Phylogenetic Relationships And Interspecific Hybridization Studies Of The Chinese Grass Lizards

Grass lizards of the oviparous genus Takydromus are a group of lacertids that have slender bodies and long tails, with tail length accounting for 200-400% of snout-vent length. Takydromus species are widespread in East Asia across the Oriental and Palearctic regions, southwards to Indochina, India and Malaysia. Of the 19 Takydromus species currently recognized, 13 can be found in China, with eight endemic to the country (three in mainland China and five in Taiwan). The systematic status and phylogeny of the genus Takydromus is still controversial until now. Earlier taxonomists split the genus into two subgenera, Takydromus and Platyplacopus.Recent studies based on molecular data all support the idea that Takydromus species form a monophyletic clade. However, these recent studies show a less clear-cut picture of the phylogenetic relationships among Takydromus species, largely because the limited number of mitochondrial genes used results in low bootstrap support values and very short internode branch lengths. Given that the characteristic of mitochondrial genome gives invaluable insights into the evolution of Takydromus, I sequenced one complete (T. wolteri) and 17 nearly complete mitochondrial genomes of other 11 Takydromus species to investigate the phylogenetic relationships among Takydromus species.The complete mitochondrial genome of T. wolteri is 18,236 bp, the remain 17 nearly complete mitochondrial genomes vary from 15,374 bp (T. stejnegeri) to 18,201 bp (T. formosanus). All 18 genomes contain two ribosomal RNAs, 22 transfer RNAs and 13 protein coding genes and a long noncoding region (D-loop). The arrangement,transcription and direction of eighteen genomes are the same as the sequence reported for T. tachydromoides.All genes in 18 mitochondrial genomes have similar A+T content from tRNA-Phe to Cyt b gene. Genes encoded by the L strand show positive AT-skew values and negative GC-skew values.Thirteen protein-coding genes (PCGs) use the standard RTG as the start code.Eleven protein-coding genes have the common stop codon TAA or the incomplete stop codons T, with an exception of ND6 using AGR as the stop codon. Among Takydromus species, the number of nucleotides in protein-coding genes differs between and within species. Nine nucleotides in COI gene and three nucleotides in ND1 gene differ between two samples of T. sexlineatus. Nucleotide differences in ND1 gene vary from 3 to 6 bp among samples of T. septentrionalis.GC-skew seems to be associated with gene direction. Such an association is not observed for AT-skew. Depending on species or individual, the AT-skew value is positive or negative for each of PCGs or the same PCGs.All 13 protein-coding mitochondrial genes of Takydromus species suffer negative selection, thus providing evidence that purifying selection plays a key role in the evolution of mtDNA. Takydromus hsuehshanesis is restricted to mountain regions higher than 2000 m, where no other Takydromus lizards can be found. Interestingly,however, there is no significant difference in selection between T. hsuehshanesis and other Takydromus species.The repetitive unit in the D-loop is longer in Takydromus species than in other lacertid lizards. The longest repetitive unit recored in this study was 201 bp.The phylogenetic relationship of Takydromus is ((((((T. viridipunctatus +T. luyeanus) + (T wolteri + T. formosanus)) ? T. hsuehshanesis) + (T. stejnegeri + T.septentrionalis)) + (T. tachydromoides + T. amutensis)) + (T. sylvaticus + T.intermedins)) + (T, sexlineatus + T. kuehnei) based on 13 protein-coding genes with high bootstrap number and posterior probability by NJ, ML, MP and BI analysis. The phylogenetic trees based on 12S + 16S rRNA, Cyt b genes show four clades of sister species existing between Takydromus species from Taiwan and mainland China,suggesting that geographic isolation plays an important role in the phyogenesis of Takydromus.The divergence time between Takydromus lizards found in mainland China and Taiwan is estinated to vary from 2.3696 to 8.2103 Mya, well before the formation of Taiwan Strait (2.0 Mya). Takydromus lizards in Taiwan can be assigned to four clades,[T. kuehnei],[T. sauteri], [T stejnegeri], and [T hsuehshanesis,T. formosanus,T. viridipunctatus, T. luyeanus], which could reflect the consequence of specieation of Takydromus lizards in Taiwan driven by dispersal and isolation.Takydromus septentrionalis and T. sexlineatus differ morphologically at hatching and during adulthood. Phylogenetically, T. septentrionalis and T. sexlineatus are not sister species, belonging to the different groups. However, hybridization is likely to occur between these two species. I collected individuals of these two species from Shaoguan where they are sympatric to conduct hybrid experiment. I used a mitochondrial gene sequnce (between COII and COIII) and six microsatellite loci to examine gene flow between the two species, aiming to examine whether the female mating with males of different species can produce offspring and whether the gene flow exist between these two species.Copulation did occur between female T. sexlineatus and male T. septentrionalis,and between female T. septentrionalis and male T. sexlineatus. Nonetheless, I did not get any hybrid offspring under the laboratory conditions, presumably because females mating with males of different species could produce offspring by using sperm of the same species stored rather than sperm obtained from the males with which they mated.In wild populations, T. sexlineatus and T. septentrionalis did not share any haplotype based on the sequence between COII and COIII genes, but I detected microsatellite introgression across species in Shaoguan, suggesting the gene flow exist in the wild.In future studies, I will continue hybrid experiments and investigate the secondary contact zone of T. sexineatus and T. septentrionalis to make sure the existence of hybridization between T. septentrionalis and T.sexlineatus.