy between

species differentiation in the genus than 16S (Roux et al., 1997); Furthermore, this is supported by

phylogenetic studies of Rickettsia, prompting a reorganisation of the genus after investigating the

16S rRNA, citrate synthase (gltA), 17 kDa antigen (17 kDa), outer membrane protein A (ompA), outer

membrane protein B (ompB) and surface cell antigen (sca4) (Roux et al., 1997; Roux and Raoult,

2000; Sekeyova et al., 2001; Fournier et al., 2003). Therefore, highlighting the requirement for

sequencing alternative genes when considering phylogenetic analysis.

1.5 Aims and hypotheses

Investigation is still needed to identify unknown microbes within the microbiome of Australian ticks.

In 2019, sequences matching Francisella, Rickettsia, and Coxiella were acquired via next-generation

sequencing (NGS) in Amblyomma albolimbatum ticks parasitising the bobtail lizard (T. rugosa) in

Western Australia. This research project will contribute to the molecular characterisation of bacterial

species within A. albolimbatum. This present study will investigate tick DNA samples previously

16

extracted in the aforementioned study and further extractions on ticks identified as being from the

same host; all of which were collected by wildlife health centres in WA. The continued molecular

characterisation of the microbial taxa associated with reptile ticks in Australia will increase our

understanding of the bacterial species these ticks harbour and assess if there is any associated

zoonotic risk.

Based on the previous bacterial community study in A. albolimbatum ticks, the first hypothesis is

additional molecular characterisation will determine how genetically related species of Francisella,

Rickettsia and Coxiella are to known pathogens and endosymbionts. Secondly, it is hypothesised

that haemoprotozoa will be identified as Hemolivia mariae within A. albolimbatum confirming Moller

(2014) morphological observations.

The aims of this thesis are two-fold: first, additional genetic information will be generated and

phylogenetic analyses will be conducted to further characterise Coxiella, Rickettsia, and Francisella

species previously detected by NGS in the tick, A. albolimbatum; and second, to investigate the

presence of haemoprotozoan parasites in A. albolimbatum.

17

CHAPTER TWO

MATERIALS AND METHODS

2.1 Sample acquisition

A total of 142 reptile ticks were collected from 67 bobtail (T. rugosa) by collaborators at Kanyana

Wildlife Rehabilitation Centre, Armadale Reptile Centre, Geovet Busselton and members of the

public, and submitted to the Vector and Waterborne Pathogen Research Group at Murdoch

University. All ticks collected directly and opportunistically from bobtails within Perth metropolitan

area in WA. Ticks were all preserved in 70% ethanol and stored at 4°C until further identification and

molecular analyses. Prior to this current project, Mckenna (2019) morphologically and molecularly

identified all ticks as Amblyomma albolimbatum. This was carried out using the morphologically and

molecular identification keys for Australian ticks to determine species, instar and sex (Roberts, 1970;

Barker and Walker, 2014); and molecular barcoding of the cytochrome C oxidase 1 (CO1) gene

(Hebert et al., 2003).

2.2 DNA extractions

Genomic DNA (gDNA) from 116 ticks were previously extracted using Qiagen DNeasy Blood and

Tissue Kit (Qiagen, Germany) following recommendations provided by manufacturers (Qiagen

Supplementary Protocol: Purification of total DNA from insects) with modifications as described by

Mckenna (2019). In addition to the DNA extracts provided by Mckenna, a further 26 ticks were

extracted during this study, using the same DNA protocols as describe by Mckenna (2019)(DNA

extraction tick instar in Table A1.1). Prior to DNA extraction, 26 ticks were individually surface-

sterilised with a 10% sodium hypochlorite, then 70% ethanol, and washed with sterile DNA-free

Phosphate-Buffered (PBS) and air dried. Each tick was then sliced into quarters using a sterile

scalpel blade and petri dish and placed into a 1.5mL safe lock tube (Eppendorf TM). Equipment was

further decontaminated with DNA AWAY (Molecular Bio-products Inc., San Diego, USA) to further

reduce contamination between samples. Buffer ATL and Proteinase K were aliquoted into the 1.5mL

18

tubes containing the ticks in the following amounts: 200μL buffer ATL and 22μL Proteinase K for

unengorged adults or nymph; 450μL buffer ATL and 50μL Proteinase K enzyme for engorged ticks.

Samples were incubated at 56°C in a headblock overnight (approximately 16 hr) shaking at 700rpm.

Following incubation, samples were spun at 15,0000 rpm for 3 minutes. 200μL of the supernatant

was transferred into a sterile 1.5mL safe lock tube (Eppendorf TM) containing a 1:1 solution of 200μL

of 96% alcohol and 200μL of buffer AL before a thorough vortex. Succeeding 500μL washes of buffer

AW1 and buffer AW2, elution buffer AE was directly added to the membrane in the following

amounts: 100μL for unengorged adults or nymph; 200μL for engorged ticks. Samples were then

incubated at room temperature for 4 minutes, and spun at 8,000rpm for 1 minute. Extracted gDNA

was put into 1.5mL safe lock tube (Eppendorf TM) with DNA ID, internal accession code: PI number,

date and initials for storage at -20°C. All 26 samples were analysed on a nanodrop to determine

nucleic acid yield and purity (Appendix Table A1.2). Extraction reagent blank was carried out to

mirror extraction process.

2.3 PCR assays

Subsets of samples relative to each taxa were analysed due to time and money constraint with

respect to the amount of assays performed. However, due to previous NGS study producing

sequences with a 100% genetic match to C. burnetii, the aetiological agent for the zoonotic disease

Q fever, all 142 samples were screened.

2.3.1 Coxiella detection using conventional PCR

Conventional PCR was used to investigate samples (n=142) for the presence of Coxiella species

within A. albolimbatum, targeting 524 bp of 16S rRNA gene (Short 16S) using primers Cox-sp434F

and Cox-sp1004R (Lalzar et al., 2012) (Table 2.1). Once sequences were obtained, samples that

tested positive using the short 16S assay, were re-tested to obtain a larger product size targeting

1.45kb of the 16S rRNA gene (Long 16S) using primers Cox-16S-1457F and Cox-16S-1457R

(Masuzawa et al., 1997) (Table 2.1). Each PCR reaction was performed in 25μl volumes containing

19

the following: 1 X KAPA buffer with dye (KAPA Biosystems, Cape Town, South Africa), 2.5mM KAPA

MgCl2, 0.25mM dNTPs (FisherBiotech, Australia), 0.4μM of each primer, 0.01mg BSA (Fisher

Biotech, Australia), 0.5 U KAPA taq (KAPA Biosystems, Massachusetts, USA) and 2μL of gDNA

template. Extraction blank, no-template and positive control was used in each assay. Thermal cycling

was performed using Applied Biosystems thermal cycler under the following conditions for the short

16S assay: 94°C for 4 minutes, then 40 cycles of 94°C for 30s, 60°C for 30s and 72°C for 45s,

followed by a final extension of 72°C for 5 minutes. In an attempt to amplify better quality bands from

samples that produced weak bands, a re-amplification step was included. The re-amplficiation step

included 1μL of purified amplicon product and an additional 10 cycles of amplification with the Cox-

sp434F and Cox-sp1004R primers. Thermal cycling conditions for the long 16S assay were 94°C for

5 minutes, then 40 cycles of 94°C for 60s, 48°C for 60s and 72°C for 120s, followed by a final

extension of 72°C for 5 minutes.

In addition to the 16S rRNA assay, a Coxiella-specific PCR assay targeting a 659 bp fragment of the

GroEL gene was performed on samples using primers Cox-660f and Cox-1320r in Table 2.1. Each

PCR reaction was performed in 25μl volumes containing the following: 1 X KAPA buffer with dye

(KAPA Biosystems, Cape Town, South Africa), 2.5mM KAPA MgCl2, 0.25mM dNTPs (FisherBiotech,

Australia), 0.4μM of each primer, 0.5 U KAPA taq (KAPA Biosystems, Massachusetts, USA) and

2μL of undiluted gDNA template. An extraction blank, no DNA-template and a positive control

(BAB33M, R. sanguineus positive for Coxiella endosymbiont) was used in each assay. Thermal

cycling was performed under the conditions shown in Table 2.1 : 95°C for 5 minutes, then 40 cycles

of 95°C for 30s, 52°C for 30s and 72°C for 1min, followed by a final extension of 72°C for 5 minutes.

20

Table 2.1. Primers used for Coxiella PCR assays.

Gene Primer Sequence (5’-3’) Size (bp) Reference

Short

16S

Cox-sp434F CCTTTTGAGCGTTGACGTTA ~524 bp Lalzar et al., 2012

Cox-sp1004R CCAAAGGCACCAAGTCATTT

Long

16S

Cox-16s-1457F ATTGAAGAGTTTGATTCTGG ~1.45 kb

Masuzawa et al.,

1997 Cox-16s-1457R CGGCTTCCCGAAGGTTAG

GroEL Cox-660f GGCGCICARATGGTTAARGA

~659 bp Angelakis et al.,

2016 Cox-1320r AACATCGCTTTACGACGA

2.3.2 Francisella detection using conventional PCR

Conventional PCR was used to investigate samples (n=21) for the presence of Francisella species

within A. albolimbatum, targeting partial regions of different genes using six different primer sets

(Ramírez-Paredes et al., 2017; Forsman et al., 1994) under the thermal cycling conditions provided

in Table 2.2. The tpiA assay was initially used for screening, with PCR amplified products confirmed

as Francisella DNA using Sanger sequencing, before retesting the samples with the other primer

sets. Each PCR reaction was performed in 25μl volumes containing the following: 1 X KAPA buffer

with dye (KAPA Biosystems, Cape Town, South Africa), 2.5mM KAPA MgCl2, 0.25mM dNTPs

(FisherBiotech, Australia), 0.4μM of each primer, 0.5 U KAPA taq (KAPA Biosystems,

Massachusetts, USA) and 2μL of undiluted gDNA template. Extraction blank, and no-DNA template

were used in each assay. No positive control was available, requiring optimisation.

21

Table 2.2. Primers and PCR cycling conditions used for Francisella PCR assays.

Gene Primer Sequence (5’ – 3’) Size (bp) PCR conditions (Temperature; °C/Time in seconds)

Reference Denaturartion Annealing Extension Cycles

tpiA

tpiA-F1 GCAAGTATTGGTGAGGATGTAATC

~674 95/60 63/30 72/60 40

Ramírez-Paredes et al., 2017

tpiA-R1 CTGCGGCACGTGGGTCATAG

rpoA

rpoA-F1 GCGACATCGAGGATATGACAT

~913 95/60 47/30 72/60 35 rpoA-R1 GGAATATTCTAGAACTACCG

prfB

prfB-F1 TTSAATCTTTACGGGACTAT

~1,009 95/60 47/30 72/60 35 prfB-R1 AACTTATCTAAATCACCATCTA

dnaA

dnaA-F1 AAAACCTTTCTACGTTTGAWT

~1,415 95/60 49/30 72/120 40 dnaA-R1 GCAACTCATAATCATCWGAT

pgm

pgm-F1 TGGCTATTCAGACTGTATCWAC

~1,613 95/60 57/30 72/120 50 pgm-R1 CAGTCATTCCTGTSASAGAT

16S

F5 CCTTTTTGAGTTTCGCTCC

~1,140 95/60 55/30 72/120 40 Forsman, et al., 1994 F11 TACCAGTTGGAAACGACTGT

22

2.3.3 Rickettsia detection using conventional and nested PCR

Conventional and nested PCR assays were used to investigate DNA samples from A. albolimbatum

(n=25) for the presence of Rickettsia using assays targeting the gltA, ompA, ompB, 17 kDa and sca4

genes using the primers under the thermal cycling conditions provided in Table 2.3. The gltA nested

assay was initially used for screening due gltA having greater phylogenetic resolution than the 16S.

with PCR amplified products confirmed as Rickettsial DNA using Sanger sequencing, before

retesting the samples with the other primer sets. Samples suggesting positive PCR products were

present were confirmed via Sanger sequencing. Each PCR reaction was performed in 25μl volumes

containing the following: 1 X KAPA buffer with dye (KAPA Biosystems, Cape Town, South Africa),

2.5mM KAPA MgCl2, 0.25mM dNTPs (FisherBiotech, Australia), 0.4μM of each primer, 0.5 U KAPA

taq (KAPA Biosystems, Massachusetts, USA) and 2μL of undiluted gDNA template. Extraction, no-

DNA template and positive control (1/1000 SFG culture from Australian Rickettsial Reference

Laboratory, Geelong) was used in each assay.

23

Table 2.3. Primers and PCR cycling conditions used for Rickettsia PCR assays.

Gene Primer Sequence (5’ – 3’) Size (bp)

PCR conditions (Temperature; °C/Time in seconds) Reference

Denaturartion Annealing Extension Cycles

gltA (1st)

gltA-F1 GCAAGTATTGGTGAGGATGTAATC ~900-1,000

95/60 63/30 72/60 40

Hii et al., 2011 gltA-R1 CTGCGGCACGTGGGTCATAG

gltA (2nd)

gltA-F2 GCGACATCGAGGATATGACAT ~650 95/30 59/30 72/60 35

gltA-R2 GGAATATTCTAGAACTACCG

gltA

RpCS.877p GGGGGCCTGCTCACGGCGG ~381 95/30 48/30 72/60 35 Roux et al., 2011

RpCS. 1258n ATTGCAAAAAGTACAGTGAACA

ompA

RR 190-70 ATGGCGAATATTTCT CCAAAA ~590 95/60 56/30 72/60 40

RR 190-701 GTTCCGTTAATGGCAGCATCT

ompB

omp B-F CGACGTTAACGGTTTCTCATTCT ~252 95/30 52/30 72/60 50 Hii et al., 2011

omp B–R ACCGGTTTCTTTGTAGTTTTCGTC

17 kDa Rr17k.1p TTTACAAAATTCTAAAAACCAT

~520 95/30 50/30 72/60 40 Ishikura et al., 2003 Rr17k.539n TCAATTCACAACTTGCCATT

Sca4 D1f ATGAGTAAAGACGGTAACCT

~910 95/60 50/60 72/60 40 Sekeyova et al., 2001 D928r AAGCTATTGCGTCATCTCCG

24

2.3.4 Piroplasm detection using nested PCR

To evaluate the presence of Piroplasms in samples, a nested PCR was used to screen tick samples

(n=45) targeting a partial region of the 18S rRNA gene of both Babesia and Theileria species using

external and internal primers (Jefferies et al., 2007) (Table 2.4). Each reaction was prepared to a

25µL volume containing: 1X KAPA buffer with dye and MgCl2 1.5 mM, an additional 1.5mM of MgCl2,

100mM dNTPs, 0.4µM of forward and reverse primers (Table 2.4). Reactions were performed under

the following thermal cycling conditions: The primary round of amplification had an initial activation

step of 94°C for 2 minutes, 58°C for 1 minute, and 72°C for 2 minutes, followed by 40 cycles of 94°C

for 30s, 58°C for 20s and 72°C for 1 minute, and a final extension of 72°C for 7 minutes. The

secondary round of amplification followed the same conditions as the primary round with following

exceptions: 1µL of primary PCR product instead of 2µL of gDNA template and the annealing

temperature was increased to 62°C. Extraction, no-DNA template and positive control (Babesia

vogeli from canine) were included with each set of PCR reactions.

Table 2.4. Primers used for Piroplasma PCR assays.

Gene Primer Sequence (5’ – 3’) Size (bp) Reference

18S (External) BTF1

GGCTCATTACAACAGTTATAG ~930 Jefferies et al., 2007

BTR1 CCCAAAGACTTTGATTTCTCTC

18S (Internal) BTF2 CCGTGCTAATTGTAGGGCTAATAC ~800

BTR2 GGACTACGACGGTATCTGATCG

2.4 Agarose Gel Electrophoresis and Sanger sequencing

All nested and conventional PCR products were separated on 1-2% agarose gel using 1 X TAE

buffer and stained using SYBR Safe (Invitrogen, Australia) to visualise under UV light. All PCR

products were visualised against a 100 bp DNA ladder (AXYGEN and Promega, Madison USA).

Amplified PCR products of the appropriate size were removed via a sterile filter tip and spun into a

25

sterilized 2.0 ml Eppendorf tube. After centrifugation the filter tip was removed using a sterile pipette

double wrapped in parafilm and decontaminated between samples (Yang et al., 2016). The purified

DNA was then used for sequencing.

The sequencing preparation of the amplified PCR products consisted of 7μl of Milli-Q water, 1μl of

either forward or reverse primer, and 4μl of PCR product. All PCR amplicons were submitted to the

Australian Genome Research Facility (AGRF) (Perth, Western Australia) for unidirectional

sequencing.

2.5 Phylogenetic analysis

Phylogenetic analyses were conducted by importing Sanger sequences obtained by AGRF into

Geneious v10 (Kearse et al., 2012) for assessment of quality and primer trimming. Sequences were

then subject to BLAST analysis using BLASTN 2.10.0 + (Altschul et al 1990) against nucleotide

collection (nt) database on the National Center for Biotechnology Information (NCBI) wesbite

(Morgulis et al., 2008), to identify most similar species and genotypes. Alignments with reference

sequences acquired from GenBank were built using MAFFT (Katoh, et al. 2002), trimmed to the

same nucldeotide length and realigned using MUSCLE alignment (Edgar, 2004). Aligned sequences

were then imported into MEGAX to determine the most suitable nucleotide substitution model based

on the Bayesian Information Score (BIC) (Kumar, et al., 2020). The best-fit substitution model for

each alignment is shown in Table 2.5. Genetic distance based on percent identity between

sequences were calculated using MEGAX (full genetic distance matrix for each alignment can be

found in appendix). A maximum-likelihood phylogenetic tree for each alignment was generated

based on 1000 bootstrap replications using MEGAX.

http://www.sciencedirect.com/science/article/pii/S0022283605803602

26

Table 2.5. Best-fit nucelotide substitution model used for each gene alignment.

Genus Gene Alignment

length BIC

score Model

Coxiella 16S 1,140 bp 10250 K2+G+I

GroEL 558 bp 7829 GTR+G+I

Concatenated 16S and GroEL 1,694 bp 13197 K2+G

Francisella 16S 1,028 bp 6801 K2+G+I

tpiA 521 bp 6093 HKY+G

prfB 900 bp 7255 T92+G

rpoA 723 bp 7265 T92+G+I

dnaA 954 bp 8508 T92+G

Concatenated (16S, tpiA, prfB, rpoA, dnaA) 4,230 bp 40774 GTR+G

Rickettsia gltA (short) 344 bp 3024 T92+G

gltA (long) 634 bp 5966 T92+G

ompA 517 bp 8651 T92+G

ompB 251 bp 3254 T92+G

17 kDa 331 bp 4400 K2+G

sca4 775 bp 8594 GTR+G+I

Concatenated (17 kDa and gltA(short)) 644 bp 5850 T92+G+I

Concatenated (ompA, ompB, and gltA(long)) 1,435 bp 16573 GTR+G+I

Concatenated (ompA, ompB, and sca4) 1,595 bp 20210 GTR+G+I

Concatenated (ompB, 17 kDa, and sca4) 1,482 bp 13722 GTR+G+I

Concatenated (ompB, and 17 kDa) 582 bp 5387 T92+G

Hemolivia 18S 791 bp 5706 T92+G

27

CHAPTER THREE

RESULTS

3.1. Molecular Characterisation of Coxiella

Amblyomma albolimbatum tick samples (n=142) were screened for the presence of Coxiella using

PCR assays targeting a short and long region of the 16S rRNA and GroEL genes (Table 3.1). All A.

albolimbatum samples failed to amplify with the short 16S PCR assay, with the exception of two

samples (C21 and C23) producing a faint 524 bp band and the Coxiella-positive control producing a

strong 524 bp band as visualised by gel electrophoresis (Figure 3.1). The faint products prompted

reamplification using an increase of cycles and from the of samples C21 and C23 which resulted in

stronger bands as shown in Figure 3.2. The no-template and extraction blank controls produced

appropriate results.

Figure 3.1. Agarose Gel Electrophoresis amplification of Coxiella 16S rRNA (short 526 bp). M:

DNA ladder Axygen 100 bp; Lane 1: SR17; Lane 2: SR18; Lane 3: SR19; Lane 4: SR20; Lane 5:

C21; Lane6: C22; Lane7: C23; Lane 8: C24; Lane 9: C25; Lane 10: C26; Lane 11: Empty well; Lane

12: extraction blank control; Lane13: Empty well; Lane 14: No-template control; Lane 15: Empty well;

Lane 16: C. burnetii positive control; Lane 17 - 18: Empty well; M: DNA ladder Axygen 100 bp.

Figure 3.2. Agarose Gel Electrophoresis reamplification of Coxiella 16S (short 526 bp). Lane

1: F6 Lane 2: C21 Lane 3: C23 Lane 4: Empty well; Lane 5: No-template control; Lane 6: Extraction

blank control; Lane 7: C. burnetii positive control; M: DNA ladder Axygen 100 bp.

28

Following successful amplification of the short 16S (529 bp), assays targeting a longer 16S (1,415

bp) and GroEL (659 bp) gene regions were performed on sample C23. Successful amplification

shown of expected product size were produced for the long 16S and GroEL, as visualised by gel

electrophoresis (Figure 3.3 and Figure 3.4 respectively). The no-template and extraction blank

controls produced appropriate results.

Figure 3.3. Agarose Gel Electrophoresis amplification of Coxiella 16S (long 1,375 bp). Lane 1:

Empty Well; Lane 2: C23 (4 µL of DNA template); Lane 3 - 5: Empty well; Lane 6: C23 (2 µL of DNA

template); Lane 7 - 8: Empty well; Lane 9: No-template Control; Lane 10 - 14: Empty well; Lane 15:

C. burnetii positive control; 16 – 18: Empty Well; M: DNA ladder Axygen 100 bp.

Figure 3.4. Agarose Gel Electrophoresis amplification of Coxiella GroEL gene. M: DNA ladder

Axygen 100 bp; Lane 1: C23; Lane 2-5: Empty well; Lane 6: Extraction blank control; Lane 7-9:

Empty well; Lane 10: C. burnetii positive control; Lane 11-12: Empty well; Lane 13: C23 (2 µL of

DNA template); Lane 14-15: Empty well; Lane 16: No-template control; Lane 17-18: Empty well; M:

DNA ladder Axygen 100 bp.

29

Table 3.1. Summary of Coxiella PCR assays.

The short and long 16S, and GroEL nucleotide sequences were compared against the GenBank

nucleotide database using nucleotide BLAST. An initial BLAST analysis against NCBI nucleotide

collection (nt) was used to determine most similar identity. BLAST results of both the short and long

16S and GroEL nucleotide sequences identified in this tick sample presented a 100% nucleotide

identity to the following strains of Coxiella burnetii: C. burnetii Schperling chromosome

(CP014563.1); C. burnetii ‘MSU Goat Q177’ (CP018150.1); C. burnetii str. Namibia genome

(CP007555.1); C. burnetii str. Ammassalik (FJ87329.1); C. burnetii CbuK Q154 (CP001020.1).

Table 3.2. BLAST results for 16S and GroEL Coxiella sequences.

Sample Internal archive code 16S Short (524 bp)

16S Long (1.45 kb)

GroEL (659 bp)

C23 PI1715 + + +

C21 PI1715 +

Sample Internal archive code

Gene Size (bp)

NCBI description E- value

Percent identity

GenBank Accession code

C23 PI1715 16S 524 Coxiella burnetii str. Schperling chromosome

0.0 100% CP014563.1

C23 PI1715 16S 1,391 Coxiella burnetii str. Schperling chromosome

0.0 100% CP014563.1

C23 PI1715 GroEL 621 Coxiella burnetii str. Schperling chromosome

0.0 100% CP014563.1

30

Phylogenetic analysis was peformed on 1,140 bp 16S sequence obtained from sample C23,

including reference sequences from GenBank via NCBI (Figure 3.5). The topology of the

phylogenetic tree shows the 16S nucleotide sequence obtained in this project clustered with strains

of Coxiella burnetii, separated from the Coxiella endosymbionts of other clades, and was

supported by 63% bootstrap confidence. The 16S sequence acquired from C23 was 100%

identical to ten strains of Coxiella burnetii. The 16S sequence generated in this project was 1-3%

distinct from endosymbionts of Ornithodoros spp. (full genetic distance matrix in Table A1.3).

31

Figure 3.5. Maximum likelihood (ML) analysis of the relationships between Coxiella at the 16S

rRNA locus (1,140 bp). Percentage support (>50%) from 1000 replicates is indicated at the left

of the supported node. The sequence generated in the present study is in bold.

32

Phylogenetic analysis was performed on 558 bp GroEL sequence from sample C23, including

refence sequences from GenBank via NCBI (Figure 3.6). The topology of the phylogenetic tree

constructed shows the GroEL sequence from this project clustered with strains of Coxiella burnetii,

seperated from the Coxiella endosymbionts of other clades and was supported by 99% bootstrap

confidence. The GroEL sequence acquired from C23 was 100% identical to ten strains of Coxiella

burnetii. The GroEL sequence generated in this project was 3-4 % distinct from endosymbitons of

Ornithodoros spp. (full genetic distance matrix in Table A1.5).

Figure 3.6. Maximum likelihood (ML) analysis of the relationships between Coxiella at the

GroEL gene locus (558 bp). Percentage support (>50%) from 1000 replicates is indicated at

the left of the supported node. The sequence generated in the present study is in bold.

33

Phylogenetic analysis based on concatenated 1,694 bp 16S and GroEL nucleotide sequences

obtained from sample C23, including reference sequences from GenBank via NCBI (Figure 3.7).

The topology of the phylogenetic tree constructed shows the concatenated sequence from this study

clustering witnah Coxiella burnetii, seperated from the Coxiella endosymbionts of other clades and

was supported by 86% bootstrap confidence. The concatenated sequences acquired from C23 were

100% identical to Coxiella burnetii. The concatenated sequence generated in this project was 2-3

% distinct from endosymbitons of Ornithodoros spp. (full genetic distance matrix in Table A1.7).

34

Figure 3.7. Maximum likelihood (ML) analysis of the relationships between Coxiella using concatenated 16S and GroEL gene loci (1,694

bp). Percentage support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequence generated in the present

study is in bold.

35

3.2. Molecular characterisation of Francisella

Conventional PCR assays were conducted on gDNA extracted from A. albolimbatum ticks (n=21) to

amplify Francisella (Table 3.3). The following Francisella genes were targeted: 16S rRNA;

chromosomal replication initiator protein alpha subunit (dnaA); phosphoglucomutase (pgm); peptide

chain release factor 2 beta subunit (prfB); DNA-directed RNA polymerase alpha subunit (rpoA); and

triose-phosphate isomerase alpha subunit (tpiA). No positive control was available for the following

assays. The no-template and extraction blank control of all Francisella PCR assays produced

appropriate results.

Table 3.3. Summary of PCR products tested positive results from Francisella assays.

sample Internal archive code

16S tpiA pgm prfB dnaA rpoA

F6 PI1744 +

F7 PI 767 +

F8 PI 1324 +

F9 PI 2743 +

R100 PI 1297 + +

R108 PI 1736 +

R20 PI 1872 + + + + +

R42 PI 982 + +

R53 PI 680 +

R55 PI 1295 + +

R64 PI 1298 + +

R67 PI 1320 + +

R68 PI 1299 + + +

R73 PI 1322 + +

Successful amplification of approximately 1,140 bp of the 16S rRNA gene was achieved in 14

samples (Figure 3.8) (Table 3.4). PCR products of expected size were obtained for rpoA (n=4), prfB

(n=6), dnaA (n=1) and tpiA (n=2) in following successful amplification and visualised under UV light

on a 1% (Table 3.4) (Figure 3.7 - 3.13 respectively). Amplification of a 1,613 bp region of pgm gene

was abandoned following an unsuccessful attempt at optimisation.

36

Figure 3.8. Agarose Gel Electrophoresis amplification of Francisella 16S rRNA. (A) 16S (1,140

bp). Lane 1-14: No sample; Lane 15: extraction blank control; Lane 16: No sample; Lane 17: no

template control; Lane 18: extra extraction blank; M: DNA ladder Axygen 100 bp. (B) 16S (1,140 bp)

M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R68; Lane 3: R42; Lane 4: No sample; Lane

5: R55; Lane 6: R73; Lane 7: R100; Lane 8: R64; Lane 9: R108; Lane 10: R53; Lane 11: R67; Lane

12: F6; Lane 13: F7; Lane 14: F8; Lane 15: F9; Lane 16: C23; Lane 17: F8 (1:10 dilution); 18: no

sample; M: DNA ladder Axygen 100 bp.

Figure 3.9. Agarose Gel Electrophoresis amplification of Francisella tpiA (674 bp) PCR

products dilutions. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R68; Lane 3: F1; Lane 4:

F2; Lane 5- 6: no sample; Lane 7: R20 (1:10 dilution); Lane 8: R68 (1:10 dilution); Lane 9: F1 (1:10

dilution); Lane 10: F2 (1:10 dilution); Lane 11: no sample; Lane 12: R20 (1:100 dilution); Lane 13;

R68 (1:100 dilution); Lane 14: F1 (1:100 dilution); Lane 15: F2 (1:100 dilution); Lane 16-18: no

sample; M: DNA ladder Axygen 100 bp.

37

Figure 3.10. Agarose Gel Electrophoresis amplification of Francisella rpoA (674 bp) PCR

products. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R108; Lane 3: R42; Lane 4: R53;

Lane 5: R67; Lane 6: F6; Lane 7: F7; Lane 8: F8; Lane 9: F9; Lane 10: NTC; Lane 11: extraction

blank; Lane 12-13: no sample; M: DNA ladder Axygen 100 bp.

Figure 3.11. Agarose Gel Electrophoresis amplification of Francisella prfB (1,009 bp) PCR

products. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R68; Lane 3: R42; Lane 4: no

sample; Lane 5: R55; Lane 6: R73; Lane 7: R100; Lane 8: R64; Lane 9: R108; Lane 10: R53; Lane

11: R67; Lane 12: F6; Lane 13: F7; Lane 14: F8; Lane 15: F9; Lane 16: C23; Lane 17: F8 (1:10

dilution); Lane 18: no sample; M: DNA ladder Axygen 100 bp.

Figure 3.12. Agarose Gel Electrophoresis amplification of Francisella dnaA (1,415 bp) PCR

products. M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2: R108; Lane 3: R42; Lane 4: R53:

Lane 5: R67; Lane 6: F6; Lane 7: F7; Lane 8: F8; Lane 9: F9; Lane 10: no template control; Lane

11: extraction blank control; M: DNA ladder Axygen 100 bp.

38

Figure 3.13. Agarose Gel Electrophoresis reamplification of Francisella dnaA PCR products.

M: DNA ladder Axygen 100 bp; Lane 1-2: No sample; Lane 3: R20: lane 4: R20; Lane 5: R20; Lane

6: extraction blank control; Lane 7: no sample; Lane 8: no template control; Lane 9-10; no sample;

M: DNA ladder Axygen 100 bp.

Sample R20 tested positive for all genes targeted (except pgm), producing clean chromatograms

used for phylogenetic analyses of individual nucleotide sequences, and a concatenated sequence.

The 16S sequences were compared against the GenBank nucleotide database using nucleotide

BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to determine

most similar identity (Table 3.4). BLAST results of the 16S Francisella sequences display a 98.85%

nucldeotide similarity to Francisella endosymbionts previously observed in the following species of

tick: Amblyomma paulopunctatum (MN998649.1), Haemaphysalis asiatica (KC170752.1), and

Dermacentor auratus (KC170749.1).

The tpiA sequence was compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the tpiA nucleotide sequence identified in this tick sample

presented a 95.37% nucleotide identity to Francisella persica ATRR VR-331 (CP013022.1).

The prfB sequence was compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the prfB nucleotide sequence identified in this tick sample


https://www.ncbi.nlm.nih.gov/nucleotide/CP013022.1?report=genbank&log$=nucltop&blast_rank=1&RID=7JEDAUST016

39

The rpoA sequence was compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the rpoA nucleotide sequence identified in this tick sample


The dnaA sequence was compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the dnaA nucleotide sequence identified in this tick sample


Table 3.4. BLAST results for 16S, tpiA, dnaA prfB and rpoA Francisella sequences.

Phylogenetic analysis was performed on 954 bp of the 16S sequences obtained from tick samples

in this study, and reference sequences retrieved from GenBank (Figure 3.14). The topology of the

phylogenetic tree shows the 16S sequences from project formed a monophyletic clade, seperated

from the Francisella spp. of other clades, and was supported by 100% boostrap confidence. The


Gene Size (bp)

NCBI description Query cover

E-value

Percent identity

Accession

F8 PI1324 16S 1,027 F. endoymbiont of A. paulopunctatum

100% 0.0 98.64% CP018093.1

F9 PI 2743 16S 1,038 F. endoymbiont of A. paulopunctatum

100% 0.0 98.36% CP018093.1

R108 PI 1736 16S 1,057 F. endoymbiont of A. paulopunctatum

100% 0.0 98.67% CP018093.1


100% 0.0 98.56% CP018093.1


100% 0.0 98.28% CP018093.1


100% 0.0 98.47% CP018093.1


100% 0.0 98.39 CP018093.1


100% 0.0 98.48% CP018093.1


100% 0.0 98.93% CP018093.1

R20 PI 1872 tpiA 626 F. persica ATCC VR-331 100% 0.0 95.37% CP013022.1

R20 PI 1872 dnaA 1,032 F. persica ATCC VR-331 100% 0.0 98.06% CP013022.1

R20 PI 1872 prfB 968 F. persica ATCC VR-331 100% 0.0 96.28% CP013022.1

R20 PI 1872 rpoA 884 F. persica ATCC VR-331 100% 0.0 96.15% CP013022.1


40

16S sequences acquired from A. albolimbatum ticks were 1-2% genetically distinct to known

Francisella endoysmbionts (full genetic distance matrix in Table A1.9).

Figure 3.14. Maximum likelihood (ML) analysis of the relationships between Francisella at the

16S rRNA gene locus (1,022 bp). Percentage support (>50%) from 1000 replicates is indicated

at the left of the supported node. The sequence generated in the present study is in bold.

41

Phylogenetic analysis was performed on 521 bp of the tpiA sequence obtained from sample R20,

and reference sequences retrieved from GenBank (Figure 3.15). The topology of the phylogenetic

tree shows the tpiA sequence from this project formed a monophyletic clade, seperated from the

Francisella spp. of other clades, and was supported by 100% boostrap confidence. The tpiA

sequence acquired from R20 was 5% distinct from Francisella persica ATRR VR-331 (CP013022.1);

11-12 % distinct from F. tularensis and F. hispaniensis; and 14-37% distinct from other Francisella

endosymbionts (full genetic distance matrix in Table A1.11).


tpiA gene locus (521bp). Percentage support (>50%) from 1000 replicates is indicated at the

left of the supported node. The sequence generated in the present study is highlighted in

bold.


42

Phylogenetic analysis was performed on 900 bp of the prfB sequence obtained from sample R20,


tree shows the prfB sequence from this project formed a monophyletic clade, seperated from the

Francisella spp. of other clades, and was supported by 98% boostrap confidence. The prfB sequence

acquired from R20 was 4% distinct from Francisella persica ATRR VR-331 (CP013022.1); 12-17 %

distinct from F. tularensis and F. hispaniensis; and 9-28% distinct from the F. philomiriga clade (full

genetic distance matrix in Table A1.13).


prfB gene locus (900 bp). Percentage support (>50%) from 1000 replicates is indicated at the


bold.

43

Phylogenetic analysis was performed on 723 bp of the rpoA sequence obtained from sample R20,


tree shows the rpoA sequence from this project formed a monophyletic clade, seperated from the

Francisella spp. of other clades, and was supported by 99% boostrap confidence. The rpoA

sequence acquired from R20 was 4% distinct from Francisella persica ATRR VR-331 (CP013022.1);

8-10 % distinct from F. tularensis and F. hispaniensis; and 8-24% distinct from the F. philomiriga

clade (full genetic distance matrix in Table A1.15).


rpoA gene locus (723 bp). Percentage support (>50%) from 1000 replicates is indicated at the


bold.

44

Phylogenetic analysis was performed on 954 bp of the dnaA sequence obtained from sample R20,


tree shows the dnaA sequence from this project formed a monophyletic clade, seperated from the

Francisella spp. of other clades, and was supported by 95% boostrap confidence. The dnA sequence

acquired from R20 was 2% distinct from Francisella persica ATRR VR-331 (CP013022.1); 7-9 %

distinct from F. tularensis and F. hispaniensis; and 13-14% distinct from the F. philomiriga clade (full

genetic distance matrix in Table A1.17).

Phylogenetic analysis was performed on a 4,230 bp concatenated sequence using 16S, tpiA, prfB,

rpoA, and dnaA sequences obtained from sample R20, including reference sequences from

GenBank via NCBI (Figure 3.19). The topology of the phylogenetic tree constructed shows the

concatenated sequence from this study forming a monophyletic clade, seperated from the

Francisella endosymbionts of other clades and supported by bootstrap value of 100%. The

concatenated sequence acquired from R20 was 3% distinct from Francisella persica ATRR VR-331

(CP013022.1) (full genetic distance matrix in Table A1.19).



45

Figure 3.18. Maximum likelihood (ML) analysis of the relationships between Francisella at the dnaA gene locus (954 bp). Percentage

support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequence generated in the present study is

highlighted in bold.

46

Figure 3.19. Maximum likelihood (ML) analysis of the relationships between Francisella using concatenated 16S, tpiA, prfB, rpoA, and

dnaA gene loci (4,230 bp). Percentage support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequence

generated in the present study is in bold.

47

3.3 Molecular characterisation of Rickettsia

Conventional and nested PCR assays were conducted on gDNA extracted from A. albolimbatum

ticks (n=26) to amplify Rickettsia. The following Rickettsia genes were targeted: citrate synthase

(gltA); outer membrane protein A (ompA); outer membrane protein B (ompB); 17-kDa protein (17

kDa); and surface cell antigen (sca4).

Table 3.5. Summary of samples producing positive results from Rickettsia assays.

Sample Internal archive code gltA ompA ompB 17 kDa sca4

SR12 PI 3329 + + + +

SR16 PI 2745 +

R106 PI 1460 + +

R52 PI 680 + +

R53 PI 680 + + + + +

R92 PI 3323 + + +

R94 PI 3329 +

R66 PI 1320 + +

R8 PI 1725 +

R12 PI 2742 +

SR15 PI 1723 +

SR14 PI 1316 +

Nested PCR on tick gDNA extractions (n=20) were unsuccessful in 19 samples. Successful

amplification of approximately of gltA gene (long 650 bp) using nested PCR assay was achieved in

one sample (SR12) (Table 3.5), and the Rickettsia-positive control. PCR product of expected size

was obtained following successful amplification and visualised under UV light on a 1% agarose gel

(Figure 3.20). Successful amplification using conventional PCR was achieved in gltA (short 380 bp),

ompA, ommB, 17 kDa, and sca4. PCR products of expected size were obtained for gltA (n=2), ompA

(n=2), ompB (n=7), 17 kDa (n=10) and sca4 (n=3) following successful amplification and visualised

under UV light on a 1% agarose gel displayed in Figure 3.21 - 3.26. The no-template and extraction

blank control of all nested and conventional Rickettsia PCR assays produced appropriate results.

48

Figure 3.20. Agarose Gel Electrophoresis amplification of Rickettsia gltA (long 650 bp) PCR

products. (A) M: no sample; Lane 1: SR11; Lane 2: SR12; Lane 3: SR13; Lane 4: SR14; Lane 5:

SR15; Lane 6: SR16; Lane 7: SR17; Lane 8: SR18; Lane 9: SR19; Lane 10: SR20; Lane 11: no

sample; Lane 12: no template control; Lane 13: no sample; Lane 14: extraction blank control; Lane

15: no sample; Lane 16: postive control; Lane 17-18: no sample; M: DNA ladder Axygen 100 bp. (B)

M: no sample; Lane 1: R69; Lane 2: R94; Lane 3: R22; Lane 4: R59; Lane 5: R10; Lane 6: R54;

Lane 7: R56; Lane 8: R82; Lane 9: R68; Lane 10: R88; Lane 11: no sample; Lane 12: DNA ladder

Axygen 100 bp; Lane 13-18: no sample; M: no sample.

Figure 3.21. Agarose Gel Electrophoresis amplification of Rickettsia gltA (short 381 bp) PCR

products. (A) M: DNA ladder Axygen 100 bp; Lane 1: no sample; Lane 2: R108; Lane 3: R42; Lane

4: R53; Lane 5: R67; Lane 6: F6; Lane 7-14 no sample; Lane 15: positive control; Lane 16-18: no

sample; M: DNA ladder Axygen 100 bp. (B) M: DNA ladder Axygen 100 bp; Lane 1: R20; Lane 2:

R68; Lane 3: R42; Lane 4: no sample; Lane 5: R55; Lane 6: R73; Lane 7: R100; Lane 8: R64; Lane

9: R108; Lane 10: R53; Lane 11: R67; Lane 12: F6; Lane 13: F7; Lane 14: F8; Lane 15: F9; Lane

16: C23; Lane 17: F8 (1:10 dilution); Lane 18: no sample; M: DNA ladder Axygen 100 bp.

49

Figure 3.22. Agarose Gel Electrophoresis amplification of ompA (590 bp) and ompB (252 bp)

Rickettsia PCR products. M: DNA ladder Axygen 100 bp; Lane 1: no sample; Lane 2: SR12; Lane

3: no sample; Lane 4: SR12 (1:10 dilution); Lane 5: no template control; Lane 6-7: no sample; Lane

8: extraction blank control; Lane 9: no sample; Lane 10: positive control (1:1000 dilution); Lane 11:

no sample; Lane 12: SR12; Lane 13: R94; Lane 14: extraction blank control; Lane 15: no template

contorl ; Lane 16: SR12 (1:10 dilution); Lane 17: no sample; Lane 18: positive control (1:1000

dilution); M: DNA ladder Axygen 100 bp.

Figure 3.23. Agarose Gel Electrophoresis amplification of ompA (590 bp) Rickettsia PCR

products. Lane 1: R53; Lane 2: no sample; M: DNA ladder Axygen 100 bp; Lane 3: no sample;

Lane 4: R53 (1:10 dilution); Lane 5:; Lane 6: R73; Lane 7: R100; Lane 8-9: no sample; Lane 10:

positive control (1:1000 dilution); Lane 11-13: no sample M: DNA ladder Axygen 100 bp.

50

Figure 3.24. Agarose Gel Electrophoresis amplification of Rickettsia ompB (252 bp) PCR

products. M: DNA ladder Axygen 100 bp; Lane 1: SR12; Lane 2: SR16; Lane 3: SR20; Lane 4:

R94; Lane 5: R106; Lane 6: R52; Lane 7: R110; Lane 8: R92; Lane 9: R53; Lane 10: extraction blank

control; Lane 11: no template control; Lane 12: no sample; Lane 13: SR12 (reamplification); Lane

14: no sample; Lane 15: R94 (reamplification): Lane 16: no sample; Lane 17: positive control (1:1000

dilution); Lane 18: no sample M: DNA ladder Axygen 100 bp.

Figure 3.25. Agarose Gel Electrophoresis amplification of Rickettsia 17 kDa (520 bp) PCR


Lane 5: R53; Lane 6: R60; Lane 7: R66 ; Lane 8: R8; Lane 9: R95; Lane 10: R92 ; Lane 11: SR12;

Lane 12: SR14; Lane 13: SR15; Lane 14: SR16; Lane 15: SR20; Lane 16: no template control; Lane

17: extraction blank control; Lane 18: positive control (1:1000 dilution); M: DNA ladder Axygen 100

bp.

Figure 3.26. Agarose Gel Electrophoresis amplification of Rickettsia sca4 (910 bp) PCR


Lane 5: R53; Lane 6: R60; Lane 7: R66 ; Lane 8: R8; Lane 9: R95; Lane 10: R92 ; Lane 11: SR12;

Lane 12: SR14; Lane 13: SR15; Lane 14: SR16; Lane 15: SR20; Lane 16: no template control; Lane

17: extraction blank control; Lane 18: positive control (1:1000 dilution); M: DNA ladder Axygen 100

bp.

