Geography and past climate changes have shaped the evolution of a widespread lizard in arid Central Asia.

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.

Dynamics behind disjunct distribution, hotspot-edge refugia, and discordant RADseq/mtDNA variability: insights from the Emei mustache toad.

BACKGROUND: The distribution of genetic diversity and the underlying processes are important for conservation planning but are unknown for most species and have not been well studied in many regions. In East Asia, the Sichuan Basin and surrounding mountains constitute an understudied region that exhibits a "ring" of high species richness overlapping the eastern edge of the global biodiversity hotspot Mountains of Southwest China. We examine the distributional history and genetic diversification of the Emei mustache toad Leptobrachium boringii, a typical "ring" element characterized by disjunct ranges in the mountains, by integrating time-calibrated gene tree, genetic variability, individual-level clustering, inference of population splitting and mixing from allele frequencies, and paleoclimatic suitability modeling. RESULTS: The results reveal extensive range dynamics, including secondary contact after long-term isolation via westward dispersal accompanied by variability loss. They allow the proposal of a model that combines recurrent contractions caused by Quaternary climatic changes and some failed expansions under suitable conditions for explaining the shared disjunct distribution pattern. Providing exceptional low-elevation habitats in the hotspot area, the eastern edge harbors both long-term refugial and young immigrant populations. This finding and a synthesis of evidence from other taxa demonstrate that a certain contributor to biodiversity, one that preserves and receives low-elevation elements of the east in this case, can be significant for only a particular part of a hotspot. By clarifying the low variability of these refugial populations, we show that discordant mitochondrial estimates of diversity can be obtained for populations that experienced admixture, which would have unlikely left proportional immigrant alleles for each locus. CONCLUSIONS: Dispersal after long-term isolation can explain much of the spatial distribution of genetic diversity in this species, while secondary contact and long-term persistence do not guarantee a large variation. The model for the formation of disjunct ranges may apply to many other taxa isolated in the mountains surrounding the Sichuan Basin. Furthermore, this study provides insights into the heterogeneous nature of hotspots and discordant variability obtained from genome-wide and mitochondrial data.

Parachute geckos free fall into synonymy: Gekko phylogeny, and a new subgeneric classification, inferred from thousands of ultraconserved elements.

Recent phylogenetic studies of gekkonid lizards have revealed unexpected, widespread paraphyly and polyphyly among genera, unclear generic boundaries, and a tendency towards the nesting of taxa exhibiting specialized, apomorphic morphologies within geographically widespread "generalist" clades. This is especially true in Australasia, where monophyly of Gekko proper has been questioned with respect to phenotypically ornate flap-legged geckos of the genus Luperosaurus, the Philippine false geckos of the genus Pseudogekko, and even the elaborately "derived" parachute geckos of the genus Ptychozoon. Here we employ sequence capture targeting 5060 ultraconserved elements (UCEs) to infer phylogenomic relationships among 42 representative ingroup gekkonine lizard taxa. We analyze multiple datasets of varying degrees of completeness (10, 50, 75, 95, and 100 percent complete with 4715, 4051, 3376, 2366, and 772 UCEs, respectively) using concatenated maximum likelihood and multispecies coalescent methods. Our sampling scheme addresses four persistent systematic questions in this group: (1) Are Luperosaurus and Ptychozoon monophyletic, and are any of these named species truly nested within Gekko? (2) Are prior phylogenetic estimates of Sulawesi's L. iskandari as the sister taxon to Melanesian G. vittatus supported by our genome-scale dataset? (3) Is the high-elevation L. gulat of Palawan Island correctly placed within Gekko? (4) And, finally, where do the enigmatic taxa P. rhacophorus and L. browni fall in a higher-level gekkonid phylogeny? We resolve these issues; confirm with strong support some previously inferred findings (placement of Ptychozoon taxa within Gekko; the sister taxon relationship between L. iskandari and G. vittatus); resolve the systematic position of unplaced taxa (L. gulat, and L. browni); and transfer L. iskandari, L. gulat, L. browni, and all members of the genus Ptychozoon to the genus Gekko. Our unexpected and novel systematic inference of the placement of Ptychozoon rhacophorus suggests that this species is not grouped with Ptychozoon or even Luperosaurus (as previously expected) but may, in fact, be most closely related to several Indochinese species of Gekko. With our resolved and strongly supported phylogeny, we present a new classification emphasizing the most inclusive, original generic name (Gekko) for these ~60 taxa, arranged into seven subgenera.

