The plateau environments are typically arid, cool, and high altitude, posing formidable challenges to wildlife survival due to resource scarcity and harsh conditions. Unraveling ecological adaptability in severe conditions requires a deeper understanding of the niche characteristics of plateau species. Trophic niche, which is a comprehensive indicator describing the energy acquisition strategy of animals, remains relatively understudied in plateau species. Here, by combining stable isotopes and morphological data, we quantified the trophic niches of two allopatric lizard species (Phrynocephalus vlangalii and P. erythrurus) that live in the hinterland of the Qinghai-Tibetan Plateau, and explored how their trophic niches correlate with morphological and environmental factors. While both trophic niche and morphological traits were similar between species, noteworthy distinctions were observed between male and female Phrynocephalus lizards. The morphological traits associated with predation (i.e. limb length and head size) and reproduction (i.e. abdomen length), annual mean temperature, and sex played influential roles in shifting trophic niches. These results imply that sexual dimorphism may facilitate inter-sex divergence in resource utilization, leading to trophic niche variations in the highland lizards. Furthermore, extreme environmental stress can constrain interspecific divergence in morphological and trophic traits. Our findings illustrate the dynamic variations of trophic niches in highland lizards, contributing to a more comprehensive understanding of the adaptation strategies employed by lizard species in plateau environments.

The complex orogenic history and structure of Central Asia, coupled with Pleistocene glacial cycles have generated its stepwise aridification. Such events would have significantly influenced the evolution of many mid-latitude species in arid Central Asia (ACA). In this study, we employed two mitochondrial genes (CO1 and ND2) and genome-wide SNPs, coupled with ecological niche modeling, to investigate the lineage diversification and historical demography within a widespread lizard Phrynocephalus helioscopus, and their associations with geography and past climate change. We obtained the mtDNA dataset for 300 individuals from 96 localities within the known range of the lizard, among which 51 individuals from 27 localities were selected for generating the SNP dataset via the genotyping-by-sequencing approach. Phylogenetic analyses of the concatenated mtDNA dataset revealed eight geographically correlated lineages that diverged by 4.21-10.41% for the CO1 gene, which were estimated to have coalesced ∼4.47 million years ago. However, we observed mito-nuclear discordance pattern regarding the population of Clade V (P. helioscopus sergeevi) from Tajikistan. Ancestral area estimations suggested that P. helioscopus originated from the Fergana Valley and then dispersed into the adjacent areas in ACA along with a history of multiple allopatric divergence processes, suggesting that Fergana may have been the cradle of diversification of P. helioscopus. The intensification of aridification across Central Asia during the Late Pliocene may have facilitated the rapid radiation of this arid-adapted lizard throughout this vast territory. Subsequently, the geological events (e.g., uplift of the Hissar-Alay, transgressions of the Caspian Sea) and geographic barriers (e.g., Amu Darya River, Zeravshan River) during the Pleistocene triggered the progressive diversification of P. helioscopus. Interestingly, Clade VIII (P. helioscopus varius) experienced rapid population growth coupled with range expansion while Clade IV (P. helioscopus cameranoi) underwent drastic population expansion associated with range contraction during the Last Glacial Maximum. In Clade IV, but not in Clade VIII, environmental turnover contributes more to mitochondrial genetic distinctiveness than geographic distance. Overall, the SNP dataset demonstrates that geographic distance plays a greater role than environmental distance. Both the mtDNA dataset and the SNP dataset suggest local-scale genetic differentiation in Clade IV and Clade VIII, revealing potential geographic barriers in the Ili River Valley and the Junggar Basin, respectively. Twenty-seven outlier SNPs associated with environmental factors (precipitation and temperature) were identified, which supports the signature of local adaptation to the arid desert environment. Finally, our finding suggests taxonomic implications, such as support for full species status for P. saidalievi (Clade II) and P. meridionalis (Clade I). Future analyses based on further evidence and increased taxon and geographic sampling should be carried out to corroborate our findings.