51

The short gltA sequences were compared against the GenBank nucleotide database using

nucleotide BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to

determine most similar identity. BLAST results of the short gltA nucleotide sequences obtained from

R53 and SR12 presented a 98.92% nucleotide identity to Rickettsia bellii (MK697586.1). The long

gltA sequence was compared against the GenBank nucleotide database using nucleotide BLAST.

BLAST results of the long gltA nucleotide sequence obtained from SR12 presented a 99.35%

nucleotide identity to Rickettsia raoultii strain Khabarovsk (CP010969.1). The BLAST results of the

short and long gltA sequences showed a nucleotide identity of two different species of Rickettsia.

Closer inspection of the two shorter sequences showed the shorter sequences aligning closer to R.

bellii, and the longer sequence aligning closer with SFG Rickettsia. Additionally, concatenating the

short and long sequences from SR12 formed a chimera, indicating that sample SR12 had possible

mixed infection of at least 2 species of Rickettsia. Furthermore, while the short gltA sequences of

R53 and SR12 from this present study were 100% identical, closer inspection of the R53 forward

and reverse chromatograms showed signs of a mixed chromatogram at over 30 peaks throughout

the sequence suggesting dual infection; therefore, the R53 gltA sequence was left out of further

analyses.

The ompA sequences were compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the ompA nucleotide sequences obtained from R53 and SR12

presented a 97.56% and 97.96% nucleotide identity respectively to the following strains of Rickettsia

raoultii: R. raoultii strain Khabrovsk (CP010969.1); R. raoultii strain IM16 (CP019435.1); R. raoultii

isolate Tomsk (MK304548.1).

The ompB sequences were compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the ompB nucleotide sequences obtained from R52 and R106

presented a 99.20% nucleotide identity to R. monacensis strain IrR/Munich (LN794217.1), and R.

monacensis strain MT34 (JX625150.1); and ompB nucleotide sequences obtained from R53, R92,

and SR12 presented a 99.20% nucleotide identity to Rickettsia endosymbiont of Amblyomma

https://www.ncbi.nlm.nih.gov/nucleotide/MK304548.1?report=genbank&log$=nucltop&blast_rank=26&RID=AT4JZVEF013

52

coelebs (MT009186.1), followed by 98.80% nucleotide identity to three R. heilongjiangensis Sendai-

58 strain MT34 (AP019865.1); R. montanensis strain OSU 85-930 (CP003340.1); R. massiliae strain

AZT80 (CP003319.1).

The 17 kDa sequences were compared against the GenBank nucleotide database using nucleotide


most similar identity. BLAST results of the identical 17 kDa nucleotide sequences obtained from R52

and R106 presented a 99% nucleotide identity to R. monacensis (LN794217), followed by 98.80%

nucleotide identity to R. tamurae (AB812550). BLAST results of the identical 17 kDa nucleotide

sequences obtained from R92 and R8 presented a 99% nucleotide identity R. raoultii strain IM16

(CP019435.1) and R. raoultii strain Khabarovsk (CP010969.1). BLAST results of the identical 17

kDa nucleotide sequences obtained from BLAST results of the R53, SR12, SR14, SR15, and SR16

presented a 99.18% nucleotide identity to Rickettsia endosymbiont of Ornithodoros (MH780988.1).

The identical sca4 sequences were compared against the GenBank nucleotide database using

nucleotide BLAST. An initial BLAST analysis against NCBI nucleotide collection (nt) was used to

determine most similar identity. BLAST results of the sca4 nucleotide sequence identified in this tick

sample presented a 99.75% nucleotide identity to Uncultured Rickettsia sp. clone ARRL2016-159

(MN431845.1) , followed by a 96.81% nucleotide identity to the closest confirmed Rickettsia species,

R. heilongjiangensis (AY331396.1).

53

Table 3.6. BLAST results for gltA, ompA, ompB, 17 kDa, and sca4 Rickettsia sequences.


Gene Size (bp)


E-value Percent identity

Accession

SR12 PI 3329 gltA(short) 342 Rickettsia bellii 100% 8e-136 98.92% MK697586.1

R53 PI 680 gltA(short) 342 Rickettsia bellii 100% 8e-136 98.92% MK697586.1

SR12 PI 3329 gltA(long) 612 Rickettsia raoultii strain Khabarovsk 100% 0.0 99.35% CP010969.1

R53 PI 680 ompA 578 Rickettsia raoultii strain Khabarovsk 99% 0.0 97.56% CP010969.1

SR12 PI 3329 ompA 587 Rickettsia raoultii strain Khabarovsk 100% 0.0 97.96% CP010969.1

R52 PI 680 ompB 251 Rickettsia monacensis strain IrR/Munich 100% 3.00e-123 99.20% LN794217.1

R53 PI 680 ompB 251 Rickettsia endosymbiont of Amblyomma coelebs 100% 3.00e-123 99.20% MT009186.1

R92 PI 3323 ompB 251 Rickettsia endosymbiont of Amblyomma coelebs 100% 3.00e-123 99.20% MT009186.1

R106 PI 1460 ompB 251 Rickettsia monacensis strain IrR/Munich 100% 3.00e-123 99.20% LN794217.1

SR12 PI 3329 ompB 251 Rickettsia endosymbiont of Amblyomma coelebs 100% 3.00e-123 99.20% MT009186.1

R8 PI 680 17 kDa 498 Rickettsia raoultii strain Khabarovsk 100% 0.0 99% CP010969.1

R52 PI 680 17 kDa 498 Rickettsia monacensis strain IrR/Munich 100% 0.0 99% LN794217.1

R53 PI 680 17 kDa 544 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1

R92 PI 3323 17 kDa 494 Rickettsia raoultii strain Khabarovsk 100% 0.0 99% CP010969.1

R106 PI 1460 17 kDa 498 Rickettsia monacensis strain IrR/Munich 100% 0.0 99% LN794217.1

SR12 PI 3329 17 kDa 500 Rickettsia endosymbiont of Ornithodoros rietcorreai 89% 0.0 99.18% MH780988.1




R53 PI 680 sca4 889 Uncultured R. sp. ARRL2015-159 91% 0.0 99.75% MN431845.1



54

Phylogenetic analysis was peformed on 344 bp gltA sequence obtained from sample SR12, including

reference sequences from GenBank via NCBI (Figure 3.29). The topology of the phylogenetic tree

shows the gltA sequence from this project formed a monophyletic clade, seperated from the

Rickettsia species of other clades, and was supported by 99% boostrap confidence. The shorter gltA

sequence generated in this project was 5% distinct from R. bellii RML369-C (NC007940), followed

by SFG (Genetic distance = 13-17%), TG (Genetic distance = 16-17%). The gltA sequence acquired

from SR12 was 14-17% distinct to transitional group species of Rickettsia (full genetic distance matrix

in Table A1.21).

55

Figure 3.27. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the gltA gene locus (344 bp). Percentage support

(>50%) from 1000 replicates is indicated at the left of the supported node. The sequences generated in the present study are highlighted

in bold.

56

Phylogenetic analysis was peformed on 634 bp gltA sequence obtained from sample SR12, including


shows the gltA nucleotide sequence from this project clustered with R. gravesii BWI-1 (DQ269435)

among the SFG, seperated from the Rickettsia species of other clades, however shows low

bootstrap confidence. The gltA sequence generated in this project was 2% distinct from R. gravesii

BWI-1 (DQ269435), followed by SFG (Genetic distance = 1-3%), TG (Genetic distance = 8-9%).

These sequences were 7-8% distinct to transitional group species of Rickettsia (full genetic distance

matrix in Table A1.23).

Figure 3.28. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the

gltA gene locus (long 634 bp). Percentage support (>50%) from 1000 replicates is indicated

at the left of the supported node. The sequences generated in the present study are


57

Phylogenetic analysis was peformed on 517 bp ompA sequence obtained from sample SR12,


phylogenetic tree shows the ompA nucleotide sequence from this project clustered with R. gravesii

BWI-1 (DQ269437) and Uncultured Rickettsia sp. clone ARRL2016-159 (MN431847), seperated

from the Rickettsia species of other clades, however shows low bootstrap confidence. The ompA

sequence generated in this project was 1-% distinct from R. gravesii BWI-1 (DQ269437) and

Uncultured Rickettsia sp. clone ARRL2016-159 (MN431847), followed by SFG (Genetic distance =

3-15%), TG (Genetic distance = 47-78%). These sequences were 5-10% distinct to transitional group

species of Rickettsia (full genetic distance matrix in Table A1.25).

58


ompA gene locus (517 bp). Percentage support (>50%) from 1000 replicates is indicated at

the left of the supported node. The sequences generated in the present study are highlighted

in bold.

Phylogenetic analysis was peformed on 251 bp ompB sequences obtained from this study, including


shows the ompB nucleotide sequences from this project clustered in two individual clades, seperated

by a genetic distance of 3%. The R53, R92, and SR12 ompB sequences were identical to each other

and formed a monophyletic clade, seperated from the Rickettsia species of other clades, and was

supported by 86% bootstrap confidence. The ompB sequences acquired from R53, R92, and SR12

were closest to related to R. gravesii BWI-1 (DQ269438) and R. amblyommatis (CP003334) (Genetic

distance = 1-2%), followed by SFG (Genetic distance = 1-4%), and TG (Genetic distance = 13-15%).

59

The sequences were 4-10% distinct to transitional Group species of Rickettsia. The R52 and R106

ompB sequences were identical to each other and formed a monophyletic clade, supported by a

high boostrap value (91%), seperated from other species of Rickettsia. The sequences were

genetically distinct but closest related to R. monacensis (LN794217) and R. tamurae (DQ113911)

(Genetic distance = 1-2%), followed by SFG (Genetic distance = 1-2%), TG (Genetic distance = 12-

13%). These sequences were 5-10% distinct to Transitional Group species of Rickettsia (full genetic

distance matrix in Table A1.27).


ompB gene locus (251 bp). Percentage support (>50%) from 1000 replicates is indicated at

the left of the supported node. The sequences generated in the present study are highlighted

in bold.

60

Phylogenetic analysis was peformed on 394 bp 17 kDa sequences obtained from this study,


phylogenetic tree shows the 17 kDa nucleotide sequences from this project clustered in three

separated clades. The identical R52 and R106 sequences, and the identical R92 and R8 sequences

were 6% distinct from each other; and the R53, SR12, SR14, SR15, and SR16 sequences were 33%

and 36% distinct from the R92 and R8 sequences, and the R52 and R106 sequences respectively.

The R52 and R106 17 kDa sequences were identical and formed a monophyletic clade, supported

by a high boostrap value (96%), seperated from other species of Rickettsia. The sequences were

genetically distinct but closest related to R. monacensis (LN794217) and R. tamurae (AB812550)

(Genetic distance = 1%), followed by SFG (Genetic distance = 6-8%), TG (Genetic distance = 15-

37%). These sequences were 4-9% distinct to transitional Group species of Rickettsia. The R92

AND R8 17 kDa sequences clustered with Uncultured R.sp. clone ARRL2016-149 (MN431847);

Uncultured R.sp. clone ARRL2016-156 (MN431848); Uncultured R.sp. clone ARRL2016-159

(MN431849); and R. raoultii strain Khabrovsk (CP010969.1), supported by a high boostrap value

(94%), however the branches at this are short and phylogenetic resolution is weak. The sequences

were 100% nudelotide identity to Uncultured R.sp. clone ARRL2016-149 (MN431847); Uncultured

R.sp. clone ARRL2016-156 (MN431848); Uncultured R.sp. clone ARRL2016-159 (MN431849); and

R. raoultii strain Khabrovsk (CP010969.1). The R92 and R8 sequences were 1-5% distinct from

SFG, 16-33% distinct from TG, and 4-8% distinct from transitional group Rickettsia. The R53, SR12,

SR14, SR15, and SR16 17 kDa sequences were identical and formed a monophyletic clade,

supported by a high boostrap value (96%), seperated from other species of Rickettsia. The

sequences were 1% distinct from R. endosymbiont of O. rietorrecai (MH780988.1), followed by SFG

(genetic distance = 33-35%), TG (genetic distance = 40-43%). These sequences were 35-37%

distinct to Transitional Group species (full genetic distance matrix in Table A1.29).

61

Figure 3.31. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the 17 kDa gene locus (394bp). Percentage

support (>50%) from 1000 replicates is indicated at the left of the supported node. The sequences generated in the present study are


62

Phylogenetic analysis was performed on 775 bp sca4 sequence obtained from sample R53, R66,

and R92, including reference sequences from GenBank via NCBI (Figure 3.32). The topology of the

phylogenetic tree shows the sca4 nucleotide sequence from this project clustered with Uncultured

Rickettsia sp. clone ARRL2016-149 (MN431843); Uncultured Rickettsia sp. clone ARRL2016-156

(MN431844); Uncultured Rickettsia sp. clone ARRL2016-159 (MN431845), seperated from the

Rickettsia species of other clades, and was supported by 99% bootstrap confidence. The sca4

sequences generated in this project were 100% idenitcal to ARRL2016-149 (MN431843); Uncultured

Rickettsia sp. clone ARRL2016-156 (MN431844); Uncultured Rickettsia sp. clone ARRL2016-159

(MN431845), and 2% distinct from R. gravesii BWI-1 (DQ269437), followed by and 2-6% distinct

from SFG, 28% distinct from TG, and 11-15% distinct from transitional group Rickettsia (full genetic

distance matrix in Table A1.31).

63

Figure 3.32. Maximum likelihood (ML) analysis of the relationships between Rickettsia at the sca4 gene locus (775 bp). Percentage support

(>50%) from 1000 replicates is indicated at the left of the supported node. The sequences generated in the present study are highlighted

in bold.

64

Phylogenetic analysis was performed on a 644 bp concatenated sequence using 17 kDa and gltA

(short) sequences obtained from sample SR12, including reference sequences from GenBank via

NCBI (Figure 3.33). The topology of the phylogenetic tree constructed shows the concatenated

sequence from this study forming a monophyletic clade, seperated from the Rickettsia species of

other clades and supported by bootstrap value of 100%. The concatenated 17-kda and gltA

sequence acquired from SR12 was 6% distinct from R. bellii RML369-C (NC 007940), followed by

24-26% distinct from SFG, 27-28% distinct from TG, and 23-27% distinct from transitional group

Rickettsia (full genetic distance matrix in Table A1.33).

Figure 3.33. Maximum likelihood (ML) analysis of the relationships between Rickettsia using

concatenated 17 kDa and gltA gene loci (644 bp). Percentage support (>50%) from 1000

replicates is indicated at the left of the supported node. The sequence generated in the

present study is in bold.

65

Phylogenetic analysis was performed on a 1,435 bp concatenated sequence using ompA, ompB,

and gltA (long) sequences obtained from sample SR12, including reference sequences from

GenBank via NCBI (Figure 3.34). The topology of the phylogenetic tree constructed shows the

concatenated sequence from this study forming a monophyletic clade, seperated from the Rickettsia

species of other clades and supported by bootstrap value of 90%. The concatenated ompA, ompB,

and gltA sequence acquired from SR12 was 2-9% distinct from R. gravesii BWI-1 (DQ269438),

followed by 2% distinct from SFG, 18-21% distinct from TG, and 13-18% distinct from transitional

group Rickettsia (full genetic distance matrix in Table A1.35).


concatenated ompA, ompB, and gltA gene loci (1,435 bp). Percentage support (>50%) from

1000 replicates is indicated at the left of the supported node. The sequence generated in the


66

Phylogenetic analysis was performed on a 1,649 bp concatenated sequence using ompA, ompB,

and sca4 sequences obtained from sample R53, including reference sequences from GenBank via



other clades and supported by bootstrap value of 99%. The concatenated ompA, ompB, and sca4

sequences acquired from R53 was 2% distinct from R. gravesii BWI-1 (DQ269438), followed by 2-

12% distinct from SFG, 29-33% distinct from TG, and 17-21% distinct from transitional group

Rickettsia (full genetic distance matrix in Table A1.37).


concatenated ompA, ompB, and sca4 gene loci (1,649 bp). Percentage support (>50%) from



67

Phylogenetic analysis was performed on a 1,482 bp concatenated sequence using ompB, 17 kDa,

and sca4 sequences obtained from sample R92, including reference sequences from GenBank via



other clades and supported by bootstrap value of 99%. The concatenated ompB, 17 kDa, and sca4

sequences acquired from R92 was 1% distinct from R. gravesii BWI-1 (DQ269438), followed by 2-

6% distinct from SFG, 20-21% distinct from TG, and 8-9% distinct from transitional group Rickettsia

(full genetic distance matrix in Table A1.39).


concatenated ompB, 17 kDa, and sca4 gene loci (1,482 bp). Percentage support (>50%) from



68

Phylogenetic analysis was performed on a 633 bp concatenated sequence using 17 kDa, and ompB

sequences obtained from samples R106 and R52, including reference sequences from GenBank

via NCBI (Figure 3.37). The topology of the phylogenetic tree constructed shows the concatenated


other clades and supported by bootstrap value of 98%. The concatenated 17 kDa and ompB

sequences acquired from R106 and R52 were 1% distinct from R. monacensis (LN794217) and R.

tamurae (AF394896), followed by 4-6% distinct from SFG, 12-14% distinct from TG, and 5-7%

distinct from transitional group Rickettsia (full genetic distance matrix in Table A1.41).


concatenated ompB and 17 kDa (582 bp). Percentage support (>50%) from 1000 replicates is

indicated at the left of the supported node. The sequence generated in the present study is

in bold.

69

3.4. Molecular characterisation of Hemaprotozoa

Nested PCR assays were conducted on gDNA extracted from A. albolimbatum ticks (n=45) targeting

the 18S rRNA of piroplasms. PCR products of expected size were obtained for 18S (n=13) following

successful amplification and visualised under UV light on a 1% agarose gel displayed in Figure 3.38

and Table 3.7. The no-template and extraction blank controls produced appropriate results.

Table 3.7. Summary of positive samples from haemoprotozoa assays.

The forward and reverse sequences obtained from the A. albolimbatum tick samples produced clean

chromatograms and were aligned in Geneious to produce a consensus sequence. The 18S

nucleotide sequences were compared against the GenBank nucleotide database using nucleotide

BLAST (BLASTN 2.11.0+ (Zhang et al. 2000, Morgulis et al. 2008)). An initial BLAST analysis against

NCBI nucleotide collection (nt) was used to determine most similar identity. BLAST results of the

18S nucleotide sequence identified in this tick sample presented a 100% nucleotide identity to

Hemolivia mariae isolate 4903 (KF992711.1), and Hemolivia mariae isolate 4955 (KF992712.1)

(Table 3.8).

Table 3.8. BLAST results 18S haemoprotozoa sequences.

sample Internal archive code 18S (850bp)

SR14 PI1316 +

R6 PI1735 +


Gene Size (bp)


E value

Percent identity

Accession

SR14 PI1316 18S 782 Hemolivia mariae isolate 4903

100% 0.0 100% KF992711.1

R6 PI1735 18S 782 Hemolivia mariae isolate 4955

100% 0.0 100% KF992712.1

70

Figure 3.38. Agarose Gel Electrophoresis amplification of haemoprotozoa 18S rRNA PCR

products. (A) M: DNA ladder Axygen 100 bp; Lane 1: R1; Lane 2: R2; Lane 3: R3; Lane 4: R4; Lane

5: R5; Lane 6: R6; Lane7: R7; Lane 8: R8; Lane 9: R9; Lane 10: R10; Lane 11: R11; Lane 12: R12;

Lane 13: R13; Lane 14: R14; Lane 15: R15; Lane 16: R16; Lane 17: R17; Lane 18: R18; M: DNA

ladder Axygen 100 bp. (B) M: DNA ladder Axygen 100 bp; Lane 1: R19; Lane 2: R20; Lane 3: R21;

Lane 4: R22; Lane 5: R23; Lane 6: R24; Lane7: R25; Lane 8: R26; Lane 9: R27; Lane 10: R28; Lane

11: R29; Lane 12: R30; Lane 13: R32; Lane 14: R33; Lane 15: R34 ; Lane 16: R35; Lane 17: R36;

Lane 18: R37; M: DNA ladder Axygen 100 bp. (C) M: DNA ladder Axygen 100 bp; Lane 1: R38; Lane

2: no sample; Lane 3: R39; Lane 4: R40; Lane 5: no sample; Lane 6: R41; Lane7: no sample; Lane

8: 42; Lane 9: 443; Lane 10: R44; Lane 11: no template control; Lane 12: no sample; Lane 13:

extraction blank control; Lane 14: no sample; Lane 15: positive control; Lane 16-18: no sample; M:

DNA ladder Axygen 100 bp.

71

Phylogenetic analysis was performed on 723 bp of the 18S nucleotide sequence obtained from

sample SR14, including reference sequences from GenBank via NCBI (Figure 3.29). The topology

of the phylogenetic tree constructed shows the 18S sequence from this study clustering with other

Hemolivia species, closest to H. mariae isolate 4903 (KF992711.1), and H. mariae isolate 4955

(KF992712.1), seperated from other clades, supported by 84% bootstrap confidence. The 18S

sequences acquired from SR14, and R6 were 100% identical to Hemolivia mariae (KF992711.1);

99.87% nucleotide to Hemolivia mariae (KF992712.1) and Hemolivia stellata (KP881349.1); and

98% similar to Hemolivia mauritanica isolate TR-8-08 (KF992698.1) (full genetic distance matrix in

Table A1.43).

Figure 3.39. Maximum likelihood (ML) analysis of the relationships between haemoprotozoa

at the 18S rRNA gene locus (723 bp). Percentage support (>50%) from 1000 replicates is

indicated at the left of the supported node. The sequence generated in the present study is

highlighted in bold


72

CHAPTER FOUR

DISCUSSION

4.1 Microbial Species of Interest

This study was prompted by a preliminary study into the microbiome of A. albolimbatum removed

from T. rugosa in 2019 (Mckenna, 2019). Previous results observed a diverse range of

microorganisms, including tick-associated genera Coxiella, Francisella and Rickettsia. However, one

limitation of microbiome analysis is the sequences generated rarely provide valuable taxonomic

resolution to a species level (Gofton et al. 2015). Here in the current study, the main aim was to

provide greater insight into the species of tick-associated microorganisms within A. albolimbatum by

generating more informative gene fragments to determine the genetic relatedness to known tick-

borne pathogens.

4.1.1 Coxiella burnetii confirmed within Amblyomma albolimbatum

Coxiella burnetii is the aetiological agent for the zoonotic disease Q fever, recognised as a global

health concern and a notifiable TBD in Australia (Australian Government Department of Health). Q

fever, or Coxiellosis in animals, is highly infectious to both humans and livestock (Maurin and Raoult,

1999). This study screened 142 A. albolimbatum ticks for the presence and molecular

characterisation of Coxiella at the 16S and GroEL gene loci. An initial 16S (short 524 bp) gene

sequence from Coxiella, genetically similar to C. burnetii, was successfully generated from one A.

albolimbatum tick gDNA sample, warranting further investigation; this fragment was longer than the

original NGS sequence generated by Mckenna (2019). Consistent with the shorter 16S, the longer

16S (long 1,391 bp) and GroEL (621 bp) gene sequences were produced from the tick sample

producing a 100% nucleotide identity of C. burnetii, confirming the presence of this pathogenic

bacterium within A. albolimbatum ticks. The 16S and GroEL sequences produced in this project were

consistent with the 16S (429 bp) sequence generated from NGS by Mckenna (2019). In addition,

further similarities exist between the results here and those reported by Mckenna; the A.

73

albolimbatum ticks gDNA was extracted from during both studies testing positive for C. burnetii,

supported by BLAST analysis, were acquired from the same T. rugosa host (ID 1715).

Phylogenetic analyses were performed on the 16S and GroEL sequences obtained from this study,

which presented 99-100% nucleotide similarity to several strains of C. burnetii. The high sequence

homology between the C. burnetii sequences from this study and available reference C. burnetii

sequences suggests a low genetic diversity of C. burnetii strains within the tick. This is contrasted

by higher genetic diversity among Coxiella endosymbionts of Ornithodoros, Argas, Rhipicephalus

and Dermacentor tick genera. The sequences from this study consistently clustered in a subclade

with previously sequenced C. burnetii strains (Figure 3.3 and 3.4), in a clade that showed the closest

relatives to be Coxiella endosymbionts of soft tick genera Ornithodoros and Argas. This topology is

congruous with earlier observations by Duron et al. (2015), suggesting that the common ancestor of

C. burnetii emanated from a Coxiella species within soft ticks. This is consistent with the evolutionary

history of Coxiella outlined by Duron et al. (2015), reporting a higher genetic diversity among Coxiella

strains of ticks and a lower genetic diversity of C. burnetii.

The bobtail host (ID 1715) ticks implicated to harbour C. burnetii was documented as being admitted

to the Kanyana Wildlife Centre due to an upper respiratory tract infection, infested with nine ticks

and was pregnant. Coxiellosis can lead to abortions, premature delivery and stillbirth in mammals

(Agerholm, 2013). Interestingly, C. burnetii transmission through shedding is at its highest during

birth and causing abortions in infected wildlife (Rousset et al., 2009). As the pregnancy resulted in a

healthy birth, the presence of C. burnetii positive ticks attached to the pregnant bobtail may not have

had any influence on this animal. However, this finding is intriguing. Furthermore, no further samples

associated with the bobtail host were available to determine if C. burnetii shedding occurred. The

potential shedding of C. burnetii in this animal is of a one health concern because other case studies

have shown veterinary staff have acquired Q fever following exposure to infected parturient material

(Kopecny et al. 2013).

74

Predominantly, Q fever is acquired through the inhalation of contaminated aerosols and/or contact

with tissue from infected domestic ruminants and domestic/companion animals (Mathews., et al

2020). While ticks are rarely implicated in the transmission of C. burnetii to humans, the presence of

C. burnetii within this bobtail tick is of great concern, because the first report of A. albolimbatum

parasitising a human was recently observed in WA (S. Egan, Pers. Comm.). Without any substantial

evidence that the bobtail was infected with C. burnetii, these findings alone cannot confirm the origin

of this bacterium within A. albolimbatum ticks, i.e., obtained from parasitising an infected bobtail.

Nevertheless, the presence of C. burnetii within A. albolimbatum ticks in Australia has potential

repercussions for the health of bobtails and their wildlife rehabilitators, necessitating further

investigation. Over the past decade, Q fever notifications without documented exposure sources

have increased. A recent study identified Australian wildlife rehabilitators were two times more likely

to acquiring Q fever than the general public (Mathews et al., 2020). The presence of C burnetii within

the bobtail tick highlights the bobtail as a potential exposure source for humans, placing wildlife

rehabilitators of these lizards at increased risk of exposure to C. burnetii and of Q fever.

While large mammals, mainly cows, were traditionally considered the vertebrate reservoir for C.

burnetii with ticks aiding in the sylvatic lifecycle, there is growing evidence for the role of companion

animals and rodents as smaller vertebrate maintenance hosts. Despite the expansion of research

into the ecology of C. burnetii, there has been a dearth of research into the presence of C. burnetii

within reptiles and their ticks. To date, C. burnetii has only been reported in turtles (Kachuga sp.);

tortoises (Gopherus flavomarginatus); snakes: rat snakes (Ptyas mucosus and Ptyas korros),

Indian/Southeast Asian rock python (Python molurus), water snakes (Natrix natrix); and skinks

(Mabuya carinata) (Stephan and Rao, 1979; Široký, 2010; Medoza-Roldan, 2021). While C. burnetii

has not been identified in Australian reptiles, the NGS study by Mckenna (2019) observed Coxiella

in 13.79% of ticks, while a separated NGS study into Bothriocroton undatum ticks from goannas,

observed C. burnetii in 1.2% of the total reads (Panetta et al. 2017). Importantly, both NGS studies

were unsuccessful in attempts to amplify C. burnetii.

75

4.1.2 Francisella sp. nov. identified within Amblyomma albolimbatum

Ticks are known to contain pathogenic and endosymbiont Francisella. Close genetic relatives of the

pathogenic species are Francisella endosymbionts, which are known to aid in blood meal digestion

(Narasimhan and Fikrig, 2015). Francisella tularensis ssp. tularensis, is the causative agent of

Tularaemia which is a highly infectious zoonosis affecting mammals, birds, and arthropods in the

Northern Hemisphere (Ellis et al., 2002; Sjödin et al., 2012). Tularaemia is a notifiable disease in

Australia1, and to date, no cases of the virulent F. tularensis ssp. tularensis or Tularaemia

transmission by ticks has been documented in Australia. In 2011, a pathogen closely resembling

the Northern hemisphere tick borne pathogen F. tularensis ssp. holartica, was reported in a woman

following a bite from the ringtail possum (Pseudocheirus peregrinus) in Tasmania, Australia (Jackson

et al., 2012). While the bacterium was not associated with a tick in the Tasmanian case, it does

highlight the importance of monitoring ticks and potential wildlife reservoirs, such as reptiles, for

potential human disease causing Francisella. A follow up study identify the presence of F. tularensis

ssp. holartica within ringtail possums (Eden et al. 2017).

Fourteen A. Albolimbatum ticks examined in this study successfully amplified a 1,120 bp region of

Francisella 16S in all samples (100%). The high sequence homology among 16S sequences from

this study (99.50%-100%), in addition to rpoA, tpiA, prfB and dnaA sequences, indicates a single

Francisella species was present among the tick samples. Consistent with Ramírez-Paredes et al.

(2017), the pgm gene failed to amplify despite attempts at optimisation. The 16S, rpoA, tpiA, prfB

and dnaA gene loci were successfully amplified arbitrarily before and after attempts to amplify pgm;

suggesting failure to amplify the gene is unlikely to be attributed to inadequate gene quantity in the

sample. Unsuccessful amplification of pgm assay was likely due to insufficient optimisation of the

1,613 bp pgm assay; analysis of the primers in situ revealed primers should bind to the pgm gene

(data not shown).

1 https://www.health.nsw.gov.au/Infectious/factsheets/Pages/tularaemia.aspx retrieved May 2021.

https://www.health.nsw.gov.au/Infectious/factsheets/Pages/tularaemia.aspx

76

Phylogenetic analyses of the Francisella sequences generated in this study consistently exhibit a

monophyletic clade, supported by high bootstrap support. Furthermore, the sequences generated

through this study did not cluster with known pathogenic or endosymbiont Francisella species;

however they consistently show a close, but genetically distant relationship of 2-5% to F. persica

ATCC 331 (CP013022.1), an endosymbiont of Argus (Persicargas) arboreus, first isolated from the

malpighian soft tubules of the tick species removed from a buff-backed heron (Bubulcus ibis) in

Egypt (Larson et al., 2016). Phylogenetic analysis of the concatenated 16S, tpiA, prfB, rpoA, and

dnaA sequence, clustered in a monophyletic clade, supported by a high bootstrap consensus value

(100%). In addition, phylogenetic analysis of the 16S sequences obtained in this study are separated

from endosymbionts of Amblyomma, Ixodes, Dermacentor, Hyalomma, Ornithodoros,

Haemaphysalis, supported by high bootstrap confidence (100%). The topology among Francisella

trees is consistent, and supports that observed by Ramírez-Paredes et al. (2017) and Gerhart et al.

(2018).

The present study identified a single Francisella species within A. albolimbatum ticks which was

most closely related (3%) to F. persica ATCC 331 (CP013022.1). Francisella persica which is a tick

endosymbiont of Argus (Persicargas) arboreus was recently reclassified from the genus Wolbachia;

a group of bacteria commonly associated with being an insect endosymbiont. The present study was

based on the presence of a 16S Francisella sequence generated from NGS by Mckenna, which had

a genetic identity of 99% to F. hispaniensis (KT281843) (Mckenna, 2019). However, further

investigation of the longer 16S and the additonal housekeeping gene sequences generated in this

study, reveal the species is not related to F. hispaniensis. Instead, this is a novel species most

closely related to F. persica VR 331(CP013022).

The genetically distinct relationship presented among all sequences generated from this study

indicate the singular Francisella species present is a novel Francisella species; and importantly, not

the aforementioned pathogen strain, F. hispaniensis. In accordance with the current criteria for

77

classifying a new bacterial species: A threshold of 98.7% of 16S rRNA gene sequence similarity with

the phylogenetically closest species with standing in nomenclature, to classify a new bacterial

species (Lagier et al., 2018). The 16S sequence generated from this study from, A. albolimbatum, is

outside the threshold at 98.56% nucleotide identity, with 100% query coverage, to F. persica ATCC

331 (CP013022.1).

In Australia, a recent report of a Francisella related bacteraemia during 2015, which F. hispaniensis

was the causative agent (Aravena-Román et al., 2015). The bacterium was isolated from blood

culture from an immunocompromised human following hospital admission, becoming the first

instance of a Francisella-caused bacteraemia reported in Western Australia. hospitalised in Perth,

with the route of Francisella transmission relating to this case remaining unknown (Aravena-Román

et al., 2015). However, it was suggested infection occurred from a Francisella contaminated air

conditioning unit, and no presence of a tick was reported (Aravena-Román et al., 2015).

The results from this study were not the first to identify potential Francisella endosymbionts within

Australia. Francisella DNA has previously been identified within Amblyomma ticks. Previously,

Vilcins et al. (2009) identified Francisella DNA in A. fimbriatum (the tropical reptile tick) ticks removed

from reptiles in the Northern Territory of Australia, reporting the first detection of Francisella DNA

associated with a tick within Australia. Furthermore, a recent study by Egan et al. (2020), reported a

Francisella endosymbiont of high prevalence and abundance in A. triguttatum (the ornate kangaroo

tick) ticks found on red kangaroos from Western Australia. While this study is not the first to identify

potential Francisella endosymbionts within Australian ticks, it is the first to provide sufficient

molecular characterisation of Francisella to establish it is a novel species. Importantly, Francisella

related illness following a tick bite has not currently been documented in Australia. Furthermore, this

study provides additional molecular information towards the Francisella genus within Australian

Amblyomma ticks. Importantly, Francisella related illness following a tick bite has not currently been

documented in Australia.

78

4.1.3 Rickettsia spp. identified within Amblyomma albolimbatum

To provide greater understanding of the genetic composition of rickettsial sequences observed within

the previous NGS study, tick samples (n=26) were screened with five phylogenetically informative

gene assays. Ten (38.5%) samples successfully amplified and were identified as Rickettsia,

providing molecular data for regions of the gltA, ompA, ompB, 17 kDa and sca4 genes. While regions

of all genes were successfully amplified and sequenced, not all sequences were produced from any

single sample (Table 2.3). Rickettsia sequences in this present study indicate the presence of at

least 3 Rickettsia species among the samples producing amplicons. This is consistent with the

observation of 3 ZOTUs identified as Rickettsia spp. from sequences generated from NGS by

Mckenna (2019). In addition, due to expected co-infection of Rickettsia within some ticks, resulting

in either co-amplification with mixed chromatograms, and/or preferential and stochastic amplification

of one Rickettsia sp. over another, thus making interpretation of the results challenging. This was

observed with samples R53 and SR12, whereby for the longer gltA, ompB, sca4 and ompA genes

the generated sequences clustered more with Uncultured Rickettsia sp. ARRL2016, while for shorter

gltA and 17 kDa genes, the generated sequences clustered with Rickettsia endosymbiont of

Ornithodors rietcorreai.

Two separate but overlapping gltA sequences were obtained from the same tick sample (SR12).

Initially, the two sequences were thought to come from the same species. However, sequence

analysis showed amplification of two distinct Rickettsia species. One with genetic similarity close to

SFG R. raoultii observed in ompA; and another more basal species, more closely related to R. bellii

and Rickettsia endosymbiont of O. rietcorreai (Figure 3.27 and 3.28). This observation is similar to

that in 17 kDa, which shows five identical sequences clustering in a similar phylogenetic position to

the gltA sequences, clustering with Rickettsia endosymbiont of O. rietcorreai.

100% sequence homology was present between the shorter gltA sequences from samples SR12

and R53. However, further analysis of the R53 chromatogram revealed the presence of dual peaks

79

at over 30 positions throughout the sequence. The R53 gltA sequence with a mixed chromatogram

from this study, suggesting a co-infected tick, is supported by evidence of previous studies that

observed mixed Rickettsia spp. in ticks. Co-infection of R. bellii and R. rhipicephali was reported in

a D. variabilis tick removed from vegetation in Arkansas, USA (Wikswo et al., 2008); R. bellii, R.

montanensis, and R. rickettsii were found in a D. variabilis tick collected from nature in Ohio, USA

(Carmichael and Fuerst, 2010); and R. bellii and R. parkeri was observed in A. ovale removed from

dogs in Brazil (Szabó et al., 2013). All three cases intially identified co-infection by observing dual

peaks at multiple positions in sequencing electropherograms produced from PCR products of genus-

specific assays i.e. gltA. Furthermore, Abreu et al., (2019), reported two cases of co-infection in

Amblyomma ticks: R. bellii and R. amblyommatis in A. calcaratum ticks; and with R. bellii and a R.

parkeri-like bacterium wihin Amblyomma sp. haplotype Nazaré ticks.

Phylogenetic analyses of the Rickettsia sequences obtained in this study show 3 different groupings

of Rickettsia species. One group was clustered with Uncultured Rickettsia sp. ARRL2016

sequences, and are consistent with the recent findings reported by Tadepalli et al. (2021), however

potentially new strains. Importantly, the grouping identified in the present study is closely related,

however genetically distinct to R. gravesii in A. triguttatum. This was supported by the topology of

the phylogenetic trees built with concatenated sequences (Figure 3.34 - 3.36), which the ARRL

sequences were not included; based on different regions of the ompB gene sequenced.

Another group showed a topology and genetic distance between 1-2% close to SFG Rickettsia, R.

monacensis and R. tamurae. Rickettsia monacensis was first isolated from I. ricinus in Germany

(Simser et al., 2002), and subsequently in the same tick species in Hungary, Portugal, Slovakia,

Czech Republic, and Poland (Thu et al., 2019). Additionally, I, nipponensis and I senensis, are

considered the primary vector of R monacensis in China and Korea respectively; and I. nipponensis

has been documented to contain R. monacensis in Japan (Thu et al., 2019). This Rickettsia has

caused symptoms resembling those of Mediterranean spotted fever in in Italy, and Spain (Jado et

80

al., 2007); and documented in Ixodes genus ticks in a variety of European and Asian countries.

Interestingly, I. ricinus has been reported to parasitise several Lacertidae genera lizards

(Tomassone et al., 2017); however not present in Australia. Vectored by Amblyomma testudinarium,

SFG R. tamurae was identified to cause disease in humans in Japan in 1993 (Imaoka et al., 2011).

Recently in Australia, a Rickettsia sp. resembling R. tamurae , Rickettsia cf. tamurae, was identified

in the goanna tick, B. undatum from Sydney, Australia (Panetta et al., 2017). Intriguingly, 17 kDa

and ompB sequences from this present study were 99% and 98% genetically similar to R. tamurae

(AT-1), and demonstrate a topology similar to that presented by Panetta et al. (2017) at the gltA

gene. However, gltA sequences for the Rickettsia grouping close to R tamurae, and R. monacensis,

in this present study were not obtained. Lastly, another grouping was basal and presented a 1%

genetic distance to Rickettisa endosymbiont of O. rietocorreai, and 9% distinct from R. bellii.

Rickettsia bellii, the most basal diverging species of Rickettsia, is likely to be non-pathogenic to

humans.

In Australia, Rickettsia spp. are one of two recognised tick-borne pathogens, along with Coxiella,

currently acknowledged as causing disease in humans. There have been concerns surrounding

Rickettsia spp. in Australian ticks since Rickettsia honei, the SFG pathogen that causes Flinders

Island spotted fever (FISF), was identified in reptile tick B. hydrosauri, documented to parasitise

humans (Stenos et al., 2003). Furthermore, Rickettsia species were identified in B. hydrosauri ticks

parasitising T. rugosa from South Australia, however T. rugosa hosts tested negative to serological

testing for Rickettsia. Therefore, it cannot currently be determined whether T. rugosa is a reservoir,

though further research investigating presence of the bacteria in the tissues or organs (Whiley et al.,

2016). Close monitoring of these pathogens and hosts is paramount to assess the potential risks to

human and animal health (Tadepalli et al., 2021; Whiley et al., 2016).

In addition, Rickettsia australis causing Queensland tick typhus. More recently, an increase of

screening using molecular techniques has led to recent discovery of infectious bacteria. For

81

example: R. gravesii, removed from A. triguttatum (the ornate kangroo tick), on Barrow Island in

Western Australia; similarly, a closely related bacteria to R honei, R. honei subsp. marmionii causing

Australian spotted fever. Intriguing that Rickettsia spp. identified in this study are related to known

SFG Rickettsia spp., however the pathogenicity of those identified in this study are unknown.

4.1.4 Hemolivia mariae confirmed within Amblyomma albolimbatum

In 2014, oocysts proposed to be Hemolivia were identified for the first time from gut smears of A.

albolimbatum (Moller, 2014). The observation was made using light microscopy, based on a unique

morphological characteristic shared by H. mariae or H. stellata, and unlikley to be H. setllata based

on a difference in host specificity and differing geographical distribution. Moreover, no molecular

tests were available at the time for the identification of the hypothesised Hemolivia. Here in this

present study, we have molecularly confirmed the presence of Hemolivia mariae within two A.

albolimbatum ticks from separate bobtail hosts.

Forty-five tick samples collected previously from T. rugosa at Kanyana Wildlife Centre were screened

for the presence of piroplasm, targeting the 18S gene of these organisms. Amplification was

successful in 13 samples, of which two generated 18S sequences of Hemolivia mariae. Phylogenetic

analysis of the 18S sequences acquired from reptile ticks, revealed the 18S sequences clustered

with sequences associated with known Hemolivia spp. and were genetically identical to H. mariae

sequences (Genbank accession numbers KF992711.1, and KF992712.1). Importantly, the 18S

sequences obtained from this study were genetically identical to H. mariae, while showing a genetic

distance of 2.00% to H. mauritanica and H. stellata. These two species of Hemolivia are known to

infect tortoises and cane toads respectively.

While this is the first molecular identification of Hemolivia mariae within A. albolimbatum, these

haemogregarines have previously been associated with T. rugosa and other ticks that parasitise this

host, A. limbatum and B. hydrosauri (Smallridge and Paperna, 1997; Barker and Walker, 2014). In

82

1997, observations were made using electron microscopy and transmission electron microscopy of

smears from a tick gut of an engorged A. limbatum tick; the engorged tick was removed from T.

rugosa with haemogregarines present in the circulating erythrocytes. Lifecycle of the

haemogregarine oocysts and sporogonic changes resembling those H. stellata were reported. In

addition to the sporokinetes which spread into the gut cells before transforming to sporocysts, were

sporokinetes found spreading throughout the tick tissue and extra-intestinal locations (Smallridge

and Paperna, 1997).

Development of H. mariae occurs by sexual and asexual reproduction phases in the gut epithelium

and lumen of the tick host (Smallridge and Paperna, 1997). Primary sexual reproduction occurs

producing oocysts; progeny of the oocysts then undergo secondary asexual production, to produce

parasite stages contained in oval cysts (Smallridge and Paperna, 1997; Smallridge and Bull (2001).

Lizards are infected following ingestion of infected ticks or transmission by tick during a blood meal,

ticks are infected from parasitising on infectious lizards (Smallridge and Paperna, 1997). In addition,

both tick hosts are susceptible to H. mariae, however susceptibility to infection and burden once

infected, is greater in A. limbatum than B. hydrosauri (Smallridge and Bull, 2001).

Ticks are essential in maintaining the life cycle of H. mariae, where parasite development within the

tick is required before it can be transmitted to the vertebrate host; an indirect life cycle involving

definitive invertebrate hosts; either A. limbatum or B. hydrosauri; and an intermediate host, T. tugosa.

Regarding the geographical distributions of T. rugosa and those of A. albolimbatum, A limbatum,

and B. hydrosauri (Smyth, 1973), when considering the greater susceptibility in A. limbatum ticks,

there is likely a correlation to the distribution with H. mariae. Moreover, the susceptibility to infection

and any burden for A albolimbatum potentially affecting its contribution to H. mariae distribution

remains unexplored.

