Molecular Phylogeography Of The Genus Gloydius

Phylogeography is a relatively young field that investigates the historical andcontemporary processes that affect the geographic distribution of genealogicallineages. Phylogeography occupies a place between micro evolutionary and macroevolutionary elds. As a branch of biogeography, system geography has not limited toexplain the current population distribution, but further explores the cause distribution,it illustrates the evolution of regional groups, analysis in time and space, andreconstruction of the development and change of the history of biology divisions.With the method of systematic geography development and molecular biologyexperiment technology penetration, appeared a new interdiscipline subject-Molecular Phylogeography. Molecular Phylogeography mainly research thepopulation genetic structure and genetic lineage by the molecular biology technologyin molecular level, discusses the form mechanism of geographical pattern. inferevolution events of existing genetic lineage spatial distribution patterns form this,such as diffusion, gene flow, fragment in population and interpopulation, and based onthe existing geology and climate change material to expound it with geological historyreason, at the same time, verify the climate and geological events by estimatingmolecular clock.Gloydius is established based on Gloydius halys as modle by Hoge&Romano-Hoge (1981), and mainly distributed in east Asia, west Asia and north Asia.This genus currently include twelve species. Gloydius is widely distributed in theterritory of China except Guangdong, Hong Kong, Taiwan and Hainan provinces.Their habitats are complicated, comeprise plain, shallow, low mountain, island, thedesert and semi-desert, even mountain plateau grassland. They are the ideal mode inmolecular geography research.Previous research has focused on the taxonomies, ecology and evolution of thistaxon.Guo et al.(1998) reviewed the systematics of this genus. On the basis of skullcomparisons, Guo et al.(1999) discussed the status and validity of its several species. A phylogenetic hypothesis based on morphological characteristics suggested thatGloydius could be divided into three groups. However, other authors have proposeddifferent taxonomic arrangements. Despite of these researches, the taxonomicarrangement of the species within Gloydius varies greatly depending upon authors.Here we explored the systematics and interspecific relationships of Gloydius,discussesed the molecular system geographical hypothesis, using both mtDNA andnDNA loci, focusing on the species that occur in China. Total genomic DNA wasextracted from liver, muscle or blood of19samples representing eight species usingthe phenol/chloroform method. The entire gene sequences for cytochrome b (Cytb),partial gene sequences of NADH dehydrogenase subunit4(ND4) and the nucleargene cmos were amplified via polymerase chain reaction (PCR) using the followingprimers: L14910/H16064, ND4/Leu and S77/S78, respectively. Protocol foramplification followed Burbrink et al.(2000), Arevalo et al.(1994) and Lawson et al.(2005). Double-stranded product was sequenced using an ABI3730Genetic Analyzer(Applied Biosystems) following manufacturer’s protocols. All mtDNA sequenceswere aligned using ClusterW in Mega4.0with default settings and checked by eye toensure pseudogenes were not amplified. Generated sequence data were analyzed withfour additional samples representing G. blomhoffii, G. brevicaudius, G. shedaoensisand G. halys, whose sequences are from Genbank. Three taxa (Protobothropsmangshanensis, Bothriechis schlegelii, Viridovipera stejnegeri) were chosen asoutgroups on the basis of previous work. Phylogenetic analyses were performed usingthree different methods: Bayesian inference (BI), maximum parsimony (MP) andmaximum likelihood (ML). The program BEAST v1.5.1(Drummond and Rambaut,2007) was used to estimate the date of origin of Gloydius under the assumption of arelaxed molecular clock using BI (Drummond et al.,2006). The conditions of thisanalysis were identical to those of the BI phylogenetic inference above. For thisanalysis, we download six species as fossil calibration point and one as outgroup fromGenBank.The main research results are as follows:1, In the BI tree, all representatives of Gloydius were shown to be monophyleticwith strong support (100%; Figure1). Within Gloydius, G. brevicaudus, G. ussuriensisand G. blomhoffi formed a highly supported monophyletic group, of which G.ussuriensis showed a sister relationship with G. blomhoffi, then both sisterly related toG. brevicaudus. G. inrtermedius, G. halys, G. saxatilis, G. shedaoensis, G. strauchi andG. liupanensis formed another monophyletic group with strong support (99%). Withinthis clade, G. inrtermedius, G. halys, G. saxatilis, G. shedaoensis formed a subcladewith high support value (100%), and G. saxatilis showed a sister relationship with G.shedaoensis. Interestingly, the two samples representing G. intermedius are notrecovered as sister taxa. The MP and ML trees topologies are generally consistentwith the BI tree. The main differences are indicated by the position of G. qinlingensis, G. strauchi and G. liupanensis.2, Phylogeny reconstruction using three different methods generally producedcongruent results, in which all the representatives of Gloydius analyzed formed a wellsupport group. The interspecific relationships presented here are consistent with thoseof previous results based on morphological comparisons to some extent (Guo et al.,2002). The monophyly including species G. brevicaudus, G. ussuriensis and G.blomhoffi generally exhibit21rows of dorsal scales at middle of body and have4palatine teeth, while those including G. inrtermedius, G. halys, G. saxatilis and G.shedaoensis possess23rows of dorsal scales on middle body and have3palatineteeth. However, another species group defined previously, which includes the threemountane species (G. strauchi, G. qinlingensis, G. liupanensis), was not recovered inour analysis。Considering both the morphological and molecular data, we suggestedthat both G. qinlingensis and G. liupanensis are valid species. However, the unsolvedsystematic relationships need to be further examined with both more intensivespecimen sampling and the application of multiple unlinked loci.3, Main branches’ divergence times by Beast software analysis as follow: thedivergent date estimation indicated that Gloydius originated in15Ma and thatintra-genus differentiation began in9.89Ma. The differentiation time of G. ussuriensisand G. blomhoffi is about3.3895in one million (HPB,1.0516to7.3285), Thedifferentiation time of both and G. brevicaudusis about7.0874in one million (HBP,3.4216to12.6626); Gloydius saxatilis and Gloydius shedaoensis’ is about0.8589inone million (HBP,0.26to1.9854), Both and G. halys’ is about2.1366in one million(HBP,0.8678to4.29). Above and G. inrtermedius’ is about2.7672in one million(HBP,1,2225to5.3621). G. strauchi, G. qinlingensis, G. liupanensis’s is form5.774one million to7.7796one million.4, The divergent date estimation indicated that Gloydius originated in themid-Miocene (about15Ma) and that intra-genus differentiation began in the lateMiocene (about9.89Ma), which is consistent with previous analyses (Guo et al.,2002). The Qinghai-Xizang Plateau began its slow uplift during the Miocene (about25–10Ma) with a rapid uplift at about3.4Ma in the mid-Pliocene (Sun,1997). It isnotable that the divergence of the genus Gloydius occurred during the uplifting of theQinghai-Xizang Plateau. The uplifting of the Plateau, which would produce physicalbarriers for gene exchanges, coupling with the climate fluctuation and glacial cycles,which would lead to population extraction and expand, could be responsible for thespeciation of each group.