Evolutionary Studies Of Phrynocephalus (Agamidae) On Qinghai-Xizang (Tibetan) Plateau

The Qinghai-Xizang(Tibetan)Plateau is an immense upland(approximately 2.4 million km~2),averaging more than 4000 m above sea level in altitude.Environmental heterogeneity in internal and adjacent areas of Tibetan Plateau had been greatly shaped by the uplifting of plateau since Pliocene,which had great impact on the formation of biological diversity in the plateau and drove the plateau overlapping with the southwestern China mountains' biodiversity hotspot.Moreover,the plateau is becoming a model region for the researches of biodiversity.Distributing almost the whole Tibetan Plateau,Phrynocephalus lizards have been paid attention and their tremendous populations provide good model organisms for analyzing evolutionary biology.Because of continuing uplifting and environmental changing,Tibetan Plateau had unique environmental diversity and biological diversity. Studies on those endemic species have great and irreplaceable theoretical value.As model organisms,a number of researches concerning evolutionary biology on Phrynocephalus in Tibetan Plateau have been done,including morphology,ecology, molecular biology,ecological genetics,phylogeography,phylogeny and taxonomy.We investigated the evolution on morphology and reproductive characteristics in P.vlangalii along elevational and climatic gradients.The morphological variation pattern follows the converse to Bergmann's rule and it suggests that this body size pattern is driven by temperature,air pressure,length of the activity season and available food resources.However,P.vlangalii has shorter relative limb length which mismatches the Alien's rule and it is suggested to strengthen the ability of activity in high altitude with hypoxia environment.Besides these morphological variations mentioned above,P.vlangalii has fewer tail vertebrae and larger body size than oviparous Phrynocephalus lizard in low elevations,which indicates that these variations among species of Phrynocephalus are affected mainly by the genetic and phylogenetic factors.As a result,these variations match the Bergmann's rule and mismatch the Jordan's rule.However,P.theobaldi in colder and more arid environments was often found with brown-red patch and/or black plaque in abdomen,but could not be found in warmer and wetter environments, e.g.,valleys in southern Tibet.It could be concluded that the reproductive strategies of P.vlangalii fit into the common pattern of animals in higher elevation,which have smaller clutches of larger offspring and lower reproductive output.At higher altitudinal environments,the lizards have lower coefficient of litter size variation.The decreased litter size cline along elevational gradients might also be correlated with more anatomical constraints at higher elevations.This strategy of females showed the shifting from r-strategy to K-strategy along with increasing altitude.The genetic diversity of P.vlangalii decreased with increasing elevation, increasing rainfall and decreasing environmental temperature.There was a higher genetic diversity in temperature and rainfall stable regions.These were similar with the results of P.vlangalii' evolution on morphology and reproduction,which suggests that fewer genotypes of P.vlangalii are selected in higher elevation and extreme environments,e.g.lower temperature,hypoxia,stronger solar radiation.The test on nucleotide substitution indicates that the evolution of mtDNA has not reached saturation.Moreover,P.erythrurus has the highest nucleotide nonsynonymous substitution/synonymous substitution value in Phrynocephalus lizards in Tibet.The results indicate that P.erythrurus is still in the process of adaptive evolution under higher altitudinal environments.Phylogenetic analyses revealed that P.vlangalii and two other proposed species (P.erythrurus and P.putjatia)together form a monophyletic mtDNA clade,which in contrast with previous studies does not include P.theobaldi and P.zetangensis.The main P.vlangalli clade comprises seven well-supported lineages that correspond to distinct geographical areas with little or no overlap,and share a most recent common ancestor at 5.06±0.68 million years ago(mya).Analyses of molecular variance indicated that most of the observed genetic variations occurred among populations/regions,which implys the long-term interruption of maternal gene flow. A combined approach based on tests of population expansion,estimation of node dates,and significance tests on clade areas indicated that phylogeographical structuring has been primarily shaped by three main periods of plateau uplift during the Pliocene and Pleistocene,specifically 3.4 mya,2.5 mya and 1.7 mya.These periods corresponded to the appearance of several mountain ranges that formed physical barriers between lineages.Populations from the Qaidam Basin are shown to have undergone major demographic and range expansions in the early Pleistocene. This is consistent with colonization of areas previously covered by the huge Qaidam paleo-lake,which desiccated at this time.It was the role of uplifting of Tibetan Plateau(e.g.,Qingzang Movement),Kunlun-Yellow Movement,Gonghe Movement, the desiccating of paleo-lakes(e.g.,Qaidam and Gonghe paleolakes)that remolded the quite different geomorphology in Tibetan Plateau.These events shaped current patterns of diversity.Analyses on phylogeography of Phrynocephalus in Tibetan Plateau discovered two older evolutionary lineages than the clade corresponding to P.putjatia.One earlier diverged clade corresponding to valleys in southern Tibet,and another corresponding to the west of southern Tibet valleys between Himalayas and Gangdise mountains.The third but latest diverged clade of P.theobaldi nested as a sister clade with P.putjatia,its distributions not only covered the Ali area in Tibet,but reached into Galleys in southern Tibet.Interestingly,our results showed that the earliest diverged clade was completely overlapped with the latest diverged clade in the valleys of southern Tibet.There were obviously morphological differences between specimens from valleys in southern Tibet and from Ali region in western Tibet. Moreover,we propose that lizards with similar morphology of these two geographically overlapped clades might have been undergoing introgression since second contact occurred.However,besides hybridization on driving morphological convergence,it is still not safe to exclude the role of morphological convergent evolution under similar condition.MtDNA hybridization between P.vlangalii and P.putjatia was firstly reported in Phrynocephalus,which occurred in the sympatric region(Shazhuyu village,Gonghe county,Qinghai Plateau)and was considered as reciprocal introgression of mtDNA. The hybridization between the two lizard species was estimated at about 0.19-0.16 mya,which was coincided with the time of disappearance of the Gonghe paleo-lake at 0.15 mya,suggesting that the two species experienced second contact.Genetic introgression might occur after these two lizard species dispersed into the contact region when the Gonghe paleolake disappeared.We provided good evidence to resolve the molecular taxonomic chaos for Phrynocephalus complex in Tibetan Plateau with complete lineage sorting.There are five monotypic species including P.forsythii,P.putjatia,P.theobaldi,P.zetangensis, P.orientalis and two polytypic species including P.vlangalii and P.erythrurus in the whole plateau.P.erythrurus diverged into P.erythrurus erythrurus and P.erythrurus pylzowi because of intervening of Tanggula mountains.P.vlanglaii diverged into P. vlangalii vlangalii,P.vlangalii pylzowi and P.vlangalii nanschanica.These three subspecies have discontinuous distribution and have been undergone long-term interruption of gene flow.In summary,uplift of the Tibetan Plateau since Miocene is considered to be one of the most important geological events on shaping the diversity of Phrynocephalus. In more detail,many tectonic and geomorphical changing events e.g.,Qingzang Movement,Kun-Huang movement,Gonghe movement,evolution of paleo-lakes,had driven the formation of species and subspecies and shaped the geographical phylogenetic patterns of Phrynocephalus.Environmental heterogeneity,especially the low temperature,hypoxia,lower food availability and shorter seasonal activity, determined the evolutionary strategies on morphology and reproduction of P. vlangalii.