83

4.2 Limitations and future directions

The results from this study obtained molecular data for genes of Coxiella burnetii, a novel Francisella

sp., two SFG Rickettsia, a Rickettsia endosymbiont, and the blood parasite Hemolivia mariae. The

16S and GroEL gene sequences obtained which showed a 100% genetic match to Coxiella burnetii

warrants further investigation. Future research into the surveillance of C. burnetii within the stump

tailed lizard tick (T. rugosa) comparing blood and tissue to determine if the lizards are a potential

reservoir for the bacterium; moreover, the presence of C. burnetii in the stump-tailed lizard tick, A.

albolimbatum, provides information for surveillance of Q fever in Australia. Previous unsuccessful

amplification attempts may have been attributed to nucleated reptile blood contributing to inhibition

of the PCR assay (Panetta et al. 2017; Mckenna, 2019).

The presence of three Rickettsia was highlighted across five phylogenetically informative gene

regions. The mixed chromatogram of the gltA gene sequence suggesting two species of Rickettsia

in one sample highlights the sensitivity of PCR, while drawing attention to the limitation regarding

differentiation between a potential dual amplification. In addition, two species with genetic similarity

to spotted fever group Rickettsia were present, however the extent of pathogenicity, if any, cannot

be determined by the results of this study. Future research involving cloning strategies of Rickettsia

genes amplified from ticks would be beneficial to make more confident assumptions about Rickettsia

spp. present in A. Albolimbatum ticks (Ishikura et al., 2003). In addition, NGS investigation using the

Rickettsia genes targeted in this study, with emphasis on the gltA and ompA genes, to assess the

degree of similarity between the Rickettsia species within A. albolimbatum in this current study that

was genetically similiar to R. tamurae, and the R. cf. tamurae documented in the goanna tick B.

undatum (Panetta et al., 2017) and to differentiate between SFG and TG Rickettsia.

This study generated sufficient molecular data to establish a novel Francisella species. Further

investigation into Francisella sp. within Australian tick species such as A. triguttatum, and A.

fimbriatum targeting 16S and additional genes, such as those sequenced in this study should be

considered to more accurately identity the Francisella species within Australian ticks.

84

Lastly, future research is necessary to determine the presence of H. mariae within questing A.

albolimbatum ticks to understand the prevalence of this haemogregarine.

4.3 Conclusion

This study successfully achieved all aims. The primary aim of the current study was to generate

genetic information for Coxiella, Rickettsia, and Francisella taxa that were initially identified via NGS.

This study provided vital new sequences confirming the presence of Coxiella burnetii, a novel

Francisella sp. related but genetically distinct to a Francisella endosymbiont, and evidence for two

SFG Rickettsia spp. and a Rickettsia sp. endosymbiont. In addition, this study achieved its secondary

aim of investigating the presence of haemoprotozoan parasites in A. albolimbatum confirming, for

the first time, the presence of Hemolivia mariae within A. albolimbatum ticks in WA. Despite these

novel findings, further research is required to determine the medical and veterinary significance of

these microorganisms within A. albolimbatum.

85

References

Abdad, M. Y., Abdallah, R. A., Karkouri, K. E., Beye, M., Stenos, J., Owen, H., Unsworth, N.,

Robertson, I., Blacksell, S. D., Nguyen, T.-T., Nappez, C., Raoult, D., Fenwick, S., &

Fournier, P.-E. (2017). Rickettsia gravesii sp. nov.: A novel spotted fever group Rickettsia in

Western Australian Amblyomma triguttatum triguttatum ticks. International Journal of

Systematic and Evolutionary Microbiology, 67(9), 3156–3161.

https://doi.org/10.1099/ijsem.0.001865

Abreu, D. P. B. de, Peixoto, M. P., Luz, H. R., Zeringóta, V., Santolin, Í. D. A. C., Famadas, K. M.,

Faccini, J. L. H., & McIntosh, D. (2019). Two for the price of one: Co-infection with

Rickettsia bellii and spotted fever group Rickettsia in Amblyomma (Acari: Ixodidae) ticks

recovered from wild birds in Brazil. Ticks and Tick-Borne Diseases, 10(6), 101266.

https://doi.org/10.1016/j.ttbdis.2019.101266

Agerholm, J. S. (2013). Coxiella burnetii associated reproductive disorders in domestic animals-a

critical review. Acta Veterinaria Scandinavica, 55(1), 13. https://doi.org/10.1186/1751-0147-

55-13

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment

search tool. Journal of molecular biology, 215(3), 403–410. https://doi.org/10.1016/S0022-

2836(05)80360-2

Angelakis, E., Mediannikov, O., Jos, S.-L., Berenger, J.-M., Parola, P., & Raoult, D. (2016).

Candidatus Coxiella massiliensis Infection. Emerging Infectious Diseases, 22(2), 285–288.

https://doi.org/10.3201/eid2202.150106

Apanaskevich, D.A., Oliver, J.H. (2014). Life cycles and natural history of ticks, in: Sonenshine,

D.E., Roe, R.M. (Eds.), Biology of ticks, (2nd ed). Oxford University Press, New York, USA.

Aravena-Román, M., Merritt, A., & Inglis, T. J. J. (2015). First case of Francisella bacteraemia in

Western Australia. New Microbes and New Infections, 8, 75–77.

https://doi.org/10.1016/j.nmni.2015.10.004

Arthur, D. R. (1962) "Ticks and Disease." Pergamon Press, Oxford.

Ash, A., Elliot, A., Godfrey, S., Burmej, H., Abdad, M. Y., Northover, A., Wayne, A., Morris, K.,

Clode, P., Lymbery, A., & Thompson, R. C. A. (2017). Morphological and molecular

description of Ixodes woyliei n. Sp. (Ixodidae) with consideration for co-extinction with its

critically endangered marsupial host. Parasites & Vectors, 10.

https://doi.org/10.1186/s13071-017-1997-8

Babudieri, B. (1959). Q fever: A zoonosis. Adv. Vet. Sci., 5, 81–154.

Barker, D. (2019). Ixodes barkeri n. sp. (Acari: Ixodidae) from the short-beaked echidna,

Tachyglossus aculeatus, with a revised key to the male Ixodes of Australia, and list of the

subgenera and species of Ixodes known to occur in Australia. Zootaxa, 4658(2),

zootaxa.4658.2.7. https://doi.org/10.11646/zootaxa.4658.2.7

86

Barker, S. C., & Walker, A. R. (2014). Ticks of Australia. The species that infest domestic animals

and humans. Zootaxa, 3816, 1–144. https://doi.org/10.11646/zootaxa.3816.1.1

Barker, S. C., Walker, A. R., & Campelo, D. (2014). A list of the 70 species of Australian ticks;

diagnostic guides to and species accounts of Ixodes holocyclus (paralysis tick), Ixodes

cornuatus (southern paralysis tick) and Rhipicephalus australis (Australian cattle tick); and

consideration of the place of Australia in the evolution of ticks with comments on four

controversial ideas. International Journal for Parasitology, 44(12), 941–953.

https://doi.org/10.1016/j.ijpara.2014.08.008

Bonnet, S. I., Binetruy, F., Hernández-Jarguín, A. M., & Duron, O. (2017). The Tick Microbiome:

Why Non-pathogenic Microorganisms Matter in Tick Biology and Pathogen Transmission.

Frontiers in Cellular and Infection Microbiology, 7. https://doi.org/10.3389/fcimb.2017.00236

Belan, I., & Bull, C. M. (1995). Host-seeking behaviour by Australian ticks (Acari: Ixodidae) with

differing host specificities. Experimental & applied acarology, 19(4), 221–232.

https://doi.org/10.1007/BF00130825

Blanc, G. (1961) Comportement de Rickettsia burneti Derrick chez la tique Hyalomma aegyptium

(LIN) et la tortue terrestre Testudo graeca LIN. Path Microbiol 24(Suppl):21–26

Bull, C. M. (Flinders U. of S. A., & King, D. R. (Agriculture P. B. of W. A. (1981). A parapatric

boundary between two species of reptile ticks in the Albany area, Western Australia

[Amblyomma albolimbatum, Aponomma hydrosauri]. Transactions of the Royal Society of

South Australia (Australia). https://agris.fao.org/agris-

search/search.do?recordID=AU8200563

Carmichael, J. R., & Fuerst, P. A. (2010). Molecular detection of Rickettsia bellii, Rickettsia

montanensis, and Rickettsia rickettsii in a Dermacentor variabilis tick from nature. Vector

Borne and Zoonotic Diseases (Larchmont, N.Y.), 10(2), 111–115.

https://doi.org/10.1089/vbz.2008.0083

Carreno, R. A., Martin, D. S., & Barta, J. R. (1999). Cryptosporidium is more closely related to the

gregarines than to coccidia as shown by phylogenetic analysis of apicomplexan parasites

inferred using small-subunit ribosomal RNA gene sequences. Parasitology

research, 85(11), 899–904. https://doi.org/10.1007/s004360050655

Chilton, N. B., & Bull, C. M. (1991). A comparison of the reproductive parameters of females of two

reptile tick species. International Journal for Parasitology, 21(8), 907–911.

https://doi.org/10.1016/0020-7519(91)90165-4

Clarridge, J. E. (2004). Impact of 16S rRNA Gene Sequence Analysis for Identification of Bacteria

on Clinical Microbiology and Infectious Diseases. Clinical Microbiology Reviews, 17(4),

840–862. https://doi.org/10.1128/CMR.17.4.840-862.2004

87

Colwell, D. D., Dantas-Torres, F., & Otranto, D. (2011). Vector-borne parasitic zoonoses: Emerging

scenarios and new perspectives. Veterinary Parasitology, 182(1), 14–21.

https://doi.org/10.1016/j.vetpar.2011.07.012

Cooper, A., Stephens, J., Ketheesan, N., & Govan, B. (2013). Detection of Coxiella burnetii DNA in

wildlife and ticks in northern Queensland, Australia. Vector borne and zoonotic diseases

(Larchmont, N.Y.), 13(1), 12–16. https://doi.org/10.1089/vbz.2011.0853

Derrick, E. H. (1937). “Q” Fever, a New Fever Entity: Clinical Features, Diagnosis and Laboratory

Investigation. Medical Journal of Australia, 2(8), 281–299. https://doi.org/10.5694/j.1326-

5377.1937.tb43743.x

Dias, V. H. G., Gomes, P. da S. F. C., Azevedo-Martins, A. C., Cabral, B. C. A., Woerner, A. E.,

Budowle, B., Moura-Neto, R. S., & Silva, R. (2020). Evaluation of 16S rRNA Hypervariable

Regions for Bioweapon Species Detection by Massively Parallel Sequencing. International

Journal of Microbiology, 2020. https://doi.org/10.1155/2020/8865520

Duron, O., Noël, V., McCoy, K. D., Bonazzi, M., Sidi-Boumedine, K., Morel, O., Vavre, F., Zenner,

L., Jourdain, E., Durand, P., Arnathau, C., Renaud, F., Trape, J. F., Biguezoton, A. S.,

Cremaschi, J., Dietrich, M., Léger, E., Appelgren, A., Dupraz, M., Gómez-Díaz, E., …

Chevillon, C. (2015). The Recent Evolution of a Maternally-Inherited Endosymbiont of Ticks

Led to the Emergence of the Q Fever Pathogen, Coxiella burnetii. PLoS pathogens, 11(5),

e1004892. https://doi.org/10.1371/journal.ppat.1004892

Eden, J.-S., Rose, K., Ng, J., Shi, M., Wang, Q., Sintchenko, V., & Holmes, E. C. (2017).

Francisella tularensis ssp. Holarctica in Ringtail Possums, Australia. Emerging Infectious

Diseases, 23(7), 1198–1201. https://doi.org/10.3201/eid2307.161863

Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high

throughput. Nucleic Acids Res. 32(5), 1792-1797.

Egan, S. L., Loh, S.-M., Banks, P. B., Gillett, A., Ahlstrom, L., Ryan, U. M., Irwin, P. J., & Oskam,

C. L. (2020). Bacterial community profiling highlights complex diversity and novel

organisms in wildlife ticks. Ticks and Tick-Borne Diseases, 11(3), 101407.

https://doi.org/10.1016/j.ttbdis.2020.101407

Ellis, J., Oyston, P. C. F., Green, M., & Titball, R. W. (2002). Tularemia. Clinical Microbiology

Reviews, 15(4), 631–646. https://doi.org/10.1128/CMR.15.4.631-646.2002

Forsman, M., Sandström, G., & Sjöstedt, A. (1994). Analysis of 16S ribosomal DNA sequences of

Francisella strains and utilization for determination of the phylogeny of the genus and for

identification of strains by PCR. International journal of systematic bacteriology, 44(1), 38–

46. https://doi.org/10.1099/00207713-44-1-38

Fournier, P.-E., Dumler, J. S., Greub, G., Zhang, J., Wu, Y., & Raoult, D. (2003). Gene Sequence-

Based Criteria for Identification of New Rickettsia Isolates and Description of Rickettsia

88

heilongjiangensis sp. Nov. Journal of Clinical Microbiology, 41(12), 5456–5465.

https://doi.org/10.1128/JCM.41.12.5456-5465.2003

Gerhart, J. G., Auguste Dutcher, H., Brenner, A. E., Moses, A. S., Grubhoffer, L., & Raghavan, R.

(2018). Multiple Acquisitions of Pathogen-Derived Francisella Endosymbionts in Soft Ticks.

Genome Biology and Evolution, 10(2), 607–615. https://doi.org/10.1093/gbe/evy021

Gerhart, J. G., Moses, A. S., & Raghavan, R. (2016). A Francisella-like endosymbiont in the Gulf

Coast tick evolved from a mammalian pathogen. Scientific Reports, 6.

https://doi.org/10.1038/srep33670

Godfrey, S., & Gardner, M. (2017). Lizards, ticks and contributions to Australian parasitology: C.

Michael Bull (1947–2016). International Journal for Parasitology: Parasites and Wildlife,

6(3), 295-298. https://doi.org/10.1016/j.ijppaw.2017.08.010

Gofton, A. W., Oskam, C. L., Lo, N., Beninati, T., Wei, H., McCarl, V., Murray, D. C., Paparini, A.,

Greay, T. L., Holmes, A. J., Bunce, M., Ryan, U., & Irwin, P. (2015). Inhibition of the

endosymbiont "Candidatus Midichloria mitochondrii" during 16S rRNA gene profiling

reveals potential pathogens in Ixodes ticks from Australia. Parasites & vectors, 8, 345.

https://doi.org/10.1186/s13071-015-0958-3

Graves, S. R., & Stenos, J. (2017). Tick-borne infectious diseases in Australia. Medical Journal of

Australia, 206(7), 320–324. https://doi.org/10.5694/mja17.00090

Greay, T. L., Gofton, A. W., Paparini, A., Ryan, U. M., Oskam, C. L., & Irwin, P. J. (2018). Recent

insights into the tick microbiome gained through next-generation sequencing. Parasites &

Vectors, 11(1), 12. https://doi.org/10.1186/s13071-017-2550-5

Gregory, A. E., van Schaik, E. J., Russell-Lodrigue, K. E., Fratzke, A. P., & Samuel, J. E. (2019).

Coxiella burnetii Intratracheal Aerosol Infection Model in Mice, Guinea Pigs, and Nonhuman

Primates. Infection and Immunity, 87(12). https://doi.org/10.1128/IAI.00178-19

Guglielmone, A. A., Robbins, R. G., Apanaskevich, D. A., Petney, T. N., Estrada-Peña, A., Horak,

I. G., Shao, R., & Barker, S. C. (2010). The Argasidae, Ixodidae and Nuttalliellidae (Acari:

Ixodida) of the world: a list of valid species names. Zootaxa, 2528(1), 1–28.

https://doi.org/10.11646/zootaxa.2528.1.1

Hebert, P. D., Cywinska, A., Ball, S. L., & deWaard, J. R. (2003). Biological identifications through

DNA barcodes. Proceedings. Biological sciences, 270(1512), 313–321.

https://doi.org/10.1098/rspb.2002.2218

Hii, S.-F., Kopp, S.R., Thompson, M.F., O'Leary, C. A., Rees, R. L., & Traub, R. J. (2011).

Molecular evidence of Rickettsia felis infection in dogs from northern territory,

Australia. Parasites Vectors, 4(198). https://doi.org/10.1186/1756-3305-4-198

Hooper, L. V., & Gordon, J. I. (2001). Commensal host-bacterial relationships in the gut. Science

(New York, N.Y.), 292(5519), 1115–1118. https://doi.org/10.1126/science.1058709

89

Imaoka, K., Kaneko, S., Tabara, K., Kusatake, K., & Morita, E. (2011). The First Human Case of

Rickettsia tamurae Infection in Japan. Case reports in dermatology, 3(1), 68–73.

https://doi.org/10.1159/000326941

Ishikura, M., Ando, S., Shinagawa, Y., Matsuura, K., Hasegawa, S., Nakayama, T., Fujita, H., &

Watanabe, M. (2003). Phylogenetic Analysis of Spotted Fever Group Rickettsiae Based on

gltA, 17-kDa, and rOmpA Genes Amplified by Nested PCR from Ticks in Japan.

Microbiology and Immunology, 47(11), 823–832. https://doi.org/10.1111/j.1348-

0421.2003.tb03448.x

Izzard, L., Graves, S., Cox, E., Fenwick, S., Unsworth, N., & Stenos, J. (2009). Novel rickettsia in

ticks, Tasmania, Australia. Emerging infectious diseases, 15(10), 1654–1656.

https://doi.org/10.3201/eid1510.090799

Jackson, J., McGregor, A., Cooley, L., Ng, J., Brown, M., Ong, C. W., Darcy, C., & Sintchenko, V.

(2012). Francisella tularensis Subspecies holarctica, Tasmania, Australia, 2011. Emerging

Infectious Diseases, 18(9), 1484–1486. https://doi.org/10.3201/eid1809.111856

Jado, I., Oteo, J. A., Aldámiz, M., Gil, H., Escudero, R., Ibarra, V., Portu, J., Portillo, A., Lezaun, M.

J., García-Amil, C., Rodríguez-Moreno, I., & Anda, P. (2007). Rickettsia monacensis and

Human Disease, Spain. Emerging Infectious Diseases, 13(9), 1405–1407.

https://doi.org/10.3201/eid1309.060186

Jefferies, R., Ryan, U. M., & Irwin, P. J. (2007). PCR-RFLP for the detection and differentiation of

the canine piroplasm species and its use with filter paper-based technologies. Veterinary

parasitology, 144(1-2), 20–27. https://doi.org/10.1016/j.vetpar.2006.09.022

Jongejan, F., & Uilenberg, G. (2004). The global importance of ticks. Parasitology, 129 Suppl, S3-

14. https://doi.org/10.1017/s0031182004005967

Katoh, K., Misawa, K., Kuma, K. and Miyata, T. (2002). MAFFT: a novel method for rapid multiple

sequence alignment based on fast Fourier transform. Nucleic Acids Research 30, 3059–

3066. doi: 10.1093/nar/gkf436.

Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., Buxton, S., Cooper,

A., Markowitz, S., Duran, C., Thierer, T., Ashton, B., Meintjes, P., Drummond, A., 2012.

Geneious Basic: An integrated and extendable desktop software platform for the

organization and analysis of sequence data. Bioinformatics 28(12), 1647-1649.

Kerr, G. D., & Bull, C. M. (2006). Interactions between climate, host refuge use, and tick population

dynamics. Parasitology Research, 99(3), 214–222. https://doi.org/10.1007/s00436-005-

0110-y

Kim, S., Guirgis, S., Harris, D., Keelan, T., & Mayer, M. (1978) Q fever—New York, Center for

Disease Control MMWR 27:321–323

90

Klappenbach, J. A., Saxman, P. R., Cole, J. R., & Schmidt, T. M. (2001). rrndb: The Ribosomal

RNA Operon Copy Number Database. Nucleic Acids Research, 29(1), 181–184.

https://doi.org/10.1093/nar/29.1.181

Kopecny, L., Bosward, K. L., Shapiro, A., & Norris, J. M. (2013). Investigating Coxiella burnetii

infection in a breeding cattery at the centre of a Q fever outbreak. Journal of feline medicine

and surgery, 15(12), 1037–1045. https://doi.org/10.1177/1098612X13487360

Kumar, S., Stecher, G., Li M., Knyaz, C., and Tamura, K. (2018). MEGA X: Molecular Evolutionary

Genetics Analysis across computing platforms. Molecular Biology and Evolution, (35),

1547-1549.

Kwak, M. L., Madden, C., & Wicker, L. (2018). Ixodes heathi n. sp. (Acari: Ixodidae), a co-

endangered tick from the critically endangered mountain pygmy possum (Burramys

parvus), with notes on its biology and conservation. Experimental & Applied Acarology,

76(3), 413–419. https://doi.org/10.1007/s10493-018-0312-5

Lagier, J.-C., Bilen, M., Cadoret, F., Drancourt, M., Fournier, P.-E., La Scola, B., & Raoult, D.

(2018). Naming microorganisms: The contribution of the IHU Méditerranée Infection,

Marseille, France. New Microbes and New Infections, 26, S89–S95.

https://doi.org/10.1016/j.nmni.2018.08.006

Lalzar, I., Harrus, S., Mumcuoglu, K. Y., & Gottlieb, Y. (2012). Composition and seasonal variation

of Rhipicephalus turanicus and Rhipicephalus sanguineus bacterial communities. Applied

and environmental microbiology, 78(12), 4110–4116. https://doi.org/10.1128/AEM.00323-

12

Larson, M. A., Nalbantoglu, U., Sayood, K., Zentz, E. B., Cer, R. Z., Iwen, P. C., Francesconi, S.

C., Bishop-Lilly, K. A., Mokashi, V. P., Sjöstedt, A., & Hinrichs, S. H. (2016).

Reclassification of Wolbachia persica as Francisella persica comb. nov. and emended

description of the family Francisellaceae. International journal of systematic and

evolutionary microbiology, 66(3), 1200–1205. https://doi.org/10.1099/ijsem.0.000855

Larson, M. A., Sayood, K., Bartling, A. M., Meyer, J. R., Starr, C., Baldwin, J., & Dempsey, M. P.

(2020). Differentiation of Francisella tularensis Subspecies and Subtypes. Journal of

Clinical Microbiology, 58(4). https://doi.org/10.1128/JCM.01495-19

Leclerque, A., & Kleespies, R. G. (2008). 16S rRNA-, GroEL- and MucZ-based assessment of the

taxonomic position of “Rickettsiella melolonthae” and its implications for the organization of

the genus Rickettsiella. International Journal of Systematic and Evolutionary Microbiology,

58(4), 749–755. https://doi.org/10.1099/ijs.0.65359-0

Li, A. Y., Adams, P. J., Abdad, M. Y., & Fenwick, S. G. (2010). High prevalence of Rickettsia

gravesii sp. Nov. In Amblyomma triguttatum collected from feral pigs. Veterinary

Microbiology, 146(1), 59–62. https://doi.org/10.1016/j.vetmic.2010.04.018

91

Marrie, T. J., Minnick, M. F., Textoris, J., Capo, C., & Mege, J.-L. (2015). Coxiella. In Molecular

Medical Microbiology (pp. 1941–1972). Elsevier. https://doi.org/10.1016/B978-0-12-397169-

2.00106-2

Masuzawa, T., Sawaki, K., Nagaoka, H., Akiyama, M., Hirai, K., & Yanagihara, Y. (1997).

Identification of rickettsiae isolated in Japan as Coxiella burnetii by 16S rRNA

sequencing. International journal of systematic bacteriology, 47(3), 883–884.

https://doi.org/10.1099/00207713-47-3-883

Mathews, K. O., Toribio, J. A., Norris, J. M., Phalen, D., Wood, N., Graves, S. R., Sheehy, P. A., &

Bosward, K. L. (2020). Coxiella burnetii seroprevalence and Q fever in Australian wildlife

rehabilitators. One health (Amsterdam, Netherlands), 12, 100197.

https://doi.org/10.1016/j.onehlt.2020.100197.

Maurin, M., & Raoult, D. (1999). Q Fever. Clinical Microbiology Reviews, 12(4), 518–553.

Mendoza-Roldan, J. A., Modry, D., & Otranto, D. (2021). Zoonotic Parasites of Reptiles: A

Crawling Threat. Trends in parasitology, 36(8), 677–687.

https://doi.org/10.1016/j.pt.2020.04.014

Mckenna, R. (2019). Bacterial community profiling of Western Australia Bobtail Lizard (Tiliqua

Rugosa) ticks. [Honours thesis, Murdoch University]. Murdoch University Research

Repository.

Moller, C. (2014). The haematology of bobtail lizards (Tiliqua rugosa) in Western Australia:

reference intervals, blood cell morphology, cytochemistry and ultrastructure. [Masters

thesis, Murdoch University]. Murdoch University Research Repository.

Mollet, C., Drancourt, M., & Raoult, D. (1997). RpoB sequence analysis as a novel basis for

bacterial identification. Molecular Microbiology, 26(5), 1005–1011.

https://doi.org/10.1046/j.1365-2958.1997.6382009.x

Mörner T., & Addison E. (2001). “Tularemia,” in Infectious Diseases of Wild Animals, 3rd Edn., ed.

Williams B. I. (Ames, Iowa: Iowa State University; ), 303–312.

Mullis, K. B., & Faloona, F. A. (1987). Specific synthesis of DNA in vitro via a polymerase-

catalyzed chain reaction. Methods in Enzymology, 155, 335–350.

https://doi.org/10.1016/0076-6879(87)55023-6

Narasimhan, S., & Fikrig, E. (2015). Tick microbiome: The force within. Trends in Parasitology,

31(7), 315–323. https://doi.org/10.1016/j.pt.2015.03.010

Nardi, T., Olivieri, E., Kariuki, E., Sassera, D., & Castelli, M. (2021). Sequence of a Coxiella

Endosymbiont of the Tick Amblyomma nuttalli Suggests a Pattern of Convergent Genome

Reduction in the Coxiella Genus. Genome Biology and Evolution, 13(1), evaa253.

https://doi.org/10.1093/gbe/evaa253

https://researchrepository.murdoch.edu.au/view/author/Mckenna,%20Ruby.html

https://researchrepository.murdoch.edu.au/id/eprint/55210/

https://researchrepository.murdoch.edu.au/id/eprint/55210/

92

Natusch, D. J. D., Lyons, J. A., Dubey, S., & Shine, R. (2018). Ticks on snakes: The ecological

correlates of ectoparasite infection in free-ranging snakes in tropical Australia. Austral

ecology, 43(5), 534-546. https://doi.org/10.1111/aec.12590

Norval, G., & Gardner, M. (2019) The natural history of the sleepy lizard, Tiliqua rugosa (Gray,

1825) – Insight from chance observations and long-term research on a common Australian

skink species. Austral Ecology, 45(4), 410-417. https://doi.org/10.1111/aec.12715

O’Donoghue, P. (2017). Haemoprotozoa: Making biological sense of molecular phylogenies.

International Journal for Parasitology: Parasites and Wildlife, 6(3), 241–256.

https://doi.org/10.1016/j.ijppaw.2017.08.007

Ogata, H., Scola, B. L., Audic, S., Renesto, P., Blanc, G., Robert, C., Fournier, P.-E., Claverie, J.-

M., & Raoult, D. (2006). Genome Sequence of Rickettsia bellii Illuminates the Role of

Amoebae in Gene Exchanges between Intracellular Pathogens. PLOS Genetics, 2(5), e76.

https://doi.org/10.1371/journal.pgen.0020076

Ogrzewalska, M., Nieri-Bastos, F. A., Marcili, A., Nava, S., González-Acuña, D., Muñoz-Leal, S.,

Ruiz-Arrondo, I., Venzal, J. M., Mangold, A., & Labruna, M. B. (2016). A novel spotted fever

group Rickettsia infecting Amblyomma parvitarsum (Acari: Ixodidae) in highlands of

Argentina and Chile. Ticks and tick-borne diseases, 7(3), 439–442.

https://doi.org/10.1016/j.ttbdis.2016.01.003

Olsufiev, N. G., Eemelvanova, O. S., & Dunayeva, T. N. (1959). Comparative study of strains of B.

tularense in the old and new world and their taxonomy. Journal of hygiene, epidemiology,

microbiology, and immunology, 3, 138–149.

Palmer, N. C., Kierstead, M., Key, D. W., Williams, J. C., Peacock, M. G., & Vellend, H. (1983).

Placentitis and Abortion in Goats and Sheep in Ontario Caused by Coxiella burnetii. The

Canadian Veterinary Journal = La Revue Veterinaire Canadienne, 24(2), 60–61.

Panetta, J. L., Šíma, R., Calvani, N. E. D., Hajdušek, O., Chandra, S., Panuccio, J., & Šlapeta, J.

(2017). Reptile-associated Borrelia species in the goanna tick (Bothriocroton undatum) from

Sydney, Australia. Parasites & Vectors, 10(1), 616. https://doi.org/10.1186/s13071-017-

2579-5

Parola, P., & Raoult, D. (2001). Ticks and Tickborne Bacterial Diseases in Humans: An Emerging

Infectious Threat. Clinical Infectious Diseases, 32(6), 897–928.

https://doi.org/10.1086/319347

Parola, Philippe, Musso, D., & Raoult, D. (2016). Rickettsia felis: The next mosquito-borne

outbreak? The Lancet Infectious Diseases, 16(10), 1112–1113.

https://doi.org/10.1016/S1473-3099(16)30331-0

Parola, Philippe, Paddock, C. D., & Raoult, D. (2005). Tick-Borne Rickettsioses around the World:

Emerging Diseases Challenging Old Concepts. Clinical Microbiology Reviews, 18(4), 719–

756. https://doi.org/10.1128/CMR.18.4.719-756.2005

93

Petney, T. N., Andrews, R. H., & Bull, C. M. (1983). Movement and Host Finding by Unfed Nymphs

of Two Australian Reptile Ticks. Australian Journal of Zoology, 31(5), 717–721.

https://doi.org/10.1071/zo9830717

Petney, T. N., & Bull, C. M. (1981). A non-specific aggregation pheromone in two Australian reptile

ticks. Animal Behaviour, 29(1), 181–185. https://doi.org/10.1016/S0003-3472(81)80164-9

Pierce, B. A. (2017) Genetics: A Conceptual Approach (6Th ed.). MAP.

Ramírez-Paredes, J. G., Thompson, K. D., Metselaar, M., Shahin, K., Soto, E., Richards, R. H.,

Penman, D. J., Colquhoun, D. J., & Adams, A. (2017). A Polyphasic Approach for

Phenotypic and Genetic Characterization of the Fastidious Aquatic Pathogen Francisella

noatunensis subsp. orientalis. Frontiers in Microbiology, 8.

https://doi.org/10.3389/fmicb.2017.02324

Rousset, E., Berri, M., Durand, B., Dufour, P., Prigent, M., Delcroix, T., Touratier, A., & Rodolakis,

A. (2009). Coxiella burnetii Shedding Routes and Antibody Response after Outbreaks of Q

Fever-Induced Abortion in Dairy Goat Herds. Applied and Environmental Microbiology,

75(2), 428–433. https://doi.org/10.1128/AEM.00690-08

Roux, V., & Raoult, D. (2000). Phylogenetic analysis of members of the genus Rickettsia using the

gene encoding the outer-membrane protein rOmpB (ompB). International Journal of

Systematic and Evolutionary Microbiology, 50 Pt 4, 1449–1455.

https://doi.org/10.1099/00207713-50-4-1449

Roux, V., Rydkina, E., Eremeeva, M., & Raoult, D. (1997). Citrate synthase gene comparison, a

new tool for phylogenetic analysis, and its application for the Rickettsiae. International

Journal of Systematic Bacteriology, 47(2), 252–261. https://doi.org/10.1099/00207713-47-

2-252

Sekeyova, Z., Roux, V., & Raoult, D. (2001). Phylogeny of Rickettsia spp. Inferred by comparing

sequences of “gene D”, which encodes an intracytoplasmic protein. International Journal of

Systematic and Evolutionary Microbiology, 51(Pt 4), 1353–1360.

https://doi.org/10.1099/00207713-51-4-1353

Sharrad, R. D., & King, D. R. (1981). The Geographical Distribution of Reptile Ticks in Western

Australia. Australian Journal of Zoology, 29(6), 861 - 873.

https://doi.org/10.1071/ZO9810861

Simser, J. A., Palmer, A. T., Fingerle, V., Wilske, B., Kurtti, T. J., & Munderloh, U. G. (2002).

Rickettsia monacensis sp. Nov., a Spotted Fever Group Rickettsia, from Ticks (Ixodes

ricinus) Collected in a European City Park. Applied and Environmental Microbiology, 68(9),

4559–4566. https://doi.org/10.1128/AEM.68.9.4559-4566.2002

Široký, P., Petrželková, K. J., Kamler, M., Mihalca, A. D., & Modrý, D. (2007). Hyalomma

aegyptium as dominant tick in tortoises of the genus Testudo in Balkan countries, with

94

notes on its host preferences. Experimental and Applied Acarology, 40(3–4), 279–290.

https://doi.org/10.1007/s10493-006-9036-z

Sjödin, A., Svensson, K., Öhrman, C., Ahlinder, J., Lindgren, P., Duodu, S., Johansson, A.,

Colquhoun, D. J., Larsson, P., & Forsman, M. (2012). Genome characterisation of the

genus Francisella reveals insight into similar evolutionary paths in pathogens of mammals

and fish. BMC Genomics, 13, 268. https://doi.org/10.1186/1471-2164-13-268

Šlapeta, J., Chandra, S., & Halliday, B. (2021). The "tropical lineage" of the brown dog tick

Rhipicephalus sanguineus sensu lato identified as Rhipicephalus linnaei (). International

journal for parasitology, 51(6), 431–436. https://doi.org/10.1016/j.ijpara.2021.02.001

Smallridge, C., & Bull, C. (2001). Infection dynamics of Hemolivia mariae in the sleepy lizard

Tiliqua rugosa. Parasitology Research, 87(8), 657–661.

https://doi.org/10.1007/s004360100430

Smallridge, Catherine, & Paperna, I. (1997). The tick-transmitted haemogregarinid of the Australian

sleepy lizard Tiliqua rugosa belongs to the genus Hemolivia. Parasite, 4, 359–363.

https://doi.org/10.1051/parasite/1997044359

Smyth, M. (1973). The distribution of three species of reptile ticks, Aponomma hydrosauri (Denny),

Amblyomma albolimbatum Neumann, and Amb. limbatum Neumann I. Distribution and

hosts. Australian Journal of Zoology, 21(1), 91–101. https://doi.org/10.1071/zo9730091

Spratt, D. M., & Beveridge, I. (2018). Wildlife parasitology in Australia: Past, present and future.

Australian Journal of Zoology, 66(4), 286. https://doi.org/10.1071/ZO19017

Springer, A., Glass, A., Probst, J., & Strube, C. (2021). Tick-borne zoonoses and commonly used

diagnostic methods in human and veterinary medicine. Parasitology Research.

https://doi.org/10.1007/s00436-020-07033-3

Stephen, S., & Rao, K. N. (1979). Coxiellosis in reptiles of South Kanara district, Karnataka. The

Indian journal of medical research, 70, 937–941.

Stewart, A., Armstrong, M., Graves, S., & Hajkowicz, K. (2017). Rickettsia australis and

Queensland Tick Typhus: A Rickettsial Spotted Fever Group Infection in Australia. The

American Journal of Tropical Medicine and Hygiene, 97(1), 24–29.

https://doi.org/10.4269/ajtmh.16-0915

Sumrandee, C., Hirunkanokpun, S., Grubhoffer, L., Baimai, V., Trinachartvanit, W., & Ahantarig, A.

(2014). Phylogenetic relationships of Francisella-like endosymbionts detected in two

species of Amblyomma from snakes in Thailand. Ticks and Tick-Borne Diseases, 5(1), 29–

32. https://doi.org/10.1016/j.ttbdis.2013.08.001

Szabó, M. P. J., Nieri-Bastos, F. A., Spolidorio, M. G., Martins, T. F., Barbieri, A. M., & Labruna, M.

B. (2013). In vitro isolation from Amblyomma ovale (Acari: Ixodidae) and ecological aspects

of the Atlantic rainforest Rickettsia, the causative agent of a novel spotted fever rickettsiosis

in Brazil. Parasitology, 140(6), 719–728. https://doi.org/10.1017/S0031182012002065

https://link.springer.com/article/10.1007/s00436-020-07033-3#auth-Antje-Glass

https://link.springer.com/article/10.1007/s00436-020-07033-3#auth-Julia-Probst

95

Tadepalli, M., Vincent, G., Hii, S. F., Watharow, S., Graves, S., & Stenos, J. (2021). Molecular

Evidence of Novel Spotted Fever Group Rickettsia Species in Amblyomma albolimbatum

Ticks from the Shingleback Skink (Tiliqua rugosa) in Southern Western Australia.

Pathogens, 10(1). https://doi.org/10.3390/pathogens10010035

Tan, C. K., & Owens, L. (2000). Infectivity, transmission and 16S rRNA sequencing of a Rickettsia,

Coxiella cheraxi sp. Nov., from the freshwater crayfish Cherax quadricarinatus. Diseases of

Aquatic Organisms, 41(2), 115–122. https://doi.org/10.3354/dao041115

Thu, M. J., Qiu, Y., Matsuno, K., Kajihara, M., Mori-Kajihara, A., Omori, R., Monma, N., Chiba, K.,

Seto, J., Gokuden, M., Andoh, M., Oosako, H., Katakura, K., Takada, A., Sugimoto, C.,

Isoda, N., & Nakao, R. (2019). Diversity of spotted fever group Rickettsiae and their

association with host ticks in Japan. Scientific Reports, 9. https://doi.org/10.1038/s41598-

018-37836-5

Tomassone, L., Ceballos, L. A., Ragagli, C., Martello, E., De Sousa, R., Stella, M. C., & Mannelli,

A. (2017). Importance of Common Wall Lizards in the Transmission Dynamics of Tick-

Borne Pathogens in the Northern Apennine Mountains, Italy. Microbial Ecology, 74(4), 961–

968. https://doi.org/10.1007/s00248-017-0994-y

Unsworth, N. B., Stenos, J., McGregor, A. R., Dyer, J. R., & Graves, S. R. (2005). Not only

'Flinders Island' spotted fever. Pathology, 37(3), 242–245.

https://doi.org/10.1080/00313020500099247

van Nunen, S. (2015). Tick-induced allergies: Mammalian meat allergy, tick anaphylaxis and their

significance. Asia Pacific Allergy, 5(1), 3–16. https://doi.org/10.5415/apallergy.2015.5.1.3

Větrovský, T., & Baldrian, P. (2013). The Variability of the 16S rRNA Gene in Bacterial Genomes

and Its Consequences for Bacterial Community Analyses. PLoS ONE, 8(2).

https://doi.org/10.1371/journal.pone.0057923

Vilcins, I.-M. E., Fournier, P.-E., Old, J. M., & Deane, E. (2009). Evidence for the Presence of I

Francisella /I and Spotted Fever Group I Rickettsia /I DNA in the Tick I Amblyomma

fimbriatum /I (Acari: Ixodidae), Northern Territory, Australia. Journal of Medical

Entomology, 46(4), 926–933. https://doi.org/10.1603/033.046.0427

Vilcins, I.-M. E., Old, J. M., & Deane, E. (2009). Molecular detection of Rickettsia, Coxiella and

Rickettsiella DNA in three native Australian tick species. Experimental and Applied

Acarology, 49(3), 229–242. https://doi.org/10.1007/s10493-009-9260-4

Waldman, J. D., Rose, G., Turner, S. W., & Pappelbaum, S. J. (1978). Patency of the interatrial

septum in children undergoing cardiac catheterization. Catheterization and cardiovascular

diagnosis, 4(4), 427–431. https://doi.org/10.1002/ccd.1810040411

Whiley, H., Custance, G., Graves, S., Stenos, J., Taylor, M., Ross, K., & Gardner, M. G. (2016).

Rickettsia Detected in the Reptile Tick Bothriocroton hydrosauri from the Lizard Tiliqua

rugosa in South Australia. Pathogens, 5(2), 41. https://doi.org/10.3390/pathogens5020041

96

Whipp, M. J., Davis, J. M., Lum, G., de Boer, J., Zhou, Y., Bearden, S. W., Petersen, J. M., Chu,

M. C., & Hogg, G. (2003). Characterization of a novicida-like subspecies of Francisella

tularensis isolated in Australia. Journal of Medical Microbiology, 52(9), 839–842.

https://doi.org/10.1099/jmm.0.05245-0

Wikswo, M. E., Hu, R., Dasch, G. A., Krueger, L., Arugay, A., Jones, K., Hess, B., Bennett, S.,

Kramer, V., & Eremeeva, M. E. (2008). Detection and identification of spotted fever group

Rickettsiae in Dermacentor species from southern California. Journal of Medical

Entomology, 45(3), 509–516. https://doi.org/10.1603/0022-

2585(2008)45[509:daiosf]2.0.co;2

Woese, C. R., & Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: The primary

kingdoms. Proceedings of the National Academy of Sciences of the United States of

America, 74(11), 5088–5090.

Yadav, M. P., & Sethi, M. S. (1979). Poikilotherms as reservoirs of Q-fever (Coxiella burnetii) in

Uttar Pradesh. Journal of Wildlife Diseases, 15(1), 15–17. https://doi.org/10.7589/0090-

3558-15.1.15

Yang, B., Wang, Y., & Qian, P. Y. (2016). Sensitivity and correlation of hypervariable regions in

16S rRNA genes in phylogenetic analysis. BMC bioinformatics, 17, 135.

https://doi.org/10.1186/s12859-016-0992-y

97

Appendix

Table A1.1. Nanodrop results of 26 samples and extraction blank.

Sample ID ng/uL A260 260/280 260/230 Constant 2ul 3ul 4ul

F1 2.12 0.042 1.11 0.41 50 4.24 6.36 8.48

F2 4.39 0.088 1.24 0.25 50 8.78 13.17 17.56

F3 29.92 0.598 1.52 1.14 50 59.84 89.76 119.68

F4 5.21 0.104 1.37 0.2 50 10.42 15.63 20.84

F5 8.43 0.169 1.32 0.86 50 16.86 25.29 33.72

F6 161.06 3.221 1.98 1.83 50 322.12 483.18 644.24

F7 236.9 4.738 2.13 1.96 50 473.8 710.7 947.6

F8 271.58 5.432 2.01 2.13 50 543.16 814.74 1086.32

F9 312.18 6.244 1.91 1.8 50 624.36 936.54 1248.72

F10 30.89 0.618 2.03 1.84 50 61.78 92.67 123.56

SR11 297.27 5.945 2.13 2.25 50 594.54 891.81 1189.08

SR12 122.74 2.455 2.04 1.92 50 245.48 368.22 490.96

SR13 344.54 6.891 2.04 2.03 50 689.08 1033.62 1378.16

SR14 111.18 2.224 1.99 1.57 50 222.36 333.54 444.72

SR15 110.16 2.203 2.05 1.95 50 220.32 330.48 440.64

SR16 115.4 2.308 2.06 1.87 50 230.8 346.2 461.6

SR17 240.05 4.801 1.97 2.1 50 480.1 720.15 960.2

SR18 76.69 1.534 1.77 0.78 50 153.38 230.07 306.76

SR19 281.18 5.624 2.09 2.27 50 562.36 843.54 1124.72

SR20 136.28 2.726 2 2.1 50 272.56 408.84 545.12

C21 108.83 2.177 2.03 1.81 50 217.66 326.49 435.32

C22 85.54 1.711 1.82 1.55 50 171.08 256.62 342.16

C23 131.61 2.632 2.05 2.06 50 263.22 394.83 526.44

C24 209.9 4.198 2.07 2.15 50 419.8 629.7 839.6

C25 127.13 2.543 2.09 2.16 50 254.26 381.39 508.52

C26 34.91 0.698 1.97 1.7 50 69.82 104.73 139.64

E 0.79 0.016 -26.55 0.1 50 1.58 2.37 3.16

98

Table A1.2. 1,140 bp Coxiella 16S Maximum Likelihood fits of 24 different nucleotide substitution models.