Complete mitochondrial genome of a blue-tailed skink Plestiodon capito (Reptilia, Squamata, Scincidae) and comparison with other Scincidae lizards.

Vertebrate mitochondrial genomes (mitogenomes) are valuable for studying phylogeny, evolutionary genetics and genomics. To date, however, compared to other vertebrate groups, our knowledge about the mitogenomes of skinks (the family Scincidae), even of reptile, has been relatively limited. In the present study, we determined the complete mitogenome of a blue-tailed skink Plestiodon capito for the first time, and compared it with other skinks available in GenBank. The circular genome is 17,344 bp long, showing a typical vertebrate pattern with 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and one control region (CR). The gene organization, nucleotide composition, and codon usage are similar to those from skinks previously published. Twelve out of 13 PCGs initiates with canonical start codon (ATG), while COX1 starts with GTG. The codon usage analysis revealed a preferential use of the LeuCUN (Leu1), Pro, and Thr codons with the A/U ending. All tRNAs in P. capito were predicted to fold into typical clover-leaf secondary structure, except tRNA-Ser AGY. The secondary structures of 12S rRNA and 16S rRNA comprises 34 helices and 56 helices, respectively. The alignment of the Plesitodon species CRs exhibited high genetic variability and rich A + T content. Besides, variable types and numbers of tandem repeat units were also identified in the CR of Plestiodon. Phylogenetic analyses recovered P. capito as the sister species to P. tunganus; monophyly of the Scincidae is well supported. Our results will help to better understand structure and evolution of the mitochondrial DNA control region in reptiles as well as the evolutionary status of P. capito, and to lay foundation for further phylogenetic study of skinks in a mitogenomic framework.

Diversification and historical demography of the rapid racerunner (Eremias velox) in relation to geological history and Pleistocene climatic oscillations in arid Central Asia.

Late Cenozoic stepwise aridification has transformed Central Asia into an arid environment, and the Pleistocene climatic oscillations exerted further ecological impact. Therefore, both aridification and glaciation would have considerably influenced the evolution of many midlatitude species in arid Central Asia (ACA). However, strong biotic evidence supporting this role is still lacking. Here, we test this perspective using a phylogeographic study of Eremias velox, which is an arid-adapted lizard, across ACA using sequences from mitochondrial cytochrome b and 12S rRNA genes. Phylogenetic analyses of the concatenated data, including 595 specimens from 107 localities, revealed ten geographically correlated lineages that diverged by 1.1-15.4% for the cytochrome b gene and 1.0-10.3% for the 12S rRNA gene, which were estimated to have coalesced ∼6.23 million years ago. Ancestral area estimations suggested that E. velox originated from the Iranian Plateau and then dispersed into Central Asia. 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 Kopet-Dagh, Tianshan and Greater Caucasus Mountains) and glacial oscillations during the Pleistocene triggered the progressive diversification of E. velox. The most recent common ancestor of the Caucasus-Central Asia clade was dated to approximately 2.05 Ma. Specifically, the diversification between the Caucasus clade (VI, VII) and the Central Asia clade (VIII, IX, X), and within the Central Asia clade may have been established and partially maintained by repeated transgressions of the Caspian Sea during the Pleistocene and Holocene. In contrast to demographic and/or range contractions in response to climatic changes during the Last Glacial Maximum (LGM) of the populations (Clades VI and X) from the Caucasus-Central Asia clade, mitochondrial evidence and ecological niche modeling support the signature of demographic and range expansions during the LGM for the Clade V populations (E. v. roborowskii, being endemic to the Turpan Depression). The effect of Pleistocene climatic changes on the historical demography of this arid-adapted species may be lineage-specific, depending predominantly on animal physiology and geography. Finally, we discuss the taxonomic implications, such as the appearance of the Turkmenistan populations as a distinct species, and E. v. roborowskii deserving a full species status.

Pathogenic Leishmania spp. detected in lizards from Northwest China using molecular methods.

