Nematology 16 (2014) 847-862 brill.com/nemy

Morphological and molecular characterisation of the Saratovpopulation of the European dagger nematode, Xiphinemadiversicaudatum (Nematoda: Dorylaimida), with notes on

phylogeography of the species

Vladimir N. CHIZHOV 1, Mikhail V. PRIDANNIKOV 1,Vlada PENEVA 2 and Sergei A. SUBBOTIN 1,3,∗

1 Center of Parasitology of A.N. Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences,Leninskii Prospect 33, Moscow 117071, Russia

2 Institute of Biodiversity and Ecosystem Research, Bulgarian Academy of Sciences,2 Gagarin Street, 1113 Sofia, Bulgaria

3 Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road, Sacramento,CA 95832, USA

Received: 16 April 2014; revised: 28 May 2014Accepted for publication: 28 May 2014; available online: 7 July 2014

Summary – Plesiotype specimens of the European dagger nematode, Xiphinema diversicaudatum, were collected in Saratov, Russia,and morphologically, morphometrically and molecularly characterised. PCR with specific primer was developed for rapid diagnosticsof this species. Phylogenetic relationships of X. diversicaudatum with other Xiphinema species, as inferred from the analyses of theD2-D3 expansion segments of 28S rRNA, ITS rRNA and coxI mtDNA gene sequences, are also provided. The study revealed thatthe clade X. diversicaudatum, together with X. bakeri (North America) and X. chengi (Asia), is related to Xiphinema species from theMediterranean region and likely originated from a common ancestor inhabiting this area. The phylogenetic reconstructions with coxImtDNA were used to describe the pattern of present genetic diversity of X. diversicaudatum and infer its biogeographical history inEurope. Haplotype diversity of X. diversicaudatum populations from Central Europe was significantly higher than those from south-eastern Europe, central, south-eastern and southern European Russia. It has been hypothesised that, during the Last Glacial Maximum,X. diversicaudatum populations probably persisted in refuge areas in the Carpathian mountains or the Alps, the species subsequentlyexpanding from these areas and colonising other regions in Europe.

Keywords – biogeography, morphology, morphometrics, mtDNA, PCR, phylogeny, Russia, Xiphinema bakeri.

The European dagger nematode was described by Mi-coletzky (1927) as Dorylaimus (Longidorus) diversicau-datum based on a single female collected in the Volga rivernear Saratov, Russia, and two males and one juvenile col-lected in two locations, the first being from the Obva riverand the second from the Vjatka river, both of which aretributaries of the Kama river. Since the first description ofXiphinema diversicaudatum (Micoletzky, 1927) Thorne,1939, morphometrics of adults from different populationshave been reported by several authors (Brown & Topham,1985; Roca & Bravo, 1997; Barsi & Lamberti, 2000), in-cluding a redescription of the species made by Goodey et

∗ Corresponding author, e-mail: subbotin@ucr.edu

al. (1960) and Pitcher et al. (1974). In 1974, M.R. Siddiqidesignated a lectotype male and a paralectotype juvenilefrom the Micoletzky slide collection (Pitcher et al., 1974)and defined the type locality for this species as being theObva river near its junction with the Kama river, the PermKrai, Russia, which is nearly 990 km from the place wherethe X. diversicaudatum female used by Micoletzky for theoriginal species description was found. In June 2012, nu-merous X. diversicaudatum females and males were foundin the vicinity of Saratov city in the rhizosphere of ash-leaf maple, Acer negundo L. These specimens might beconsidered as geographically closest to where the single

© Koninklijke Brill NV, Leiden, 2014 DOI:10.1163/15685411-00002813

V.N. Chizhov et al.

female of X. diversicaudatum was collected by Micolet-zky, and are referred to here as plesiotypes.