Model #Param BIC AICc lnL (+I) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C

K2+G+I 94 10250 9416 -4614 0.478 0.408 4.537 0.25 0.25 0.25 0.25 0.02 0.02 0.2 0.02 0.2 0.02 0.02 0.2 0.02 0.2 0.02 0.02

HKY+G+I 97 10263 9402 -4604 0.486 0.407 4.825 0.26 0.212 0.22 0.308 0.02 0.02 0.25 0.02 0.18 0.03 0.02 0.17 0.03 0.21 0.02 0.02

T92+G+I 95 10268 9425 -4617 0.482 0.41 4.588 0.236 0.236 0.264 0.264 0.02 0.02 0.22 0.02 0.22 0.02 0.02 0.19 0.02 0.19 0.02 0.02

TN93+G+I 98 10270 9400 -4602 0.478 0.405 4.646 0.26 0.212 0.22 0.308 0.02 0.02 0.22 0.02 0.21 0.03 0.02 0.2 0.03 0.19 0.02 0.02

GTR+G+I 101 10275 9379 -4588 0.493 0.421 4.477 0.26 0.212 0.22 0.308 0.03 0.02 0.22 0.04 0.21 0.03 0.03 0.2 0 0.19 0.02 0

K2+G 93 10337 9511 -4662 n/a 0.374 3.634 0.25 0.25 0.25 0.25 0.03 0.03 0.2 0.03 0.2 0.03 0.03 0.2 0.03 0.2 0.03 0.03

HKY+G 96 10356 9504 -4656 n/a 0.37 3.776 0.26 0.212 0.22 0.308 0.02 0.02 0.24 0.03 0.17 0.03 0.03 0.17 0.03 0.2 0.02 0.02

T92+G 94 10357 9522 -4667 n/a 0.374 3.65 0.236 0.236 0.264 0.264 0.03 0.03 0.21 0.03 0.21 0.03 0.03 0.19 0.03 0.19 0.03 0.03

TN93+G 97 10358 9497 -4651 n/a 0.378 3.655 0.26 0.212 0.22 0.308 0.02 0.02 0.2 0.03 0.22 0.03 0.03 0.21 0.03 0.17 0.02 0.02

GTR+G 100 10367 9479 -4639 n/a 0.374 3.598 0.26 0.212 0.22 0.308 0.04 0.03 0.19 0.04 0.21 0.04 0.03 0.21 0.01 0.16 0.03 0.01

K2+I 93 10592 9766 -4790 0.373 n/a 3.298 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

TN93+I 97 10610 9749 -4777 0.373 n/a 3.312 0.26 0.212 0.22 0.308 0.02 0.03 0.19 0.03 0.22 0.04 0.03 0.21 0.04 0.16 0.02 0.03

T92+I 94 10613 9779 -4795 0.373 n/a 3.307 0.236 0.236 0.264 0.264 0.03 0.03 0.2 0.03 0.2 0.03 0.03 0.18 0.03 0.18 0.03 0.03

HKY+I 96 10616 9764 -4786 0.373 n/a 3.373 0.26 0.212 0.22 0.308 0.02 0.03 0.24 0.03 0.17 0.04 0.03 0.16 0.04 0.2 0.02 0.03

GTR+I 100 10638 9750 -4775 0.373 n/a 2.823 0.26 0.212 0.22 0.308 0.04 0.03 0.19 0.04 0.2 0.04 0.03 0.19 0.03 0.16 0.03 0.02

JC+G+I 93 10739 9914 -4864 0.462 0.513 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G 92 10801 9984 -4900 n/a 0.409 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

K2 92 10862 10045 -4930 n/a n/a 3.121 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

TN93 96 10879 10026 -4917 n/a n/a 3.122 0.26 0.212 0.22 0.308 0.03 0.03 0.18 0.03 0.21 0.04 0.03 0.21 0.04 0.16 0.03 0.03

T92 93 10884 10058 -4936 n/a n/a 3.122 0.236 0.236 0.264 0.264 0.03 0.03 0.2 0.03 0.2 0.03 0.03 0.18 0.03 0.18 0.03 0.03

HKY 95 10892 10049 -4929 n/a n/a 3.12 0.26 0.212 0.22 0.308 0.03 0.03 0.23 0.03 0.17 0.04 0.03 0.16 0.04 0.2 0.03 0.03

GTR 99 10907 10029 -4915 n/a n/a 2.686 0.26 0.212 0.22 0.308 0.03 0.03 0.18 0.04 0.2 0.04 0.03 0.19 0.04 0.16 0.03 0.03

JC+I 92 11023 10206 -5011 0.373 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 91 11280 10472 -5145 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

99

Table A2.3. 1,140 bp Coxiella 16S genetic distance matrix.

Sample C23

ex.

A. A

lbolim

batum

(PI 1

715)

C. b

urne

tii str. N

amibia gen

ome (C

P0075

55.1 )

C. b

urne

tii Dug

way

5J1

08-1

11 (C

P0007

33.1 )

C. b

urne

tii Cb1

75 G

uyan

a (H

G82

5990

.3)

C. b

urne

tii RSA 3

31 (C

P0008

90.1)

C. b

urne

tii Cbu

K Q15

4 (C

P0010

20.1 )

C. b

urne

tii RSA 4

93 (A

E0168

28.3)

C. b

urne

tii M

SU G

oat Q

177 (C

P0181

50.1 )

C. b

urne

tii str. nine

mile

pha

se II (C

P0351

12.1)

C. b

urne

tii str. Hen

zerlin

g (C

P0145

59.1)

C. b

urne

tii str. Sch

perling

(CP01

4563

)

Legion

ella pne

umop

hila

(EU22

1243

)

Can

dida

tus C. m

udro

wiae

(CP02

4961

.1)

C. e

ndos

ymbion

t ex.

R. tur

anicu

s (J

Q48

0822

.1)

C. e

ndos

ymbion

t ex.

R. s

angu

ineu

s (K

U89

2220

)

C. en

dosy

mbion

t ex.

R. g

eigy

i (KP99

4837

)

C. e

ndos

ymbion

t ex.

R. e

verts

i (KP99

4835

)

C. e

ndos

ymbion

t ex.

R. d

ecolor

atus

(KP99

4833

)

C. en

dosy

mbion

t ex. R. b

ursa

(KP99

4831

)

C. en

dosy

mbion

t ex. R. a

ustra

lis (K

P9948

29)

C. en

dosy

mbion

t ex. R. a

nnulatus

(KP99

4827

)

C. en

dosy

mbion

t ex. O. s

phen

iscus

(KP99

4799

)

C. en

dosy

mbion

t ex. O. s

onra

i (K

P9947

97)

C. en

dosy

mbion

t ex. O. ros

tratus

(KP99

4791

)

C. en

dosy

mbion

t ex. O. p

eruv

ianu

s (K

P9947

89)

C. e

ndos

ymbion

t ex.

O. o

cciden

talis

(KP99

4787

)

C. en

dosy

mbion

t ex. O. m

aritim

us (K

P9947

83)

C. e

ndos

ymbion

t ex.

O. k

airo

uane

nsis

(KP99

4779

)

C. e

ndos

ymbion

t ex.

O. e

rratic

us (K

P9947

77)

C. e

ndos

ymbion

t ex. O. d

enm

arki

(KP99

4776

)

C. en

dosy

mbion

t ex. O. c

apen

sis (K

P9947

74)

C. e

ndos

ymbion

t ex.

O. b

rasil

iens

is (K

P9947

72)

C. en

dosy

mbion

t ex. O. a

mblus

(KP99

4770

)

C. e

ndos

ymbion

t ex.

I. ricin

us (K

P9948

25)

C. en

dosy

mbion

t ex. H. s

him

oga

(KC17

0760

.1)

C. en

dosy

mbion

t ex. H. o

besa

(KC17

0759

.1)

C. e

ndos

ymbion

t ex.

H. la

gran

gei (K

C17

0756

.1)

C. e

ndos

ymbion

t ex. H. lo

ngico

rnis

(AY34

2035

.1)

C. e

ndos

ymbion

t e

x. A

rg. m

onac

hus

(KP98

5446

)

C. e

ndos

ymbion

t ex.

Am. tho

lloni

(MN99

5410

)

C. en

dosy

mbion

t ex. Am

. splen

didu

m (M

N99

5413

)

C. en

dosy

mbion

t ex. Am

. nap

onen

se (M

N99

5401

)

C. en

dosy

mbion

t ex. Am

. latep

unctatum

(MN99

5399

)

C. en

dosy

mbion

t ex. Am

. coe

lebs

(MN99

5392

)

C. en

dosy

mbion

t ex.

A.C

ajen

nens

e (K

P9854

82.1)

C. e

ndos

ymbion

t ex.

Am. a

mer

icanu

m (A

Y9398

24)

C. c

hera

xi (N

R 116

014)

Sample C23 ex. A. Albolimbatum (PI 1715)

C. burnetii str. Namibia genome (CP007555.1 ) 0.00

C. burnetii Dugway 5J108-111 (CP000733.1 ) 0.00 0.00

C. burnetii Cb175 Guyana (HG825990.3) 0.00 0.00 0.00

C. burnetii RSA 331 (CP000890.1) 0.00 0.00 0.00 0.00

C. burnetii CbuK Q154 (CP001020.1 ) 0.00 0.00 0.00 0.00 0.00

C. burnetii RSA 493 (AE016828.3) 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii MSU Goat Q177 (CP018150.1 ) 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii str. nine mile phase II (CP035112.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii str. Henzerling (CP014559.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii str. Schperling (CP014563) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Legionella pneumophila (EU221243) 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15

Candidatus C. mudrowiae (CP024961.1) 0.04 0.04 0.03 0.03 0.03 0.04 0.03 0.04 0.03 0.03 0.04 0.17

C. endosymbiont ex. R. turanicus (JQ480822.1) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.16 0.00

C. endosymbiont ex. R. sanguineus (KU892220) 0.04 0.04 0.04 0.04 0.03 0.04 0.04 0.04 0.04 0.03 0.04 0.17 0.00 0.00

C. endosymbiont ex. R. geigyi (KP994837) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03

C. endosymbiont ex. R. evertsi (KP994835) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03 0.01

C. endosymbiont ex. R. decoloratus (KP994833) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03 0.01 0.00

C. endosymbiont ex. R. bursa (KP994831) 0.05 0.05 0.04 0.05 0.04 0.05 0.04 0.05 0.04 0.04 0.05 0.17 0.04 0.04 0.04 0.02 0.02 0.02

C. endosymbiont ex. R. australis (KP994829) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 0.04 0.04 0.04 0.01 0.01 0.02 0.03

C. endosymbiont ex. R. annulatus (KP994827) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.03 0.03 0.03 0.00 0.01 0.01 0.02 0.01

C. endosymbiont ex. O. spheniscus (KP994799) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.16 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05

C. endosymbiont ex. O. sonrai (KP994797) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04

C. endosymbiont ex. O. rostratus (KP994791) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.04 0.04 0.04 0.04 0.05 0.04 0.05 0.04 0.02 0.03

C. endosymbiont ex. O. peruvianus (KP994789) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.05 0.05 0.04 0.04 0.04 0.05 0.05 0.04 0.00 0.03 0.02

C. endosymbiont ex. O. occidentalis (KP994787) 0.03 0.03 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.15 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04 0.00 0.03 0.03

C. endosymbiont ex. O. maritimus (KP994783) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.01 0.04 0.02 0.01 0.04

C. endosymbiont ex. O. kairouanensis (KP994779) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.04 0.04 0.04 0.05 0.05 0.05 0.03 0.05 0.05 0.04 0.04 0.04 0.04 0.04 0.04

C. endosymbiont ex. O. erraticus (KP994777) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04 0.00 0.03 0.03 0.00 0.03 0.04

C. endosymbiont ex. O. denmarki (KP994776) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.01 0.03 0.02 0.01 0.03 0.01 0.04 0.03

C. endosymbiont ex. O. capensis (KP994774) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.01 0.03 0.02 0.01 0.03 0.01 0.04 0.03 ?

C. endosymbiont ex. O. brasiliensis (KP994772) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.04 0.04 0.05 0.04 0.05 0.05 0.05 0.05 0.02 0.03 0.02 0.02 0.03 0.02 0.04 0.03 0.02 0.02

C. endosymbiont ex. O. amblus (KP994770) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.01 0.03 0.02 0.01 0.04 0.01 0.04 0.03 0.01 0.01 0.02

C. endosymbiont ex. I. ricinus (KP994825) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.16 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.05 0.04 0.04 0.03 0.03 0.03 0.03 0.04 0.02 0.03 0.03 0.03 0.03 0.03

C. endosymbiont ex. H. shimoga (KC170760.1) 0.05 0.05 0.04 0.04 0.05 0.05 0.04 0.05 0.04 0.05 0.05 0.17 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.04 0.05 0.04 0.05 0.05 0.03 0.05 0.05 0.05 0.05 0.05 0.03

C. endosymbiont ex. H. obesa (KC170759.1) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.05 0.04 0.05 0.04 0.04 0.04 0.04 0.05 0.03 0.04 0.04 0.04 0.04 0.04 0.02 0.02

C. endosymbiont ex. H. lagrangei (KC170756.1) 0.05 0.05 0.04 0.04 0.05 0.05 0.04 0.05 0.04 0.05 0.05 0.18 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.04 0.05 0.05 0.04 0.05 0.04 0.04 0.05 0.05 0.05 0.05 0.03 0.02 0.02

C. endosymbiont ex. H. longicornis (AY342035.1) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.17 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.04 0.04 0.04 0.04 0.05 0.03 0.04 0.04 0.04 0.05 0.05 0.02 0.02 0.01 0.00

C. endosymbiont ex. Arg. monachus (KP985446) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.16 0.04 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.02 0.03 0.02 0.01 0.03 0.02 0.04 0.03 0.02 0.02 0.02 0.02 0.03 0.04 0.04 0.05 0.04

C. endosymbiont ex. Am. tholloni (MN995410) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.16 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.05 0.04 0.05 0.03 0.04 0.04 0.03 0.04 0.03 0.03 0.04 0.04 0.04 0.04 0.02 0.03 0.03 0.03 0.03 0.04

C. endosymbiont ex. Am. splendidum (MN995413) 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.17 0.06 0.06 0.06 0.07 0.06 0.07 0.06 0.07 0.07 0.06 0.05 0.06 0.06 0.05 0.06 0.04 0.05 0.06 0.06 0.06 0.06 0.04 0.05 0.05 0.05 0.05 0.05 0.04

C. endosymbiont ex. Am. naponense (MN995401) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.16 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.03 0.04 0.02 0.04 0.04 0.02 0.04 0.04 0.02 0.04 0.04 0.04 0.04 0.03 0.05 0.04 0.05 0.04 0.04 0.04 0.06

C. endosymbiont ex. Am. latepunctatum (MN995399) 0.04 0.04 0.03 0.03 0.03 0.04 0.03 0.04 0.03 0.03 0.04 0.16 0.03 0.03 0.03 0.04 0.03 0.04 0.04 0.04 0.04 0.05 0.02 0.04 0.04 0.02 0.04 0.04 0.02 0.04 0.04 0.04 0.04 0.03 0.05 0.04 0.05 0.05 0.04 0.04 0.06 0.03

C. endosymbiont ex. Am. coelebs (MN995392) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.15 0.04 0.04 0.03 0.04 0.03 0.04 0.04 0.04 0.04 0.05 0.03 0.04 0.04 0.03 0.05 0.04 0.03 0.04 0.04 0.05 0.05 0.04 0.05 0.04 0.04 0.04 0.04 0.04 0.05 0.03 0.03

C. endosymbiont ex. A.Cajennense (KP985482.1) 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.17 0.07 0.07 0.07 0.06 0.06 0.06 0.07 0.06 0.06 0.07 0.06 0.07 0.07 0.06 0.07 0.06 0.06 0.06 0.06 0.07 0.07 0.06 0.07 0.07 0.07 0.07 0.07 0.06 0.07 0.07 0.06 0.05

C. endosymbiont ex. Am. americanum (AY939824) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.18 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.06 0.07 0.08 0.06 0.07 0.07 0.06 0.08 0.07 0.06 0.08 0.08 0.08 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.06 0.06 0.03

C. cheraxi (NR 116014) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.14 0.05 0.05 0.05 0.06 0.06 0.06 0.07 0.06 0.06 0.05 0.04 0.05 0.05 0.04 0.05 0.06 0.04 0.04 0.04 0.05 0.05 0.05 0.06 0.05 0.06 0.05 0.04 0.05 0.07 0.05 0.05 0.05 0.08 0.08

100

Table A3.4. Coxiella GroEL Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 64 7785 7287 -3579 n/a 0.327 3.164 0.286 0.286 0.214 0.214 0.03 0.03 0.16 0.03 0.16 0.03 0.03 0.22 0.03 0.22 0.03 0.03

T92+G+I 65 7791 7285 -3577 0.248 0.573 3.142 0.286 0.286 0.214 0.214 0.03 0.03 0.16 0.03 0.16 0.03 0.03 0.22 0.03 0.22 0.03 0.03

HKY+G 66 7794 7280 -3574 n/a 0.327 3.238 0.299 0.273 0.171 0.258 0.03 0.02 0.2 0.04 0.13 0.03 0.04 0.21 0.03 0.23 0.03 0.02

HKY+G+I 67 7799 7278 -3572 0.262 0.598 3.223 0.299 0.273 0.171 0.258 0.03 0.02 0.2 0.04 0.13 0.03 0.04 0.21 0.03 0.23 0.03 0.02

TN93+G 67 7800 7279 -3572 n/a 0.328 3.184 0.299 0.273 0.171 0.258 0.03 0.02 0.17 0.04 0.15 0.03 0.04 0.24 0.03 0.2 0.03 0.02

TN93+G+I 68 7806 7277 -3570 0.243 0.566 3.168 0.299 0.273 0.171 0.258 0.03 0.02 0.17 0.04 0.15 0.03 0.04 0.24 0.03 0.2 0.03 0.02

K2+G 63 7814 7324 -3599 n/a 0.332 3.073 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

K2+G+I 64 7821 7323 -3597 0.239 0.563 3.053 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

GTR+G 70 7822 7278 -3569 n/a 0.327 3.197 0.299 0.273 0.171 0.258 0.02 0.03 0.17 0.02 0.15 0.03 0.05 0.24 0.03 0.2 0.04 0.02

GTR+G+I 71 7829 7276 -3567 0.242 0.565 3.178 0.299 0.273 0.171 0.258 0.02 0.03 0.17 0.02 0.15 0.03 0.05 0.24 0.03 0.2 0.04 0.02

T92+I 64 7857 7359 -3615 0.497 n/a 2.684 0.286 0.286 0.214 0.214 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.21 0.03 0.21 0.04 0.03

HKY+I 66 7865 7351 -3609 0.497 n/a 2.763 0.299 0.273 0.171 0.258 0.04 0.02 0.19 0.04 0.13 0.03 0.04 0.2 0.03 0.22 0.04 0.02

TN93+I 67 7873 7352 -3609 0.497 n/a 2.737 0.299 0.273 0.171 0.258 0.04 0.02 0.18 0.04 0.13 0.03 0.04 0.21 0.03 0.21 0.04 0.02

K2+I 63 7888 7398 -3636 0.497 n/a 2.598 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

GTR+I 70 7897 7352 -3606 0.496 n/a 2.734 0.299 0.273 0.171 0.258 0.02 0.03 0.18 0.03 0.13 0.04 0.05 0.21 0.03 0.21 0.05 0.02

JC+G 62 8167 7684 -3780 n/a 0.389 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 63 8172 7682 -3778 0.279 0.782 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 62 8219 7737 -3806 0.491 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

T92 63 8253 7763 -3818 n/a n/a 2.207 0.286 0.286 0.214 0.214 0.04 0.03 0.15 0.04 0.15 0.03 0.04 0.2 0.03 0.2 0.04 0.03

HKY 65 8265 7760 -3815 n/a n/a 2.216 0.299 0.273 0.171 0.258 0.04 0.03 0.18 0.05 0.12 0.04 0.05 0.19 0.04 0.21 0.04 0.03

TN93 66 8266 7752 -3810 n/a n/a 2.215 0.299 0.273 0.171 0.258 0.04 0.03 0.15 0.05 0.14 0.04 0.05 0.22 0.04 0.18 0.04 0.03

K2 62 8272 7789 -3833 n/a n/a 2.181 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

GTR 69 8292 7755 -3808 n/a n/a 2.216 0.299 0.273 0.171 0.258 0.04 0.03 0.15 0.04 0.14 0.05 0.06 0.22 0.03 0.18 0.05 0.02

JC 61 8573 8099 -3988 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

101

Table A4.5. Coxiella GroEL genetic distance matrix.

C. b

urne

tii Cbu

K Q15

4 (C

P0010

20.1 )

C. b

urne

tii str. N

amibia gen

ome (C

P0075

55.1 )

C. b

urne

tii M

SU G

oat Q

177 (C

P0181

50.1 )

C. b

urne

tii str. Sch

perling

(CP01

4563

)

C. b

urne

tii str. nine

mile

pha

se II (C

P0351

12.1)

C. b

urne

tii str. Hen

zerlin

g (C

P0145

59.1)

C. b

urne

tii RSA 3

31 (C

P0008

90.1)

C. b

urne

tii RSA 4

93 (A

E0168

28.3)

C. b

urne

tii Dug

way

5J1

08-1

11 (C

P0007

33.1 )

C. b

urne

tii Cb1

75 G

uyan

a (H

G82

5990

.3)

C. en

dosy

mbion

t ex.

A.C

ajen

nens

e (K

P9854

82.1)

C. e

ndos

ymbion

t e

x. A

rg. m

onac

hus

(KP98

5446

)

C. en

dosy

mbion

t ex.

D. m

argina

tus

(KP98

5488

.1)

C. en

dosy

mbion

t ex

. D. s

ilvar

um

(KP98

5490

)

C. en

dosy

mbion

t ex.

H. p

unctata

(KP98

5492

)

C. en

dosy

mbion

t ex.

O. a

mblus

(KP98

5447

)

C. en

dosy

mbion

t ex. O. b

rasil

iens

is (K

P9854

49)

C. en

dosy

mbion

t ex. O.C

apen

sis

(KP98

5451

)

C. en

dosy

mbion

t ex.

O. e

rratic

us

(KP98

5454

)

C. e

ndos

ymbion

t e

x. O

. kairo

uane

nsis

(KP98

5457

)

C. en

dosy

mbion

t ex.

O. m

aritim

us (K

P9854

59)

C. en

dosy

mbion

t ex.

O. m

aroc

anus

(KP98

5462

)

C. e

ndos

ymbion

t ex.

O. o

cciden

talis

(KP98

5464

)

C. en

dosy

mbion

t ex. O. p

eruv

ianu

s (K

P9854

66)

C. e

ndos

ymbion

t ex.

O. ros

tratus

(KP98

5468

)

C. e

ndos

ymbion

t ex. O. rup

estris

(KP98

5472

)

C. en

dosy

mbion

t ex. O. s

onra

i (K

P9854

74)

C. en

dosy

mbion

t ex.

R. p

usillu

s (K

P9855

18)

C. en

dosy

mbion

t ex.

R. s

angu

ineu

s (M

K1192

08)

C. en

dosy

mbion

t ex. R. tur

anicu

s (K

P9855

22)

Sample C23

ex.

A. A

lbolim

batum

(PI 1

715)

Legion

ella pne

umop

hila

(EU22

1243

)

C. burnetii CbuK Q154 (CP001020.1 )

C. burnetii str. Namibia genome (CP007555.1 ) 0.00

C. burnetii MSU Goat Q177 (CP018150.1 ) 0.00 0.00

C. burnetii str. Schperling (CP014563) 0.00 0.00 0.00

C. burnetii str. nine mile phase II (CP035112.1) 0.00 0.00 0.00 0.00

C. burnetii str. Henzerling (CP014559.1) 0.00 0.00 0.00 0.00 0.00

C. burnetii RSA 331 (CP000890.1) 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii RSA 493 (AE016828.3) 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii Dugway 5J108-111 (CP000733.1 ) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii Cb175 Guyana (HG825990.3) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. endosymbiont ex. A.Cajennense (KP985482.1)0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39 0.39

C. endosymbiont ex. Arg. monachus (KP985446) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.39

C. endosymbiont ex. D. marginatus (KP985488.1)0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.24 0.32

C. endosymbiont ex. D. silvarum (KP985490) 0.29 0.29 0.29 0.29 0.30 0.29 0.29 0.30 0.30 0.30 0.27 0.31 0.04

C. endosymbiont ex. H. punctata (KP985492) 0.32 0.32 0.32 0.32 0.33 0.33 0.33 0.33 0.33 0.33 0.28 0.34 0.20 0.22

C. endosymbiont ex. O. amblus (KP985447) 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.04 0.38 0.03 0.32 0.32 0.33

C. endosymbiont ex. O. brasiliensis (KP985449)0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.42 0.08 0.32 0.32 0.35 0.07

C. endosymbiont ex. O.Capensis (KP985451) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.39 0.04 0.33 0.33 0.34 0.02 0.07

C. endosymbiont ex. O. erraticus (KP985454) 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.29 0.29 0.17 0.18 0.18 0.28 0.30 0.29

C. endosymbiont ex. O. kairouanensis (KP985457)0.29 0.29 0.29 0.29 0.28 0.29 0.29 0.28 0.28 0.28 0.38 0.30 0.32 0.35 0.31 0.30 0.31 0.31 0.28

C. endosymbiont ex. O. maritimus (KP985459) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.37 0.03 0.32 0.32 0.31 0.03 0.07 0.02 0.28 0.30

C. endosymbiont ex. O. marocanus (KP985462) 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.29 0.30 0.17 0.18 0.18 0.28 0.30 0.29 0.00 0.27 0.28

C. endosymbiont ex. O. occidentalis (KP985464)0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.37 0.29 0.21 0.22 0.23 0.29 0.30 0.29 0.08 0.28 0.28 0.09

C. endosymbiont ex. O. peruvianus (KP985466) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.38 0.04 0.31 0.32 0.32 0.03 0.07 0.03 0.28 0.29 0.02 0.28 0.28

C. endosymbiont ex. O. rostratus (KP985468) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.38 0.04 0.33 0.31 0.31 0.04 0.07 0.03 0.28 0.30 0.02 0.28 0.28 0.03

C. endosymbiont ex. O. rupestris (KP985472) 0.26 0.26 0.26 0.26 0.25 0.26 0.26 0.25 0.25 0.25 0.40 0.27 0.33 0.36 0.30 0.25 0.28 0.26 0.28 0.06 0.25 0.28 0.29 0.26 0.26

C. endosymbiont ex. O. sonrai (KP985474) 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.27 0.29 0.30 0.18 0.18 0.18 0.28 0.30 0.29 0.01 0.27 0.28 0.00 0.09 0.28 0.28 0.29

C. endosymbiont ex. R. pusillus (KP985518) 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.30 0.29 0.32 0.17 0.19 0.17 0.30 0.32 0.32 0.09 0.29 0.30 0.09 0.15 0.31 0.32 0.30 0.08

C. endosymbiont ex. R. sanguineus (MK119208) 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.29 0.31 0.18 0.19 0.17 0.28 0.31 0.30 0.07 0.30 0.28 0.07 0.14 0.30 0.30 0.30 0.07 0.02

C. endosymbiont ex. R. turanicus (KP985522) 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.28 0.29 0.31 0.17 0.18 0.18 0.29 0.32 0.31 0.07 0.30 0.29 0.07 0.14 0.30 0.31 0.29 0.07 0.02 0.01

Sample C23 ex. A. Albolimbatum (PI 1715) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.39 0.04 0.30 0.29 0.32 0.04 0.07 0.03 0.27 0.29 0.03 0.27 0.28 0.03 0.03 0.26 0.27 0.30 0.28 0.28

Legionella pneumophila (EU221243) 0.39 0.39 0.39 0.39 0.38 0.39 0.39 0.38 0.38 0.38 0.46 0.40 0.38 0.41 0.43 0.38 0.40 0.39 0.37 0.36 0.38 0.37 0.37 0.39 0.39 0.37 0.37 0.35 0.36 0.36 0.39

102

Table A1.6. Coxiella concatenated 16S and GroEL Maximum Likelihood fits of 24 different nucleotide substitution models.


K2+G 49 13197 12773 -6337 n/a 0.203 2.603 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

K2+G+I 50 13197 12765 -6332 0.384 0.446 2.651 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

HKY+G 52 13201 12752 -6324 n/a 0.201 2.708 0.27 0.231 0.205 0.294 0.03 0.03 0.21 0.04 0.15 0.04 0.04 0.17 0.04 0.2 0.03 0.03

HKY+G+I 53 13202 12743 -6319 0.39 0.449 2.753 0.27 0.231 0.205 0.294 0.03 0.03 0.21 0.04 0.15 0.04 0.04 0.17 0.04 0.2 0.03 0.03

TN93+G+I 54 13202 12735 -6314 0.381 0.444 2.67 0.27 0.231 0.205 0.294 0.03 0.03 0.17 0.04 0.18 0.04 0.04 0.21 0.04 0.16 0.03 0.03

TN93+G 53 13203 12745 -6319 n/a 0.206 2.62 0.27 0.231 0.205 0.294 0.03 0.03 0.18 0.04 0.18 0.04 0.04 0.2 0.04 0.16 0.03 0.03

T92+G 50 13207 12774 -6337 n/a 0.203 2.604 0.251 0.251 0.249 0.249 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

T92+G+I 51 13207 12766 -6332 0.384 0.446 2.651 0.251 0.251 0.249 0.249 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

GTR+G+I 57 13216 12723 -6304 0.379 0.446 2.64 0.27 0.231 0.205 0.294 0.03 0.04 0.18 0.04 0.18 0.05 0.05 0.21 0.02 0.16 0.04 0.01

GTR+G 56 13237 12752 -6320 n/a 0.213 2.22 0.27 0.231 0.205 0.294 0.04 0.04 0.18 0.04 0.16 0.05 0.05 0.18 0.04 0.17 0.04 0.03

K2+I 49 13250 12827 -6364 0.661 n/a 2.398 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

HKY+I 52 13254 12804 -6350 0.663 n/a 2.511 0.27 0.231 0.205 0.294 0.03 0.03 0.21 0.04 0.14 0.04 0.04 0.16 0.04 0.19 0.03 0.03

TN93+I 53 13260 12802 -6348 0.66 n/a 2.437 0.27 0.231 0.205 0.294 0.03 0.03 0.19 0.04 0.16 0.04 0.04 0.19 0.04 0.17 0.03 0.03

T92+I 50 13260 12828 -6364 0.661 n/a 2.399 0.251 0.251 0.249 0.249 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

GTR+I 56 13298 12814 -6351 0.658 n/a 2.048 0.27 0.231 0.205 0.294 0.04 0.04 0.19 0.04 0.14 0.05 0.05 0.16 0.05 0.17 0.04 0.03

JC+G 48 13570 13155 -6529 n/a 0.242 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 49 13574 13150 -6526 0.351 0.505 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 48 13613 13197 -6551 0.648 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

TN93 52 13777 13327 -6612 n/a n/a 1.805 0.27 0.231 0.205 0.294 0.04 0.04 0.15 0.05 0.17 0.05 0.05 0.19 0.05 0.14 0.04 0.04

K2 48 13779 13364 -6634 n/a n/a 1.802 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

T92 49 13789 13366 -6634 n/a n/a 1.802 0.251 0.251 0.249 0.249 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

HKY 51 13791 13350 -6624 n/a n/a 1.803 0.27 0.231 0.205 0.294 0.04 0.04 0.19 0.05 0.13 0.05 0.05 0.15 0.05 0.17 0.04 0.04

GTR 55 13809 13334 -6612 n/a n/a 1.555 0.27 0.231 0.205 0.294 0.05 0.04 0.15 0.06 0.15 0.06 0.05 0.17 0.05 0.14 0.05 0.04

JC 47 14078 13672 -6789 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

103

Table A1.7. Coxiella concatenated 16S and GroEL genetic distance matrix.

C. b

urne

tii M

SU G

oat Q

177 (C

P0181

50.1)

C. b

urne

tii C

b175

Guy

ana (H

G82

5990

.3)

C. b

urne

tii Cbu

K Q15

4 (C

P0010

20.1)

C. b

urne

tii D

ugway

5J1

08-1

11 (C

P0007

33.1)

C. b

urne

tii RSA 4

93 (A

E0168

28.3)

C. b

urne

tii str. N

amibia gen

ome (C

P0075

55.1)

C. b

urne

tii str. H

eizb

erg (C

P0145

61.1)

C. b

urne

tii str. H

enze

rling

(CP01

4559

.1)

C. b

urne

tii str. nine

mile

pha

se II (C

P0351

12.1)

C. en

dosy

mbion

t ex.

Am. c

ajen

nens

e(K

P9854

82.1)

C. e

ndos

ymbion

t ex.

Argas

mon

achu

s

(KP98

5446

)

C. en

dosy

mbion

t ex.

O. a

mblus

(KP99

4770

)

C. en

dosy

mbion

t ex.

O. b

rasil

iens

is (K

P9947

72)

C. en

dosy

mbion

t ex.

O. c

apen

sis (K

P9947

74)

C. e

ndos

ymbion

t ex.

O. e

rratic

us (K

P9947

77)

C. en

dosy

mbion

t ex.

O. k

airo

uane

nsis

(KP99

4779

)

C. e

ndos

ymbion

t ex.

O. m

aritim

us (K

P9854

59)

C. e

ndos

ymbion

t ex.

O. o

cciden

talis

(KP98

5464

)

C. en

dosy

mbion

t ex.

O. p

eruv

ianu

s (K

P9854

66)

C. e

ndos

ymbion

t ex.

O. ros

tratus

(KP99

4791

)

C. en

dosy

mbion

t ex.

O. s

onra

i (KP99

4797

)

C. e

ndos

ymbion

t ex.

R. s

angu

ineu

s (K

U89

2220

)

C. e

ndos

ymbion

t ex.

R. tur

anicus

(JQ48

0822

.1)

Legion

ella pne

umop

hila

230

0/99

(EU22

1243

)

Sample C23

ex.

Am

. albolim

batum

(PI 1

715)

C. burnetii MSU Goat Q177 (CP018150.1)

C. burnetii Cb175 Guyana (HG825990.3) 0.00

C. burnetii CbuK Q154 (CP001020.1) 0.00 0.00

C. burnetii Dugway 5J108-111 (CP000733.1) 0.00 0.00 0.00

C. burnetii RSA 493 (AE016828.3) 0.00 0.00 0.00 0.00

C. burnetii str. Namibia genome (CP007555.1) 0.00 0.00 0.00 0.00 0.00

C. burnetii str. Heizberg (CP014561.1) 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii str. Henzerling (CP014559.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. burnetii str. nine mile phase II (CP035112.1) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

C. endosymbiont ex. Am. cajennense (KP985482.1) 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.15

C. endosymbiont ex. Argas monachus (KP985446) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.16

C. endosymbiont ex. O. amblus (KP994770) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02

C. endosymbiont ex. O. brasiliensis (KP994772) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.17 0.04 0.04

C. endosymbiont ex. O. capensis (KP994774) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.01 0.04

C. endosymbiont ex. O. erraticus (KP994777) 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.13 0.10 0.10 0.10 0.10

C. endosymbiont ex. O. kairouanensis (KP994779) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.15 0.11 0.11 0.11 0.11 0.10

C. endosymbiont ex. O. maritimus (KP985459) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.02 0.04 0.02 0.10 0.11

C. endosymbiont ex. O. occidentalis (KP985464) 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.15 0.10 0.10 0.10 0.10 0.03 0.10 0.10

C. endosymbiont ex. O. peruvianus (KP985466) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.01 0.04 0.01 0.10 0.11 0.01 0.10

C. endosymbiont ex. O. rostratus (KP994791) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.15 0.02 0.03 0.04 0.03 0.10 0.11 0.02 0.10 0.02

C. endosymbiont ex. O. sonrai (KP994797) 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.13 0.10 0.10 0.10 0.10 0.00 0.10 0.10 0.03 0.10 0.10

C. endosymbiont ex. R. sanguineus (KU892220) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.13 0.11 0.11 0.11 0.11 0.04 0.11 0.11 0.05 0.11 0.11 0.03

C. endosymbiont ex. R. turanicus (JQ480822.1) 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.13 0.11 0.11 0.11 0.12 0.04 0.11 0.11 0.06 0.12 0.11 0.04 0.00

Legionella pneumophila 2300/99 (EU221243) 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.22 0.25 0.23 0.22 0.23 0.22 0.21 0.22 0.22 0.21 0.22 0.22 0.21 0.22 0.22

Sample C23 ex. Am. albolimbatum (PI 1715) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.15 0.02 0.02 0.03 0.02 0.09 0.10 0.02 0.09 0.02 0.02 0.09 0.10 0.10 0.22

104

Table A1.8. Francisella 16S Maximum Likelihood fits of 24 different nucleotide substitution models.


K2+G 103 6791 5877 -2835 n/a 0.35 2.41 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

K2+G+I 104 6801 5879 -2835 0.28 0.64 2.45 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

HKY+G 106 6802 5862 -2825 n/a 0.34 2.47 0.268 0.217 0.207 0.309 0.03 0.03 0.22 0.04 0.15 0.05 0.04 0.15 0.05 0.19 0.03 0.03

T92+G 104 6803 5880 -2836 n/a 0.35 2.42 0.242 0.242 0.258 0.258 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.17 0.04 0.17 0.04 0.04

TN93+G 107 6803 5855 -2820 n/a 0.35 2.44 0.268 0.217 0.207 0.309 0.03 0.03 0.16 0.04 0.2 0.04 0.04 0.21 0.04 0.14 0.03 0.03

K2+I 103 6806 5893 -2843 0.4 n/a 2.22 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

HKY+G+I 107 6813 5864 -2825 0.29 0.63 2.51 0.268 0.217 0.207 0.309 0.03 0.03 0.22 0.04 0.15 0.05 0.04 0.15 0.05 0.19 0.03 0.03

T92+G+I 105 6813 5882 -2836 0.28 0.64 2.45 0.242 0.242 0.258 0.258 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.17 0.04 0.17 0.04 0.04

TN93+G+I 108 6814 5856 -2820 0.27 0.64 2.44 0.268 0.217 0.207 0.309 0.03 0.03 0.16 0.04 0.2 0.04 0.04 0.21 0.04 0.14 0.03 0.03

T92+I 104 6818 5896 -2844 0.4 n/a 2.22 0.242 0.242 0.258 0.258 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.17 0.04 0.17 0.04 0.04

HKY+I 106 6819 5879 -2833 0.4 n/a 2.23 0.268 0.217 0.207 0.309 0.03 0.03 0.21 0.04 0.14 0.05 0.04 0.15 0.05 0.18 0.03 0.03

TN93+I 107 6819 5870 -2828 0.4 n/a 2.22 0.268 0.217 0.207 0.309 0.03 0.03 0.16 0.04 0.19 0.05 0.04 0.2 0.05 0.14 0.03 0.03

K2 102 6826 5922 -2859 n/a n/a 2.1 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

GTR+G 110 6832 5856 -2818 n/a 0.36 2.37 0.268 0.217 0.207 0.309 0.05 0.02 0.16 0.06 0.2 0.05 0.02 0.21 0.04 0.14 0.04 0.03

T92 103 6838 5925 -2859 n/a n/a 2.11 0.242 0.242 0.258 0.258 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.16 0.04 0.16 0.04 0.04

TN93 106 6839 5899 -2843 n/a n/a 2.1 0.268 0.217 0.207 0.309 0.04 0.03 0.16 0.04 0.18 0.05 0.04 0.19 0.05 0.14 0.04 0.03

HKY 105 6839 5908 -2849 n/a n/a 2.1 0.268 0.217 0.207 0.309 0.04 0.03 0.21 0.04 0.14 0.05 0.04 0.14 0.05 0.18 0.04 0.03

GTR+G+I 111 6841 5856 -2817 0.27 0.65 2.38 0.268 0.217 0.207 0.309 0.05 0.02 0.16 0.06 0.2 0.05 0.02 0.21 0.04 0.14 0.04 0.03

GTR+I 110 6848 5873 -2826 0.4 n/a 1.98 0.268 0.217 0.207 0.309 0.05 0.03 0.16 0.06 0.17 0.06 0.04 0.18 0.04 0.14 0.04 0.03

GTR 109 6868 5902 -2842 n/a n/a 1.87 0.268 0.217 0.207 0.309 0.05 0.03 0.16 0.06 0.17 0.06 0.04 0.18 0.05 0.14 0.04 0.03

JC+G 102 6902 5997 -2896 n/a 0.42 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 103 6912 5999 -2896 0.23 0.71 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 102 6914 6010 -2903 0.4 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 101 6931 6036 -2917 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

105

Table A1.9. Francisella 16S genetic distance matrix.

Sample R68 ex.

A albolimbatum

(PI 1

299)

Sample R55 ex.

A albolimbatum

(PI 1

295)

Sample R64 ex.

A albolimbatum

(PI 1

298)

Sample R108 ex.

A albolimbatum

(PI 1

736)

Sample R53 ex.

A albolimbatum

(PI 6

80)

Sample R67 ex.

A albolimbatum

(PI 1

320)

Sample F8 ex. A albolim

batum (P

I 1324)

Sample F9 ex. A albolim

batum (P

I 2743)

Sample R20 ex.

A. albolim

batum (PI 1

872)

F. cf. n

ovicida

3523 (CP002558)

F. cf. n

ovicida

Fx1 (C

P002557)

F. endosymbiont ex.

Am. geoemyd

ae (K

T382871.1)

F. endosy

mbiont ex.

Am. goeldii (

MW287918.1)

F. endosy

mbiont ex.

Am. humerale (M

W287921.1)

F. endosy

mbiont ex.

Am. latum

(MW287937.1)

F. endosy

mbiont ex.

Am. oblongogutta

tum (M

W287912.1)

F. endosymbiont e

x. Am. p

acae (M

W287930.1)

F. endosy

mbiont ex.

Am. longiro

stre

(MW287926.1)

F. endosy

mbiont ex.

Am. paulopuncta

tum (M

W287939.1)

F. endosy

mbiont ex.

Am. scu

lptum (M

W287940.1)

F. endosy

mbiont ex.

D. atro

signatus

(KC170748.1)

F. endosy

mbiont ex.

Am. variu

m (M

W287934.1)

F. endosy

mbiont ex. D. o

ccidentalis

(MW287945.1)

F. endosy

mbiont ex.

D. varia

bilis st

r. MV (A

Y805307.1)

F. endosy

mbiont ex.

D. anderso

ni (MT712191.1)

F. endosy

mbiont ex.

D. a

uratus (KC170749.1)

F. endosy

mbiont ex.

D. c

ompactus

(KC170750.1)

F. endosy

mbiont ex.

D. retic

ulatus (M

G859281.1)

F. endosy

mbiont ex.

Hyl. Aegyp

tium

(MH645203.1)

F. endosy

mbiont ex.

Hyl.

Asiatic

um (K

X852464.1)

F. endosymbiont e

x. Hyl.

trunca

tum (J

F290387.1)

F. endosy

mbiont ex.

Hyl.

excava

tum (M

W287952.1)

F. endosy

mbiont ex.

Hyl. Lusit

anicum

(MW287950.1)

F. endosy

mbiont ex.

Hae. fl

ava (K

Y797658)

F. endosy

mbiont ex.

Hae. hys

tricis

(KC170753.1)

F. endosy

mbiont ex.

Hae. shim

oga (K

C170755.1)

F. endosy

mbiont ex.

Hae. papuana (K

C170754.1)

F. endosy

mbiont ex.

R. h

aemaphysaloides

(KC170751.1)

F. endosy

mbiont ex.

I. ric

inus (MW287984.1)

F. endosy

mbiont ex.

I. sca

pularis (M

W287955.1)

F. endosy

mbiont ex.

O. p

orcinus

(MW287943.1)

F. endosy

mbiont ex.

