study of thai tarantulas: a molecular phylogenetic...

Study of Thai Tarantulas: a Molecular Phylogenetic Approach

NATAPOT WARRITDepartment of Biology

Chulalongkorn University

• Globally included approximately 40,000+ described species (Platnick, 2008)

• Estimated number 60,000-170,000 species(Coddington and Levi, 1991)

pidersS

pidersS

Spiders are the most diverse and abundant invertebrate predators in terrestrial ecosystems (Wise, 1993)

SPIDER CLASSIFICATION

0.3%

• Segmented spider

• 1 family

• 8 genera, 96 species

Mesothelae

• Mygalomorph spiders

and Tarantulas

• 16 families

• 335 genera, 2,600 species

Mygalomorphae

• True spider

• 95 families

• 37,000 species

Araneomorphae

93.2%

6.5%

Mygalomorphae

Mecicobothriidae

Microstigmatidae

Hexathelidae

Dipluridae

Nemesiidae

Paratropididae

Barychelidae

Atypidae

Antrodiaetidae

Cyrtaucheniidae

Idiopidae

Ctenizidae

Migidae

Actinopodidae

Euctenizidae

Theraphosidae Tarantulas> 900 species

Chilobrachys Karsch, 1891

Orphnaecus Simon, 1892

Phlogiellus Pocock, 1897

Poecilotheria Simon, 1885

Coremiocnemis Simon, 1892

Lyrognathus Pocock, 1895

Psednocnemis West, 2012

Selenocosmia Ausserer, 1871

Selenotholus Hogg, 1902

Selenotypus Pocock, 1895

Family

Theraphosidae

Acanthopelminae

Aviculariinae

Ornithoctoninae

Eumenophorinae

Harpactirinae

Ischnocolinae

Poecilotheriinae

Spelopelminae

Stromatopelminae

Theraphosinae

Selenogyrinae

Selenocosmiinae

Thrigmopoeinae

10 Subfamilies, 931 species

World Spider Catalog (2017)

Citharognathus Pocock, 1895

Omothymus Thorell, 1891

Cyriopagopus Simon, 1887

Lampropelma Simon, 1892

Ornithoctonus Pocock, 1892

Phormingochilus Pocock, 1895

Cyriopagopus

Simon, 1887

• The range of this genus includes :

Myanmar, southeastern China,

Cambodia, Thailand, Vietnam,

Malaysia, Singapore, and Borneo

• Cyriopagopus is a popular genus of Southeast Asia tarantula

in exotic pet trade.

Cyriopagopus in Thailand

Thai Zebra

Cobalt Blue

Vietnamese Earth Tiger

Thai BlackC. minax (Thorell, 1897)

C. albostriatus (Simon, 1886)

C. lividus (Smith, 1996)

C. longipes (Von Wirth & Striffler, 2005)

C. vonwirthi (Schmidt, 2005) Silver back

How many species?

114 specimens

MolecularMorphology and

geographic distribution

Methods

External

morphology

stridulating organs, plumose setae on outer face

of chelicerae clypeus, fringe of hair, carapace

Geographic

distribution

specimens localities

distribution range

type localities

Sex organs

Female receptacle

Pedipalp

External morphology

clypeus, fringe of hair, carapace

stridulating organs, plumose setae on outer face of chelicerae

All Character depends by age and

cannot delimit Cyriopagopus in

species-level.

Sex organs

not consistent

high homogeneity

C.albostriatus

albostriatus

C. minax C. lividus C. longipes

Morphology and

geographic distribution Molecular

Molecular Protocol

• 25 mg of the tissue

• extracted using Dneasy Tissue Kit (Qiagen)

• DNA were quantified

- spectrophotometer

- agarose gel electrophoresis

Mitochondrial gene cytochrome

oxidase I (COI) was amplified

PCR Sequencing

Analysed with AB 3130xl

Genetic Analyzer

Idaho State University, USA

PCR products were purified using ExoSAP-IT®

All sequences were initially aligned by eyes

Checked peak of nucleotide positions and

protein translation

MEGA7 (Kumar et al. 2016)

DNA alignment

Sequences were aligned again with

CLUSTAL X v.2.1

Molecular

analyses

Phylogenetic analysis

Species

delimitation

DNA barcoding gap

Molecular analyses

sp. Asp. Bsp. Csp. D

TCS

parsimony

network

PopART

Haplotype

network

(http://popart.otago.ac.nz)

Phylogenetic analysis

Bayesian inference (BI)Maximum Likelihood (ML)

• Markov Chain Monte Carlo

(MCMC) run for 5,000,000

• saved to file every 100

generations

• 25% discarded as burn-in

MrBayes ver. 3.2.6 RAxML-HPC BlackBox

(ver 8.2.9)

(Miller et al. 2010)

JModelTest v.2.1

GTR+G+I

Phylogenetic analysis

Species

delimitation

DNA barcoding gap

Molecular analyses

sp. Asp. Bsp. Csp. D

TCS

parsimony

network

PopART

Haplotype

network

(http://popart.otago.ac.nz)

http://mptp.h-its.org/#/tree

Multi-rate Poisson Tree Processes

http://species.h-its.org/ptp

Bayesian variant Poisson Tree Processes

http://www.r-project.org/)

Generalized Mixed Yule Coalescent

http://wwwabi.snv.jussieu.fr/public/abgd

Automatic Barcode Gap Discovery

sp. Asp. Bsp. Csp. D

Species

delimitation

ABGD

GMYC

bPTPMPTP

Ultrametric trees

BEAST v. 2.4.5

(CIPRES Science Gateway v3.3)

• relaxed clock log normal

• 1.0 fixed clock rate

• 50,000,000 generations

• sampling 2,500 generations

• 25% burn-in

(ESS) exceeded 200

TRACER 1.6

Distance method

Barcode Gap

• Pmin = 0.001

• Pmax = 0.100

• Steps = 10

• X = 1.0

JC69 and K80

Phylogenetic analysis

Species

delimitation

DNA barcoding gap

Molecular analyses

sp. Asp. Bsp. Csp. D

TCS

parsimony

network

PopART

Haplotype

network

(http://popart.otago.ac.nz)

uncorrected p-

distances

(mean = 3.31)

K-2 parameter

(mean = 3.48)

C minax clade A 4.33 4.54

C minax clade B 1.43 1.45

C minax clade C 2.50 2.65

C. lividus 3.11 3.22

C. longipes 4.26 4.49

C. schmidti 7.99 8.52

C. albostriatus +

vonwirthi5.10 5.41

O. aureotibialis 3.98 4.17

O. costalis 0.00 0.00

“Citharognathus sp.”0.37 0.37

Omothymus sp. n/c n/c

DNA barcoding gap

Intraspecific variation (within species)

Phylogenetic analysis

Species

delimitation

DNA barcoding gap

Molecular analyses

sp. Asp. Bsp. Csp. D

TCS

parsimony

network

PopART

Haplotype

network

(http://popart.otago.ac.nz)

Haplotype

Network

Results

Phylogenetic analysis

C. longipes

C. lividus

“C. minax”

C. albostriatusC. vonworthi

(junior synonym)

C. minax s. str.

Chomphuphung et al. (in prep.)

Phylogenetic analysis

Citharognathus sp.

New species

Omothymus sp.

New species

Acknowledgements

Center of Excellence in Biodiversity

Scholarship Foundation

Chulalongkorn University

VaratSivayyapram

NarinChomphuphung

Chawakorn Kunsete

Deborah Smith

CU BEE&SPIDERRESEARCH UNIT

Thank You !

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