Xiphinema diversicaudatum is polyphagous and par-asitises various woody and herbaceous plants. In sev-eral countries this nematode has a quarantine status asit may induce severe root galling on several plants andalso vectors both Arabis mosaic virus and strawberry la-tent ringspot virus (Pitcher et al., 1974). Xiphinema di-versicaudatum is widely distributed in many Europeancountries (Brown & Taylor, 1987; Anon., 2001), the Eu-ropean part of Russia, including the North Caucasus re-gion (Brown et al., 1990), New Zealand (Sturhan et al.,1997) and South Africa (Heyns & Coomans, 1984). Al-though X. diversicaudatum was reported in Canada (Mul-vey, 1961) and several states of the USA (Pitcher etal., 1974), Robbins & Brown (1991) suggested that thisspecies was relatively rare in North America, probablyhaving been imported with plant material from Europe,viz. roses, and was introduced into residential propertieswith plant material from infested nurseries. Also, it islikely that some of the earlier reports of this species inNorth America (Norton et al., 1984), Asia (Bhatt, 1967;Acharya et al., 1988; Mulawarman, 2008; Talezari et al.,2010) and Africa (Morkini et al., 2014) refer to other in-digenous species. Computer modelling using meteorolog-ical data associated with the recorded distribution of X.diversicaudatum in Europe, however, revealed that thisspecies could become established in North America, someregions of South America, Australia and Asia (Boag et al.,1997).

Mitochondrial DNA analysis has proved to be a power-ful tool for assessing intraspecific genetic structure pat-terns and phylogeography in plant-parasitic nematodes.Recently, Gutiérrez-Gutiérrez et al. (2011) used mito-chondrial DNA variation to study genetic structure andCOI haplotype distribution for X. pachtaicum and X. in-dex populations from grapevines across Spain and Italy,although this approach has never been explored for X. di-versicaudatum.

Several authors have molecularly characterised variouspopulations of X. diversicaudatum using ribosomal RNAgenes (De Giorgi et al., 1994; Wang et al., 2003; He etal., 2005) and mtDNA genes (Kumari et al., 2010; Kumari& Di Cesare, 2013). PCR-RFLP diagnostic profile (Lam-berti et al., 1999) and PCR with species-specific primerwere also developed for diagnostics of this species (Wanget al., 2003; Hübschen et al., 2004). However, these meth-ods have not been validated and tested with closely re-lated species. Thus, re-evaluation of previously published

molecular data and characterisation of the type populationof X. diversicaudatum are topical.

The main goals of our study were to: i) provide morpho-logical and molecular characterisation of X. diversicauda-tum from Saratov suburb; ii) develop PCR with specificprimer for rapid diagnostics of this species; and iii) study aphylogeographical structure of X. diversicaudatum acrossEurope using the coxI gene marker.

Materials and methods

NEMATODE POPULATIONS

The plesiotypes of X. diversicaudatum were collectedin the area of northern suburb of the Saratov city, leftbank of the Guselka river, right tributary of the Volgariver, 1.5 km away from its junction. Coordinates ofthe sampling location are: 51°35.845′N 45°08.413′E.Several other populations of this species were collectedin locations in the Moscow and Voronezh regions, theAdygeya, Stavropol and Krasnodar Territories of Russia,and in two locations in Bulgaria. Three populations of X.bakeri from two USA states, Washington and California,and an unidentified Xiphinema sp. from Azores, Portugal,were also included in this study (Table 1). The nematodeswere extracted from soil by a sieving and decantingmethod (Brown & Boag, 1988) followed by inspection inPetri dishes under a MBS-10 stereomicroscope.

MORPHOLOGICAL STUDY

Plesiotype specimens from the Saratov population werekilled by gentle heat, fixed with 4% TAF solution bywarming to 56°C and mounted in anhydrous glycerin formorphological examination. Specimens were measuredand photographed with a Carl Zeiss polarising microscopeAxio Imager Z2.

DNA EXTRACTION, PCR AND SEQUENCING

DNA was extracted from single nematode individuals.Protocols of DNA extraction with proteinase K, PCR andsequencing were described by Tanha Maafi et al. (2003).The primer sets for amplification of the nuclear ribosomalRNA (D2-D3 expansion segments of 28S rRNA, ITSrRNA) and mitochondrial (coxI mtDNA) genes are givenin Table 2. Two μl of the PCR product was run on a 1%TAE buffered agarose gel. PCR products were purifiedafter amplification with QIAquick (Qiagen) gel extractionkit and used for direct sequencing. The newly obtained

848 Nematology

Characterisation and phylogeography of Xiphinema diversicaudatumTa

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Vol. 16(7), 2014 849

V.N. Chizhov et al.