R. sanguineus

(MH645201.1)

F. hisp

aniensis FSC454 (C

P018093)

F. hisp

aniensis st

r. 2008651S (K

T281843)

F. noatunensis

subsp

. noatunensis

FSC774 (NZ C

P053850)

F. noatunensis

subsp

. orie

ntalis st

r. Toba 04 (C

P003402)

F. orie

ntalis FNO12 (C

P011921)

F. persi

ca ATCC VR-331 (C

P012505)

F. philo

miragia str

. 1951 (A

Y496933)

F. philo

miragia

str. G

A01-2794 (CP009440)

F. philo

miragia

subsp

. noatunensis

str. 2

006/09/130 (EF490217)

F. philo

miragia

subsp

. philo

miragia ATCC 25015 (C

P010019)

F. opportu

nistica

str. 1

4-2155 (CP022375)

F. sp. M

A067296 (CP016930)

F. philo

miragia

subsp

. philo

miragia

ATCC 25017 (CP000937.1)

F. salin

a (F7308 R0007 - 1

0861471)

F. tularensis

str. 3

523 (AY243028.1)

F. tularensis

str. S

chu4 (C

P013853)

F. tularensis

subsp

. holarct

ica FSC200 (F

TS 1142 - 14009524)

F. halio

ticida

str. S

himane-1 (J

F290376)

F. tularensis

subsp

. holarct

ica LVS (A

M233362.1)

F. tularensis

subsp

. mediasia

tica

FSC147 (CP000915)

F. tularensis

subsp

. novic

ida F6168 (CP000915)

F. tularensis

subsp

. novic

ida U112 (FTN 0472 - 4

548231)

F. tularensis

subsp

. tularensis

(NE061598)

F. tularensis

subsp

. tularensis

str. W

Y96 (CP012037)

P. salm

onis str

. LF-89 (C

P011849)

Sample R68 ex. A albolimbatum (PI 1299)

Sample R55 ex. A albolimbatum (PI 1295) 0.00

Sample R64 ex. A albolimbatum (PI 1298) 0.00 0.00

Sample R108 ex. A albolimbatum (PI 1736) 0.00 0.00 0.00

Sample R53 ex. A albolimbatum (PI 680) 0.00 0.00 0.00 0.00

Sample R67 ex. A albolimbatum (PI 1320) 0.00 0.00 0.00 0.00 0.00

Sample F8 ex. A albolimbatum (PI 1324) 0.00 0.00 0.00 0.00 0.00 0.00

Sample F9 ex. A albolimbatum (PI 2743) 0.00 0.00 0.00 0.00 0.00 0.00 0.00

Sample R20 ex. A. albolimbatum (PI 1872) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

F. cf. novicida 3523 (CP002558) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02

F. cf. novicida Fx1 (CP002557) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00

F. endosymbiont ex. Am. geoemydae (KT382871.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01

F. endosymbiont ex. Am. goeldii (MW287918.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00

F. endosymbiont ex. Am. humerale (MW287921.1) 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00

F. endosymbiont ex. Am. latum (MW287937.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

F. endosymbiont ex. Am. oblongoguttatum (MW287912.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00

F. endosymbiont ex. Am. pacae (MW287930.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00

F. endosymbiont ex. Am. longirostre (MW287926.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.00

F. endosymbiont ex. Am. paulopunctatum (MW287939.1) 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00

F. endosymbiont ex. Am. sculptum (MW287940.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.01 0.00 0.01 0.00

F. endosymbiont ex. D. atrosignatus (KC170748.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01

F. endosymbiont ex. Am. varium (MW287934.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

F. endosymbiont ex. D. occidentalis (MW287945.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01

F. endosymbiont ex. D. variabilis str. MV (AY805307.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.02 0.01 0.00

F. endosymbiont ex. D. andersoni (MT712191.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.01 0.00 0.00

F. endosymbiont ex. D. auratus (KC170749.1) 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.00 0.01 0.02 0.02 0.02

F. endosymbiont ex. D. compactus (KC170750.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00

F. endosymbiont ex. D. reticulatus (MG859281.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02

F. endosymbiont ex. Hyl. Aegyptium (MH645203.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01

F. endosymbiont ex. Hyl. Asiaticum (KX852464.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.01 0.00 0.01 0.01 0.02 0.01 0.00

F. endosymbiont ex. Hyl. truncatum (JF290387.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.00 0.00

F. endosymbiont ex. Hyl. excavatum (MW287952.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.00 0.00 0.00

F. endosymbiont ex. Hyl. Lusitanicum (MW287950.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.00 0.00 0.00 0.00

F. endosymbiont ex. Hae. flava (KY797658) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.00 0.01 0.01 0.01

F. endosymbiont ex. Hae. hystricis (KC170753.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02

F. endosymbiont ex. Hae. shimoga (KC170755.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00

F. endosymbiont ex. Hae. papuana (KC170754.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01

F. endosymbiont ex. R. haemaphysaloides (KC170751.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.00 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01

F. endosymbiont ex. I. ricinus (MW287984.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02

F. endosymbiont ex. I. scapularis (MW287955.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.02 0.02 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.02 0.02 0.02 0.02 0.01

F. endosymbiont ex. O. porcinus (MW287943.1) 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.01 0.00 0.01 0.00 0.00 0.01 0.01 0.01 0.00 0.01 0.01 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.01 0.02 0.02 0.02 0.02 0.01 0.00

F. endosymbiont ex. R. sanguineus (MH645201.1) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0 0.00 0.00 0.00 0.00 0.01 0.02 0.02 0.02 0.02 0.01 0.00 0.00

F. hispaniensis FSC454 (CP018093) 0.01 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01

F. hispaniensis str. 2008651S (KT281843) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00

F. noatunensis subsp. noatunensis FSC774 (NZ CP053850) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02

F. noatunensis subsp. orientalis str. Toba 04 (CP003402) 0.03 0.04 0.04 0.03 0.04 0.04 0.03 0.04 0.04 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.02 0.03 0.01

F. orientalis FNO12 (CP011921) 0.03 0.04 0.04 0.03 0.04 0.04 0.03 0.04 0.04 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.02 0.03 0.01 0.00

F. persica ATCC VR-331 (CP012505) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02

F. philomiragia str. 1951 (AY496933) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02

F. philomiragia str. GA01-2794 (CP009440) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00

F. philomiragia subsp. noatunensis str. 2006/09/130 (EF490217)0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.02 0.02 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00 0.00

F. philomiragia subsp. philomiragia ATCC 25015 (CP010019) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00 0.00 0.00

F. opportunistica str. 14-2155 (CP022375) 0.03 0.04 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03

F. sp. MA067296 (CP016930) 0.03 0.04 0.03 0.03 0.04 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.00

F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1)0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.01 0.02 0.00 0.00 0.00 0.00 0.02 0.02

F. salina (F7308 R0007 - 10861471) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.02 0.02 0.03 0.03 0.03 0.03 0.04 0.03 0.04 0.04 0.03 0.03 0.03 0.02 0.02 0.03 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.03 0.03 0.01

F. tularensis str. 3523 (AY243028.1) 0.01 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.00 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

F. tularensis str. Schu4 (CP013853) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00

F. tularensis subsp. holarctica FSC200 (FTS 1142 - 14009524)0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.03 0.03 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.01 0.00

F. halioticida str. Shimane-1 (JF290376) 0.03 0.04 0.03 0.03 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.01 0.02 0.02 0.03 0.01 0.01 0.01 0.01 0.04 0.04 0.01 0.01 0.03 0.03 0.03

F. tularensis subsp. holarctica LVS (AM233362.1) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01 0.01 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.03 0.03 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.01 0.00 0.00 0.03

F. tularensis subsp. mediasiatica FSC147 (CP000915) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00

F. tularensis subsp. novicida F6168 (CP000915) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00 0.00

F. tularensis subsp. novicida U112 (FTN 0472 - 4548231) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00 0.00 0.00

F. tularensis subsp. tularensis (NE061598) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0 0.00 0.03 0.00 0 0 0

F. tularensis subsp. tularensis str. WY96 (CP012037) 0.02 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 0.02 0.01 0.02 0.02 0.02 0.02 0.03 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.01 0.01 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.01 0.00 0.00 0.03 0.00 0.00 0.00 0.00 0.00

P. salmonis str. LF-89 (CP011849) 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.17 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.17 0.17 0.17 0.16 0.15 0.16 0.16 0.16 0.15 0.15 0.15 0.16 0.16 0.15 0.15 0.15 0.15 0.15 0.15 0.15 0.16 0.16 0.15 0.15 0.16 0.16 0.16 0.16 0.16 0.16 0.15 0.16

106

Table A1.10. Francisella tpiA Maximum Likelihood fits of 24 different nucleotide substitution models.


HKY+G 46 6093 5757 -2832 n/a 0.49 2.2 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.09 0.03 0.05 0.23 0.03 0.23 0.05 0.02

HKY+G+I 47 6100 5756 -2831 0.24 1.08 2.31 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.1 0.03 0.05 0.23 0.03 0.23 0.05 0.02

TN93+G 47 6102 5758 -2832 n/a 0.49 2.22 0.325 0.321 0.134 0.22 0.05 0.02 0.17 0.05 0.09 0.03 0.05 0.21 0.03 0.25 0.05 0.02

T92+G 44 6106 5784 -2848 n/a 0.5 2.19 0.323 0.323 0.177 0.177 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

TN93+G+I 48 6108 5757 -2830 0.24 1.05 2.33 0.325 0.321 0.134 0.22 0.05 0.02 0.17 0.05 0.09 0.03 0.05 0.21 0.03 0.25 0.05 0.02

HKY+I 46 6111 5774 -2841 0.35 n/a 2.22 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.09 0.03 0.05 0.23 0.03 0.23 0.05 0.02

T92+G+I 45 6112 5783 -2846 0.24 1.1 2.3 0.323 0.323 0.177 0.177 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.23 0.03 0.23 0.05 0.03

TN93+I 47 6119 5776 -2841 0.35 n/a 2.22 0.325 0.321 0.134 0.22 0.05 0.02 0.16 0.05 0.09 0.03 0.05 0.21 0.03 0.24 0.05 0.02

T92+I 44 6122 5800 -2856 0.35 n/a 2.24 0.323 0.323 0.177 0.177 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.23 0.03 0.23 0.05 0.03

GTR+G 50 6125 5759 -2829 n/a 0.51 2.17 0.325 0.321 0.134 0.22 0.06 0.02 0.16 0.06 0.09 0.02 0.04 0.21 0.05 0.24 0.03 0.03

GTR+G+I 51 6132 5759 -2828 0.23 1.07 2.27 0.325 0.321 0.134 0.22 0.06 0.02 0.17 0.06 0.09 0.02 0.04 0.21 0.04 0.25 0.03 0.03

GTR+I 50 6142 5776 -2838 0.35 n/a 2.23 0.325 0.321 0.134 0.22 0.06 0.01 0.16 0.06 0.09 0.02 0.04 0.22 0.04 0.24 0.03 0.02

K2+G 43 6161 5846 -2880 n/a 0.56 1.94 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+G+I 44 6168 5846 -2879 0.25 1.31 2.04 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

K2+I 43 6173 5858 -2886 0.35 n/a 2.07 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

HKY 45 6235 5906 -2908 n/a n/a 1.45 0.325 0.321 0.134 0.22 0.06 0.03 0.13 0.06 0.08 0.04 0.06 0.2 0.04 0.2 0.06 0.03

TN93 46 6243 5907 -2907 n/a n/a 1.46 0.325 0.321 0.134 0.22 0.06 0.03 0.14 0.06 0.07 0.04 0.06 0.18 0.04 0.21 0.06 0.03

T92 43 6248 5933 -2924 n/a n/a 1.45 0.323 0.323 0.177 0.177 0.06 0.03 0.11 0.06 0.11 0.03 0.06 0.2 0.03 0.2 0.06 0.03

GTR 49 6264 5906 -2904 n/a n/a 1.46 0.325 0.321 0.134 0.22 0.08 0.02 0.14 0.08 0.07 0.03 0.06 0.18 0.05 0.21 0.04 0.03

K2 42 6287 5979 -2948 n/a n/a 1.43 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC+G 42 6291 5984 -2950 n/a 0.69 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 43 6299 5985 -2949 0.02 0.73 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 42 6307 5999 -2958 0.33 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 41 6389 6090 -3004 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

107

Table A1.11. Francisella tpiA genetic distance matrix.

F. philo

miragia subsp. p

hilomira

gia str.

1951 (EU683025)

F. philo

miragia

subsp. noatunensis str.

DSM 18777 (EU683027)

Sample R20 ex.

A. albolim

batum (P

I 1872)

F. halio

ticida str.

Shimane-1 (J

F290385)

F. philo

miragia subsp. p

hilomira

gia ATCC 25015 (C

P010019.1)

F. philo

miragia

str. GA01-2794 (C

P009440.1)

P. salm

onis (CP011849)

F. tularensis

subsp. mediasiatic

a FSC147 (CP000915.1)

F. noatunensis

subsp. orie

ntalis str.

Toba 04 (CP003402.1)

F. orie

ntalis FNO12 (C

P011921.2)

F. tularensis

subsp. tularensis W

Y96-3418 (CP000608.1)

F. opportu

nistica str.

14-2155 (CP022375.1)

F. noatunensis

subsp. noatunensis FSC774 (C

P053850.1)

F. cf. n

ovicida Fx1 (CP002557.1)

F. tularensis

str. Schu4 (C

P013853.1)

F. cf. n

ovicida 3523 (CP002558.1)

F. philo

miragia

subsp. philo

miragia

ATCC 25017 (CP000937.1)

F. persica ATCC VR-331 (C

P012505.1)

F. hispaniensis FSC454 (C

P018093.1)

F. sp. MA067296 (C

P016930.1)

F. tularensis

subsp. holarctic

a LVS (AM233362.1 )

F. tularensis

subsp. novicida F6168 (C

P009353.1)

F. philomiragia subsp. philomiragia str. 1951 (EU683025)

F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.06

Sample R20 ex. A. albolimbatum (PI 1872) 0.33 0.34

F. halioticida str. Shimane-1 (JF290385) 0.33 0.35 0.36

F. philomiragia subsp. philomiragia ATCC 25015 (CP010019.1) 0.05 0.04 0.35 0.34

F. philomiragia str. GA01-2794 (CP009440.1) 0.06 0.02 0.37 0.35 0.06

P. salmonis (CP011849) 1.09 1.08 1.04 1.07 1.13 1.07

F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.31 0.31 0.11 0.35 0.33 0.34 1.06

F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.07 0.02 0.36 0.35 0.05 0.03 1.09 0.34

F. orientalis FNO12 (CP011921.2) 0.07 0.02 0.36 0.35 0.05 0.03 1.09 0.34 0.00

F. tularensis subsp. tularensis WY96-3418 (CP000608.1) 0.32 0.31 0.11 0.36 0.33 0.35 1.06 0.00 0.34 0.34

F. opportunistica str. 14-2155 (CP022375.1) 0.30 0.34 0.14 0.37 0.37 0.35 1.02 0.16 0.38 0.38 0.16

F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.06 0.00 0.34 0.35 0.04 0.02 1.08 0.31 0.02 0.02 0.31 0.34

F. cf. novicida Fx1 (CP002557.1) 0.32 0.32 0.11 0.36 0.34 0.35 1.07 0.01 0.35 0.35 0.01 0.16 0.32

F. tularensis str. Schu4 (CP013853.1) 0.32 0.31 0.11 0.36 0.33 0.35 1.06 0.00 0.34 0.34 0.00 0.16 0.31 0.01

F. cf. novicida 3523 (CP002558.1) 0.31 0.34 0.11 0.35 0.37 0.37 1.02 0.05 0.38 0.38 0.05 0.16 0.34 0.05 0.05

F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.01 0.05 0.34 0.34 0.05 0.06 1.10 0.32 0.07 0.07 0.33 0.32 0.05 0.33 0.33 0.33

F. persica ATCC VR-331 (CP012505.1) 0.32 0.31 0.05 0.35 0.33 0.34 1.06 0.11 0.34 0.34 0.11 0.16 0.31 0.11 0.11 0.13 0.33

F. hispaniensis FSC454 (CP018093.1) 0.32 0.34 0.12 0.36 0.37 0.37 1.03 0.05 0.38 0.38 0.05 0.16 0.34 0.05 0.05 0.00 0.34 0.13

F. sp. MA067296 (CP016930.1) 0.30 0.34 0.14 0.37 0.37 0.35 1.02 0.16 0.38 0.38 0.16 0.00 0.34 0.16 0.16 0.16 0.32 0.16 0.16

F. tularensis subsp. holarctica LVS (AM233362.1 ) 0.31 0.30 0.11 0.36 0.32 0.34 1.08 0.01 0.33 0.33 0.01 0.17 0.30 0.02 0.01 0.05 0.32 0.12 0.05 0.17

F. tularensis subsp. novicida F6168 (CP009353.1) 0.31 0.32 0.11 0.35 0.34 0.34 1.06 0.01 0.35 0.35 0.01 0.16 0.32 0.01 0.01 0.06 0.32 0.12 0.06 0.16 0.02

108

Table A1.12. Francisella prfB Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 44 7255 6920 -3416 n/a 0.58 2.03 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

T92+G+I 45 7265 6922 -3416 0.09 0.73 2.05 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

HKY+G 46 7271 6921 -3414 n/a 0.58 2.03 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02

T92+I 44 7275 6940 -3426 0.33 n/a 2.02 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

TN93+G 47 7277 6920 -3413 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.2 0.05 0.02

HKY+G+I 47 7281 6923 -3414 0.08 0.71 2.05 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02

TN93+G+I 48 7287 6922 -3413 0.05 0.67 2.04 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.21 0.05 0.02

HKY+I 46 7291 6941 -3425 0.32 n/a 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02

TN93+I 47 7299 6941 -3423 0.32 n/a 2 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.25 0.03 0.21 0.05 0.02

GTR+G 50 7299 6919 -3409 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02

GTR+G+I 51 7308 6920 -3409 0.1 0.77 2.05 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02

GTR+I 50 7317 6936 -3418 0.33 n/a 2.07 0.346 0.317 0.136 0.202 0.07 0.02 0.13 0.07 0.11 0.02 0.05 0.25 0.02 0.22 0.03 0.02

K2+G 43 7345 7018 -3466 n/a 0.67 1.8 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+G+I 44 7355 7020 -3466 0 0.67 1.81 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+I 43 7361 7033 -3474 0.33 n/a 1.82 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

T92 43 7361 7034 -3474 n/a n/a 1.46 0.331 0.331 0.169 0.169 0.06 0.03 0.1 0.06 0.1 0.03 0.06 0.21 0.03 0.21 0.06 0.03

HKY 45 7377 7034 -3472 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.12 0.07 0.08 0.04 0.07 0.19 0.04 0.21 0.06 0.03

TN93 46 7383 7033 -3470 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.11 0.07 0.1 0.04 0.07 0.22 0.04 0.19 0.06 0.03

GTR 49 7403 7030 -3466 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.07 0.02 0.11 0.08 0.1 0.02 0.06 0.22 0.05 0.19 0.04 0.03

K2 42 7430 7111 -3513 n/a n/a 1.42 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC+G 42 7480 7160 -3538 n/a 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 43 7490 7162 -3538 0 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 42 7494 7175 -3545 0.3 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 41 7545 7233 -3575 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

109

Table A1.13. Francisella prfB genetic distance matrix.

Sample R20

ex. A. a

lbolim

batum

(PI 1

872)

F. tu

lare

nsis

sub

sp.

holarctic

a LVS (A

M23

3362

.1 )

F. tu

lare

nsis

sub

sp.

med

iasiatica

FSC

147 (C

P0009

15.1)

F. philom

iragia

sub

sp.

philo

mira

gia

ATC

C 250

17 (C

P0009

37.1)

F. cf. no

vicida

Fx1

(CP00

2557

.1)

F. cf. no

vicida

352

3 (C

P0025

58.1)

F. noa

tune

nsis

sub

sp. or

ientalis

str. Tob

a 04

(CP00

3402

.1)

F. tu

lare

nsis

sub

sp.

novic

ida

F61

68 (C

P0093

53.1)

F. philom

iragia

str. G

A01-2

794 (C

P0094

40.1)

F. orie

ntalis

FNO12

(CP01

1921

.2)

F. tu

lare

nsis

sub

sp. tu

lare

nsis

WY96

(CP01

2037

.1)

F. per

sica

ATCC V

R-3

31 (C

P0125

05.1)

F. tu

lare

nsis

str. S

chu4

(CP01

3853

.1)

F. sp

. MA06

7296

(CP01

6930

.1)

F. hispa

nien

sis

FSC45

4 (C

P0180

93.1)

F. noa

tune

nsis

sub

sp.

noatun

ensis

FSC

774 (C

P0538

50.1)

F. philom

iragia

subs

p. ph

ilom

iragia

ATC

C 250

15 (CP01

0019

.1)

F. philom

iragia

subs

p. ph

ilom

iragia

str. 195

1 (E

U68

3025

)

F. philom

iragia

sub

sp.

noatun

ensis

str. DSM

187

77 (E

U68

3027

)

F. haliotic

ida

str. S

him

ane-

1 (JF2

9038

5)

F. opp

ortunistica

str. 14-

2155

(CP02

2375

.1)

P. salm

onis

(C

P0118

49)

Sample R20 ex. A. albolimbatum (PI 1872)

F. tularensis subsp. holarctica LVS (AM233362.1 ) 0.15

F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.16 0.01

F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.22 0.18 0.18

F. cf. novicida Fx1 (CP002557.1) 0.17 0.04 0.04 0.15

F. cf. novicida 3523 (CP002558.1) 0.14 0.08 0.09 0.19 0.10

F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.23 0.19 0.19 0.06 0.17 0.20

F. tularensis subsp. novicida F6168 (CP009353.1) 0.17 0.05 0.05 0.15 0.01 0.11 0.17

F. philomiragia str. GA01-2794 (CP009440.1) 0.22 0.16 0.16 0.05 0.15 0.18 0.05 0.16


F. tularensis subsp. tularensis WY96 (CP012037.1) 0.16 0.00 0.00 0.18 0.04 0.09 0.19 0.05 0.16 0.19

F. persica ATCC VR-331 (CP012505.1) 0.04 0.13 0.14 0.22 0.15 0.12 0.24 0.15 0.22 0.24 0.14

F. tularensis str. Schu4 (CP013853.1) 0.16 0.00 0.00 0.18 0.04 0.09 0.19 0.05 0.16 0.19 0.00 0.14

F. sp. MA067296 (CP016930.1) 0.09 0.14 0.14 0.21 0.16 0.12 0.23 0.16 0.20 0.23 0.14 0.09 0.14

F. hispaniensis FSC454 (CP018093.1) 0.12 0.09 0.10 0.19 0.11 0.03 0.20 0.12 0.18 0.20 0.10 0.11 0.10 0.13

F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.24 0.17 0.17 0.07 0.16 0.20 0.06 0.16 0.06 0.06 0.17 0.24 0.17 0.22 0.20

F. philomiragia subsp. philomiragia ATCC 25015 (CP010019.1) 0.22 0.18 0.18 0.01 0.15 0.19 0.06 0.15 0.06 0.06 0.18 0.22 0.18 0.21 0.18 0.07

F. philomiragia subsp. philomiragia str. 1951 (EU683025) 0.22 0.17 0.18 0.02 0.14 0.19 0.07 0.15 0.07 0.07 0.17 0.22 0.17 0.21 0.19 0.07 0.02

F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.24 0.17 0.17 0.07 0.16 0.20 0.06 0.16 0.06 0.06 0.17 0.24 0.17 0.22 0.20 0.00 0.07 0.07

F. halioticida str. Shimane-1 (JF290385) 0.28 0.28 0.29 0.23 0.28 0.25 0.25 0.28 0.25 0.25 0.29 0.26 0.29 0.25 0.24 0.23 0.23 0.24 0.23

F. opportunistica str. 14-2155 (CP022375.1) 0.09 0.14 0.14 0.21 0.16 0.12 0.23 0.16 0.20 0.23 0.14 0.09 0.14 0.00 0.13 0.22 0.21 0.21 0.22 0.25

P. salmonis (CP011849) 0.59 0.55 0.55 0.56 0.54 0.57 0.56 0.53 0.56 0.56 0.54 0.60 0.54 0.54 0.58 0.56 0.57 0.56 0.56 0.53 0.54

110

Table A1.14. Francisella rpoA Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 44 7255 6920 -3416 n/a 0.58 2.03 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

T92+G+I 45 7265 6922 -3416 0.09 0.73 2.05 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

HKY+G 46 7271 6921 -3414 n/a 0.58 2.03 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02

T92+I 44 7275 6940 -3426 0.33 n/a 2.02 0.331 0.331 0.169 0.169 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.23 0.03 0.23 0.05 0.03

TN93+G 47 7277 6920 -3413 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.2 0.05 0.02

HKY+G+I 47 7281 6923 -3414 0.08 0.71 2.05 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02

TN93+G+I 48 7287 6922 -3413 0.05 0.67 2.04 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.26 0.03 0.21 0.05 0.02

HKY+I 46 7291 6941 -3425 0.32 n/a 2.02 0.346 0.317 0.136 0.202 0.05 0.02 0.14 0.05 0.09 0.03 0.05 0.22 0.03 0.24 0.05 0.02

TN93+I 47 7299 6941 -3423 0.32 n/a 2 0.346 0.317 0.136 0.202 0.05 0.02 0.12 0.05 0.11 0.03 0.05 0.25 0.03 0.21 0.05 0.02

GTR+G 50 7299 6919 -3409 n/a 0.58 2.02 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02

GTR+G+I 51 7308 6920 -3409 0.1 0.77 2.05 0.346 0.317 0.136 0.202 0.06 0.02 0.12 0.07 0.11 0.02 0.05 0.26 0.03 0.21 0.03 0.02

GTR+I 50 7317 6936 -3418 0.33 n/a 2.07 0.346 0.317 0.136 0.202 0.07 0.02 0.13 0.07 0.11 0.02 0.05 0.25 0.02 0.22 0.03 0.02

K2+G 43 7345 7018 -3466 n/a 0.67 1.8 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+G+I 44 7355 7020 -3466 0 0.67 1.81 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+I 43 7361 7033 -3474 0.33 n/a 1.82 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

T92 43 7361 7034 -3474 n/a n/a 1.46 0.331 0.331 0.169 0.169 0.06 0.03 0.1 0.06 0.1 0.03 0.06 0.21 0.03 0.21 0.06 0.03

HKY 45 7377 7034 -3472 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.12 0.07 0.08 0.04 0.07 0.19 0.04 0.21 0.06 0.03

TN93 46 7383 7033 -3470 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.06 0.03 0.11 0.07 0.1 0.04 0.07 0.22 0.04 0.19 0.06 0.03

GTR 49 7403 7030 -3466 n/a n/a 1.46 0.346 0.317 0.136 0.202 0.07 0.02 0.11 0.08 0.1 0.02 0.06 0.22 0.05 0.19 0.04 0.03

K2 42 7430 7111 -3513 n/a n/a 1.42 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC+G 42 7480 7160 -3538 n/a 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 43 7490 7162 -3538 0 0.82 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 42 7494 7175 -3545 0.3 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 41 7545 7233 -3575 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

111

Table A1.15. Francisella rpoA genetic distance matrix.

F. per

sica

ATCC V

R-3

31 (C

P0125

05.1)

Sample R20

ex. A. a

lbolim

batum

(PI 1

872)

F. cf. no

vicida

352

3 (C

P0025

58.1)

F. cf. no

vicida

Fx1

(CP00

2557

.1)

F. haliotic

ida

str. S

him

ane-

1 (JF2

9038

5)

F. hispa

nien

sis

FSC45

4 (C

P0180

93.1)

F. noa

tune

nsis

sub

sp.

noatun

ensis

FSC

774 (C

P0538

50.1)

F. noa

tune

nsis

sub

sp. or

ientalis

str. Tob

a 04

(CP00

3402

.1)

F. opp

ortunistica

str. 14-

2155

(CP02

2375

.1)

F. orie

ntalis

FNO12

(CP01

1921

.2)

F. philom

iragia

str. G

A01-2

794 (C

P0094

40.1)

F. philom

iragia

sub

sp.

noatun

ensis

str. DSM

187

77 (E

U68

3027

)

F. p

hilom

iragia

sub

sp. p

hilom

iragia ATC

C 250

15 (C

P0100

19.1)

F. philom

iragia

sub

sp.

philo

mira

gia

ATC

C 250

17 (C

P0009

37.1)

F. philom

iragia su

bsp.

philo

mira

gia

str. 195

1 (E

U68

3001

)

F. sp

. MA06

7296

(CP01

6930

.1)

F. tu

lare

nsis

str. S

chu4

(CP01

3853

.1)

F. tu

lare

nsis

sub

sp.

holarctic

a LVS (A

M23

3362

.1 )

F. tu

lare

nsis

sub

sp.

med

iasiatica

FSC

147 (C

P0009

15.1)

F. tu

lare

nsis

sub

sp.

novic

ida

F61

68 (C

P0093

53.1)

F. tu

lare

nsis

sub

sp. tu

lare

nsis

WY96

-341

8 (C

P0006

08.1)

P. salm

onis

(CP01

1849

)

F. persica ATCC VR-331 (CP012505.1)

Sample R20 ex. A. albolimbatum (PI 1872) 0.04

F. cf. novicida 3523 (CP002558.1) 0.10 0.11

F. cf. novicida Fx1 (CP002557.1) 0.08 0.09 0.07

F. halioticida str. Shimane-1 (JF290385) 0.24 0.25 0.24 0.21

F. hispaniensis FSC454 (CP018093.1) 0.10 0.11 0.00 0.08 0.25

F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.23 0.23 0.23 0.20 0.26 0.23

F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.24 0.25 0.23 0.21 0.26 0.23 0.01

F. opportunistica str. 14-2155 (CP022375.1) 0.09 0.10 0.10 0.09 0.25 0.10 0.25 0.26


F. philomiragia str. GA01-2794 (CP009440.1) 0.23 0.23 0.22 0.20 0.27 0.23 0.02 0.02 0.26 0.02

F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.23 0.23 0.23 0.20 0.26 0.23 0 0.01 0.25 0.01 0.02

F. philomiragia subsp. philomiragia ATCC 25015 (CP010019.1) 0.24 0.24 0.25 0.22 0.28 0.25 0.08 0.09 0.27 0.09 0.07 0.08

F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.24 0.24 0.24 0.21 0.27 0.24 0.08 0.09 0.27 0.09 0.07 0.08 0.01

F. philomiragia subsp. philomiragia str. 1951 (EU683001) 0.24 0.24 0.25 0.22 0.28 0.25 0.08 0.09 0.27 0.09 0.07 0.08 0.01 0.01

F. sp. MA067296 (CP016930.1) 0.09 0.10 0.10 0.09 0.25 0.10 0.25 0.26 0.00 0.26 0.26 0.25 0.27 0.27 0.27

F. tularensis str. Schu4 (CP013853.1) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09

F. tularensis subsp. holarctica LVS (AM233362.1 ) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.00

F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.00 0.00

F. tularensis subsp. novicida F6168 (CP009353.1) 0.08 0.09 0.08 0.00 0.22 0.08 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.01 0.01 0.01

F. tularensis subsp. tularensis WY96-3418 (CP000608.1) 0.08 0.09 0.07 0.00 0.21 0.07 0.21 0.21 0.09 0.21 0.20 0.21 0.23 0.22 0.22 0.09 0.00 0.00 0.00 0.01

P. salmonis (CP011849) 1.02 1.01 1.00 0.91 1.12 1.01 1.04 1.03 1.03 1.03 1.05 1.04 1.07 1.03 1.03 1.03 0.92 0.92 0.92 0.90 0.92

112

Table A1.16. Francisella dnaA Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 44 8508 8159 -4035 n/a 0.17 2.93 0.335 0.335 0.165 0.165 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02

T92+G+I 45 8518 8161 -4035 0 0.17 2.93 0.335 0.335 0.165 0.165 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02

TN93+G 47 8528 8155 -4031 n/a 0.19 2.91 0.367 0.304 0.138 0.191 0.03 0.02 0.1 0.04 0.15 0.02 0.04 0.33 0.02 0.19 0.03 0.02

GTR+G 50 8531 8135 -4017 n/a 0.19 2.92 0.367 0.304 0.138 0.191 0.06 0.01 0.11 0.07 0.15 0.01 0.03 0.34 0 0.2 0.02 0

HKY+G 46 8534 8169 -4038 n/a 0.17 3.02 0.367 0.304 0.138 0.191 0.04 0.02 0.15 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.04 0.02

TN93+G+I 48 8536 8155 -4030 0.34 0.44 3.1 0.367 0.304 0.138 0.191 0.03 0.01 0.1 0.04 0.16 0.02 0.04 0.34 0.02 0.19 0.03 0.01

GTR+G+I 51 8537 8132 -4015 0.36 0.47 3.15 0.367 0.304 0.138 0.191 0.06 0.01 0.1 0.07 0.16 0.01 0.02 0.35 0 0.2 0.02 0

HKY+G+I 47 8544 8171 -4038 0 0.17 3.03 0.367 0.304 0.138 0.191 0.04 0.02 0.15 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.04 0.02

T92+I 44 8582 8233 -4073 0.47 n/a 2.52 0.335 0.335 0.165 0.165 0.04 0.02 0.12 0.04 0.12 0.02 0.04 0.25 0.02 0.25 0.04 0.02

TN93+I 47 8591 8218 -4062 0.47 n/a 2.55 0.367 0.304 0.138 0.191 0.04 0.02 0.09 0.05 0.15 0.02 0.05 0.32 0.02 0.18 0.04 0.02

GTR+I 50 8600 8204 -4052 0.47 n/a 2.06 0.367 0.304 0.138 0.191 0.06 0.01 0.1 0.08 0.14 0.04 0.03 0.3 0 0.19 0.06 0

HKY+I 46 8611 8246 -4077 0.47 n/a 2.51 0.367 0.304 0.138 0.191 0.04 0.02 0.14 0.05 0.1 0.03 0.05 0.22 0.03 0.27 0.04 0.02

K2+G 43 8738 8397 -4156 n/a 0.23 2.2 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

K2+G+I 44 8748 8399 -4155 0.14 0.31 2.22 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

K2+I 43 8790 8449 -4182 0.48 n/a 2.17 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

T92 43 8824 8483 -4198 n/a n/a 1.42 0.335 0.335 0.165 0.165 0.06 0.03 0.1 0.06 0.1 0.03 0.06 0.21 0.03 0.21 0.06 0.03

GTR 49 8829 8441 -4171 n/a n/a 1.44 0.367 0.304 0.138 0.191 0.08 0.03 0.09 0.1 0.12 0.02 0.07 0.26 0.01 0.18 0.04 0

TN93 46 8838 8473 -4190 n/a n/a 1.43 0.367 0.304 0.138 0.191 0.06 0.03 0.09 0.07 0.11 0.04 0.07 0.25 0.04 0.17 0.06 0.03

HKY 45 8849 8493 -4201 n/a n/a 1.42 0.367 0.304 0.138 0.191 0.06 0.03 0.12 0.07 0.08 0.04 0.07 0.19 0.04 0.22 0.06 0.03

JC+G 42 8912 8579 -4247 n/a 0.32 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 43 8922 8581 -4247 0 0.32 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 42 8962 8629 -4272 0.45 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

K2 42 8972 8639 -4277 n/a n/a 1.39 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC 41 9102 8777 -4347 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

113

Table A1.17. Francisella dnaA genetic distance matrix.

F. tu

lare

nsis

sub

sp.

holarctic

a LVS (A

M23

3362

.1 )

F. tu

lare

nsis

sub

sp.

med

iasiatica

FSC

147 (C

P0009

15.1)

F. philom

iragia

sub

sp.

philo

mira

gia

ATC

C 250

17 (C

P0009

37.1)

F. cf. no

vicida

Fx1

(CP00

2557

.1)

F. cf. no

vicida

352

3 (C

P0025

58.1)

F. noa

tune

nsis

sub

sp. or

ientalis

str. Tob

a 04

(CP00

3402

.1)

F. tu

lare

nsis

sub

sp.

novic

ida

F61

68 (C

P0093

53.1)

F. philom

iragia

str. G

A01-2

794 (C

P0094

40.1)

F. philom

iragia

subs

p. ph

ilom

iragia

ATC

C 250

15

F. orie

ntalis

FNO12

(CP01

1921

.2)

F. tu

lare

nsis

sub

sp. tu

lare

nsis

WY96

-341

8 (C

P0006

08.1)

F. per

sica

ATCC V

R-3

31 (C

P0125

05.1)

F. tu

lare

nsis

str. S

chu4

(CP01

3853

.1)

F. sp

. MA06

7296

(CP01

6930

.1)

F. hispa

nien

sis

FSC45

4 (C

P0180

93.1)

F. opp

ortunistica

str. 14-

2155

(CP02

2375

.1)

F. noa

tune

nsis

sub

sp.

noatun

ensis

FSC

774 (C

P0538

50.1)

P. salm

onis

LF-

89 A

TCC V

R-1

361 (2

9882

744)

F. philom

iragia

subs

p. ph

ilom

iragia

str. 195

1 (E

U68

3025

)

F. philom

iragia

sub

sp.

noatun

ensis

str. DSM

187

77 (E

U68

3027

)

F. haliotic

ida

str. S

him

ane-

1 (JF2

9038

5)

Sample R20

ex. A. a

lbolim

batum

(PI 1

872)

F. tularensis subsp. holarctica LVS (AM233362.1 )

F. tularensis subsp. mediasiatica FSC147 (CP000915.1) 0.00

F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1) 0.14 0.14

F. cf. novicida Fx1 (CP002557.1) 0.01 0.01 0.13

F. cf. novicida 3523 (CP002558.1) 0.07 0.06 0.14 0.06

F. noatunensis subsp. orientalis str. Toba 04 (CP003402.1) 0.14 0.14 0.03 0.13 0.14

F. tularensis subsp. novicida F6168 (CP009353.1) 0.01 0.01 0.13 0.01 0.07 0.14

F. philomiragia str. GA01-2794 (CP009440.1) 0.15 0.15 0.05 0.14 0.15 0.03 0.15

F. philomiragia subsp. philomiragia ATCC 25015 0.13 0.13 0.02 0.12 0.12 0.04 0.13 0.06


F. tularensis subsp. tularensis WY96-3418 (CP000608.1) 0.00 0.00 0.14 0.01 0.06 0.14 0.01 0.15 0.13 0.14

F. persica ATCC VR-331 (CP012505.1) 0.06 0.07 0.15 0.07 0.09 0.15 0.06 0.16 0.14 0.15 0.06

F. tularensis str. Schu4 (CP013853.1) 0.00 0.00 0.14 0.01 0.06 0.14 0.01 0.15 0.13 0.14 0.00 0.06

F. sp. MA067296 (CP016930.1) 0.08 0.08 0.15 0.08 0.10 0.15 0.08 0.17 0.14 0.15 0.08 0.06 0.08

F. hispaniensis FSC454 (CP018093.1) 0.07 0.06 0.13 0.06 0.00 0.14 0.07 0.16 0.12 0.14 0.06 0.09 0.06 0.09

F. opportunistica str. 14-2155 (CP022375.1) 0.08 0.08 0.15 0.08 0.10 0.15 0.08 0.17 0.14 0.15 0.08 0.06 0.08 0.00 0.09

F. noatunensis subsp. noatunensis FSC774 (CP053850.1) 0.14 0.13 0.04 0.13 0.14 0.01 0.14 0.03 0.04 0.01 0.13 0.15 0.14 0.15 0.14 0.15

P. salmonis LF-89 ATCC VR-1361 (29882744) 0.68 0.69 0.71 0.68 0.72 0.72 0.68 0.69 0.71 0.72 0.68 0.66 0.68 0.71 0.71 0.71 0.70

F. philomiragia subsp. philomiragia str. 1951 (EU683025) 0.13 0.13 0.02 0.12 0.12 0.03 0.13 0.05 0.00 0.03 0.13 0.14 0.13 0.14 0.12 0.14 0.04 0.71

F. philomiragia subsp. noatunensis str. DSM 18777 (EU683027) 0.14 0.14 0.04 0.13 0.15 0.01 0.14 0.03 0.04 0.01 0.14 0.15 0.14 0.15 0.15 0.15 0.00 0.71 0.04

F. halioticida str. Shimane-1 (JF290385) 0.16 0.15 0.15 0.16 0.16 0.15 0.16 0.15 0.15 0.15 0.15 0.17 0.15 0.17 0.16 0.17 0.15 0.68 0.15 0.15

Sample R20 ex. A. albolimbatum (PI 1872) 0.07 0.07 0.14 0.07 0.09 0.15 0.07 0.17 0.14 0.15 0.07 0.02 0.07 0.06 0.09 0.06 0.15 0.68 0.14 0.15 0.17

114

Table A1.18. Francisella concatenated 16S, tpiA, prfB, rpoA and dnaA Maximum Likelihood fits of 24 different nucleotide substitution

models.


GTR+G 48 40774 40325 -20114 n/a 0.295 1.948 0.323 0.28 0.158 0.239 0.06 0.03 0.13 0.07 0.14 0.02 0.06 0.24 0.03 0.17 0.03 0.02

GTR+G+I 49 40779 40321 -20111 0.181 0.425 1.981 0.323 0.28 0.158 0.239 0.06 0.03 0.13 0.07 0.14 0.02 0.06 0.24 0.03 0.17 0.03 0.02

TN93+G 45 40804 40384 -20147 n/a 0.294 1.947 0.323 0.28 0.158 0.239 0.05 0.03 0.13 0.05 0.13 0.04 0.05 0.24 0.04 0.17 0.05 0.03

TN93+G+I 46 40810 40380 -20144 0.175 0.418 1.979 0.323 0.28 0.158 0.239 0.05 0.03 0.13 0.05 0.14 0.04 0.05 0.24 0.04 0.17 0.05 0.03

HKY+G 44 40836 40424 -20168 n/a 0.291 1.965 0.323 0.28 0.158 0.239 0.05 0.03 0.16 0.05 0.11 0.04 0.05 0.19 0.04 0.21 0.05 0.03

HKY+G+I 45 40841 40421 -20165 0.171 0.41 1.997 0.323 0.28 0.158 0.239 0.05 0.03 0.16 0.05 0.11 0.04 0.05 0.19 0.04 0.22 0.05 0.03

T92+G 42 40854 40461 -20189 n/a 0.296 1.945 0.301 0.301 0.199 0.199 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03

T92+G+I 43 40860 40458 -20186 0.167 0.412 1.975 0.301 0.301 0.199 0.199 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03

GTR+I 48 41097 40648 -20276 0.491 n/a 1.928 0.323 0.28 0.158 0.239 0.07 0.03 0.13 0.08 0.13 0.03 0.05 0.23 0.03 0.18 0.03 0.02

TN93+I 45 41133 40712 -20311 0.49 n/a 1.9 0.323 0.28 0.158 0.239 0.05 0.03 0.13 0.05 0.13 0.04 0.05 0.23 0.04 0.18 0.05 0.03

HKY+I 44 41164 40752 -20332 0.491 n/a 1.908 0.323 0.28 0.158 0.239 0.05 0.03 0.16 0.05 0.1 0.04 0.05 0.18 0.04 0.21 0.05 0.03

T92+I 42 41181 40788 -20352 0.491 n/a 1.898 0.301 0.301 0.199 0.199 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03

K2+G 41 41230 40847 -20382 n/a 0.317 1.824 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+G+I 42 41237 40845 -20380 0.146 0.423 1.842 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

K2+I 41 41535 41151 -20535 0.488 n/a 1.782 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

JC+G 40 42072 41698 -20809 n/a 0.343 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 41 42083 41699 -20809 0.051 0.378 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 40 42367 41993 -20956 0.479 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

GTR 47 42418 41978 -20942 n/a n/a 1.401 0.323 0.28 0.158 0.239 0.08 0.03 0.12 0.09 0.11 0.03 0.07 0.2 0.04 0.17 0.04 0.02

TN93 44 42475 42064 -20988 n/a n/a 1.399 0.323 0.28 0.158 0.239 0.06 0.03 0.12 0.07 0.11 0.05 0.07 0.2 0.05 0.16 0.06 0.03

HKY 43 42505 42103 -21008 n/a n/a 1.398 0.323 0.28 0.158 0.239 0.06 0.03 0.14 0.07 0.09 0.05 0.07 0.16 0.05 0.19 0.06 0.03

T92 41 42519 42135 -21027 n/a n/a 1.4 0.301 0.301 0.199 0.199 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.18 0.04 0.18 0.06 0.04

K2 40 42757 42383 -21152 n/a n/a 1.395 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC 39 43469 43104 -21513 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

115

Table A1.19. Francisella concatenated 16S, tpiA, prfB, rpoA and dnaA genetic distance matrix.