BACKGROUND: Leishmaniosis, a disease caused by pathogenic Leishmania parasites, remains an unresolved health problem in the New World and the Old World. It is well known that lizards can be infected by a subgenus of Leishmania parasites, i.e. Sauroleishmania, which is non-pathogenic to humans. However, evidence suggests that lizards may also harbor pathogenic Leishmania species including the undetermined Leishmania sp., discovered in our previous work. Leishmania DNA in lizard blood can be detected by using molecular methods, such as the polymerase chain reaction (PCR). RESULTS: Three hundred and sixteen lizards, representing 13 species of four genera, were captured for blood samples collection in Northwest China. Two reliable molecular markers (cytochrome b and heat shock protein 70 genes) were used for detection in the lizard blood samples, to confirm a widespread presence of pathogenic Leishmania parasites and the distribution pattern of Leishmania spp. in lizards from Northwest China. The PCR data indicated positive detection rate for Leishmania in all the tested lizards with an overall prevalence of 57.91% (183/316). Apart from lizard parasites like Leishmania tarentolae and Leishmania sp., several pathogenic Leishmania including L. turanica, L. tropica and L. donovani complex were identified by using phylogenetic analysis. Co-existence of different haplotypes was observed in most Leishmania DNA-positive lizards with an overall rate of 77.6% (142/183). Even mixed infections with different Leishmania species appeared to occur in the lizards with an overall rate of 37.7% (69/183). CONCLUSIONS: Lizards can harbor pathogenic Leishmania spp. Co-existence of different haplotypes or even species of Leishmania indicates mixed infections in natural lizard host. Lizards may contribute to the spread of Leishmania parasites. The pathogenic Leishmania species detected in lizards from Northwest China may be of great eco-epidemiological importance.

Comparative analyses of phenotypic methods and 16S rRNA, khe, rpoB genes sequencing for identification of clinical isolates of Klebsiella pneumoniae.

The present work aimed to evaluate 16S rRNA, khe and rpoB gene sequencing for the identification of Klebsiella pneumoniae in comparison with phenotypic methods. Fifteen clinical isolates were examined, which were initially identified as K. pneumoniae subsp. pneumoniae using the automated VITEK 32 system in two hospitals in Enshi City, China. Their identity was further supported by conventional phenotypic methods on the basis of morphological and biochemical characteristics. Using Bayesian phylogenetic analyses and haplotypes network reconstruction, 13 isolates were identified as K. pneumoniae, whereas the other two isolates (K19, K24) were classified as Shigella sp. and Enterobacter sp., respectively. Of the three genes, 16S rRNA and khe gene could discriminate the clinical isolates at the genus level, whereas rpoB could discriminate Klebsiella at the species and even subspecies level. Overall, the gene tree based on rpoB is more compatible with the currently accepted classification of Klebsiella than those based on 16S rRNA and khe genes, showing that rpoB can be a powerful tool for identification of K. pneumoniae isolates. Above all, our study challenges the utility of khe as a species-specific marker for identification of K. pneumoniae.

Transcriptomic profiling revealed immune-related signaling pathways in response to experimental infection of Leishmania donovani in two desert lizards from Northwest China.

Little is known about the immune response of lizards to Leishmania parasties. In this study, we conducted the first liver transcriptome analysis of two lizards (Phrynocephalus przewalskii and Eremias multiocellata) challenged with L. donovani, endemic to the steppe desert region of northwestern China. Our results revealed that multiple biological processes and immune-related signaling pathways are closely associated with the immune response to experimental L. donovani infection in the two lizards, and that both lizards show similar changes to mammals in terms of immunity to Leishmania. However, the interspecific divergence of the two lizards leads to different transcriptomic changes. In particular, in contrast to P. przewalskii, the challenged E. mutltiocellata was characterized by the induction of down-regulation of most DEGs. These findings will contribute to the scarce resources on lizard immunity and provide a reference for further research on immune mechanisms in reptiles.

Unraveling molecular mechanistic disparities in pathogenic visceral Leishmania resistance between reptiles and mammals through comparative transcriptomic analyses.

