study of thai tarantulas: a molecular phylogenetic...
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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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