Table 2. Primer sets used in the present study.

Primer code Sequence (5′ → 3′) Amplified gene Amplicon length (bp) Reference

TW81 GTT TCC GTA GGT GAA CCT GC ITS1-rRNA approx. 990-1010 Tanha Maafi et al. (2003)Xip5.8S CAC CGC TTA GAA TGG AAT CGC This study

D2A ACA AGT ACC GTG AGG GAA AGT TG D2-D3 of 28S rRNA approx. 855 Subbotin et al. (2006)D3B TCG GAA GGA ACC AGC TAC TA

TW81 GTT TCC GTA GGT GAA CCT GC ITS1-rRNA approx. 864 This studyXip_diver_ITS GAA TAA ACA CCT TTC AAC GCT C

COIF GAT TTT TTG GKC ATC CWG ARG COI of mtDNA 435 Kumari et al. (2010)XIPHR2 GTA CAT AAT GAA AAT GTG CCA C

sequences were submitted to the GenBank database underaccession numbers: KF292276-KF292306 as indicated inTable 1 and in the phylogenetic trees.

PCR WITH SPECIES-SPECIFIC PRIMER

A species-specific primer for X. diversicaudatum (Ta-ble 2) was designed using the sequence alignment of ITS-rRNA gene. The PCR mixture was prepared as describedby Tanha Maafi et al. (2003). The PCR amplification pro-file consisted of 4 min at 94°C; 30 cycles of 1 min at94°C, 45 s at 57°C and 45 s at 72°C, followed by a fin-al step of 10 min at 72°C. Two μl of the PCR productswere run on a 1.4% TAE buffered agarose gel, stained andphotographed. Several X. diversicaudatum and X. bakerisamples were used to test the specificity of PCR with thenewly designed species-specific primer.

PHYLOGENETIC ANALYSIS

The newly obtained sequences of the ITS1 rRNA, theD2-D3 of 28S rRNA and coxI genes were aligned us-ing ClustalX 1.83 with default parameters with their cor-responding published gene sequences (He et al., 2005;Robbins et al., 2009; Kumari et al., 2010; Gutiérrez-Gutiérrez et al., 2013). Outgroup taxa for each datasetwere chosen according to the results of previous publisheddata. Sequence datasets were analysed with Bayesian in-ference (BI) using MrBayes 3.1.2 (Huelsenbeck & Ron-quist, 2001) and with Maximum Likelihood using PAUP∗4b10 (Swofford, 2003). The best fit model of DNA evolu-tion was obtained using the program JModeltest v. 0.1.1(Posada, 2008) with the Akaike Information Criterion. BIanalysis for each gene was initiated with a random start-ing tree and was run with four chains for 1.0 × 106 gen-erations. The Markov chains were sampled at intervals of

100 generations. Two runs were performed for each anal-ysis. After discarding burn-in samples and evaluating con-vergence, the remaining samples were retained for furtheranalysis. The topologies were used to generate a 50% ma-jority rule consensus tree. Posterior probabilities (PP) aregiven on appropriate clades. In ML analysis the estimationof the support for each node was obtained by bootstrapanalysis with 100 fast-step replicates.

All coxI sequences of X. diversicaudatum includedin the present study were obtained from populationscollected in four geographical regions: Central Europe(Czech Republic, Slovakia and Austria), south-easternEurope (Bulgaria), central and south-eastern Europeanparts of Russia (Moscow, Voronezh and Saratov regions)and the southern European part of Russia (Krasnodar andStavropol Territories, Adygeya). The coxI sequence align-ment was used to construct phylogenetic network estima-tion using statistical parsimony with the TSC software(Clement et al., 2000).

Results

Xiphinema diversicaudatum (Micoletzky, 1927)Thorne, 1939

Saratov population, Russia(Fig. 1)

MEASUREMENTS

See Table 3.