Intraspecific rearrangements of mitochondrial genomes are rarely reported in reptiles, even in vertebrates. The sunwatcher toad-headed agama, Phryncoephalus helioscopus, can serve as an excellent model for investigating the dynamic mitogenome structure at intraspecific level. To date, seven subspecies of P. helioscopus are well recognized, but little is known about the mitogenomic evolution among different subspecies. In this study, complete mitogenomes of subspecies P. helioscopus varius II and P. helioscopus cameranoi were determined by next-generation sequencing, and another P. helioscopus varius I retrieved from GenBank was compiled for comparative analysis. The nucleotide composition and the codon usage are similar to those previously published from toad-headed agamas. P. helioscopus varius II and P. helioscopus cameranoi have 23 tRNA genes, including standard 22 tRNA genes and one extra tRNA-Phe (tRNA-Phe duplication). Gene order and phylogenetic analyses in the genus Phrynocephalus support prevalent intraspecific gene rearrangement in P. helioscopus and other congener species including P. erythrurus, P. vlangalii, and P. forsythii. Six different mitochondrial gene arrangements are observed in Phrynocephalus. Overall, the occurrence of rearrangements may result from multiple independent structural dynamic events. The split of the two subspecies in P. helioscopus was dated at approximately 2.34 million years ago (Ma). Two types of gene rearrangements are found in the three mitogenomes of P. helioscopus, and this intraspecific rearrangement phenomenon can be explained by the tandem duplication/random loss (TDRL) model. Post duplication, the alternative loss types can occur in 0.23-0.72 Ma, suggesting that the duplication and fixation of these rearrangements can occur quite quickly. These findings highlight the need for more mitogenomes at the population level in order to better understand the potentially rampant intraspecific mitogenomic reorganization in Phrynocephalus.

Due to their integral roles in oxidative phosphorylation, mitochondrially encoded proteins represent common targets of selection in response to altitudinal hypoxia across high-altitude taxa. While previous studies revealed evidence of positive selection on mitochondrial genomes of high-altitude Phrynocephalus lizards, their conclusions were restricted by out-of-date phylogenies and limited taxonomic sampling. Using topologies derived from both nuclear and mitochondrial DNA phylogenies, we re-assessed the evidence of positive selection on the mitochondrial genomes of high-altitude Phrynocephalus. We sampled representative species from all four main lineages and sequenced the mitochondrial genome of P. maculatus, a putative sister taxon to the high-altitude group. Positive selection was assessed through two widely used branch-site tests: the branch-site model in PAML and BUSTED in HyPhy. No evidence of positive selection on mitochondrial genes was detected on branches leading to two most recent common ancestors of high-altitude species; however, we recovered evidence of positive selection on COX1 on the P. forsythii branch, which represents a reversal from high- to low-elevation environments. A positively selected site therein marked a threonine to valine substitution at position 419. We suggest this bout of selection occurred as the ancestors of P. forsythii re-colonized lower altitude environments north of the Tibetan Plateau. Despite their role in oxidative phosphorylation, we posit that mitochondrial genes are unlikely to have represented historical targets of selection for high-altitude adaptation in Phrynocephalus. Consequently, future studies should address the roles of nuclear genes and differential gene expression.

The complete mitochondrial genome was sequenced from the toad-headed lizard, Phrynocephalus vlangalii vlangalii. The overall length of mitogenome is 16,319 bp, including 22 tRNA, 13 protein coding genes, 2 rRNA genes, and 2 control regions. The gene order and content were same with the published congeneric mitogenomes, besides the small portion between tRNA-Pro and tRNA-Phe.

The Qinghai-Tibetan Plateau (QTP) is the world\'s highest and largest plateau, but the role of its uplift in the evolution of species or biotas still remains poorly known. Toad-headed lizards of the reproductively bimodal genus Phrynocephalus are a clade of agamids, with all viviparous species restricted to the QTP and adjacent regions. The eastern part of the range of the viviparous taxa is occupied by three closely related but taxonomically controversial species, P. guinanensis, P. putjatia and P. vlangalii. Here, we combined genetic (mitochondrial ND4 gene and nine microsatellite loci), morphological (11 mensural and 11 meristic variables), and ecological (nine climatic variables) data to explore possible scenarios that may explain the discordance between genetic and morphological patterns, and to test whether morphological divergence is associated with local adaptation.
We found weak genetic differentiation but pronounced morphological divergence, especially between P. guinanensis and P. vlangalii. Genetically, the species boundary was not so clear between any species pair. Morphologically, the species boundary was clear between P. guinanensis and P. vlangalii but not between other two species pairs. Body size and scale characters accounted best for morphological divergence between species. Morphological divergence was related to habitat types that differ climatically.
Our study provides evidence for genetic and morphological divergence among the three closely related viviparous species of Phrynocephalus lizards, and supports the idea that natural selection in spatially heterogeneous environments can lead to population divergence even in the presence of gene flow. Our study supports the hypothesis that the evolutionary divergence between viviparous Phrynocephalus species was a consequence of environmental change after the uplift of the QTP.