P. s

almon

is

str.

LF-8

9 (C

P0118

49)

F. haliotic

ida

str.

Shim

ane-

1 (J

F290

376)

F. hisp

aniens

is

FSC45

4 (C

P01

8093

)

F. cf. no

vicida

352

3 (C

P00

2558

)

F. tular

ensis

sub

sp. ho

larc

tica

LVS (A

M23

3362

.1)

F. tular

ensis

sub

sp. m

ediasiat

ica F

SC14

7 (C

P00

0915

)

F. tular

ensis

str.

Sch

u4 (C

P01

3853

)

F. tular

ensis

sub

sp. tu

lare

nsis

str.

WY96

(CP01

2037

)

F. cf. no

vicida

Fx1

(CP00

2557

)

F. tular

ensis

sub

sp. no

vicida

F61

68 (C

P00

0915

)

F. sp

. MA06

7296

(CP01

6930

)

F. opp

ortunistica

str.

14-

2155

(CP02

2375

)

Sam

ple

R20

ex. A

. albolim

batum

(PI 1

872)

F. per

sica

ATC

C V

R-3

31 (C

P01

2505

)

F. noa

tune

nsis

subs

p. n

oatune

nsis

FSC77

4 (N

Z CP05

3850

)

F. philom

iragia

sub

sp. ph

ilom

iragia

ATC

C 250

15 (C

P01

0019

)

F. philom

iragia

str.

1951

(AY49

6933

)

F. philom

iragia

sub

sp. ph

ilom

iragia

ATC

C 250

17 (C

P00

0937

.1)

F. noa

tune

nsis

subs

p. o

rient

alis

str.

Tob

a 04

(CP00

3402

)

F. orie

ntalis

FNO12

(CP01

1921

)

F. philom

iragia

str.

GA01

-279

4 (C

P00

9440

)

P. salmonis str. LF-89 (CP011849)

F. halioticida str. Shimane-1 (JF290376) 0.58

F. hispaniensis FSC454 (CP018093) 0.60 0.17

F. cf. novicida 3523 (CP002558) 0.60 0.17 0.01

F. tularensis subsp. holarctica LVS (AM233362.1) 0.58 0.17 0.05 0.05

F. tularensis subsp. mediasiatica FSC147 (CP000915) 0.58 0.17 0.05 0.05 0.00

F. tularensis str. Schu4 (CP013853) 0.58 0.17 0.05 0.05 0.00 0.00

F. tularensis subsp. tularensis str. WY96 (CP012037) 0.58 0.17 0.05 0.05 0.00 0.00 0.00

F. cf. novicida Fx1 (CP002557) 0.58 0.17 0.06 0.05 0.02 0.02 0.01 0.01

F. tularensis subsp. novicida F6168 (CP000915) 0.58 0.17 0.06 0.06 0.02 0.01 0.01 0.01 0.01

F. sp. MA067296 (CP016930) 0.58 0.18 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09

F. opportunistica str. 14-2155 (CP022375) 0.58 0.18 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.09 0.00

Sample R20 ex. A. albolimbatum (PI 1872) 0.59 0.18 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.07

F. persica ATCC VR-331 (CP012505) 0.59 0.17 0.08 0.08 0.07 0.07 0.07 0.07 0.08 0.08 0.07 0.07 0.03

F. noatunensis subsp. noatunensis FSC774 (NZ CP053850) 0.64 0.21 0.21 0.21 0.19 0.19 0.19 0.19 0.19 0.19 0.22 0.22 0.22 0.21

F. philomiragia subsp. philomiragia ATCC 25015 (CP010019) 0.59 0.16 0.15 0.15 0.14 0.14 0.14 0.14 0.13 0.14 0.16 0.16 0.16 0.16 0.10

F. philomiragia str. 1951 (AY496933) 0.59 0.16 0.14 0.14 0.14 0.14 0.14 0.14 0.13 0.13 0.16 0.16 0.16 0.15 0.10 0.01

F. philomiragia subsp. philomiragia ATCC 25017 (CP000937.1)0.59 0.16 0.15 0.15 0.14 0.14 0.14 0.14 0.13 0.14 0.16 0.16 0.16 0.16 0.10 0.01 0.01

F. noatunensis subsp. orientalis str. Toba 04 (CP003402) 0.59 0.17 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.14 0.11 0.11 0.14 0.13 0.12 0.07 0.07 0.07

F. orientalis FNO12 (CP011921) 0.59 0.17 0.15 0.16 0.15 0.15 0.15 0.15 0.14 0.14 0.17 0.17 0.17 0.16 0.09 0.04 0.05 0.04 0.04

F. philomiragia str. GA01-2794 (CP009440) 0.59 0.16 0.15 0.15 0.14 0.14 0.14 0.14 0.14 0.14 0.16 0.16 0.17 0.16 0.09 0.05 0.05 0.04 0.05 0.03

116

Table A1.20. 344 bp Rickettsia gltA Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 62 3024 2574 -1224 n/a 0.267 3.221 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02

T92+I 62 3029 2579 -1227 0.644 n/a 3.148 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02

T92+G+I 63 3031 2574 -1224 0.353 0.592 3.237 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.26 0.02 0.26 0.04 0.02

HKY+G 64 3037 2573 -1222 n/a 0.262 3.284 0.366 0.291 0.143 0.2 0.03 0.02 0.16 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.03 0.02

TN93+G 65 3041 2569 -1219 n/a 0.272 3.212 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.16 0.02 0.04 0.32 0.02 0.2 0.03 0.02

HKY+I 64 3044 2579 -1225 0.646 n/a 3.187 0.366 0.291 0.143 0.2 0.03 0.02 0.15 0.04 0.11 0.02 0.04 0.23 0.02 0.28 0.03 0.02

HKY+G+I 65 3046 2574 -1222 0.351 0.574 3.309 0.366 0.291 0.143 0.2 0.03 0.02 0.16 0.04 0.11 0.02 0.04 0.23 0.02 0.29 0.03 0.02

TN93+I 65 3046 2574 -1222 0.639 n/a 3.128 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.15 0.02 0.04 0.31 0.02 0.2 0.03 0.02

TN93+G+I 66 3048 2569 -1218 0.376 0.656 3.237 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.16 0.02 0.04 0.32 0.02 0.2 0.03 0.02

K2+G 61 3060 2617 -1247 n/a 0.24 3.283 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

GTR+G 68 3064 2570 -1217 n/a 0.281 2.504 0.366 0.291 0.143 0.2 0.03 0.03 0.12 0.03 0.13 0.03 0.07 0.27 0.02 0.21 0.05 0.01

K2+I 61 3065 2622 -1250 0.667 n/a 3.223 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

GTR+I 68 3067 2573 -1218 0.631 n/a 2.872 0.366 0.291 0.143 0.2 0.01 0.03 0.11 0.02 0.14 0.03 0.07 0.29 0.02 0.21 0.05 0.02

K2+G+I 62 3067 2617 -1246 0.388 0.575 3.319 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

GTR+G+I 69 3070 2569 -1215 0.355 0.637 2.652 0.366 0.291 0.143 0.2 0.02 0.03 0.12 0.03 0.14 0.03 0.07 0.28 0.02 0.21 0.05 0.01

T92 61 3092 2649 -1263 n/a n/a 2.899 0.329 0.329 0.171 0.171 0.04 0.02 0.13 0.04 0.13 0.02 0.04 0.25 0.02 0.25 0.04 0.02

TN93 64 3108 2643 -1257 n/a n/a 2.894 0.366 0.291 0.143 0.2 0.03 0.02 0.11 0.04 0.15 0.02 0.04 0.3 0.02 0.2 0.03 0.02

HKY 63 3108 2651 -1262 n/a n/a 2.89 0.366 0.291 0.143 0.2 0.04 0.02 0.15 0.04 0.11 0.02 0.04 0.22 0.02 0.28 0.04 0.02

GTR 67 3132 2645 -1255 n/a n/a 2.141 0.366 0.291 0.143 0.2 0.03 0.03 0.11 0.04 0.12 0.04 0.07 0.25 0.02 0.21 0.05 0.02

K2 60 3135 2699 -1289 n/a n/a 2.865 0.25 0.25 0.25 0.25 0.03 0.03 0.19 0.03 0.19 0.03 0.03 0.19 0.03 0.19 0.03 0.03

JC+G 60 3151 2716 -1298 n/a 0.259 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 60 3157 2721 -1300 0.659 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 61 3160 2717 -1297 0.366 0.6 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 59 3222 2793 -1337 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

117

Table A1.21. 344 bp Rickettsia gltA genetic distance matrix.

Sample SR

12 ex.

A. a

lbolim

batum

(P

I 332

9)

Unc

ulture

d R. sp

. clone

ARRL2

016-

156 (M

N43

1835

)

Unc

ulture

d R. s

p. clone

ARRL2

015-

159 (M

N43

1836

)

R. tam

urae

str. A

T 1 (A

F394

896 )

R. s

lova

ca is

olate Xin

jiang

(MF0

0252

8)

R. s

ibirica

246

(AABW

0100

0001

)

R. rick

ettsii

str. A

rizon

a (C

P0033

07)

R. rhipice

phali

str. H

J 5 (C

P0131

33)

R. rao

ultii

str. K

haba

rovs

k (C

P0109

69)

R. p

arke

ri str. P

ortsm

outh (NC 017

044)

R. m

ontane

nsis

str. O

SU 85 93

0 (C

P0033

40)

R. m

onac

ensis

str. IrR/M

unich (L

N79

4217

)

R. m

assil

iae

str. A

ZT80

(CP00

3319

)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. h

onei

RB (N

Z AJT

T010

0000

4)

R. h

eilong

jiang

ensis

Sen

dai 5

8 DNA (A

P0198

65)

R. g

rave

sii B

WI 1

(DQ26

9435

)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. c

onor

ii str. M

alish 7 (A

E0069

14)

R. a

mblyo

mm

atis

str. GAT

30V (

CP00

3334

)

R. a

frica

e E

SF 5 (C

P0016

12)

Unc

ulture

d R. s

p. clone

ARRL2

016-

149 (M

N43

1834

)

R. a

esch

liman

nii

str. RH15

(HM

0502

89)

R. a

ustra

lis

str. Cutlack

(NC 017

058)

R. a

kari

str. H

artfo

rd (NC 009

881)

R. felis

URRW

XCal2 (C

P0000

53)

R. a

sem

bone

nsis

str. N

MRCii (

NZ

JWSW

0100

0078

)

R. typ

hi str. W

ilmington

(NC 006

142)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. b

ellii

RM

L369

-C (N

C 007

940)

Sample SR12 ex. A. albolimbatum (PI 3329)

Uncultured R. sp. clone ARRL2016-156 (MN431835) 0.14

Uncultured R. sp. clone ARRL2015-159 (MN431836) 0.14 0.00

R. tamurae str. AT 1 (AF394896 ) 0.15 0.04 0.04

R. slovaca isolate Xinjiang (MF002528) 0.15 0.01 0.01 0.04

R. sibirica 246 (AABW01000001) 0.14 0.00 0.00 0.04 0.00

R. rickettsii str. Arizona (CP003307) 0.14 0.01 0.01 0.04 0.01 0.01

R. rhipicephali str. HJ 5 (CP013133) 0.14 0.01 0.01 0.05 0.01 0.01 0.02

R. raoultii str. Khabarovsk (CP010969) 0.14 0.00 0.00 0.04 0.01 0.00 0.01 0.01

R. parkeri str. Portsmouth (NC 017044) 0.14 0.01 0.01 0.04 0.01 0.00 0.01 0.01 0.01

R. montanensis str. OSU 85 930 (CP003340) 0.13 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01

R. monacensis str. IrR/Munich (LN794217) 0.15 0.03 0.03 0.01 0.04 0.04 0.04 0.04 0.03 0.04 0.03

R. massiliae str. AZT80 (CP003319) 0.14 0.01 0.01 0.05 0.01 0.01 0.02 0.00 0.01 0.01 0.01 0.04

R. japonica str. M14012 (NZ AP017596) 0.14 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01

R. honei RB (NZ AJTT01000004) 0.15 0.01 0.01 0.04 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.04 0.01 0.01

R. heilongjiangensis Sendai 58 DNA (AP019865) 0.14 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01 0.00 0.01

R. gravesii BWI 1 (DQ269435) 0.15 0.01 0.01 0.05 0.02 0.02 0.02 0.02 0.01 0.01 0.02 0.05 0.02 0.02 0.02 0.02

R. helvetica C9P9 (CM001467) 0.17 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.05

R. conorii str. Malish 7 (AE006914) 0.14 0.01 0.01 0.05 0.01 0.01 0.01 0.02 0.01 0.01 0.01 0.04 0.02 0.01 0.01 0.01 0.02 0.06

R. amblyommatis str. GAT 30V (CP003334) 0.14 0.00 0.00 0.04 0.00 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01 0.01 0.01 0.01 0.02 0.05 0.01

R. africae ESF 5 (CP001612) 0.14 0.00 0.00 0.04 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.04 0.01 0.01 0.00 0.01 0.02 0.05 0.01 0.01

Uncultured R. sp. clone ARRL2016-149 (MN431834) 0.14 0.00 0.00 0.04 0.01 0.00 0.01 0.01 0.00 0.01 0.00 0.03 0.01 0.00 0.01 0.00 0.01 0.05 0.01 0.00 0.00

R. aeschlimannii str. RH15 (HM050289) 0.14 0.00 0.00 0.04 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.04 0.01 0.01 0.01 0.01 0.02 0.05 0.01 0.01 0.01 0.00

R. australis str. Cutlack (NC 017058) 0.14 0.03 0.03 0.04 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.04 0.04 0.04 0.04 0.04 0.05 0.05 0.04 0.04 0.04 0.03 0.04

R. akari str. Hartford (NC 009881) 0.16 0.05 0.05 0.03 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.03 0.06 0.05 0.05 0.05 0.06 0.06 0.06 0.05 0.05 0.05 0.05 0.03

R. felis URRWXCal2 (CP000053) 0.15 0.03 0.03 0.03 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.03 0.04 0.04 0.04 0.04 0.05 0.04 0.04 0.04 0.04 0.03 0.04 0.03 0.04

R. asembonensis str. NMRCii (NZ JWSW01000078) 0.17 0.05 0.05 0.05 0.05 0.05 0.06 0.06 0.05 0.05 0.05 0.05 0.06 0.05 0.05 0.05 0.06 0.05 0.06 0.05 0.05 0.05 0.05 0.06 0.07 0.04

R. typhi str. Wilmington (NC 006142) 0.17 0.08 0.08 0.09 0.08 0.09 0.09 0.09 0.08 0.09 0.08 0.08 0.09 0.09 0.09 0.09 0.09 0.11 0.09 0.08 0.09 0.08 0.09 0.10 0.09 0.09 0.12

R. prowazekii str. Madrid E (NC 000963) 0.16 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.10 0.09 0.08 0.08 0.08 0.08 0.10 0.10 0.09 0.11 0.04

R. bellii RML369-C (NC 007940) 0.05 0.16 0.16 0.17 0.17 0.16 0.16 0.16 0.16 0.16 0.15 0.17 0.16 0.16 0.16 0.16 0.17 0.17 0.16 0.16 0.16 0.16 0.16 0.17 0.18 0.18 0.17 0.19 0.18

118

Table A1.22. 634 bp Rickettsia gltA Maximum Likelihood fits of 24 different nucleotide substitution models.

Model #Param BIC AICc lnL (+I) (+G) R Freq AFreq TFreq CFreq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C

T92+G 56 5966 5533 -2710 n/a 0.37 2.58 0.32 0.32 0.18 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.23 0.02 0.23 0.04 0.02

K2+G 55 5973 5547 -2718 n/a 0.31 2.6 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

T92+G+I 57 5976 5534 -2710 0.12 0.47 2.66 0.32 0.32 0.18 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.23 0.02 0.23 0.04 0.02

K2+G+I 56 5982 5548 -2718 0.15 0.43 2.67 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

HKY+G 58 5986 5537 -2710 n/a 0.37 2.56 0.3 0.33 0.19 0.18 0.04 0.02 0.13 0.04 0.14 0.02 0.04 0.24 0.02 0.22 0.04 0.02

T92+I 56 5988 5554 -2721 0.41 n/a 2.91 0.32 0.32 0.18 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.24 0.02 0.24 0.04 0.02

TN93+G 59 5994 5538 -2710 n/a 0.36 2.6 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.13 0.02 0.04 0.23 0.02 0.23 0.04 0.02

HKY+G+I 59 5994 5538 -2710 0.13 0.49 2.64 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.25 0.02 0.22 0.04 0.02

K2+I 55 5999 5573 -2731 0.41 n/a 2.78 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

TN93+G+I 60 6004 5539 -2709 0.13 0.49 2.64 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.13 0.02 0.04 0.23 0.02 0.23 0.04 0.02

HKY+I 58 6006 5557 -2720 0.41 n/a 2.9 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.25 0.02 0.23 0.04 0.02

TN93+I 59 6016 5559 -2720 0.41 n/a 2.91 0.3 0.33 0.19 0.18 0.04 0.02 0.14 0.04 0.14 0.02 0.04 0.25 0.02 0.23 0.04 0.02

GTR+G 62 6016 5536 -2706 n/a 0.38 2.05 0.3 0.33 0.19 0.18 0.05 0.04 0.15 0.04 0.11 0.04 0.06 0.19 0.01 0.24 0.07 0.01

GTR+G+I 63 6021 5533 -2703 0.09 0.46 2.21 0.3 0.33 0.19 0.18 0.04 0.04 0.15 0.03 0.11 0.04 0.06 0.21 0 0.24 0.07 0

GTR+I 62 6025 5545 -2710 0.41 n/a 2.87 0.3 0.33 0.19 0.18 0.02 0.04 0.14 0.02 0.14 0.04 0.06 0.24 0 0.23 0.07 0

T92 55 6074 5648 -2769 n/a n/a 1.66 0.32 0.32 0.18 0.18 0.06 0.03 0.12 0.06 0.12 0.03 0.06 0.2 0.03 0.2 0.06 0.03

K2 54 6082 5664 -2778 n/a n/a 1.68 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

HKY 57 6092 5651 -2768 n/a n/a 1.66 0.3 0.33 0.19 0.18 0.06 0.03 0.12 0.05 0.12 0.03 0.05 0.21 0.03 0.19 0.06 0.03

TN93 58 6102 5653 -2768 n/a n/a 1.66 0.3 0.33 0.19 0.18 0.06 0.03 0.12 0.05 0.12 0.03 0.05 0.22 0.03 0.19 0.06 0.03

JC+G 54 6108 5690 -2791 n/a 0.39 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 55 6118 5692 -2791 0 0.39 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

GTR 61 6118 5646 -2762 n/a n/a 1.55 0.3 0.33 0.19 0.18 0.04 0.04 0.11 0.03 0.11 0.05 0.07 0.2 0.03 0.19 0.09 0.03

JC+I 54 6137 5719 -2805 0.39 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 53 6193 5783 -2838 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

119

Table A1.23. 634 bp Rickettsia gltA genetic distance matrix.

Sample SR

12 ex.

A. a

lbolim

batum

(P

I 332

9)

R. a

esch

liman

nii s

tr. R

H15

(HM

0502

89)

R. a

frica

e E

SF 5 (C

P0016

12)

R. a

mblyo

mm

atis

str. GAT

30V (

CP00

3334

)

R. c

onor

ii str. M

alish 7 (A

E0069

14)

R. h

eilong

jiang

ensis

Sen

dai 5

8 DNA (A

P0198

65)

R. h

onei

RB (N

Z AJT

T010

0000

4)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. m

assil

iae

str. A

ZT80

(CP00

3319

)

R. m

ontane

nsis

str. O

SU 85 93

0 (C

P0033

40)

R. p

arke

ri str. P

ortsm

outh (NC 017

044)

R. p

eaco

ckii

str. Rus

tic (NC 012

730)

R. rao

ultii

str. Kha

baro

vsk (C

P0109

69)

R. rhipice

phali s

tr. H

J 5 (C

P0131

33)

R rick

ettsii

str. A

rizon

a (C

P0033

07)

R. s

ibirica

246

(AABW

0100

0001

)

R. s

lova

ca is

olate Xin

jiang

(MF0

0252

8)

R. g

rave

sii B

WI 1

(DQ26

9435

)

R. m

onac

ensis

str. IrR/M

unich

(LN79

4217

)

R. tam

urae

str. A

T 1 (A

F394

896 )

R. a

ustra

lis

str. Cutlack

(NC 017

058)

R. a

sem

bone

nsis

str. N

MRCii (

NZ

JWSW

0100

0078

)

R. felis

URRW

XCal2 (C

P0000

53)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. a

kari

str. H

artfo

rd (NC 009

881)

R. typ

hi str. W

ilmington

(NC 006

142)

R. b

ellii

RM

L369

-C (N

C 007

940)


R. aeschlimannii str. RH15 (HM050289) 0.01

R. africae ESF 5 (CP001612) 0.01 0.01

R. amblyommatis str. GAT 30V (CP003334) 0.01 0.01 0.01

R. conorii str. Malish 7 (AE006914) 0.01 0.01 0.01 0.01

R. heilongjiangensis Sendai 58 DNA (AP019865) 0.01 0.01 0.00 0.01 0.01

R. honei RB (NZ AJTT01000004) 0.01 0.01 0.00 0.01 0.00 0.01

R. japonica str. M14012 (NZ AP017596) 0.01 0.01 0.00 0.01 0.01 0 0.01

R. massiliae str. AZT80 (CP003319) 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01

R. montanensis str. OSU 85 930 (CP003340) 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.01

R. parkeri str. Portsmouth (NC 017044) 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01

R. peacockii str. Rustic (NC 012730) 0.01 0.01 0.00 0.01 0.00 0.01 0.00 0.01 0.01 0.01 0.00

R. raoultii str. Khabarovsk (CP010969) 0.01 0.00 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01

R. rhipicephali str. HJ 5 (CP013133) 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.02 0.00 0.01 0.01 0.01 0.01

R rickettsii str. Arizona (CP003307) 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.00 0.01 0.01 0.02

R. sibirica 246 (AABW01000001) 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.01 0.00

R. slovaca isolate Xinjiang (MF002528) 0.01 0.01 0.00 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.01 0.01 0.01 0.00

R. gravesii BWI 1 (DQ269435) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

R. monacensis str. IrR/Munich (LN794217) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.02 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04

R. tamurae str. AT 1 (AF394896 ) 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.01

R. australis str. Cutlack (NC 017058) 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.06 0.07 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.07

R. asembonensis str. NMRCii (NZ JWSW01000078) 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.08 0.07 0.07 0.08 0.07 0.07 0.05

R. felis URRWXCal2 (CP000053) 0.08 0.07 0.08 0.07 0.08 0.08 0.08 0.08 0.08 0.07 0.07 0.08 0.07 0.07 0.08 0.07 0.08 0.08 0.08 0.07 0.06 0.01

R. prowazekii str. Madrid E (NC 000963) 0.08 0.08 0.07 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.09 0.08 0.08 0.10 0.11 0.12

R. akari str. Hartford (NC 009881) 0.08 0.08 0.08 0.07 0.08 0.08 0.08 0.08 0.08 0.07 0.08 0.08 0.07 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.03 0.06 0.07 0.10

R. typhi str. Wilmington (NC 006142) 0.09 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.09 0.09 0.08 0.08 0.09 0.08 0.08 0.11 0.12 0.13 0.03 0.10

R. bellii RML369-C (NC 007940) 0.17 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.16 0.17 0.17 0.16 0.16 0.18 0.16 0.16 0.17 0.15 0.16 0.17 0.17 0.16

120

Table A1.24. Rickettsia ompA Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 58 8651 8237 -4060 n/a 3.25 1.21 0.2949 0.2949 0.2051 0.2051 0.07 0.05 0.11 0.07 0.11 0.05 0.07 0.16 0.05 0.16 0.07 0.05

TN93+G 61 8656 8220 -4049 n/a 3.46 0.92 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.13 0.07 0.07 0.06 0.07 0.12 0.06 0.16 0.08 0.05

T92 57 8657 8250 -4068 n/a n/a 0.89 0.2949 0.2949 0.2051 0.2051 0.08 0.05 0.1 0.08 0.1 0.05 0.08 0.14 0.05 0.14 0.08 0.05

HKY+G 60 8666 8238 -4059 n/a 3.28 1.21 0.2877 0.3021 0.1835 0.2267 0.07 0.04 0.13 0.06 0.1 0.05 0.06 0.17 0.05 0.16 0.07 0.04

HKY 59 8669 8248 -4065 n/a n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.11 0.08 0.09 0.06 0.08 0.14 0.06 0.14 0.08 0.05

T92+I 58 8671 8257 -4070 0.05 n/a 1.19 0.2949 0.2949 0.2051 0.2051 0.07 0.05 0.11 0.07 0.11 0.05 0.07 0.16 0.05 0.16 0.07 0.05

T92+G+I 59 8671 8250 -4066 0 3.08 1.35 0.2949 0.2949 0.2051 0.2051 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.17 0.04 0.17 0.06 0.04

TN93 60 8674 8246 -4063 n/a n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.12 0.08 0.08 0.06 0.08 0.13 0.06 0.15 0.08 0.05

TN93+I 61 8675 8240 -4059 0.05 n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.08 0.05 0.12 0.07 0.08 0.06 0.07 0.12 0.06 0.16 0.08 0.05

GTR+G 64 8677 8220 -4046 n/a 3.53 0.92 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.13 0.07 0.07 0.05 0.09 0.12 0.06 0.16 0.07 0.05

TN93+G+I 62 8679 8237 -4056 0 3.35 1.15 0.2877 0.3021 0.1835 0.2267 0.07 0.04 0.12 0.07 0.1 0.05 0.07 0.16 0.05 0.16 0.07 0.04

HKY+I 60 8684 8256 -4067 0.05 n/a 1.19 0.2877 0.3021 0.1835 0.2267 0.07 0.04 0.12 0.06 0.1 0.05 0.06 0.17 0.05 0.16 0.07 0.04

HKY+G+I 61 8684 8249 -4063 0 3.11 1.35 0.2877 0.3021 0.1835 0.2267 0.06 0.04 0.13 0.06 0.11 0.05 0.06 0.18 0.05 0.17 0.06 0.04

GTR+G+I 65 8686 8222 -4046 0 3.52 0.92 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.13 0.07 0.07 0.05 0.09 0.12 0.06 0.16 0.07 0.05

GTR 63 8693 8244 -4058 n/a n/a 0.88 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.12 0.06 0.08 0.05 0.09 0.13 0.07 0.15 0.07 0.05

GTR+I 64 8696 8239 -4055 0.05 n/a 0.89 0.2877 0.3021 0.1835 0.2267 0.07 0.06 0.12 0.07 0.08 0.05 0.09 0.12 0.07 0.15 0.07 0.05

K2+G 57 8756 8349 -4117 n/a 2.68 1.25 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06

K2+G+I 58 8776 8362 -4123 0 2.53 1.39 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC+G 56 8776 8376 -4132 n/a 3.29 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

K2 56 8777 8377 -4132 n/a n/a 1.12 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06

K2+I 57 8782 8375 -4130 0.06 n/a 1.23 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06

JC+G+I 57 8785 8378 -4132 0 3.29 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 56 8799 8399 -4143 0.06 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 55 8799 8407 -4148 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

121

Table A1.25. Rickettsia ompA genetic distance matrix.

R. m

ontane

sis OSU

85-

390

(CP00

3340

)

R. p

arke

ri str. Por

tsm

outh (N

C 017

044)

R. s

ibirica

246

(AABW

0100

0001

)

R. h

eilong

jiang

ensis

Sen

dai-5

8 (A

P0198

65)

R. a

frica

e E

SF-5 (C

P0016

12)

R. rhipice

phali

HJ 5 (C

P0131

33)

R. g

rave

sii

BWI-1

(DQ26

9437

)

R. a

esch

liman

nii

str. RH15

(HM

0502

89)

R. m

assil

iae

str. AZT

80 (C

P0033

19)

R. m

onac

ensis

str. IrR

/Mun

ich (L

N79

4217

)

R. s

lova

ca is

olate Xin

jiang

(MF0

0253

5)

R. rao

ultii

str. Kha

baro

vsk (C

P1096

9)

Unc

ulture

d R. s

p. clone

ARRL2

016-

159 (M

N43

1847

)

R. p

eaco

cki

(NC 012

730)

R. c

onor

ii str. M

alish

7 (A

E0069

14)

R. rick

ettsii

str. A

rizon

a (C

P0033

07)

R. h

onei

RB (N

Z AJT

T000

0000

0.1)

Sample SR

12 ex. Am

. albolim

batum

(PI 3

329)

R. b

ellii

RM

L369

-C (N

C 007

940)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. tam

urae

str. AT-

1 (A

F394

896)

R. a

mblyo

mm

atis

str. G

AT-30

V (CP00

3334

)

R. typ

hi st

r. W

ilmington

(NC-0

0614

2)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. a

ustra

lis

str. Cutlack

(NC 017

058)

R. felis

URRW

XCal2 (C

P0000

53)

R. a

kari

(NC 009

881)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. a

sem

bone

nsis

str. NM

RCii (

NZ JW

SW00

0000

00.1)

R. montanesis OSU 85-390 (CP003340)

R. parkeri str. Portsmouth (NC 017044) 0.10

R. sibirica 246 (AABW01000001) 0.12 0.04

R. heilongjiangensis Sendai-58 (AP019865) 0.10 0.07 0.08

R. africae ESF-5 (CP001612) 0.12 0.03 0.02 0.07

R. rhipicephali HJ 5 (CP013133) 0.08 0.08 0.10 0.09 0.10

R. gravesii BWI-1 (DQ269437) 0.05 0.06 0.09 0.09 0.09 0.04

R. aeschlimannii str. RH15 (HM050289) 0.07 0.08 0.10 0.10 0.10 0.04 0.04

R. massiliae str. AZT80 (CP003319) 0.07 0.07 0.09 0.09 0.09 0.02 0.03 0.03

R. monacensis str. IrR/Munich (LN794217) 0.12 0.20 0.22 0.20 0.22 0.17 0.14 0.16 0.16

R. slovaca isolate Xinjiang (MF002535) 0.10 0.03 0.02 0.07 0.01 0.09 0.07 0.09 0.08 0.20

R. raoultii str. Khabarovsk (CP10969) 0.06 0.07 0.08 0.08 0.09 0.04 0.04 0.04 0.03 0.16 0.07

Uncultured R. sp. clone ARRL2016-159 (MN431847) 0.08 0.08 0.11 0.11 0.10 0.06 0.03 0.06 0.05 0.18 0.09 0.05

R. peacocki (NC 012730) 0.15 0.10 0.09 0.13 0.08 0.15 0.13 0.14 0.14 0.24 0.07 0.15 0.15

R. conorii str. Malish 7 (AE006914) 0.11 0.05 0.04 0.07 0.04 0.09 0.07 0.09 0.08 0.18 0.03 0.07 0.09 0.07

R. rickettsii str. Arizona (CP003307) 0.11 0.05 0.03 0.07 0.03 0.10 0.09 0.10 0.09 0.23 0.03 0.09 0.10 0.10 0.04

R. honei RB (NZ AJTT00000000.1) 0.14 0.04 0.03 0.09 0.02 0.12 0.10 0.12 0.11 0.24 0.03 0.10 0.12 0.10 0.05 0.04

Sample SR12 ex. Am. albolimbatum (PI 3329) 0.05 0.06 0.09 0.09 0.09 0.03 0.01 0.04 0.03 0.15 0.07 0.03 0.02 0.13 0.07 0.09 0.10

R. bellii RML369-C (NC 007940) 0.74 0.81 0.89 0.86 0.88 0.73 0.70 0.76 0.70 0.94 0.86 0.75 0.78 0.90 0.88 0.91 0.97 0.73

R. japonica str. M14012 (NZ AP017596) 0.12 0.09 0.11 0.04 0.10 0.11 0.11 0.11 0.11 0.20 0.09 0.09 0.14 0.16 0.08 0.10 0.12 0.12 0.90

R. tamurae str. AT-1 (AF394896) 0.10 0.16 0.16 0.16 0.15 0.12 0.09 0.12 0.11 0.08 0.14 0.11 0.13 0.18 0.13 0.15 0.16 0.10 0.91 0.18

R. amblyommatis str. GAT-30V (CP003334) 0.07 0.07 0.08 0.10 0.08 0.04 0.04 0.05 0.04 0.15 0.06 0.04 0.06 0.11 0.08 0.07 0.10 0.03 0.77 0.12 0.09

R. typhi str. Wilmington (NC-006142) 0.53 0.61 0.60 0.65 0.56 0.54 0.51 0.45 0.49 0.67 0.54 0.51 0.50 0.56 0.56 0.56 0.64 0.47 1.45 0.67 0.63 0.41

R. prowazekii str. Madrid E (NC 000963) 0.81 0.86 0.88 0.87 0.86 0.74 0.79 0.75 0.72 0.95 0.86 0.79 0.82 0.97 0.83 0.86 0.91 0.78 1.62 0.92 0.93 0.85 1.02

R. australis str. Cutlack (NC 017058) 0.77 0.84 0.84 0.87 0.82 0.72 0.73 0.75 0.72 0.87 0.84 0.80 0.73 0.90 0.79 0.85 0.86 0.74 1.80 0.86 0.88 0.67 1.31 1.53


R. akari (NC 009881) 0.78 0.76 0.76 0.89 0.79 0.76 0.75 0.74 0.71 0.98 0.77 0.77 0.73 0.82 0.78 0.82 0.86 0.74 1.42 0.99 0.86 0.66 1.12 1.65 1.06 0.12

R. helvetica C9P9 (CM001467) 0.55 0.52 0.50 0.53 0.51 0.53 0.51 0.51 0.53 0.59 0.51 0.49 0.55 0.50 0.47 0.46 0.56 0.50 1.69 0.57 0.55 0.50 1.29 1.16 1.71 1.36 1.75

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.57 0.57 0.61 0.65 0.62 0.58 0.60 0.57 0.59 0.64 0.62 0.54 0.67 0.69 0.61 0.57 0.68 0.59 1.77 0.69 0.65 0.56 1.59 1.69 1.70 1.52 1.57 0.29

122

Table A1.26. Rickettsia ompB Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 66 3254 2791 -1329 n/a 1.24 1.71 0.304 0.304 0.196 0.196 0.05 0.04 0.13 0.05 0.13 0.04 0.05 0.19 0.04 0.19 0.05 0.04

T92+G+I 67 3261 2792 -1328 0 1.22 1.71 0.304 0.304 0.196 0.196 0.05 0.04 0.13 0.05 0.13 0.04 0.05 0.2 0.04 0.2 0.05 0.04

T92 65 3265 2809 -1339 n/a n/a 1.42 0.304 0.304 0.196 0.196 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.18 0.04 0.18 0.06 0.04

T92+I 66 3266 2803 -1335 0.1789 n/a 1.58 0.304 0.304 0.196 0.196 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.19 0.04 0.19 0.06 0.04

HKY+G 68 3272 2795 -1329 n/a 1.24 1.71 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.19 0.04 0.2 0.05 0.03

K2+G 65 3275 2819 -1344 n/a 1.17 1.64 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

TN93+G 69 3279 2796 -1328 n/a 1.26 1.7 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.2 0.04 0.19 0.05 0.03

HKY+G+I 69 3280 2796 -1328 1E-05 1.24 1.71 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.19 0.04 0.2 0.05 0.03

HKY 67 3282 2812 -1339 n/a n/a 1.42 0.305 0.303 0.183 0.21 0.06 0.04 0.13 0.06 0.11 0.04 0.06 0.18 0.04 0.18 0.06 0.04

HKY+I 68 3283 2806 -1335 0.1777 n/a 1.59 0.305 0.303 0.183 0.21 0.06 0.03 0.13 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.03

K2+G+I 66 3283 2821 -1344 0 1.15 1.65 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

K2+I 65 3287 2831 -1350 0.1962 n/a 1.54 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

K2 64 3288 2839 -1355 n/a n/a 1.38 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05

TN93+G+I 70 3288 2798 -1328 1E-05 1.23 1.72 0.305 0.303 0.183 0.21 0.05 0.03 0.13 0.05 0.12 0.04 0.05 0.21 0.04 0.19 0.05 0.03

GTR+G 72 3290 2785 -1320 n/a 1.22 1.81 0.305 0.303 0.183 0.21 0.01 0.05 0.13 0.01 0.12 0.07 0.09 0.21 0.02 0.19 0.09 0.02

TN93 68 3291 2815 -1339 n/a n/a 1.42 0.305 0.303 0.183 0.21 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.2 0.04 0.17 0.06 0.04

TN93+I 69 3292 2808 -1335 0.1765 n/a 1.59 0.305 0.303 0.183 0.21 0.06 0.03 0.13 0.06 0.12 0.04 0.06 0.2 0.04 0.18 0.06 0.03

GTR+G+I 73 3299 2787 -1320 0 1.18 1.83 0.305 0.303 0.183 0.21 0.01 0.05 0.13 0.01 0.12 0.07 0.09 0.21 0.02 0.19 0.09 0.02

GTR+I 72 3301 2796 -1326 0.1924 n/a 1.72 0.305 0.303 0.183 0.21 0.01 0.05 0.13 0.01 0.12 0.07 0.09 0.19 0.02 0.19 0.1 0.02

GTR 71 3314 2816 -1336 n/a n/a 1.13 0.305 0.303 0.183 0.21 0.05 0.05 0.12 0.05 0.09 0.06 0.09 0.16 0.04 0.17 0.09 0.03

JC+G 64 3317 2868 -1370 n/a 1.52 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 63 3324 2882 -1378 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 65 3325 2869 -1369 1E-05 1.52 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 64 3327 2879 -1375 0.1573 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

123

Table A1.27. Rickettsia ompB genetic distance matrix.

Sample R52

ex. A a

lbolim

batum

(PI 6

80)

Sample R92

ex. A a

lbolim

batum

(PI 3

323)

Sample R10

6 ex

. A a

lbolim

batum

(P

I 146

0)

Sample SR

12 ex. A a

lbolim

batum

(PI 3

329)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. a

frica

e E

SF-5 (C

P0016

12)

R. a

mblyo

mm

atis

str. G

AT-30

V (CP00

3334

)

R. a

kari

Har

tford

(NC 009

881)

R. a

ustra

lis str. C

utlack

(NC 017

058)

R. felis

URRW

XCal2 (C

P0000

53)

R. a

sem

bone

nsis

str. NM

RCii (

NZ JW

SW00

0000

00.1)

R. c

onor

ii str. M

alish 7 (A

E0069

14.1)

R. g

rave

sii

BWI-1

(DQ26

9436

)

R. h

eilong

jiang

ensis

Sen

dai-5

8 (A

P0198

65)

R. h

onei R

B (NZ

AJTT0

0000

000.1)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. m

assil

iae

str. A

ZT80

(CP00

3319

)

R. m

onac

ensis

str. IrR

/Mun

ich (L

N79

4217

)

R. m

ontane

nsis

OSU

85-

390 (C

P0033

40)

R. p

arke

ri str. Por

tsm

outh (N

C 017

044)

R. rao

ultii

str. K

haba

rovs

k (C

P1096

9)

R. rhipice

phali

HJ 5 (C

P0131

33)

R. rick

ettsii

str. Ariz

ona (C

P0033

07)

R. s

ibirica

24

6 (A

ABW01

0000

01)

R. s

lova

ca iso

late X

injia

ng (M

F002

535)

R. tam

urae

str. A

T-1

R. a

esch

liman

nii

str. RH15

(HM

0502

89)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. typ

hi str. W

ilmington

(NC-0

0614

2)

Sample R53

ex. A. a

lbolim

batum

(PI 6

80)

R. b

ellii

RM

L369

-C (N

C 007

940)

Sample R52 ex. A albolimbatum (PI 680)

Sample R92 ex. A albolimbatum (PI 3323) 0.03

Sample R106 ex. A albolimbatum (PI 1460) 0.00 0.03

Sample SR12 ex. A albolimbatum (PI 3329) 0.04 0.00 0.04

R. helvetica C9P9 (CM001467) 0.03 0.04 0.03 0.04

R. africae ESF-5 (CP001612) 0.02 0.02 0.02 0.03 0.02

R. amblyommatis str. GAT-30V (CP003334) 0.04 0.02 0.04 0.03 0.04 0.02

R. akari Hartford (NC 009881) 0.10 0.10 0.10 0.09 0.08 0.08 0.09

R. australis str. Cutlack (NC 017058) 0.07 0.07 0.07 0.07 0.05 0.05 0.07 0.06

R. felis URRWXCal2 (CP000053) 0.05 0.04 0.05 0.04 0.03 0.03 0.03 0.05 0.03

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.05 0.04 0.05 0.05 0.04 0.04 0.05 0.07 0.04 0.01

R. conorii str. Malish 7 (AE006914.1) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04

R. gravesii BWI-1 (DQ269436) 0.03 0.01 0.03 0.02 0.03 0.02 0.02 0.09 0.06 0.03 0.04 0.02

R. heilongjiangensis Sendai-58 (AP019865) 0.02 0.01 0.02 0.02 0.03 0.01 0.02 0.08 0.05 0.02 0.03 0.01 0.01

R. honei RB (NZ AJTT00000000.1) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01

R. japonica str. M14012 (NZ AP017596) 0.03 0.02 0.03 0.03 0.03 0.02 0.03 0.09 0.06 0.03 0.04 0.02 0.02 0.01 0.02

R. massiliae str. AZT80 (CP003319) 0.02 0.01 0.02 0.02 0.03 0.01 0.02 0.08 0.05 0.02 0.03 0.01 0.01 0.00 0.01 0.01

R. monacensis str. IrR/Munich (LN794217) 0.01 0.02 0.01 0.03 0.03 0.02 0.03 0.10 0.07 0.04 0.04 0.02 0.02 0.01 0.02 0.02 0.01

R. montanensis OSU 85-390 (CP003340) 0.02 0.01 0.02 0.02 0.03 0.01 0.02 0.08 0.05 0.02 0.03 0.01 0.01 0.00 0.01 0.01 0.00 0.01

R. parkeri str. Portsmouth (NC 017044) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01

R. raoultii str. Khabarovsk (CP10969) 0.03 0.02 0.03 0.03 0.03 0.02 0.02 0.09 0.06 0.03 0.04 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.01 0.02

R. rhipicephali HJ 5 (CP013133) 0.03 0.02 0.03 0.02 0.03 0.01 0.02 0.09 0.06 0.03 0.03 0.01 0.01 0.00 0.01 0.01 0.00 0.02 0.00 0.01 0.01

R. rickettsii str. Arizona (CP003307) 0.02 0.02 0.02 0.03 0.03 0.00 0.03 0.09 0.06 0.04 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01 0.00 0.02 0.02

R. sibirica 246 (AABW01000001) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01 0.00 0.02 0.01 0.00

R. slovaca isolate Xinjiang (MF002535) 0.02 0.02 0.02 0.03 0.02 0.00 0.02 0.08 0.05 0.03 0.04 0.00 0.02 0.01 0.00 0.02 0.01 0.02 0.01 0.00 0.02 0.01 0.00 0.00

R. tamurae str. AT-1 (AF394896) 0.02 0.04 0.02 0.04 0.03 0.03 0.03 0.11 0.07 0.05 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.01 0.03 0.03 0.03 0.03 0.04 0.03 0.03

R. aeschlimannii str. RH15 (HM050289) 0.03 0.02 0.03 0.03 0.03 0.02 0.03 0.09 0.06 0.03 0.04 0.02 0.02 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.01 0.01 0.02 0.02 0.03

R. prowazekii str. Madrid E (NC 000963) 0.13 0.15 0.13 0.16 0.14 0.13 0.14 0.20 0.17 0.14 0.16 0.13 0.15 0.14 0.13 0.15 0.14 0.13 0.14 0.13 0.15 0.14 0.13 0.13 0.13 0.14 0.13

R. typhi str. Wilmington (NC-006142) 0.12 0.13 0.12 0.14 0.10 0.11 0.12 0.12 0.11 0.09 0.11 0.11 0.13 0.11 0.11 0.13 0.11 0.12 0.11 0.11 0.13 0.11 0.12 0.11 0.11 0.12 0.12 0.08

Sample R53 ex. A. albolimbatum (PI 680) 0.03 0 0.03 0.00 0.04 0.02 0.02 0.10 0.07 0.04 0.04 0.02 0.01 0.01 0.02 0.02 0.01 0.02 0.01 0.02 0.02 0.02 0.02 0.02 0.02 0.04 0.02 0.15 0.13

R. bellii RML369-C (NC 007940) 0.36 0.36 0.36 0.36 0.37 0.37 0.35 0.38 0.36 0.33 0.36 0.37 0.35 0.35 0.37 0.37 0.35 0.36 0.35 0.37 0.37 0.34 0.36 0.37 0.37 0.38 0.33 0.41 0.38 0.36

124

Table A1.28. Rickettsia 17 kDa Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 76 4395 3834 -1840 n/a 0.81 2.24 0.269 0.269 0.231 0.231 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.19 0.04 0.19 0.04 0.04

K2+G 75 4400 3846 -1848 n/a 0.81 2.23 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

T92+G+I 77 4405 3836 -1840 0 0.8 2.24 0.269 0.269 0.231 0.231 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.19 0.04 0.19 0.04 0.04

K2+G+I 76 4410 3848 -1848 0 0.83 2.21 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

TN93+G 79 4411 3827 -1834 n/a 0.97 2.21 0.295 0.243 0.18 0.282 0.04 0.03 0.13 0.05 0.18 0.04 0.05 0.24 0.04 0.14 0.04 0.03

HKY+G 78 4414 3838 -1841 n/a 0.78 2.33 0.295 0.243 0.18 0.282 0.04 0.03 0.19 0.05 0.12 0.04 0.05 0.17 0.04 0.2 0.04 0.03

TN93+G+I 80 4421 3830 -1834 0 0.95 2.21 0.295 0.243 0.18 0.282 0.04 0.03 0.13 0.05 0.18 0.04 0.05 0.24 0.04 0.14 0.04 0.03

HKY+G+I 79 4424 3840 -1841 0 0.79 2.32 0.295 0.243 0.18 0.282 0.04 0.03 0.19 0.05 0.12 0.04 0.05 0.17 0.04 0.2 0.04 0.03

T92+I 76 4427 3866 -1856 0.2 n/a 2.03 0.269 0.269 0.231 0.231 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.18 0.04 0.18 0.04 0.04

K2+I 75 4432 3877 -1863 0.19 n/a 2.02 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

T92 75 4434 3880 -1864 n/a n/a 1.8 0.269 0.269 0.231 0.231 0.05 0.04 0.15 0.05 0.15 0.04 0.05 0.17 0.04 0.17 0.05 0.04

GTR+G 82 4434 3828 -1832 n/a 1.02 1.6 0.295 0.243 0.18 0.282 0.05 0.05 0.14 0.06 0.14 0.04 0.08 0.19 0.04 0.15 0.04 0.03

K2 74 4438 3891 -1871 n/a n/a 1.8 0.25 0.25 0.25 0.25 0.04 0.04 0.16 0.04 0.16 0.04 0.04 0.16 0.04 0.16 0.04 0.04

TN93+I 79 4439 3855 -1848 0.16 n/a 2.01 0.295 0.243 0.18 0.282 0.04 0.03 0.13 0.05 0.17 0.05 0.05 0.23 0.05 0.14 0.04 0.03

GTR+G+I 83 4440 3827 -1830 0 1 1.66 0.295 0.243 0.18 0.282 0.05 0.05 0.14 0.06 0.14 0.04 0.08 0.19 0.04 0.15 0.03 0.03

TN93 78 4440 3864 -1854 n/a n/a 1.85 0.295 0.243 0.18 0.282 0.04 0.03 0.12 0.05 0.17 0.05 0.05 0.23 0.05 0.13 0.04 0.03

HKY+I 78 4445 3869 -1856 0.2 n/a 2.1 0.295 0.243 0.18 0.282 0.04 0.03 0.19 0.05 0.12 0.05 0.05 0.16 0.05 0.2 0.04 0.03

HKY 77 4453 3884 -1865 n/a n/a 1.81 0.295 0.243 0.18 0.282 0.04 0.03 0.18 0.05 0.11 0.05 0.05 0.15 0.05 0.19 0.04 0.03

GTR+I 82 4460 3854 -1845 0.16 n/a 1.52 0.295 0.243 0.18 0.282 0.05 0.05 0.13 0.06 0.14 0.04 0.08 0.19 0.04 0.14 0.04 0.03

GTR 81 4463 3865 -1851 n/a n/a 1.39 0.295 0.243 0.18 0.282 0.05 0.05 0.13 0.06 0.14 0.05 0.08 0.18 0.05 0.13 0.04 0.03

JC+G 74 4501 3955 -1903 n/a 0.88 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 75 4511 3957 -1903 0 0.89 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 74 4529 3982 -1917 0.17 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 73 4529 3990 -1922 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

125

Table A1.29. Rickettsia 17 kDa genetic distance matrix.