DESCRIPTION

Female

Body C-shaped on heat fixation, with sharper curvein posterior part. Cuticle thickness at middle of body

850 Nematology

Characterisation and phylogeography of Xiphinema diversicaudatum

Fig. 1. Xiphinema diversicaudatum. Saratov population. A: Anterior region of female; B: Pharynx base of female; C: Vulval region; D:Z-differentiation; E: Egg in uterus; F, G: Tail region of females; I, J: Tail region of males; H: Tail region of female J4; K: Tail region ofmale J4; L: Vulval region with part of uterus.

Vol. 16(7), 2014 851

V.N. Chizhov et al.

Table 3. Morphometrics of the Saratov population of Xiphinema diversicaudatum. All measurements are in μm and in the form: mean ±s.d. (range).

Character Female Male

n 25 23L 4291 ± 317 (3711-4708) 4188 ± 277 (3729-4696)a 75.6 ± 5.8 (63.9-86.8) 85.8 ± 5.9 (73.9-95.2)b 9.5 ± 0.9 (7.3-10.7) 8.7 ± 0.9 (6.8-10.2)c 85.4 ± 10.7 (69.4-108.3) 87.4 ± 11.6 (58.4-112.2)c′ 1.1 ± 0.1 (0.9-1.3) 1.1 ± 0.2 (0.8-1.6)V 42 ± 2.1 (39-50) –Spicules – 63 ± 3.2 (58-68)Odontostyle 126 ± 4.5 (114-132) 128 ± 4.4 (117-135)Odontophore 76 ± 3.7 (67-82) 77 ± 3.0 (70-82)Total spear length 203 ± 6.1 (190-211) 205 ± 5.6 (190-214)Lip diam. 14 ± 0.8 (12-15) 13.5 ± 0.9 (12-15)Anterior end to guide ring 110 ± 14.9 (91-135) 111 ± 14.8 (70-132)Anterior end to nerve ring 239 ± 21.0 (201-277) 246 ± 26.3 (183-283)Pharynx length 455 ± 40.8 (389-525) 485 ± 45.9 (423-578)Pharyngeal bulb diam. 25 (22-30) 25 (20-30)Pharyngeal bulb length 97 (85-108) 106 (99-114)Body diam. 57 ± 5.4 (47-65) 49 ± 4.1 (41-60)Tail length 51 ± 5.3 (41-65) 49 ± 6.4 (35-68)Diam. at anus level 46 ± 3.0 (41-50) 43 ± 2.2 (38-47)Anterior gonad 591 ± 79.5 (395-775) –Posterior gonad 675 ± 77.8 (484-856) –Anterior end to cloaca – 1843 ± 195 (1357-2148)Rectum length 37 ± 4.4 (30-47) –

2-3 μm. Lip region slightly convex, 3-4 μm high, some-what less than body diam. at lip base, usually not offset,seldom set off from rest of body by a weak depression.Amphidial fovea stirrup-shaped. Odontophore tripartite,with mace-like flanges, 11-13 μm wide. Two nerve ringspresent, wider one located posterior to odontophore base,oblique, pointing ventrally to second one which is thin-ner and located at some distance posterior. Pharyngealbulb four times basal diam. long. Ventral and lateral hy-podermal gland pores located along body length in thepharyngeal area, their number varying from 4-5 and 9-11, respectively. Dorsal pores poorly detectable. Nuclei ofdorsal and ventrosublateral glands situated at 7.7-12.5%and 61.1-63.6% (n = 5) of distance from anterior endof bulb, respectively. Glandularium 115-135 μm long.Gonoducts paired, with reflexed ovaries, posterior genitalbranch usually slightly exceeding anterior one in length.Nuclei of oogonia in germinative part of gonad 14-17 μm,nucleoli 5 μm in diam. Uterus differentiated, tripartite, Z-differentiation as 8-14, usually compactly arranged, semi-transparent rose-like bodies 4-6 μm in diam., pars distalis

vaginae 23-26 μm long, pars proximalis vaginae 7-8 μmlong. Distal part of vagina sclerotised, proximal part non-sclerotised. Prerectum divided from intestine by a clearboundary of taller cells. Anus and rectum prominent. Tailconvex-conoid, dorsally rounded with a ventral peg, bear-ing three pairs of caudal pores. Occasionally, peg may betotally absent, or appearing as a small projection. Individ-uals with a deformed peg often observed.