Viviparous (live-bearing) vertebrates have evolved repeatedly within otherwise oviparous (egg-laying) clades. Over two-thirds of these changes in vertebrate reproductive parity mode happened in squamate reptiles, where the transition has happened between 98 and 129 times. The transition from oviparity to viviparity requires numerous physiological, morphological, and immunological changes to the female reproductive tract, including eggshell reduction, delayed oviposition, placental development for supply of water and nutrition to the embryo by the mother, enhanced gas exchange, and suppression of maternal immune rejection of the embryo. We performed genomic and transcriptomic analyses of a closely related oviparous-viviparous pair of lizards (Phrynocephalus przewalskii and Phrynocephalus vlangalii) to examine these transitions. Expression patterns of maternal oviduct through reproductive development of the egg and embryo differ markedly between the two species. We found changes in expression patterns of appropriate genes that account for each of the major aspects of the oviparity to viviparity transition. In addition, we compared the gene sequences in transcriptomes of four oviparous-viviparous pairs of lizards in different genera (Phrynocephalus, Eremias, Scincella, and Sphenomorphus) to look for possible gene convergence at the sequence level. We discovered low levels of convergence in both amino acid replacement and evolutionary rate shift. This suggests that most of the changes that produce the oviparity-viviparity transition are changes in gene expression, so occasional reversals to oviparity from viviparity may not be as difficult to achieve as has been previously suggested.

Although many cases of genetic adaptations to high elevations have been reported, the processes driving these modifications and the pace of their evolution remain unclear. Many high-elevation adaptations (HEAs) are thought to have arisen in situ as populations rose with growing mountains. In contrast, most high-elevation lineages of the Qinghai-Tibetan Plateau appear to have colonized from low-elevation areas. These lineages provide an opportunity for studying recent HEAs and comparing them with ancestral low-elevation alternatives. Herein, we compare four frogs (three species of Nanorana and a close lowland relative) and four lizards (Phrynocephalus) that inhabit a range of elevations on or along the slopes of the Qinghai-Tibetan Plateau. The sequential cladogenesis of these species across an elevational gradient allows us to examine the gradual accumulation of HEA at increasing elevations. Many adaptations to high elevations appear to arise gradually and evolve continuously with increasing elevational distributions. Numerous related functions, especially DNA repair and energy metabolism pathways, exhibit rapid change and continuous positive selection with increasing elevations. Although the two studied genera are distantly related, they exhibit numerous convergent evolutionary changes, especially at the functional level. This functional convergence appears to be more extensive than convergence at the individual gene level, although we found 32 homologous genes undergoing positive selection for change in both high-elevation groups. We argue that species groups distributed along a broad elevational gradient provide a more powerful system for testing adaptations to high-elevation environments compared with studies that compare only pairs of high-elevation versus low-elevation species.

Animals living at high altitude must adapt to environments with hypoxia and low temperatures, but relatively little is known about underlying genetic changes. Toad-headed lizards of the genus Phrynocephalus cover a broad altitudinal gradient of over 4000 m and are useful models for studies of such adaptive responses. In one of the first studies to have considered selection on mitochondrial protein-coding regions in an ectothermic group distributed over such a wide range of environments, we analysed nineteen complete mitochondrial genomes from all Chinese Phrynocephalus (including eight genomes sequenced for the first time). Initial analyses used site and branch-site model (program: PAML) approaches to examine nonsynonymous: synonymous substitution rates across the mtDNA tree.
Ten positively selected sites were discovered, nine of which corresponded to subunits ND2, ND3, ND4, ND5, and ND6 within the respiratory chain enzyme mitochondrial Complex I (NADH Coenzyme Q oxidoreductase). Four of these sites showed evidence of general long-term selection across the group while the remainder showed evidence of episodic selection across different branches of the tree. Some of these branches corresponded to increases in altitude and/or latitude. Analyses of physicochemical changes in protein structures revealed that residue changes at sites that were under selection corresponded to major functional differences. Analyses of coevolution point to coevolution of selected sites within the ND4 subunit, with key sites associated with proton translocation across the mitochondrial membrane.
Our results identify mitochondrial Complex I as a target for environment-mediated selection in this group of lizards, a complex that frequently appears to be under selection in other organisms. This makes these lizards good candidates for more detailed future studies of molecular evolution.

In this study, we report the complete mitochondrial genome of Phrynocephalus forsythii (Reptilia, Squamata, Agamidae), which is a circular molecule of 16,143 bp in size and consists of 13 protein-coding genes, 22 transfer RNAs, 2 ribosomal RNAs and 2 non-coding sequence (D-loop). The mitogenome of P. forsythii was similar to the typical mtDNA of vertebrates in gene arrangement and composition. The control region composed of two parts: one (348 bp) between tRNA(Phe) and the other (636 bp) between tRNA(Pro) and 12S rRNA. The A + T content of overall base of the composition of H-strand is 62.0% (T: 25.6%, C: 25.7%, A: 36.3% and G: 12.3%). The whole mitogenomic sequence of P. forsythii provides powerful data to study of its phylogenetic position within toad-headed lizards.