Sample SR

16 ex. A. a

lbolim

batum

(P

I 274

5)

Sample SR

15 ex. A

. albolim

batum

(PI 1

723)

Sample SR

14 ex. A. a

lbolim

batum

(PI 1

316)

Sample SR

12 ex. A. a

lbolim

batum

(PI 3

329)

R. b

ellii

RM

L369

-C (N

C 007

940)

Sample R92

ex.

A. a

lbolim

batum

(PI 3

323)

Sample R8 ex

. A. a

lbolim

batum

(PI 1

725)

Sample R10

6 ex

. A. a

lbolim

batum

(PI 1

460)

Sample R52

ex. A. a

lbolim

batum

(PI 6

80)

Unc

ulture

d R. s

p. clone

ARRL2

016-

156 (M

N43

1838

)

Sample R53

ex. A. a

lbolim

batum

(PI 6

80)

R. typ

hi st

r. W

ilmington

(NC-0

0614

2)

R. tam

urae

str. AT-

1 (A

F394

896)

R. s

lova

ca is

olate Xin

jiang

(MF0

0253

5)

R. s

ibirica

246

(AABW

0100

0001

)

R. rick

ettsii

str. A

rizon

a (C

P0033

07)

R. rhipice

phali

HJ 5 (C

P0131

33)

R. rao

ultii

str. Kha

baro

vsk (C

P1096

9)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. p

arke

ri str. Por

tsm

outh (N

C 017

044)

R. m

ontane

sis

OSU

85-

390 (C

P0033

40)

R. m

onac

ensis

str. IrR

/Mun

ich (L

N79

4217

)

R. m

assil

iae

str. AZT

80 (C

P0033

19)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. h

onei

RB (N

Z AJT

T000

0000

0.1)

R. h

eilong

jiang

ensis

Sen

dai-5

8 (A

P0198

65)

R. g

rave

sii

BWI-1

(DQ26

9437

)

R. felis

URRW

XCal2 (C

P0000

53)

R. c

onor

ii str. M

alish

7 (A

E0069

14)

R. a

ustra

lis

str. Cutlack

(NC 017

058)

R. a

sem

bone

nsis

str. NM

RCii (

NZ JW

SW00

0000

00.1)

R. a

mblyo

mm

atis

str. G

AT-30

V (CP00

3334

)

R. a

frica

e E

SF-5 (C

P0016

12)

R. a

esch

liman

nii

str. RH15

(HM

0502

89)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. a

kari

str.

Har

tford

(NC 009

881)

Unc

ulture

d R. s

p. clone

ARRL2

016-

159 (M

N43

1849

)

Unc

ulture

d R. s

p. clone

ARRL2

016-

149 (M

N43

1837

)


Sample SR15 ex. A. albolimbatum (PI 1723) 0.00

Sample SR14 ex. A. albolimbatum (PI 1316) 0.00 0.00

Sample SR12 ex. A. albolimbatum (PI 3329) 0.00 0.00 0.00

R. bellii RML369-C (NC 007940) 0.08 0.08 0.08 0.08

Sample R92 ex. A. albolimbatum (PI 3323) 0.31 0.31 0.31 0.31 0.30

Sample R8 ex. A. albolimbatum (PI 1725) 0.31 0.31 0.31 0.31 0.30 0.00

Sample R106 ex. A. albolimbatum (PI 1460) 0.34 0.34 0.34 0.34 0.31 0.06 0.06

Sample R52 ex. A. albolimbatum (PI 680) 0.34 0.34 0.34 0.34 0.31 0.06 0.06 0.00

Uncultured R. sp. clone ARRL2016-156 (MN431838) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06

Sample R53 ex. A. albolimbatum (PI 680) 0.00 0.00 0.00 0.00 0.08 0.31 0.31 0.34 0.34 0.31

R. typhi str. Wilmington (NC-006142) 0.37 0.37 0.37 0.37 0.32 0.12 0.12 0.11 0.11 0.12 0.37

R. tamurae str. AT-1 (AF394896) 0.33 0.33 0.33 0.33 0.30 0.05 0.05 0.01 0.01 0.05 0.33 0.11

R. slovaca isolate Xinjiang (MF002535) 0.33 0.33 0.33 0.33 0.30 0.01 0.01 0.07 0.07 0.01 0.33 0.14 0.06

R. sibirica 246 (AABW01000001) 0.33 0.33 0.33 0.33 0.30 0.01 0.01 0.07 0.07 0.01 0.33 0.14 0.06 0.01

R. rickettsii str. Arizona (CP003307) 0.32 0.32 0.32 0.32 0.28 0.01 0.01 0.07 0.07 0.01 0.32 0.14 0.06 0.01 0.01

R. rhipicephali HJ 5 (CP013133) 0.31 0.31 0.31 0.31 0.30 0.01 0.01 0.05 0.05 0.01 0.31 0.12 0.05 0.02 0.02 0.02

R. raoultii str. Khabarovsk (CP10969) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01

R. prowazekii str. Madrid E (NC 000963) 0.40 0.40 0.40 0.40 0.35 0.15 0.15 0.14 0.14 0.15 0.40 0.05 0.13 0.16 0.16 0.16 0.14 0.15


R. montanesis OSU 85-390 (CP003340) 0.32 0.32 0.32 0.32 0.30 0.01 0.01 0.06 0.06 0.01 0.32 0.12 0.05 0.01 0.01 0.01 0.02 0.01 0.15 0.01

R. monacensis str. IrR/Munich (LN794217) 0.33 0.33 0.33 0.33 0.30 0.05 0.05 0.01 0.01 0.05 0.33 0.11 0.00 0.06 0.06 0.06 0.05 0.05 0.13 0.05 0.05

R. massiliae str. AZT80 (CP003319) 0.32 0.32 0.32 0.32 0.31 0.01 0.01 0.06 0.06 0.01 0.32 0.14 0.06 0.02 0.02 0.02 0.01 0.01 0.16 0.02 0.02 0.06

R. japonica str. M14012 (NZ AP017596) 0.32 0.32 0.32 0.32 0.30 0.01 0.01 0.07 0.07 0.01 0.32 0.14 0.06 0.01 0.01 0.01 0.02 0.01 0.16 0.01 0.02 0.06 0.02

R. honei RB (NZ AJTT00000000.1) 0.32 0.32 0.32 0.32 0.30 0.00 0.00 0.06 0.06 0.00 0.32 0.13 0.05 0.01 0.01 0.01 0.02 0.00 0.15 0.00 0.01 0.05 0.02 0.01

R. heilongjiangensis Sendai-58 (AP019865) 0.33 0.33 0.33 0.33 0.31 0.01 0.01 0.07 0.07 0.01 0.33 0.14 0.06 0.02 0.02 0.02 0.03 0.01 0.17 0.01 0.02 0.06 0.03 0.01 0.01

R. gravesii BWI-1 (DQ269437) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01 0.00 0.15 0.01 0.01 0.05 0.01 0.01 0.00 0.01

R. felis URRWXCal2 (CP000053) 0.34 0.34 0.34 0.34 0.33 0.07 0.07 0.08 0.08 0.07 0.34 0.14 0.07 0.08 0.07 0.08 0.07 0.07 0.16 0.08 0.08 0.07 0.09 0.08 0.07 0.08 0.07

R. conorii str. Malish 7 (AE006914) 0.32 0.32 0.32 0.32 0.28 0.01 0.01 0.07 0.07 0.01 0.32 0.14 0.06 0.01 0.01 0.00 0.02 0.01 0.16 0.00 0.01 0.06 0.02 0.01 0.01 0.02 0.01 0.08

R. australis str. Cutlack (NC 017058) 0.32 0.32 0.32 0.32 0.30 0.03 0.03 0.06 0.06 0.03 0.32 0.12 0.06 0.04 0.04 0.04 0.04 0.03 0.15 0.04 0.04 0.06 0.05 0.04 0.04 0.05 0.03 0.06 0.04

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.31 0.31 0.31 0.31 0.29 0.05 0.05 0.03 0.03 0.05 0.31 0.12 0.03 0.06 0.06 0.05 0.05 0.05 0.14 0.05 0.05 0.03 0.06 0.06 0.05 0.06 0.05 0.05 0.05 0.04

R. amblyommatis str. GAT-30V (CP003334) 0.32 0.32 0.32 0.32 0.32 0.01 0.01 0.08 0.08 0.01 0.32 0.14 0.06 0.02 0.02 0.02 0.03 0.01 0.16 0.02 0.02 0.06 0.03 0.03 0.02 0.03 0.01 0.08 0.02 0.04 0.06

R. africae ESF-5 (CP001612) 0.32 0.32 0.32 0.32 0.29 0.01 0.01 0.07 0.07 0.01 0.32 0.13 0.06 0.01 0.01 0.01 0.03 0.01 0.16 0.01 0.02 0.06 0.03 0.02 0.01 0.02 0.01 0.09 0.01 0.05 0.06 0.02

R. aeschlimannii str. RH15 (HM050289) 0.32 0.32 0.32 0.32 0.32 0.01 0.01 0.06 0.06 0.01 0.32 0.14 0.06 0.02 0.02 0.02 0.02 0.01 0.15 0.02 0.02 0.06 0.02 0.03 0.02 0.03 0.01 0.08 0.02 0.04 0.06 0.03 0.03

R. helvetica C9P9 (CM001467) 0.34 0.34 0.34 0.34 0.30 0.04 0.04 0.06 0.06 0.04 0.34 0.12 0.06 0.05 0.05 0.05 0.05 0.04 0.14 0.05 0.05 0.06 0.06 0.05 0.04 0.05 0.04 0.09 0.05 0.06 0.06 0.06 0.05 0.06

R. akari str. Hartford (NC 009881) 0.34 0.34 0.34 0.34 0.32 0.05 0.05 0.07 0.07 0.05 0.34 0.13 0.07 0.06 0.06 0.06 0.06 0.05 0.16 0.06 0.06 0.07 0.07 0.06 0.05 0.06 0.05 0.08 0.06 0.03 0.06 0.07 0.06 0.06 0.06

Uncultured R. sp. clone ARRL2016-159 (MN431849) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01 0.00 0.15 0.01 0.01 0.05 0.01 0.01 0.00 0.01 0.00 0.07 0.01 0.03 0.05 0.01 0.01 0.01 0.04 0.05

Uncultured R. sp. clone ARRL2016-149 (MN431837) 0.31 0.31 0.31 0.31 0.30 0.00 0.00 0.06 0.06 0.00 0.31 0.12 0.05 0.01 0.01 0.01 0.01 0.00 0.15 0.01 0.01 0.05 0.01 0.01 0.00 0.01 0.00 0.07 0.01 0.03 0.05 0.01 0.01 0.01 0.04 0.05 0.00

126

Table A1.30. Rickettsia sca4 Maximum Likelihood fits of 24 different nucleotide substitution models.


GTR+G+I 73 8594 8004 -3929 0.16 1.725 1.192 0.38 0.261 0.18 0.179 0.04 0.07 0.1 0.06 0.11 0.05 0.14 0.15 0.01 0.2 0.07 0.01

GTR+G 72 8604 8022 -3939 n/a 1.001 1.08 0.38 0.261 0.18 0.179 0.04 0.07 0.1 0.06 0.09 0.05 0.14 0.14 0.03 0.2 0.07 0.03

HKY+G 68 8607 8057 -3960 n/a 0.893 1.407 0.38 0.261 0.18 0.179 0.05 0.04 0.11 0.08 0.11 0.04 0.08 0.16 0.04 0.23 0.05 0.04

TN93+G 69 8609 8051 -3956 n/a 0.919 1.391 0.38 0.261 0.18 0.179 0.05 0.04 0.09 0.08 0.13 0.04 0.08 0.18 0.04 0.2 0.05 0.04

GTR+I 72 8610 8027 -3941 0.312 n/a 1.057 0.38 0.261 0.18 0.179 0.04 0.06 0.1 0.06 0.09 0.05 0.14 0.14 0.03 0.2 0.07 0.03

HKY+I 68 8610 8060 -3962 0.33 n/a 1.411 0.38 0.261 0.18 0.179 0.05 0.04 0.11 0.08 0.11 0.04 0.08 0.16 0.04 0.23 0.05 0.04

HKY+G+I 69 8613 8055 -3958 0.177 1.719 1.419 0.38 0.261 0.18 0.179 0.05 0.04 0.11 0.08 0.11 0.04 0.08 0.16 0.04 0.23 0.05 0.04

TN93+I 69 8614 8056 -3959 0.327 n/a 1.402 0.38 0.261 0.18 0.179 0.05 0.04 0.09 0.08 0.13 0.04 0.08 0.19 0.04 0.19 0.05 0.04

TN93+G+I 70 8618 8052 -3956 0.2 2.048 1.404 0.38 0.261 0.18 0.179 0.05 0.04 0.09 0.08 0.13 0.04 0.08 0.18 0.04 0.2 0.05 0.04

T92+G 66 8620 8086 -3977 n/a 0.921 1.389 0.32 0.32 0.18 0.18 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.04

T92+I 66 8624 8090 -3979 0.326 n/a 1.397 0.32 0.32 0.18 0.18 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.04

T92+G+I 67 8626 8084 -3975 0.146 1.531 1.398 0.32 0.32 0.18 0.18 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.19 0.04 0.19 0.06 0.04

GTR 71 8659 8085 -3971 n/a n/a 1.106 0.38 0.261 0.18 0.179 0.03 0.07 0.09 0.04 0.11 0.05 0.14 0.16 0.04 0.18 0.07 0.04

HKY 67 8677 8136 -4001 n/a n/a 1.219 0.38 0.261 0.18 0.179 0.06 0.04 0.1 0.08 0.1 0.04 0.08 0.15 0.04 0.22 0.06 0.04

TN93 68 8680 8130 -3997 n/a n/a 1.225 0.38 0.261 0.18 0.179 0.06 0.04 0.09 0.08 0.12 0.04 0.08 0.18 0.04 0.18 0.06 0.04

T92 65 8687 8161 -4015 n/a n/a 1.231 0.32 0.32 0.18 0.18 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.18 0.04 0.18 0.07 0.04

K2+G 65 8738 8213 -4041 n/a 0.846 1.368 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05

K2+G+I 66 8745 8211 -4039 1E-05 0.843 1.369 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05

K2+I 65 8748 8222 -4046 0.339 n/a 1.355 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05

K2 64 8815 8297 -4084 n/a n/a 1.191 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06

JC+G 64 8842 8324 -4098 n/a 0.994 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 65 8848 8323 -4096 6E-06 0.991 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 64 8851 8333 -4102 0.313 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 63 8903 8393 -4134 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

127

Table A1.31. Rickettsia sca4 genetic distance matrix.

Sample R53 ex. A

. albolim

batum (PI 6

80)

Sample R66 ex. A

. albolim

batum (PI 1

320)

Sample R92 ex. A

. albolim

batum (PI 3

323)

R. aeschlim

annii str.

RH15 (HM050275)

R. afric

ae ESF-5 (CP001612)

R. akari H

artford (N

C 009881)

R. amblyommatis

str. GAT-30V (C

P003334)

R. asembonensis s

tr. NMRCii (

NZ JWSW00000000.1)

R. austra

lis str. Cutla

ck (NC 017058)

R. prowazekii s

tr. Madrid

E (NC 000963)

R. typhi s

t Wilm

ington (NC 006142)

R. conorii

str. Malish 7 (A

E006914.1)

R. felis U

RRWXCal2 (CP000053)

R. gravesii B

WI-1 (D

Q269439)

R. heilo

ngjiangensis Sendai-5

8 (AP019865)

R. honei R

B (NZ AJTT00000000.1)

R. japonica str.

M14012 (NZ AP017596)

R. massilia

e str. AZT80 (C

P003319)

R. monacensis s

tr. IrR

/Munich (L

N794217)

R. montanensis O

SU 85-930 (CP003340)

R. parkeri s

tr. Ports

mouth (NC 017044)

R. peacockii s

tr. Rustic

(NC_012730)

R. raoultii

str. Khabarovsk (C

P10969)

R. rhipicephali H

J 5 (CP013133)

R. ricketts

ii str.

Arizona (C

P003307)

R. sibiric

a 246 (AABW01000001)

R. slovaca is

olate Xinjiang (M

F002531)

R. tamurae s

tr. AT-1 (D

Q113911)

R. helvetic

a C9P9 (C

M001467)

Uncultured R

. sp. c

lone ARRL2016-149 (MN431843)

Uncultured R

. sp. c

lone ARRL2016-156 (MN431844)

Uncultured R

. sp. c

lone ARRL2016-159 (MN431845)

R. bellii

RML369-C (N

C 007940)

Sample R53 ex. A. albolimbatum (PI 680)

Sample R66 ex. A. albolimbatum (PI 1320) 0.00

Sample R92 ex. A. albolimbatum (PI 3323) 0.00 0.00

R. aeschlimannii str. RH15 (HM050275) 0.03 0.03 0.03

R. africae ESF-5 (CP001612) 0.03 0.03 0.03 0.02

R. akari Hartford (NC 009881) 0.15 0.15 0.15 0.15 0.15

R. amblyommatis str. GAT-30V (CP003334) 0.03 0.03 0.03 0.02 0.02 0.14

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.13 0.13 0.13 0.12 0.12 0.07 0.12

R. australis str. Cutlack (NC 017058) 0.14 0.14 0.14 0.13 0.13 0.05 0.12 0.05

R. prowazekii str. Madrid E (NC 000963) 0.28 0.28 0.28 0.27 0.28 0.32 0.28 0.29 0.30

R. typhi str. Wilmington (NC 006142) 0.29 0.29 0.29 0.28 0.29 0.31 0.28 0.28 0.30 0.07

R. conorii str. Malish 7 (AE006914.1) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.28 0.28

R. felis URRWXCal2 (CP000053) 0.11 0.11 0.11 0.10 0.10 0.08 0.10 0.04 0.07 0.29 0.29 0.11

R. gravesii BWI-1 (DQ269439) 0.02 0.02 0.02 0.03 0.03 0.15 0.03 0.14 0.14 0.28 0.28 0.03 0.12

R. heilongjiangensis Sendai-58 (AP019865) 0.02 0.02 0.03 0.02 0.01 0.14 0.02 0.12 0.13 0.27 0.27 0.01 0.10 0.02

R. honei RB (NZ AJTT00000000.1) 0.03 0.03 0.03 0.02 0.01 0.14 0.02 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.01

R. japonica str. M14012 (NZ AP017596) 0.03 0.03 0.03 0.02 0.02 0.15 0.02 0.12 0.13 0.27 0.27 0.02 0.10 0.03 0.01 0.01

R. massiliae str. AZT80 (CP003319) 0.03 0.03 0.03 0.01 0.02 0.15 0.02 0.12 0.13 0.28 0.28 0.02 0.10 0.03 0.01 0.02 0.02


R. montanensis OSU 85-930 (CP003340) 0.03 0.03 0.03 0.02 0.02 0.15 0.02 0.12 0.13 0.28 0.28 0.02 0.10 0.03 0.02 0.02 0.02 0.02 0.05

R. parkeri str. Portsmouth (NC 017044) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.27 0.28 0.01 0.10 0.03 0.01 0.01 0.02 0.02 0.05 0.02

R. peacockii str. Rustic (NC_012730) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.13 0.14 0.27 0.28 0.01 0.11 0.03 0.01 0.01 0.02 0.02 0.05 0.02 0.01

R. raoultii str. Khabarovsk (CP10969) 0.03 0.03 0.03 0.02 0.02 0.14 0.02 0.12 0.13 0.27 0.27 0.02 0.10 0.03 0.01 0.02 0.02 0.02 0.05 0.02 0.02 0.02

R. rhipicephali HJ 5 (CP013133) 0.03 0.03 0.03 0.01 0.02 0.15 0.02 0.12 0.13 0.27 0.27 0.02 0.10 0.03 0.01 0.02 0.02 0.01 0.05 0.02 0.02 0.02 0.02

R. rickettsii str. Arizona (CP003307) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.01 0.01 0.01 0.02 0.05 0.02 0.01 0.01 0.02 0.02

R. sibirica 246 (AABW01000001) 0.03 0.03 0.03 0.02 0.01 0.15 0.03 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.02 0.01 0.02 0.02 0.05 0.03 0.00 0.01 0.02 0.03 0.01

R. slovaca isolate Xinjiang (MF002531) 0.03 0.03 0.03 0.02 0.01 0.15 0.02 0.12 0.13 0.28 0.28 0.01 0.10 0.03 0.01 0.00 0.01 0.02 0.05 0.02 0.01 0.00 0.02 0.02 0.01 0.01

R. tamurae str. AT-1 (DQ113911) 0.04 0.04 0.05 0.03 0.02 0.15 0.04 0.13 0.13 0.29 0.30 0.02 0.11 0.04 0.03 0.02 0.03 0.03 0.06 0.04 0.02 0.02 0.03 0.03 0.02 0.02 0.02

R. helvetica C9P9 (CM001467) 0.09 0.09 0.09 0.09 0.09 0.13 0.08 0.10 0.11 0.28 0.28 0.08 0.09 0.09 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.09 0.08 0.09 0.09 0.09 0.08 0.10

Uncultured R. sp. clone ARRL2016-149 (MN431843) 0.00 0.00 0.00 0.03 0.03 0.15 0.03 0.13 0.14 0.28 0.28 0.03 0.11 0.02 0.02 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.09

Uncultured R. sp. clone ARRL2016-156 (MN431844) 0.00 0.00 0.00 0.03 0.03 0.15 0.03 0.13 0.14 0.28 0.29 0.03 0.11 0.02 0.02 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.09 0.00

Uncultured R. sp. clone ARRL2016-159 (MN431845) 0.00 0.00 0.00 0.03 0.03 0.15 0.03 0.13 0.14 0.28 0.28 0.03 0.11 0.02 0.02 0.03 0.03 0.03 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.09 0.00 0.00

R. bellii RML369-C (NC 007940) 0.61 0.61 0.61 0.60 0.62 0.71 0.61 0.69 0.71 0.75 0.71 0.61 0.67 0.63 0.61 0.61 0.61 0.59 0.63 0.60 0.62 0.61 0.60 0.59 0.62 0.62 0.61 0.63 0.66 0.61 0.62 0.61

128

Table A1.32. Rickettsia concatenated 17 kDa and gltA (short) Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 62 5841 5352 -2614 n/a 0.304 2.764 0.304 0.304 0.196 0.196 0.04 0.03 0.15 0.04 0.15 0.03 0.04 0.23 0.03 0.23 0.04 0.03

T92+G+I 63 5850 5352 -2613 0.253 0.508 2.778 0.304 0.304 0.196 0.196 0.04 0.03 0.15 0.04 0.15 0.03 0.04 0.23 0.03 0.23 0.04 0.03

TN93+G 65 5850 5337 -2603 n/a 0.321 2.744 0.27 0.338 0.232 0.16 0.04 0.03 0.17 0.03 0.12 0.02 0.03 0.17 0.02 0.29 0.04 0.03

HKY+G 64 5858 5352 -2612 n/a 0.298 2.883 0.27 0.338 0.232 0.16 0.04 0.03 0.12 0.03 0.17 0.02 0.03 0.25 0.02 0.2 0.04 0.03

TN93+G+I 66 5859 5338 -2603 0.247 0.536 2.757 0.27 0.338 0.232 0.16 0.04 0.03 0.17 0.03 0.12 0.02 0.03 0.17 0.02 0.29 0.04 0.03

T92+I 62 5865 5375 -2626 0.598 n/a 2.672 0.304 0.304 0.196 0.196 0.04 0.03 0.14 0.04 0.14 0.03 0.04 0.22 0.03 0.22 0.04 0.03

HKY+G+I 65 5866 5353 -2611 0.253 0.497 2.897 0.27 0.338 0.232 0.16 0.04 0.03 0.12 0.03 0.17 0.02 0.03 0.25 0.02 0.2 0.04 0.03

GTR+G 68 5871 5334 -2599 n/a 0.325 2.56 0.27 0.338 0.232 0.16 0.03 0.02 0.16 0.03 0.12 0.04 0.03 0.17 0.02 0.27 0.08 0.03

TN93+I 65 5872 5358 -2614 0.587 n/a 2.655 0.27 0.338 0.232 0.16 0.04 0.03 0.17 0.04 0.12 0.02 0.04 0.17 0.02 0.28 0.04 0.03

GTR+G+I 69 5876 5332 -2597 0.254 0.551 2.79 0.27 0.338 0.232 0.16 0.03 0.03 0.17 0.03 0.12 0.04 0.03 0.17 0.01 0.29 0.08 0.02

HKY+I 64 5882 5377 -2624 0.601 n/a 2.756 0.27 0.338 0.232 0.16 0.04 0.03 0.12 0.04 0.17 0.02 0.04 0.25 0.02 0.2 0.04 0.03

K2+G 61 5887 5406 -2642 n/a 0.296 2.768 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

GTR+I 68 5895 5358 -2611 0.583 n/a 2.385 0.27 0.338 0.232 0.16 0.03 0.03 0.16 0.03 0.12 0.04 0.03 0.17 0.02 0.27 0.08 0.03

K2+G+I 62 5895 5406 -2641 0.265 0.506 2.784 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

K2+I 61 5912 5430 -2654 0.606 n/a 2.681 0.25 0.25 0.25 0.25 0.03 0.03 0.18 0.03 0.18 0.03 0.03 0.18 0.03 0.18 0.03 0.03

TN93 64 6012 5507 -2689 n/a n/a 2.338 0.27 0.338 0.232 0.16 0.05 0.03 0.16 0.04 0.11 0.02 0.04 0.16 0.02 0.28 0.05 0.03

T92 61 6012 5531 -2704 n/a n/a 2.329 0.304 0.304 0.196 0.196 0.04 0.03 0.14 0.04 0.14 0.03 0.04 0.22 0.03 0.22 0.04 0.03

HKY 63 6034 5536 -2705 n/a n/a 2.325 0.27 0.338 0.232 0.16 0.05 0.03 0.11 0.04 0.16 0.02 0.04 0.24 0.02 0.19 0.05 0.03

GTR 67 6034 5505 -2685 n/a n/a 2.137 0.27 0.338 0.232 0.16 0.04 0.03 0.15 0.03 0.11 0.04 0.03 0.16 0.02 0.26 0.08 0.04

K2 60 6062 5588 -2734 n/a n/a 2.315 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

JC+G 60 6072 5599 -2739 n/a 0.324 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 61 6081 5599 -2738 0.246 0.533 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 60 6094 5621 -2750 0.597 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 59 6232 5766 -2824 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

129

Table A1.33. Rickettsia concatenated 17 kDa and gltA (short) genetic distance matrix.

Sample

SR12

ex

Am. a

lbolim

batum

(PI 3

329)

Uncult

ured

R.sp. cl

one A

RRL2

016-1

49 (MN43

1847

)

R. aes

chlim

annii

str. R

H15 (H

M05

0289

)

R. afric

ae ESF-

5 (C

P0016

12)

R. aka

ri str.

hartfo

rd (NC 00

9881

)

R. ambly

ommati

s

str. G

AT-30

V (CP00

3334

)

R. ase

mbo

nens

is str

. NMRC

ii (NZ JW

SW00

0000

00.1)

R. aus

tralis

str

. Cutl

ack (

NC 017

058)

R. bell

ii RM

L369

-C (N

C 00

7940

)

R. felis

URRW

XCal2 (

CP0000

53)

R. grav

esii

BWI-1

(DQ26

9439

)

R. helv

etica

C9P

9 (C

M00

1467

)

R. hon

ei R

B (NZ AJ

TT00

0000

00.1)

R. japo

nica

str. M

1401

2 (NZ

AP01

7596

)

R. mas

siliae

str. A

ZT80

(CP00

3319

)

R. mon

acen

sis

str. Ir

R/Mun

ich (L

N7942

17)

R. park

eri

str. P

ortsm

outh

(NC 01

7044

)

R. pea

cock

i (N

C 0127

30)

R. prow

azek

ii str

. Mad

rid E

(NC 00

0963

)

R. raou

ltii

str. K

haba

rovs

k (CP1

0969

)

R. rick

ettsii

str. A

rizon

a (CP0

0330

7)

R. sibi

rica

246 (

AABW01

0000

01)

R. tamurae

str

. AT-

1 (AF3

9489

6)

R. con

orii

str. M

alish

7 (A

E006

914)

R. heil

ongjian

gens

is

Sen

dai-5

8 (AP0

1986

5)

R. typh

i str

. Wilm

ington

(NC-00

6142

)

R. mon

tanes

is OSU 85

-390

(CP0

0334

0)

R. slov

aca

isola

te Xinj

iang (

MF0

0253

5)

Sample SR12 ex Am. albolimbatum (PI 3329)

Uncultured R.sp. clone ARRL2016-149 (MN431847) 0.23

R. aeschlimannii str. RH15 (HM050289) 0.24 0.01

R. africae ESF-5 (CP001612) 0.25 0.01 0.01

R. akari str. hartford (NC 009881) 0.26 0.05 0.06 0.06

R. amblyommatis str. GAT-30V (CP003334) 0.24 0.01 0.02 0.01 0.06

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.25 0.05 0.06 0.06 0.06 0.06

R. australis str. Cutlack (NC 017058) 0.24 0.04 0.04 0.04 0.03 0.04 0.05

R. bellii RML369-C (NC 007940) 0.06 0.23 0.24 0.23 0.26 0.24 0.23 0.24

R. felis URRWXCal2 (CP000053) 0.26 0.05 0.06 0.06 0.06 0.06 0.05 0.05 0.26

R. gravesii BWI-1 (DQ269439) 0.24 0.01 0.01 0.01 0.06 0.02 0.06 0.04 0.24 0.06

R. helvetica C9P9 (CM001467) 0.27 0.05 0.05 0.05 0.06 0.06 0.06 0.06 0.24 0.06 0.05

R. honei RB (NZ AJTT00000000.1) 0.24 0.00 0.01 0.00 0.05 0.01 0.05 0.04 0.24 0.06 0.01 0.05

R. japonica str. M14012 (NZ AP017596) 0.24 0.01 0.01 0.01 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.01

R. massiliae str. AZT80 (CP003319) 0.24 0.01 0.01 0.02 0.06 0.02 0.06 0.05 0.24 0.07 0.02 0.06 0.02 0.02

R. monacensis str. IrR/Munich (LN794217) 0.25 0.04 0.05 0.05 0.05 0.05 0.04 0.05 0.24 0.05 0.05 0.06 0.05 0.05 0.05

R. parkeri str. Portsmouth (NC 017044) 0.24 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.00 0.01 0.02 0.05

R. peacock i (NC 012730) 0.25 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.00 0.01 0.02 0.05 0.00

R. prowazek ii str. Madrid E (NC 000963) 0.28 0.11 0.12 0.12 0.14 0.12 0.13 0.13 0.27 0.13 0.12 0.13 0.12 0.12 0.12 0.11 0.12 0.12

R. raoultii str. Khabarovsk (CP10969) 0.23 0.00 0.01 0.01 0.05 0.01 0.05 0.04 0.23 0.05 0.01 0.05 0.00 0.01 0.01 0.04 0.01 0.01 0.11

R. rickettsii str. Arizona (CP003307) 0.24 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.01 0.01 0.02 0.05 0.00 0.00 0.12 0.01

R. sibirica 246 (AABW01000001) 0.25 0.01 0.01 0.00 0.06 0.02 0.06 0.04 0.23 0.06 0.01 0.05 0.00 0.01 0.02 0.05 0.00 0.00 0.12 0.01 0.00

R. tamurae str. AT-1 (AF394896) 0.25 0.04 0.05 0.05 0.05 0.05 0.04 0.05 0.24 0.05 0.05 0.05 0.05 0.05 0.06 0.00 0.05 0.05 0.11 0.04 0.05 0.05

R. conorii str. Malish 7 (AE006914) 0.24 0.01 0.02 0.01 0.06 0.02 0.06 0.05 0.23 0.06 0.02 0.06 0.01 0.01 0.02 0.05 0.01 0.01 0.13 0.01 0.01 0.01 0.05

R. heilongjiangensis Sendai-58 (AP019865) 0.25 0.01 0.02 0.01 0.05 0.02 0.06 0.04 0.24 0.06 0.02 0.05 0.01 0.00 0.02 0.05 0.01 0.01 0.12 0.01 0.01 0.01 0.05 0.02

R. typhi str. Wilmington (NC-006142) 0.27 0.11 0.11 0.12 0.12 0.11 0.12 0.12 0.26 0.12 0.11 0.12 0.11 0.12 0.12 0.10 0.11 0.12 0.04 0.11 0.12 0.12 0.10 0.12 0.12

R. montanesis OSU 85-390 (CP003340) 0.23 0.01 0.01 0.01 0.05 0.02 0.05 0.04 0.23 0.06 0.01 0.05 0.01 0.01 0.02 0.05 0.01 0.01 0.12 0.01 0.01 0.01 0.05 0.02 0.02 0.11

R. slovaca isolate Xinjiang (MF002535) 0.25 0.01 0.01 0.00 0.06 0.01 0.06 0.04 0.24 0.06 0.01 0.05 0.01 0.01 0.02 0.05 0.00 0.00 0.12 0.01 0.01 0.00 0.05 0.01 0.01 0.11 0.01

130

Table A1.34. Rickettsia concatenated ompA, ompB, and gltA (long) Maximum Likelihood fits of 24 different nucleotide substitution

models.


T92+G 56 16534 16063 -7975 n/a 0.36 1.379 0.309 0.309 0.191 0.191 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.18 0.04 0.18 0.06 0.04

T92+G+I 57 16540 16061 -7973 0.167 0.505 1.38 0.309 0.309 0.191 0.191 0.06 0.04 0.11 0.06 0.11 0.04 0.06 0.18 0.04 0.18 0.06 0.04

TN93+G 59 16547 16051 -7966 n/a 0.359 1.408 0.299 0.32 0.183 0.199 0.06 0.04 0.15 0.06 0.08 0.04 0.06 0.15 0.04 0.22 0.06 0.04

HKY+G 58 16552 16065 -7974 n/a 0.361 1.377 0.299 0.32 0.183 0.199 0.06 0.04 0.12 0.06 0.11 0.04 0.06 0.19 0.04 0.18 0.06 0.04

HKY+G+I 59 16559 16062 -7972 0.169 0.507 1.379 0.299 0.32 0.183 0.199 0.06 0.04 0.12 0.06 0.11 0.04 0.06 0.19 0.04 0.18 0.06 0.04

TN93+G+I 60 16565 16060 -7970 0.166 0.486 1.641 0.299 0.32 0.183 0.199 0.06 0.03 0.14 0.05 0.1 0.04 0.05 0.18 0.04 0.21 0.06 0.03

GTR+G 62 16567 16046 -7961 n/a 0.355 1.412 0.299 0.32 0.183 0.199 0.06 0.04 0.15 0.05 0.08 0.05 0.07 0.15 0.03 0.22 0.08 0.02

GTR+G+I 63 16573 16043 -7959 0.171 0.502 1.414 0.299 0.32 0.183 0.199 0.06 0.04 0.15 0.05 0.08 0.05 0.07 0.15 0.02 0.22 0.08 0.02

T92+I 56 16711 16240 -8064 0.465 n/a 1.237 0.309 0.309 0.191 0.191 0.07 0.04 0.11 0.07 0.11 0.04 0.07 0.18 0.04 0.18 0.07 0.04

TN93+I 59 16726 16230 -8056 0.465 n/a 1.246 0.299 0.32 0.183 0.199 0.07 0.04 0.13 0.06 0.09 0.04 0.06 0.15 0.04 0.2 0.07 0.04

HKY+I 58 16728 16240 -8062 0.465 n/a 1.235 0.299 0.32 0.183 0.199 0.07 0.04 0.11 0.06 0.1 0.04 0.06 0.18 0.04 0.17 0.07 0.04

GTR+I 62 16750 16228 -8052 0.466 n/a 1.244 0.299 0.32 0.183 0.199 0.07 0.05 0.13 0.06 0.09 0.05 0.08 0.15 0.03 0.2 0.07 0.03

K2+G 55 16762 16299 -8095 n/a 0.352 1.381 0.25 0.25 0.25 0.25 0.05 0.05 0.14 0.05 0.14 0.05 0.05 0.14 0.05 0.14 0.05 0.05

K2+G+I 56 16768 16297 -8093 0.174 0.499 1.384 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

K2+I 55 16938 16476 -8183 0.472 n/a 1.243 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06

JC+G 54 16979 16525 -8208 n/a 0.378 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 55 16986 16524 -8207 0.174 0.543 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 54 17138 16684 -8288 0.47 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

T92 55 17351 16888 -8389 n/a n/a 1.145 0.309 0.309 0.191 0.191 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.17 0.04 0.17 0.07 0.04

HKY 57 17366 16887 -8386 n/a n/a 1.145 0.299 0.32 0.183 0.199 0.07 0.04 0.11 0.07 0.1 0.04 0.07 0.18 0.04 0.16 0.07 0.04

TN93 58 17369 16881 -8382 n/a n/a 1.145 0.299 0.32 0.183 0.199 0.07 0.04 0.12 0.07 0.09 0.04 0.07 0.16 0.04 0.18 0.07 0.04

GTR 61 17394 16881 -8380 n/a n/a 1.146 0.299 0.32 0.183 0.199 0.07 0.05 0.12 0.06 0.09 0.05 0.08 0.16 0.04 0.18 0.08 0.04

K2 54 17606 17152 -8522 n/a n/a 1.122 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06

JC 53 17786 17340 -8617 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

131

Table A1.35. Rickettsia concatenated ompA, ompB, and gltA (long) genetic distance matrix.

Sample SR

12 ex.