Male

Smaller than female. Under heat relaxation, body C-shaped, more strongly curved in posterior part. Lip regionand pharynx similar to those in female. Nuclei of dorsaland ventrosublateral glands situated at 7.0-9.8% and 60.8-66.1% (n = 5) of distance from anterior end of bulb,respectively. Glandularium 115-142 μm long. Spiculesdorsally convex, 7-10 μm wide at distal part, 14-16 μmwide at middle part. Ventromedian supplements 4-5 ina row, located at 75-100 μm apart from adcloacal pair(each 5-6 μm in diam., located 20-25 μm anterior tocloacal aperture). Tail conoid, dorsally convex, ventrally

852 Nematology

Characterisation and phylogeography of Xiphinema diversicaudatum

straight, ventral peg of variable length, seldom absent.Caudal pores present as 3-4 pairs.

Egg

Uterine eggs (n = 8, mostly two per uterus) measuring193 ± 12 (178-213) × 43 ± 2 (40-48) μm.

VOUCHER MATERIAL

Slides were deposited at the Type Specimens Collectionof the Helminthological Museum of the Center for Parasi-tology of IPEE RAS (Moscow) under the numbers 18/40-18/51, and at the State Collection of Phytopathogenic Mi-croorganisms of All-Russian Institute for Phytopathologyof the Russian Agricultural Academy (Bolshye Vyazemy,Moscow, Russia) under the numbers C1307T0-C1307T8.

MOLECULAR CHARACTERISATION, PHYLOGENY AND

PHYLOGEOGRAPHY

D2-D3 of 28S rRNA gene

The alignment contained 38 sequences of Xiphinemaspecies including two sequences of X. index selected asoutgroup taxon and was 777 bp in length. Twelve se-quences of X. diversicaudatum were included in the anal-ysis. Intraspecific variation for X. diversicaudatum was 0-0.8% (0-6 bp), for X. bakeri 0-0.9% (0-7 bp), for X. baet-ica 0.1-0.8% (1-6 bp) and for X. coxi europaeum 0-0.9%(0-7 bp). Phylogenetic relationships of X. diversicauda-tum with related species as inferred from BI is presentedin Figure 2. Xiphinema diversicaudatum clustered withX. bakeri and X. chengi with high support value (PP =100). Xiphinema species under the number AY601624,previously identified as X. diversicaudatum, did not clus-ter with the Saratov and other X. diversicaudatum popula-tions and was related to X. coxi europaeum. On this basis,we conclude that species with sequence under the numberAY601624 was misidentified and belonged to X. coxi eu-ropaeum as proposed by Gutiérrez-Gutiérrez et al. (2013).

ITS1 rRNA gene

The alignment contained 21 sequences of Xiphinemaspecies including two X. index used as outgroup taxaand was 1367 bp in length. Intraspecific variation for X.diversicaudatum was 0.4-1.9% (4-18 bp), for X. bakeri 0-2.0% (0-19 bp), for X. bernardi 0.1-0.4% (1-4 bp) andfor X. coxi europaeum 0.0-3.2% (0-29 bp). Phylogeneticrelationships of X. diversicaudatum with related speciesas inferred from BI is presented in Figure 3. Xiphinemadiversicaudatum clustered with X. bakeri and X. chengi

with high support value (PP = 100). Based on the resultsof sequencing and phylogenetic analysis we consider thatsequence under the number AY430183 was misidentifiedand belonged to X. coxi europaeum as proposed byGutiérrez-Gutiérrez et al. (2013).

coxI of mtDNA gene

The alignment contained 29 sequences of X. diversi-caudatum and two sequences of X. bakeri used as out-group taxa and was 363 bp in length. Sequence chro-matograms of X. bakeri obtained from individual nema-todes showed multiply picks in several (three G/A and twoT/C) positions indicating that heteroplasmy or the pres-ence of a mixture of more than one type of mitochondrialDNA genome might occur in this species.