Am

. albolim

batum

(PI 3

329)

R. p

eaco

cki

(NC 012

730)

R. a

kari

(NC 009

881)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. m

ontane

sis OSU

85-

390 (C

P0033

40)

R. tam

urae

str. AT-

1 (A

F394

896)

R. a

esch

liman

nii

str. RH15

(HM

0502

89)

R. a

frica

e E

SF-5 (C

P0016

12)

R. a

mblyo

mm

atis

str. G

AT-30

V (CP00

3334

)

R. a

sem

bone

nsis

str. NM

RCii (

NZ JW

SW00

0000

00.1)

R. a

ustra

lis

str. Cutlack

(NC 017

058)

R. b

ellii

RM

L369

-C (N

C 007

940)

R. c

onor

ii str. M

alish

7 (A

E0069

14)

R. felis

URRW

XCal2 (C

P0000

53)

R. g

rave

sii

BWI-1

(DQ26

9439

)

R. h

eilong

jiang

ensis

Sen

dai-5

8 (A

P0198

65)

R. h

onei

RB (N

Z AJT

T000

0000

0.1)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. m

assil

iae

str. AZT

80 (C

P0033

19)

R. m

onac

ensis

str. IrR

/Mun

ich (L

N79

4217

)

R. p

arke

ri str. Por

tsm

outh (N

C 017

044)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. rao

ultii

str. Kha

baro

vsk (C

P1096

9)

R. rhipice

phali

HJ 5 (C

P0131

33)

R. rick

ettsii

str. A

rizon

a (C

P0033

07)

R. s

ibirica

246

(AABW

0100

0001

)

R. s

lova

ca is

olate Xin

jiang

(MF0

0253

5)

R. typ

hi st

r. W

ilmington

(NC-0

0614

2)

Sample SR12 ex. Am. albolimbatum (PI 3329)

R. peacocki (NC 012730) 0.04

R. akari (NC 009881) 0.16 0.16

R. helvetica C9P9 (CM001467) 0.09 0.08 0.17

R. montanesis OSU 85-390 (CP003340) 0.02 0.05 0.15 0.08

R. tamurae str. AT-1 (AF394896) 0.06 0.07 0.17 0.09 0.04

R. aeschlimannii str. RH15 (HM050289) 0.03 0.04 0.15 0.09 0.03 0.05

R. africae ESF-5 (CP001612) 0.03 0.02 0.16 0.08 0.04 0.07 0.04

R. amblyommatis str. GAT-30V (CP003334) 0.02 0.04 0.15 0.09 0.03 0.05 0.02 0.03

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.13 0.13 0.16 0.11 0.12 0.13 0.12 0.13 0.12

R. australis str. Cutlack (NC 017058) 0.18 0.17 0.19 0.18 0.16 0.17 0.17 0.16 0.17 0.17

R. bellii RML369-C (NC 007940) 0.29 0.29 0.35 0.30 0.29 0.30 0.27 0.28 0.27 0.31 0.39

R. conorii str. Malish 7 (AE006914) 0.03 0.02 0.15 0.09 0.03 0.06 0.03 0.01 0.03 0.12 0.17 0.28

R. felis URRWXCal2 (CP000053) 0.16 0.16 0.10 0.17 0.15 0.16 0.15 0.15 0.14 0.11 0.22 0.33 0.16

R. gravesii BWI-1 (DQ269439) 0.02 0.04 0.16 0.09 0.03 0.06 0.03 0.04 0.02 0.13 0.18 0.29 0.03 0.16

R. heilongjiangensis Sendai-58 (AP019865) 0.03 0.03 0.16 0.09 0.03 0.06 0.03 0.02 0.03 0.13 0.17 0.27 0.02 0.16 0.03

R. honei RB (NZ AJTT00000000.1) 0.03 0.03 0.16 0.09 0.04 0.07 0.04 0.01 0.03 0.12 0.17 0.28 0.01 0.16 0.04 0.02

R. japonica str. M14012 (NZ AP017596) 0.03 0.04 0.17 0.09 0.04 0.07 0.04 0.03 0.04 0.13 0.18 0.28 0.02 0.17 0.04 0.01 0.03

R. massiliae str. AZT80 (CP003319) 0.02 0.04 0.15 0.08 0.03 0.05 0.01 0.03 0.02 0.12 0.17 0.27 0.03 0.15 0.03 0.03 0.03 0.03

R. monacensis str. IrR/Munich (LN794217) 0.05 0.07 0.17 0.09 0.04 0.02 0.06 0.07 0.05 0.13 0.18 0.30 0.06 0.17 0.05 0.06 0.06 0.06 0.05

R. parkeri str. Portsmouth (NC 017044) 0.03 0.03 0.15 0.09 0.04 0.06 0.03 0.01 0.03 0.12 0.18 0.28 0.01 0.16 0.03 0.02 0.01 0.03 0.03 0.06

R. prowazekii str. Madrid E (NC 000963) 0.21 0.20 0.27 0.22 0.20 0.20 0.20 0.19 0.19 0.24 0.35 0.37 0.19 0.29 0.20 0.20 0.20 0.21 0.20 0.21 0.20

R. raoultii str. Khabarovsk (CP10969) 0.02 0.04 0.15 0.08 0.02 0.05 0.02 0.03 0.02 0.13 0.17 0.28 0.03 0.15 0.02 0.02 0.03 0.03 0.02 0.05 0.02 0.20

R. rhipicephali HJ 5 (CP013133) 0.02 0.05 0.16 0.09 0.03 0.06 0.02 0.03 0.03 0.13 0.18 0.29 0.03 0.15 0.03 0.03 0.03 0.03 0.01 0.06 0.03 0.21 0.02

R. rickettsii str. Arizona (CP003307) 0.03 0.04 0.16 0.09 0.03 0.06 0.03 0.02 0.03 0.13 0.18 0.28 0.02 0.17 0.03 0.02 0.02 0.03 0.03 0.06 0.01 0.20 0.02 0.03

R. sibirica 246 (AABW01000001) 0.03 0.03 0.15 0.09 0.03 0.06 0.03 0.01 0.03 0.13 0.17 0.28 0.01 0.16 0.03 0.02 0.01 0.02 0.03 0.06 0.01 0.20 0.02 0.03 0.01

R. slovaca isolate Xinjiang (MF002535) 0.02 0.03 0.16 0.09 0.03 0.06 0.03 0.01 0.03 0.13 0.18 0.28 0.01 0.16 0.03 0.01 0.01 0.02 0.02 0.06 0.01 0.20 0.02 0.03 0.01 0.01

R. typhi str. Wilmington (NC-006142) 0.18 0.16 0.23 0.16 0.17 0.17 0.17 0.16 0.16 0.21 0.24 0.33 0.16 0.24 0.18 0.16 0.17 0.17 0.18 0.18 0.17 0.15 0.18 0.18 0.18 0.17 0.17

132

Table A1.36. Rickettsia concatenated ompA, ompB, and sca4 Maximum Likelihood fits of 24 different nucleotide substitution models.


HKY+G 58 20185 19692 -9788 n/a 0.576 1.214 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.1 0.04 0.08 0.16 0.04 0.19 0.06 0.04

TN93+G 59 20194 19692 -9787 n/a 0.575 1.221 0.344 0.278 0.182 0.196 0.06 0.04 0.12 0.08 0.1 0.04 0.08 0.15 0.04 0.2 0.06 0.04

HKY+G+I 59 20195 19694 -9788 0 0.575 1.215 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.1 0.04 0.08 0.16 0.04 0.19 0.06 0.04

T92+G 56 20198 19722 -9805 n/a 0.579 1.211 0.311 0.311 0.189 0.189 0.07 0.04 0.11 0.07 0.11 0.04 0.07 0.18 0.04 0.18 0.07 0.04

GTR+G 62 20200 19672 -9774 n/a 0.566 1.239 0.344 0.278 0.182 0.196 0.05 0.05 0.12 0.06 0.1 0.05 0.1 0.15 0.03 0.2 0.08 0.03

TN93+G+I 60 20205 19694 -9787 1E-05 0.574 1.222 0.344 0.278 0.182 0.196 0.06 0.04 0.12 0.08 0.1 0.04 0.08 0.15 0.04 0.2 0.06 0.04

T92+G+I 57 20209 19724 -9805 1E-05 0.578 1.212 0.311 0.311 0.189 0.189 0.07 0.04 0.11 0.07 0.11 0.04 0.07 0.18 0.04 0.18 0.07 0.04

GTR+G+I 63 20210 19674 -9774 1E-05 0.565 1.24 0.344 0.278 0.182 0.196 0.05 0.05 0.12 0.06 0.1 0.05 0.1 0.15 0.03 0.2 0.08 0.03

HKY+I 58 20406 19913 -9898 0.324 n/a 1.136 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.1 0.04 0.08 0.15 0.04 0.19 0.06 0.04

TN93+I 59 20415 19913 -9898 0.325 n/a 1.14 0.344 0.278 0.182 0.196 0.06 0.04 0.11 0.08 0.09 0.04 0.08 0.14 0.04 0.2 0.06 0.04

T92+I 56 20419 19943 -9915 0.324 n/a 1.131 0.311 0.311 0.189 0.189 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.17 0.04 0.17 0.07 0.04

GTR+I 62 20428 19901 -9888 0.324 n/a 1.148 0.344 0.278 0.182 0.196 0.05 0.05 0.11 0.06 0.09 0.05 0.1 0.14 0.03 0.2 0.07 0.03

K2+G 55 20465 19998 -9944 n/a 0.552 1.212 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06

K2+G+I 56 20476 20000 -9944 0 0.552 1.212 0.25 0.25 0.25 0.25 0.06 0.06 0.14 0.06 0.14 0.06 0.06 0.14 0.06 0.14 0.06 0.06

JC+G 54 20679 20220 -10056 n/a 0.591 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 55 20690 20222 -10056 1E-05 0.591 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

K2+I 55 20701 20233 -10061 0.333 n/a 1.126 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06

HKY 57 20777 20292 -10089 n/a n/a 1.054 0.344 0.278 0.182 0.196 0.07 0.04 0.1 0.08 0.1 0.05 0.08 0.15 0.05 0.18 0.07 0.04

T92 55 20787 20319 -10104 n/a n/a 1.057 0.311 0.311 0.189 0.189 0.07 0.04 0.1 0.07 0.1 0.04 0.07 0.16 0.04 0.16 0.07 0.04

TN93 58 20788 20294 -10089 n/a n/a 1.054 0.344 0.278 0.182 0.196 0.07 0.04 0.1 0.08 0.1 0.05 0.08 0.15 0.05 0.18 0.07 0.04

GTR 61 20796 20278 -10078 n/a n/a 1.055 0.344 0.278 0.182 0.196 0.05 0.05 0.1 0.07 0.1 0.05 0.1 0.15 0.04 0.18 0.07 0.04

JC+I 54 20902 20442 -10167 0.328 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

K2 54 21094 20635 -10263 n/a n/a 1.032 0.25 0.25 0.25 0.25 0.06 0.06 0.13 0.06 0.13 0.06 0.06 0.13 0.06 0.13 0.06 0.06

JC 53 21273 20822 -10358 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

133

Table A1.37. Rickettsia concatenated ompA, ompB, and sca4 genetic distance matrix.

R. pea

cock

i (N

C 012730

)

R. aka

ri (NC 00

9881

)

R. helv

etica

C9P9 (CM

0014

67)

R. mon

tane

sis O

SU 85-3

90 (C

P0033

40)

R. tam

urae

str. A

T-1 (A

F39489

6)

R. aes

chlim

annii

str. R

H15 (H

M050

289)

R. afric

ae E

SF-5 (C

P0016

12)

R. am

blyom

matis

str.

GAT-30V

(CP00

3334)

R. ase

mbon

ensis

str. N

MRCii (

NZ JWSW

000000

00.1)

R. aus

tralis

str. C

utlack

(NC 0

17058

)

R. bell

ii RM

L369-C

(NC 0

07940

)

R. con

orii str

. Mali

sh 7

(AE006

914)

R. felis

URRWXCal2

(CP00

0053)

R. gra

vesii

BWI-1

(DQ269

439)

R. heil

ongji

ange

nsis S

endai-

58 (A

P0198

65)

R. hon

ei R

B (NZ A

JTT00

00000

0.1)

R. japon

ica str

. M14

012 (

NZ AP017

596)

R. mas

siliae

str. A

ZT80 (CP003

319)

R. mon

acensis

str. Ir

R/Mun

ich (L

N79421

7)

R. par

keri

str. P

ortsm

outh (N

C 0170

44)

R. pro

wazekii

str. M

adrid

E (N

C 000963

)

R. rao

ultii str

. Kha

baro

vsk (

CP1096

9)

R. rhip

icepha

li HJ 5

(CP013

133)

R. rick

ettsii

str.

Arizon

a (CP003

307)

R. sibi

rica

246 (A

ABW010

00001)

R. slov

aca is

olate

Xinjiang

(MF00

2535)

R. typhi

str. W

ilming

ton (N

C-006

142)

Sample

R53 ex

Am. a

lbolim

batum

(PI 6

80)

R. peacocki (NC 012730)

R. akari (NC 009881) 0.19

R. helvetica C9P9 (CM001467) 0.12 0.19

R. montanesis OSU 85-390 (CP003340) 0.05 0.19 0.12

R. tamurae str. AT-1 (AF394896) 0.06 0.21 0.12 0.05

R. aeschlimannii str. RH15 (HM050289) 0.05 0.19 0.12 0.03 0.05

R. africae ESF-5 (CP001612) 0.03 0.19 0.12 0.04 0.06 0.04

R. amblyommatis str. GAT-30V (CP003334) 0.05 0.19 0.12 0.03 0.05 0.03 0.04

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.16 0.16 0.12 0.15 0.16 0.15 0.15 0.15

R. australis str. Cutlack (NC 017058) 0.20 0.19 0.19 0.19 0.21 0.20 0.19 0.20 0.16

R. bellii RML369-C (NC 007940) 0.56 0.68 0.62 0.54 0.58 0.54 0.55 0.53 0.66 0.74

R. conorii str. Malish 7 (AE006914) 0.02 0.19 0.12 0.04 0.05 0.04 0.01 0.03 0.15 0.19 0.54

R. felis URRWXCal2 (CP000053) 0.17 0.10 0.17 0.17 0.18 0.16 0.17 0.15 0.12 0.21 0.66 0.17

R. gravesii BWI-1 (DQ269439) 0.05 0.19 0.13 0.03 0.06 0.04 0.04 0.03 0.16 0.20 0.55 0.04 0.18

R. heilongjiangensis Sendai-58 (AP019865) 0.04 0.19 0.12 0.03 0.05 0.03 0.02 0.03 0.15 0.19 0.53 0.02 0.17 0.03

R. honei RB (NZ AJTT00000000.1) 0.03 0.18 0.12 0.04 0.05 0.04 0.01 0.04 0.15 0.19 0.54 0.01 0.17 0.04 0.02


R. massiliae str. AZT80 (CP003319) 0.05 0.19 0.12 0.03 0.05 0.02 0.03 0.03 0.14 0.20 0.53 0.03 0.16 0.03 0.03 0.03 0.03



R. prowazekii str. Madrid E (NC 000963) 0.31 0.41 0.35 0.32 0.33 0.32 0.32 0.31 0.36 0.48 0.75 0.31 0.40 0.32 0.32 0.32 0.33 0.32 0.34 0.32

R. raoultii str. Khabarovsk (CP10969) 0.05 0.19 0.12 0.03 0.05 0.03 0.03 0.03 0.15 0.20 0.53 0.03 0.16 0.03 0.02 0.03 0.03 0.02 0.06 0.03 0.31

R. rhipicephali HJ 5 (CP013133) 0.05 0.19 0.12 0.03 0.05 0.02 0.04 0.03 0.15 0.20 0.54 0.04 0.17 0.03 0.02 0.03 0.03 0.01 0.06 0.04 0.32 0.02

R. rickettsii str. Arizona (CP003307) 0.04 0.19 0.12 0.04 0.05 0.04 0.02 0.03 0.15 0.20 0.55 0.02 0.18 0.03 0.02 0.01 0.03 0.03 0.06 0.02 0.32 0.03 0.03

R. sibirica 246 (AABW01000001) 0.03 0.19 0.12 0.04 0.05 0.04 0.02 0.04 0.15 0.20 0.55 0.01 0.17 0.04 0.02 0.02 0.03 0.03 0.07 0.01 0.33 0.03 0.04 0.02

R. slovaca isolate Xinjiang (MF002535) 0.03 0.19 0.12 0.03 0.05 0.03 0.01 0.03 0.15 0.20 0.55 0.01 0.17 0.03 0.02 0.01 0.02 0.03 0.06 0.01 0.32 0.02 0.03 0.01 0.01

R. typhi str. Wilmington (NC-006142) 0.27 0.37 0.29 0.29 0.30 0.29 0.28 0.29 0.31 0.37 0.69 0.28 0.35 0.29 0.28 0.28 0.29 0.29 0.31 0.29 0.18 0.29 0.29 0.30 0.30 0.29

Sample R53 ex Am. albolimbatum (PI 680) 0.05 0.19 0.12 0.03 0.06 0.04 0.04 0.03 0.15 0.21 0.55 0.04 0.17 0.02 0.03 0.04 0.04 0.03 0.06 0.04 0.33 0.03 0.03 0.03 0.03 0.03 0.29

134

Table A1.38. Rickettsia concatenated ompB, 17 kDa, and sca4 Maximum Likelihood fits of 24 different nucleotide substitution models.


GTR+G+I 63 13722 13183 -6528 0.135 0.748 1.78 0.348 0.258 0.182 0.211 0.02 0.07 0.11 0.03 0.14 0.04 0.12 0.2 0.01 0.18 0.05 0.01

GTR+G 62 13747 13217 -6546 n/a 0.582 1.39 0.348 0.258 0.182 0.211 0.04 0.06 0.12 0.05 0.11 0.04 0.12 0.16 0.03 0.19 0.05 0.03

HKY+G 58 13764 13267 -6576 n/a 0.526 1.772 0.348 0.258 0.182 0.211 0.05 0.03 0.14 0.06 0.12 0.04 0.06 0.17 0.04 0.22 0.05 0.03

TN93+G 59 13765 13260 -6571 n/a 0.543 1.739 0.348 0.258 0.182 0.211 0.05 0.03 0.11 0.06 0.14 0.04 0.06 0.2 0.04 0.19 0.05 0.03

GTR+I 62 13768 13238 -6557 0.438 n/a 1.535 0.348 0.258 0.182 0.211 0.02 0.06 0.11 0.03 0.13 0.04 0.12 0.18 0.04 0.19 0.05 0.03

T92+G 56 13772 13293 -6590 n/a 0.535 1.744 0.303 0.303 0.197 0.197 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03

HKY+G+I 59 13773 13268 -6575 0.033 0.565 1.773 0.348 0.258 0.182 0.211 0.05 0.03 0.14 0.06 0.12 0.04 0.06 0.17 0.04 0.22 0.05 0.03

TN93+G+I 60 13774 13260 -6570 0.069 0.633 1.742 0.348 0.258 0.182 0.211 0.05 0.03 0.11 0.06 0.14 0.04 0.06 0.2 0.04 0.19 0.05 0.03

T92+G+I 57 13781 13293 -6589 0.031 0.571 1.745 0.303 0.303 0.197 0.197 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.2 0.03 0.2 0.05 0.03

TN93+I 59 13796 13291 -6586 0.446 n/a 1.721 0.348 0.258 0.182 0.211 0.05 0.03 0.11 0.06 0.14 0.04 0.06 0.2 0.04 0.19 0.05 0.03

HKY+I 58 13797 13301 -6592 0.451 n/a 1.748 0.348 0.258 0.182 0.211 0.05 0.03 0.14 0.06 0.12 0.04 0.06 0.17 0.04 0.22 0.05 0.03

T92+I 56 13804 13325 -6607 0.449 n/a 1.724 0.303 0.303 0.197 0.197 0.05 0.04 0.13 0.05 0.13 0.04 0.05 0.2 0.04 0.2 0.05 0.04

K2+G 55 13902 13431 -6660 n/a 0.511 1.738 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

K2+G+I 56 13910 13431 -6659 1E-04 0.51 1.739 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

K2+I 55 13940 13469 -6680 0.458 n/a 1.711 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

GTR 61 13971 13449 -6663 n/a n/a 1.313 0.348 0.258 0.182 0.211 0.03 0.06 0.1 0.04 0.12 0.04 0.12 0.17 0.04 0.17 0.05 0.03

TN93 58 13998 13502 -6693 n/a n/a 1.455 0.348 0.258 0.182 0.211 0.05 0.04 0.1 0.07 0.13 0.04 0.07 0.19 0.04 0.17 0.05 0.04

HKY 57 14008 13520 -6703 n/a n/a 1.448 0.348 0.258 0.182 0.211 0.05 0.04 0.13 0.07 0.11 0.04 0.07 0.15 0.04 0.21 0.05 0.04

T92 55 14009 13538 -6714 n/a n/a 1.456 0.303 0.303 0.197 0.197 0.06 0.04 0.12 0.06 0.12 0.04 0.06 0.18 0.04 0.18 0.06 0.04

K2 54 14151 13689 -6790 n/a n/a 1.437 0.25 0.25 0.25 0.25 0.05 0.05 0.15 0.05 0.15 0.05 0.05 0.15 0.05 0.15 0.05 0.05

JC+G 54 14160 13698 -6795 n/a 0.59 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 55 14169 13698 -6794 7E-06 0.59 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 54 14196 13734 -6813 0.436 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 53 14373 13920 -6907 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

135

Table A1.39. Rickettsia concatenated ompB, 17 kDa, and sca4 genetic distance matrix.

Sam

ple

R92

ex. Am

. albolim

batu

m

(PI 3

323)

R. g

rave

sii

BW

I-1 (D

Q26

9437

)

R. a

esch

liman

nii

str.

RH15

(HM05

0289

)

R. m

assilia

e str.

AZT80 (C

P00

3319

)

R. rhipice

phali

HJ 5 (C

P01

3133

)

R. m

ontane

sis OSU 85-

390

(CP00

3340

)

R. p

eaco

cki

(NC 012

730)

R. a

frica

e E

SF-

5 (C

P00

1612

)

R. c

onor

ii str.

Malish 7

(AE00

6914

)

R. h

onei

RB (N

Z AJT

T000

0000

0.1)

R. p

arke

ri str.

Portsm

outh (N

C 017

044)

R. rick

ettsii

str.

Ariz

ona

(CP00

3307

)

R. s

ibiric

a 246

(AABW

0100

0001

)

R. s

lova

ca is

olat

e Xinjia

ng (M

F002

535)

R. a

mblyo

mm

atis

str.

GAT-3

0V (C

P00

3334

)

R. h

eilong

jiang

ensis

Sen

dai-5

8 (A

P01

9865

)

R. jap

onica

str.

M14

012 (N

Z AP01

7596

)

R. rao

ultii

str.

Khaba

rovs

k (C

P10

969)

R. m

onac

ensis

str.

IrR/M

unich

(LN79

4217

)

R. tam

urae

str.

AT-1 (A

F3948

96)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. a

kari

(NC 009

881)

R. felis

URRW

XCal2 (C

P00

0053

)

R. a

sem

bone

nsis

str.

NM

RCii (

NZ J

WSW

0000

0000

.1)

R. a

ustra

lis

str.

Cutlack

(NC 017

058)

R. typ

hi

str.

Wilm

ington

(NC-0

0614

2)

R. p

rowaz

ekii

str.

Mad

rid E

(NC 000

963)

R. b

ellii

RM

L369

-C (N

C 007

940)

Sample R92 ex. Am. albolimbatum (PI 3323)

R. gravesii BWI-1 (DQ269437) 0.01

R. aeschlimannii str. RH15 (HM050289) 0.02 0.02

R. massiliae str. AZT80 (CP003319) 0.02 0.02 0.01

R. rhipicephali HJ 5 (CP013133) 0.02 0.02 0.01 0.01

R. montanesis OSU 85-390 (CP003340) 0.02 0.02 0.01 0.01 0.02

R. peacocki (NC 012730) 0.02 0.02 0.02 0.02 0.02 0.02

R. africae ESF-5 (CP001612) 0.02 0.02 0.02 0.02 0.02 0.02 0.01

R. conorii str. Malish 7 (AE006914) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01

R. honei RB (NZ AJTT00000000.1) 0.02 0.02 0.01 0.01 0.02 0.01 0.01 0.01 0.01

R. parkeri str. Portsmouth (NC 017044) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.00 0.01

R. rickettsii str. Arizona (CP003307) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.00 0.01

R. sibirica 246 (AABW01000001) 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.01 0.01 0.01 0.00 0.01

R. slovaca isolate Xinjiang (MF002535) 0.02 0.02 0.02 0.02 0.02 0.02 0.00 0.01 0.00 0.00 0.00 0.00 0.01

R. amblyommatis str. GAT-30V (CP003334) 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02

R. heilongjiangensis Sendai-58 (AP019865) 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.02


R. raoultii str. Khabarovsk (CP10969) 0.02 0.02 0.01 0.01 0.02 0.01 0.02 0.02 0.02 0.01 0.02 0.01 0.02 0.01 0.02 0.01 0.01


R. tamurae str. AT-1 (AF394896) 0.04 0.04 0.04 0.04 0.04 0.04 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.04 0.03 0.03 0.04 0.03

R. helvetica C9P9 (CM001467) 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.06 0.07

R. akari (NC 009881) 0.08 0.09 0.08 0.08 0.09 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.08 0.08 0.08 0.09 0.08

R. felis URRWXCal2 (CP000053) 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.09 0.08 0.06

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.08 0.09 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.07 0.08 0.07 0.05 0.03


R. typhi str. Wilmington (NC-006142) 0.20 0.19 0.19 0.20 0.19 0.19 0.20 0.20 0.20 0.19 0.19 0.20 0.20 0.19 0.20 0.19 0.19 0.19 0.19 0.19 0.19 0.20 0.20 0.19 0.20

R. prowazekii str. Madrid E (NC 000963) 0.21 0.21 0.20 0.21 0.20 0.20 0.21 0.21 0.21 0.20 0.20 0.21 0.21 0.21 0.21 0.20 0.20 0.20 0.20 0.20 0.20 0.22 0.21 0.21 0.23 0.07

R. bellii RML369-C (NC 007940) 0.44 0.45 0.44 0.44 0.43 0.43 0.44 0.44 0.43 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.44 0.45 0.45 0.45 0.47 0.46 0.46 0.48 0.48 0.52

136

Table A1.40. Rickettsia concatenated ompB, and 17 kDa Maximum Likelihood fits of 24 different nucleotide substitution models.


T92+G 58 5387 4939 -2411 n/a 0.415 2.496 0.283 0.283 0.217 0.217 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.2 0.03 0.2 0.04 0.03

T92+I 58 5393 4945 -2414 0.531 n/a 2.475 0.283 0.283 0.217 0.217 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.2 0.03 0.2 0.04 0.03

T92+G+I 59 5396 4941 -2411 0.298 0.911 2.507 0.283 0.283 0.217 0.217 0.04 0.03 0.16 0.04 0.16 0.03 0.04 0.2 0.03 0.2 0.04 0.03

HKY+G 60 5404 4940 -2410 n/a 0.409 2.552 0.301 0.265 0.183 0.25 0.04 0.03 0.18 0.04 0.13 0.04 0.04 0.19 0.04 0.22 0.04 0.03

K2+G 57 5405 4965 -2425 n/a 0.405 2.502 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

HKY+I 60 5410 4946 -2413 0.533 n/a 2.531 0.301 0.265 0.183 0.25 0.04 0.03 0.18 0.04 0.13 0.04 0.04 0.19 0.04 0.22 0.04 0.03

K2+I 57 5410 4970 -2428 0.536 n/a 2.475 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

TN93+G 61 5411 4940 -2409 n/a 0.426 2.494 0.301 0.265 0.183 0.25 0.04 0.03 0.15 0.04 0.16 0.04 0.04 0.23 0.04 0.18 0.04 0.03

HKY+G+I 61 5413 4942 -2410 0.288 0.863 2.561 0.301 0.265 0.183 0.25 0.04 0.03 0.18 0.04 0.13 0.04 0.04 0.19 0.04 0.22 0.04 0.03

K2+G+I 58 5414 4966 -2425 0.27 0.796 2.511 0.25 0.25 0.25 0.25 0.04 0.04 0.18 0.04 0.18 0.04 0.04 0.18 0.04 0.18 0.04 0.04

GTR+G 64 5415 4920 -2396 n/a 0.424 2.502 0.301 0.265 0.183 0.25 0.02 0.06 0.15 0.02 0.16 0.04 0.1 0.23 0.01 0.18 0.04 0.01

TN93+G+I 62 5419 4940 -2408 0.328 1.066 2.51 0.301 0.265 0.183 0.25 0.04 0.03 0.15 0.04 0.16 0.04 0.04 0.23 0.04 0.18 0.04 0.03

GTR+G+I 65 5423 4921 -2395 0.4 1.567 2.523 0.301 0.265 0.183 0.25 0.02 0.06 0.15 0.02 0.16 0.04 0.1 0.23 0.01 0.18 0.04 0.01

TN93+I 61 5443 4972 -2425 0.32 n/a 2.232 0.301 0.265 0.183 0.25 0.04 0.03 0.14 0.05 0.16 0.04 0.05 0.23 0.04 0.17 0.04 0.03

GTR+I 64 5456 4961 -2416 0.32 n/a 1.952 0.301 0.265 0.183 0.25 0.02 0.06 0.14 0.03 0.14 0.04 0.1 0.2 0.04 0.17 0.04 0.03

T92 57 5482 5041 -2463 n/a n/a 2.042 0.283 0.283 0.217 0.217 0.05 0.04 0.15 0.05 0.15 0.04 0.05 0.19 0.04 0.19 0.05 0.04

HKY 59 5500 5044 -2463 n/a n/a 2.042 0.301 0.265 0.183 0.25 0.04 0.03 0.17 0.05 0.12 0.04 0.05 0.18 0.04 0.2 0.04 0.03

K2 56 5501 5069 -2478 n/a n/a 2.029 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

TN93 60 5504 5040 -2460 n/a n/a 2.051 0.301 0.265 0.183 0.25 0.04 0.03 0.14 0.05 0.15 0.04 0.05 0.22 0.04 0.17 0.04 0.03

GTR 63 5511 5024 -2449 n/a n/a 2.063 0.301 0.265 0.183 0.25 0.03 0.06 0.14 0.03 0.15 0.04 0.1 0.22 0.02 0.17 0.04 0.01

JC+G 56 5550 5117 -2502 n/a 0.461 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 57 5559 5119 -2502 0.109 0.582 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 56 5580 5147 -2517 0.32 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC 55 5634 5209 -2549 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

137

Table A1.41. Rickettsia concatenated ompB, and 17 kDa genetic distance matrix.

Sample R52

ex. Am

. albolim

batum

(PI 6

80)

Sample R10

6 ex

. Am

. albolim

batum

(PI 6

80)

R. tam

urae

str. AT-

1 (A

F394

896)

R. m

onac

ensis

str. IrR

/Mun

ich (L

N79

4217

)

R. h

onei

RB (N

Z AJT

T000

0000

0.1)

R. a

sem

bone

nsis

str. NM

RCii (

NZ JW

SW00

0000

00.1)

R. m

assil

iae

str. AZT

80 (C

P0033

19)

R. rhipice

phali

HJ 5 (C

P0131

33)

R. p

eaco

cki

(NC 012

730)

R. a

kari

(NC 009

881)

R. h

elve

tica

C9P

9 (C

M00

1467

)

R. m

ontane

sis OSU

85-

390

(CP00

3340

)

R. a

esch

liman

nii

str. RH15

(HM

0502

89)

R. a

frica

e E

SF-5 (C

P0016

12)

R. c

onor

ii str. M

alish

7 (A

E0069

14)

R. g

rave

sii

BWI-1

(DQ26

9437

)

R. h

eilong

jiang

ensis

Sen

dai-5

8 (A

P0198

65)

R. ja

ponica

str. M

1401

2 (N

Z AP01

7596

)

R. p

arke

ri str. Por

tsm

outh (N

C 017

044)

R. rao

ultii

str. Kha

baro

vsk (C

P1096

9)

R. rick

ettsii

str. A

rizon

a (C

P0033

07)

R. s

ibirica

246

(AABW

0100

0001

)

R. s

lova

ca is

olate Xin

jiang

(MF0

0253

5)

R. a

mblyo

mm

atis

str. G

AT-30

V (CP00

3334

)

R. a

ustra

lis

str. Cutlack

(NC 017

058)

R. felis

URRW

XCal2 (C

P0000

53)

R. typ

hi st

r. W

ilmington

(NC-0

0614

2)

R. p

rowaz

ekii

str. M

adrid

E (N

C 000

963)

R. b

ellii

RM

L369

-C (N

C 007

940)

Sample R52 ex. Am. albolimbatum (PI 680)

Sample R106 ex. Am. albolimbatum (PI 680) 0.00

R. tamurae str. AT-1 (AF394896) 0.01 0.01

R. monacensis str. IrR/Munich (LN794217) 0.01 0.01 0.01

R. honei RB (NZ AJTT00000000.1) 0.04 0.04 0.04 0.04

R. asembonensis str. NMRCii (NZ JWSW00000000.1) 0.04 0.04 0.04 0.04 0.05

R. massiliae str. AZT80 (CP003319) 0.04 0.04 0.05 0.04 0.01 0.05

R. rhipicephali HJ 5 (CP013133) 0.04 0.04 0.05 0.04 0.02 0.05 0.01

R. peacocki (NC 012730) 0.05 0.05 0.05 0.04 0.01 0.05 0.02 0.02

R. akari (NC 009881) 0.05 0.05 0.05 0.04 0.03 0.04 0.04 0.04 0.04

R. helvetica C9P9 (CM001467) 0.05 0.05 0.05 0.05 0.04 0.06 0.04 0.04 0.04 0.04

R. montanesis OSU 85-390 (CP003340) 0.05 0.05 0.04 0.04 0.01 0.05 0.01 0.02 0.02 0.03 0.04

R. aeschlimannii str. RH15 (HM050289) 0.05 0.05 0.05 0.04 0.01 0.05 0.01 0.02 0.02 0.04 0.04 0.01

R. africae ESF-5 (CP001612) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02

R. conorii str. Malish 7 (AE006914) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02 0.00

R. gravesii BWI-1 (DQ269437) 0.05 0.05 0.04 0.04 0.01 0.04 0.01 0.02 0.01 0.03 0.04 0.01 0.01 0.01 0.01

R. heilongjiangensis Sendai-58 (AP019865) 0.05 0.05 0.05 0.04 0.01 0.05 0.02 0.02 0.02 0.03 0.04 0.01 0.02 0.01 0.01 0.01

R. japonica str. M14012 (NZ AP017596) 0.05 0.05 0.05 0.04 0.01 0.05 0.02 0.02 0.02 0.04 0.04 0.02 0.02 0.02 0.02 0.01 0.01

R. parkeri str. Portsmouth (NC 017044) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.00 0.03 0.04 0.01 0.02 0.00 0.00 0.01 0.01 0.01

R. raoultii str. Khabarovsk (CP10969) 0.05 0.05 0.04 0.04 0.01 0.04 0.01 0.02 0.01 0.03 0.04 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

R. rickettsii str. Arizona (CP003307) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.01 0.00 0.00 0.01 0.01 0.02 0.00 0.01

R. sibirica 246 (AABW01000001) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02 0.00 0.00 0.01 0.01 0.02 0.00 0.01 0.00

R. slovaca isolate Xinjiang (MF002535) 0.05 0.05 0.05 0.04 0.00 0.05 0.02 0.02 0.01 0.03 0.04 0.01 0.02 0.00 0.00 0.01 0.01 0.02 0.00 0.01 0.00 0.00

R. amblyommatis str. GAT-30V (CP003334) 0.06 0.06 0.05 0.05 0.02 0.05 0.03 0.03 0.03 0.04 0.05 0.02 0.03 0.02 0.03 0.02 0.03 0.03 0.02 0.02 0.03 0.03 0.03



R. typhi str. Wilmington (NC-006142) 0.12 0.12 0.12 0.12 0.13 0.12 0.14 0.13 0.14 0.12 0.12 0.13 0.13 0.13 0.13 0.13 0.14 0.14 0.13 0.13 0.13 0.13 0.13 0.14 0.12 0.12

R. prowazekii str. Madrid E (NC 000963) 0.14 0.14 0.14 0.13 0.15 0.15 0.15 0.15 0.16 0.15 0.14 0.15 0.14 0.16 0.16 0.15 0.16 0.16 0.15 0.15 0.15 0.16 0.16 0.16 0.17 0.16 0.06

R. bellii RML369-C (NC 007940) 0.34 0.34 0.35 0.34 0.34 0.33 0.34 0.33 0.34 0.34 0.34 0.33 0.33 0.34 0.33 0.33 0.34 0.34 0.33 0.34 0.33 0.34 0.34 0.35 0.34 0.34 0.36 0.39

138

Table A1.42. Haemoprotozoa 18S Maximum Likelihood fits of 24 different nucleotide substitution models.

Model #Param BIC AICc lnL (+i) (+G) R Freq A Freq T Freq C Freq G A=>T A=>C A=>G T=>A T=>C T=>G C=>A C=>T C=>G G=>A G=>T G=>C

T92+G 40 5706 5400 -2660 n/a 0.21 2.13 0.315 0.315 0.185 0.185 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.22 0.03 0.22 0.05 0.03

T92+G+I 41 5714 5401 -2659 0.361 0.464 2.147 0.315 0.315 0.185 0.185 0.05 0.03 0.13 0.05 0.13 0.03 0.05 0.22 0.03 0.22 0.05 0.03

HKY+G 42 5717 5396 -2656 n/a 0.212 2.124 0.321 0.308 0.148 0.223 0.05 0.02 0.15 0.05 0.1 0.03 0.05 0.21 0.03 0.22 0.05 0.02

TN93+G 43 5725 5396 -2655 n/a 0.212 2.114 0.321 0.308 0.148 0.223 0.05 0.02 0.13 0.05 0.12 0.03 0.05 0.25 0.03 0.19 0.05 0.02

HKY+G+I 43 5725 5397 -2655 0.368 0.477 2.142 0.321 0.308 0.148 0.223 0.05 0.02 0.15 0.05 0.1 0.03 0.05 0.21 0.03 0.22 0.05 0.02

TN93+G+I 44 5733 5397 -2654 0.346 0.444 2.131 0.321 0.308 0.148 0.223 0.05 0.02 0.13 0.05 0.12 0.03 0.05 0.25 0.03 0.19 0.05 0.02

GTR+G 46 5752 5401 -2654 n/a 0.216 1.971 0.321 0.308 0.148 0.223 0.06 0.02 0.14 0.06 0.11 0.03 0.05 0.23 0.03 0.2 0.04 0.02

K2+G 39 5756 5458 -2690 n/a 0.22 2.015 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

GTR+G+I 47 5758 5399 -2652 0.346 0.445 2.144 0.321 0.308 0.148 0.223 0.06 0.03 0.14 0.06 0.12 0.03 0.06 0.25 0.02 0.2 0.04 0.01

K2+G+I 40 5765 5459 -2689 0.382 0.529 2.029 0.25 0.25 0.25 0.25 0.04 0.04 0.17 0.04 0.17 0.04 0.04 0.17 0.04 0.17 0.04 0.04

T92+I 40 5788 5482 -2701 0.374 n/a 1.788 0.315 0.315 0.185 0.185 0.05 0.03 0.12 0.05 0.12 0.03 0.05 0.21 0.03 0.21 0.05 0.03

HKY+I 42 5797 5477 -2696 0.374 n/a 1.792 0.321 0.308 0.148 0.223 0.05 0.03 0.15 0.06 0.1 0.04 0.06 0.2 0.04 0.21 0.05 0.03

TN93+I 43 5806 5477 -2696 0.374 n/a 1.785 0.321 0.308 0.148 0.223 0.05 0.03 0.13 0.05 0.11 0.04 0.05 0.22 0.04 0.19 0.05 0.03

GTR+I 46 5830 5478 -2693 0.374 n/a 1.778 0.321 0.308 0.148 0.223 0.07 0.03 0.13 0.07 0.11 0.03 0.06 0.23 0.02 0.19 0.04 0.01

K2+I 39 5831 5533 -2727 0.374 n/a 1.755 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

JC+G 38 5860 5570 -2747 n/a 0.239 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+I 38 5867 5577 -2750 0.661 n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

JC+G+I 39 5869 5571 -2746 0.347 0.527 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

T92 39 5872 5574 -2748 n/a n/a 1.674 0.315 0.315 0.185 0.185 0.06 0.03 0.12 0.06 0.12 0.03 0.06 0.2 0.03 0.2 0.06 0.03

HKY 41 5881 5568 -2743 n/a n/a 1.675 0.321 0.308 0.148 0.223 0.06 0.03 0.14 0.06 0.09 0.04 0.06 0.2 0.04 0.21 0.06 0.03

TN93 42 5890 5569 -2742 n/a n/a 1.673 0.321 0.308 0.148 0.223 0.06 0.03 0.13 0.06 0.1 0.04 0.06 0.22 0.04 0.19 0.06 0.03

K2 38 5910 5620 -2772 n/a n/a 1.679 0.25 0.25 0.25 0.25 0.05 0.05 0.16 0.05 0.16 0.05 0.05 0.16 0.05 0.16 0.05 0.05

GTR 45 5912 5568 -2739 n/a n/a 1.66 0.321 0.308 0.148 0.223 0.07 0.03 0.13 0.07 0.11 0.03 0.06 0.22 0.02 0.19 0.04 0.01

JC 37 6002 5720 -2823 n/a n/a 0.5 0.25 0.25 0.25 0.25 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08 0.08

139

Table A1.43. Haemoprotozoa 18S genetic distance matrix.

Sample

SR14

ex.

A. albo

limba

tum

(PI 1

316)

Hepato

zoon

felis

(AB7

7150

1.1)

Hepato

zoon

felis

AB771

552.1

Hepato

zoon

canis

(AF1

7683

5)

Hepato

zoon

felis

isolat

e La

CONES

/Asia

tic lio

n 01 (

HQ82

9438

.1)

Hepato

zoon

felis

isola

te La

CONES/In

dian L

eopa

rd 02

(HQ82

9444

.1)

Hepato

zoon

felis

isola

te La

CONES/B

enga

l Tiger

01 (H

Q82

9445

.1)

H. mau

ritanic

a

isola

te TR

-8-08

(KF9

9269

8.1)

H. mariae

isolate

490

3 (KF9

9271

1.1)

H. mariae

isolate

495

5 (KF9

9271

2.1)

H. sp

. ex.

R. pulc

herrima

(KF9

9271

3.1)

H. stel

lata

KP88

1349

.1

K. sp

. Bto-20

19a i

solate

B3 (

MK39

6906

.1)

K. cf. l

acaz

ei (M

K497

254.1

)

K. sp

. isolate

CR12

(MK62

1304

.1)

B. gibs

oni

(KC46

1261

)

B. stab

leri

(HQ22

4961

)

D. rana

rum

(HQ22

4957

)

K. heli

cina

(HQ22

4955

)

A. bam

baroo

niae

(AF4

9405

9)


Hepatozoon felis (AB771501.1) 0.03

Hepatozoon felis AB771552.1 0.03 0.00

Hepatozoon canis (AF176835) 0.05 0.04 0.04

Hepatozoon felis isolate LaCONES/Asiatic lion 01 (HQ829438.1)0.03 0.00 0.01 0.04

Hepatozoon felis isolate LaCONES/Indian Leopard 02 (HQ829444.1)0.03 0.01 0.01 0.04 0.00

Hepatozoon felis isolate LaCONES/Bengal Tiger 01 (HQ829445.1)0.03 0.02 0.02 0.04 0.02 0.02

H. mauritanica isolate TR-8-08 (KF992698.1) 0.02 0.03 0.04 0.04 0.04 0.04 0.04

H. mariae isolate 4903 (KF992711.1) 0 0.03 0.03 0.05 0.03 0.03 0.03 0.02

H. mariae isolate 4955 (KF992712.1) 0.00 0.03 0.03 0.05 0.03 0.03 0.03 0.02 0.00

H. sp. ex. R. pulcherrima (KF992713.1) 0.02 0.02 0.03 0.04 0.03 0.03 0.03 0.01 0.02 0.02

H. stellata KP881349.1 0.02 0.02 0.02 0.04 0.02 0.03 0.03 0.02 0.02 0.02 0.01

K. sp. Bto-2019a isolate B3 (MK396906.1) 0.04 0.02 0.02 0.05 0.02 0.02 0.03 0.04 0.04 0.04 0.03 0.03

K. cf. lacazei (MK497254.1) 0.04 0.02 0.02 0.05 0.02 0.03 0.03 0.05 0.04 0.04 0.04 0.03 0.01

K. sp. isolate CR12 (MK621304.1) 0.03 0.00 0.01 0.04 0.01 0.01 0.02 0.03 0.03 0.03 0.02 0.02 0.02 0.02

B. gibsoni (KC461261) 0.16 0.18 0.18 0.19 0.18 0.18 0.18 0.17 0.16 0.16 0.17 0.17 0.17 0.17 0.18

B. stableri (HQ224961) 0.07 0.06 0.06 0.08 0.06 0.06 0.07 0.07 0.07 0.07 0.06 0.06 0.07 0.07 0.06 0.18

D. ranarum (HQ224957) 0.07 0.07 0.07 0.08 0.07 0.07 0.07 0.07 0.07 0.07 0.06 0.06 0.07 0.07 0.07 0.18 0.01

K. helicina (HQ224955) 0.13 0.12 0.12 0.14 0.12 0.13 0.14 0.13 0.13 0.13 0.13 0.13 0.14 0.13 0.12 0.17 0.13 0.14

A. bambarooniae (AF494059) 0.12 0.12 0.12 0.12 0.12 0.12 0.13 0.11 0.12 0.12 0.11 0.11 0.13 0.12 0.12 0.15 0.12 0.12 0.07

molecular characterisation of microorganisms within the

Documents