Intraspecific sequence variation for X. diversicaudatumwas 0-3.1% (0-11 bp). Twenty-seven positions were vari-able. Fifteen coxI haplotypes were distinguished withinX. diversicaudatum samples from four main sampling ge-ographical areas (Table 4). Five haplotypes (with maxi-mal 3.1% sequence differences between these haplotypes)were found from Central Europe (Czech Republic, Slo-vakia, Austria); one (0%) haplotype from south-easternEurope (Bulgaria), five haplotypes (0.83%) from cen-tral and south-eastern European Russia, and four haplo-types (0.55%) from southern European Russia. Phyloge-netic relationships of X. diversicaudatum haplotypes asinferred from BI are presented in Figure 4A. Sequencesof two Central European haplotypes occupied basal po-sitions in the tree and the other three haplotypes fromthis region had sister relationships with either the south-eastern Europe haplotype or the haplotypes from cen-tral and south-east European Russia. Statistical parsimonynetwork showing the relationships between these haplo-types is given in Figure 4B. Distribution of coxI haplo-types across Europe is given in Figure 5.

PCR WITH SPECIES-SPECIFIC PRIMER

A fragment of the ITS1 rRNA gene sequence alignmentfor Xiphinema species with marked position for the D24primer, which was designed by Wang et al. (2003) andconsidered as the X. diversicaudatum specific primer, isgiven in Figure 6A. In silico analysis shows that the D24primer sequence matched well with the corresponding se-quence for X. bakeri. Thus, false positive results couldbe obtained if this primer is used in PCR with a samplecontaining X. bakeri. The position of the newly designedspecies-specific primer Xip_diver_ITS for X. diversicau-datum is presented in Figure 6B. Results of PCR with

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V.N. Chizhov et al.

Fig. 2. Phylogenetic relationships between Xiphinema diversicaudatum and related species: Maximum likelihood tree as inferred fromthe D2-D3 of 28S rRNA gene sequence alignment under the GTR + I + G model. Newly obtained sequences are indicated by boldletters. Originally identified as: ∗ – X. diversicaudatum, but considered as X. coxi europaeum by Gutiérrez-Gutiérrez et al. (2013).

854 Nematology

Characterisation and phylogeography of Xiphinema diversicaudatum

Fig. 3. Phylogenetic relationships between Xiphinema diversicaudatum and related species: Bayesian majority rule consensus tree fromtwo runs as inferred from the ITS1 rRNA gene sequence alignment under the GTR + I + G model. Newly obtained sequences areindicated by bold letters. Originally identified as: ∗ – X. diversicaudatum, but considered as X. coxi europaeum by Gutiérrez-Gutiérrezet al. (2013).

this new species-specific primer are given in Figure 7.The combination of the universal primer TW81 with thespecies-specific primer yielded a single PCR product ca864 bp long for all studied X. diversicaudatum. No ampli-cons were found in a sample with X. bakeri DNA or in acontrol without DNA.

Discussion

The measurements and morphology of the Saratovpopulation of X. diversicaudatum obtained in the presentstudy are within the ranges reported for X. diversi-

caudatum populations from Europe, South Africa, USAand New Zealand (Thorne, 1939; Goodey et al., 1960;Sturhan, 1963; Pitcher et al., 1974; Brown & Topham,1984, 1985; Heyns & Coomans, 1984; Liskova et al.,1993; Taylor et al., 1994; Roca & Bravo, 1997; Sturhanet al., 1997; Lamberti et al., 1999; Kumari, 2006). Themorphometric characters of the Saratov female specimensalso conform well to the data published by Micoletzky(1923, 1927), except for odontostyle length, which wasslightly shorter at 126 (114-132) vs 133 μm. The spiculelength of specimens from the Saratov population wasshorter comparing with that for the lectotype at 63 (58-68) vs 75 μm (Pitcher et al., 1974). Although X. diver-

Vol. 16(7), 2014 855

V.N. Chizhov et al.

Tabl

e4.

Var

iabl

epo

sitio

nsin

the

coxI

regi

onde

finin

g15

hapl

otyp

esin

Xip

hine

ma

dive

rsic

auda

tum

.H

aplo

type

Num

ber

ofsi

tes

Var

iabl

epo

sitio

ns(b

p)

1118

2038

4756

7710

111

013

414

014

416

417

617

918

819

720

923

330

230

531

432

633

534

134

735

01

2A

AG

CC

GT

AC

AT

CC

AA

AA

CC

AT

AG

TC

TC

23

CA

AC

TG

TA

CG

TC

CC

GA

AG

CA

TA

GT

CC

C3

1C

CG

CC

GT

GC

GT

TC

AG

AA

GC

AT

AG

TT

TC

41

CA

GC

CG

TG

CG

TT

CA

GG

AG

CA

TG

GT

TT

C5

1C

AG

CC

GT

GT

GT

TC

AG

AA

GC

AT

AG

TT

TC

62

CA

GC

CG

TG

CG

TT

CA

GA

AG

CA

TA

GT

TT

C7

3A

AG

CC

GT

GC

GT

TC

AG

AA

GC

GT

AG

TT

TC

81

CA

GC

CG

TA

CG

TT

TA

GA

AG

CA

TA

AT

TT

C9

2C

AG

TC

GT

GC

GT

CT

AG

AA

GC

AC

AA

TT

TC

101

CA

GC

CG

CG

CG

CC

CA

GA

AG

TG

TA

GT

TT

C11

1C

AG

CC

GC

GC

GC

CC

AG

AA

GT

GT

AG

TT

TT

128

CA

GC

CA

CG

CG

TC

CA

GA

AG

TG

TA

GT

TT

C13

1C

AG

CC

AC

GC

GT

CC

AG

AC

GT

GT

AG

TT

TC

141

CA

GC

CA

CG

CG

TC

CA

GA

AG

TG

TA

GG

CT

C15

1C

AG

CC

AC

GC

GT

CC

AG

AA

GT

GT

AG

GT

TC

The

num

ber

ofsi

tes

atw

hich

apa

rtic

ular

hapl

otyp

eoc

curs

isal

soin

dica

ted.

856 Nematology

Characterisation and phylogeography of Xiphinema diversicaudatum

Fig. 4. Phylogenetic relationships between coxI haplotypes of Xiphinema diversicaudatum and X. bakeri. A: Bayesian majority ruleconsensus tree from two runs as inferred from the coxI gene sequence alignment under the GTR + I + G model. Newly obtainedsequences are indicated by bold letters; B: Statistical parsimony network showing the phylogenetic relationships between haplotypesof X. diversicaudatum. Small black cycles represent missing haplotypes. Pie chart sizes are proportional to the number of samples witha particular haplotype.

sicaudatum showed high intraspecific variations in manycharacters, the Saratov population seems to be morpho-metrically similar to three populations originating fromthe Balkan Peninsula, e.g., female odontostyle length 126(114-132) μm from Saratov vs 124.9-131.1 (114-138) μmfrom Vojvodina, Montenegro and Serbia, male spiculelength 63 (58-68) μm from Saratov vs 65.9 (63-67) μmfrom Vojvodina (Barsi & Lamberti, 2000) and 51-65 μmfrom Bulgaria (Pinus nigra L.) (Peneva & Choleva, 1992).However, as Brown (1985) revealed, such morphometricvariability may be natural variation resulting from X. di-versicaudatum populations having adapted to survive inbiotopes with different environmental conditions.

Brown & Taylor (1987), Brown et al. (1990), Roca &Bravo (1997) and other authors reported X. diversicau-datum from most European countries. This species has

been considered as one of widely geographical distributedXiphinema species in Europe with findings from Nor-way to southern Spain and Italy. However, further studiesshowed that several identification of this species in south-ern Mediterranean regions and Portugal were incorrect orrequired confirmation (Roca & Bravo, 1997; Gutiérrez-Gutiérrez et al., 2011). Xiphinema diversicaudatum is atemperate species and some reports (Brown & Taylor,1987; Morkini et al., 2014) of this species in southernMediterranean regions likely belong to other, morpholog-ically similar, species. The improved PCR with species-specific primer X. diversicaudatum developed in our studywill help to diagnose this species.

The results of our phylogeographical analysis allow usto show some patterns of distribution and dispersal ofX. diversicaudatum in Europe and hypothesise a centre

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Fig. 5. Distribution of the studied Xiphinema diversicaudatum coxI haplotypes across Europe. This figure is published in colour in theonline edition of this journal, which can be accessed via http://booksandjournals.brillonline.com/content/journals/15685411.

of origin for this species. The phylogeographical patternof a species is a result of the combined influence ofcontemporary and historical processes. Several studieshave documented that the present distribution patternsof temperate species of European animals and plantsare largely determined by the extensive climatic changesduring the Pleistocene. The climatic cycles, with glacialand interglacial periods, impact on the distribution andevolution of species. During the Last Glacial Maximum(LGM) (18 000-21 000 ya), large parts of north Europewere covered by ice sheets and the major mountainranges of Europe had extensive ice caps. Between themain ice sheet and southern mountain blocks was a plainof permafrost, tundra and cold steppe, which extended

eastwards across Russia to the Urals (Hewitt, 1999).The majority of species persisted through the LGM inclimatically suitable refuge areas. Almost all Europeanspecies of Mediterranean origin had their refuges in atleast one of the three major European peninsulas ofthe Mediterranean area (Iberia, Italy and the Balkans)because of their species richness and considerable levelof endemism (Taberlet et al., 1998; Hewitt, 1999, 2000;Schmitt, 2007). It is interesting to note that the highestspecies richness for European Xiphinema species was alsoreported from the Iberian, Italian and Balkan Peninsulas(Topham & Alphey, 1985; Brown & Taylor, 1987).

The analysis of phylogenetic relationships of X. diver-sicaudatum with other species using the D2-D3 of 28S

858 Nematology

Characterisation and phylogeography of Xiphinema diversicaudatum

Fig. 6. Fragments of alignment of the ITS1-rRNA gene sequences for Xiphinema. A: Underlined sequence indicates the positionsfor the D24 primer (5′-GAG ATA TAA AGC GAA AAC CGC GAG-3′) designed by Wang et al. (2003) for diagnostics of X.diversicaudatum. Grey areas matching this primer with the X. bakeri ITS sequence; B: Underlined sequence indicates the positionsfor the new Xip_diver_ITS primer (5′-GAA TAA ACA CCT TTC AAC GCT C-3′).

rRNA and gene ITS1 rRNA, as presented by Gutiérrez-Gutiérrez et al. (2013) and our study, revealed that theX. diversicaudatum clade, together with X. bakeri (NorthAmerica) and X. chengi (Asia), related to Xiphinemaspecies (X. abrantinum, X. baetica and others) from theMediterranean region and likely originated from a com-mon ancestor inhabiting this region. We hypothesise thatX. diversicaudatum most likely survived in Central Eu-rope during the last glacial period. Based on palaeoeco-logical evidence, it has been recently concluded that therewas no forestless landscape in Central Europe during thelast glacial as had previously been thought. Some treesand other plant and animal species survived in micro-environmentally favourable areas adjoining the watershedof the high mountain systems in Central Europe, like theAlps and Carpathian mountains (Willis & Van Andel,2004; Schmitt, 2007).

Phylogeographical signatures of refuge areas includehigh level of genetic diversity and the presence of endemichaplotypes. Our analysis showed a high level of mtDNAvariation and unique genotypes in the Czech Republic.According to this scenario, the higher genetic diversityof the Czech Republic populations of X. diversicaudatumis explained by longer persistence in the refuge areasin Central Europe, probably, in the Carpathians or theAlps, whereas the homogeneity of other populations isprobably a result of the loss of genetic variation resultingfrom rapid expansion and colonisation to Eastern Europeand other territories. However, we are aware that only alimited dataset with mtDNA sequences have been used inthe present study and additional sampling, especially innorthern Italy and northern Spain, is needed to make amore comprehensive analysis and either to confirm this orto test alternative hypotheses.

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Fig. 7. Gel with specific amplicons obtained in by PCR withthe X. diversicaudatum species-specific primer. Lanes: M =100 bp DNA ladder (Promega); 1-3 = X. diversicaudatum; 1 =CD1283; 2 = CD1330; 3 = CD1314; 4 = X. bakeri, CD947;5 = control without DNA.

Acknowledgements

The work was supported by the Russian Foundation ofBasic Research, project number 14-04-00953 and ANI-DIV project of the Bulgarian Academy of Sciences. Theauthors especially thank Mr R.V. Khusainov for provid-ing numerous X. diversicaudatum samples for molecularstudy and identification of plant hosts for the project.

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