Leishmaniasis is one of the most important neglected tropical parasitic diseases, manifesting various clinical forms depending on the parasite species and the genetic background of the host. In order to elucidate the underlying mechanisms of reptilian defense against pathogenic Leishmania species and to delineate the global gene expression profile alterations during host-pathogen interaction, we established experimental animal and cell models using both heterothermic lizards (Phrynocephalus przewalskii) and homothermic mammals (BALB/c mice) infected with pathogenic Leishmania infantum (high virulence HCZ strain) and Leishmania donovani (low virulence 801 strain). Overall, the lizards didn't show any obvious clinical symptoms or immune responses in vivo. Using RNA-seq methodology, differentially expressed genes identified in the HCZ and 801-comparison groups of P. przewalskii were primarily associated with arginine biosynthesis, the MAPK signaling pathway and the PI3K-Akt signaling pathway. In contrast, higher parasite loads, exacerbated hepatic inflammatory lesions and enhanced immune responses were observed in BALB/c mice, with DEGs predominantly associated with immunological diseases, innate and adaptive immune responses. By integrating transcriptional data from reptile and mammalian hosts, we elucidated the pivotal role of amino acid metabolism and lipid metabolism in parasite control. In contrast to findings from animal experiments, Leishmania parasites effectively infected peritoneal macrophages of lizards in vitro, demonstrating a high infection rate. Furthermore, we used RT-qPCR to detect changes in cytokine expression in macrophages and found that Th1-type cytokines were significantly upregulated in lizards, facilitating the clearance of the HCZ strain 24 hours post-infection. Conversely, cytokine expression was generally suppressed in BALB/c mice, allowing immune evasion by the parasites.

Genetic Structure and Population History of the Zaisan Toad-Headed Agama (Phrynocephalus melanurus) Inferred from Mitochondrial DNA.

The agamid lizard Phrynocephalus melanurus is restricted to Northwest China (Dzungar Basin) and the adjacent Eastern Kazakhstan (Zaisan and Alakol basins). To elucidate the phylogeography of P. melanurus, we obtained the mitochondrial DNA COI segments of 175 sampled lizards from 44 localities across the whole distribution. Phylogenetic analyses revealed two main Clades comprising five geographically structured lineages (I, IIa, IIb1, IIb2, and IIb3) that fit an isolation-by-distance (IBD) model. The divergence from the most recent common ancestor was dated to ~1.87 million years ago (Ma). Demographic analyses demonstrated lineage-specific response to past climate change: stable population for Clade I, Subclade IIb1; past population expansion for IIb3 since 0.18 Ma, respectively. Bayesian phylogeographic diffusion analyses detected initial spreading at the Saur Mount vicinity, approximately 1.8 Ma. Historical species distribution model (SDM) projected expansion of the suitable habitat in the last interglacial and shift and contraction in the last glacial maximum and Holocene epochs. The SDM predicted a drastic reduction in suitable area throughout the range as a response to future climate change. Our findings suggest that the evolution of P. melanurus followed a parapatric divergence with subsequent dispersal and adaptation to cold and dry environments during the Quaternary. Overall, this work improves our understanding of the lineage diversification and population dynamics of P. melanurus, providing further insights into the evolutionary processes that occurred in Northwest China and adjacent Eastern Kazakhstan.

Range-Wide Phylogeography and Ecological Niche Modeling Provide Insights into the Evolutionary History of the Mongolian Racerunner (Eremias argus) in Northeast Asia.

The Mongolian racerunner, Eremias argus, is a small lizard endemic to Northeast Asia that can serve as an excellent model for investigating how geography and past climate change have jointly influenced the evolution of biodiversity in this region. To elucidate the processes underlying its diversification and demography, we reconstructed the range-wide phylogeographic pattern and evolutionary trajectory, using phylogenetic, population genetic, landscape genetic, Bayesian phylogeographic reconstruction and ecological niche modeling approaches. Phylogenetic analyses of the mtDNA cyt b gene revealed eight lineages that were unbounded by geographic region. The genetic structure of E. argus was mainly determined by geographic distance. Divergence dating indicated that E. argus and E. brenchleyi diverged during the Mid-Pliocene Warm Period. E. argus was estimated to have coalesced at~0.4351 Ma (Marine Isotope Stage 19). Bayesian phylogeographic diffusion analysis revealed out-of-Inner Mongolia and rapid colonization events from the end of the Last Interglacial to the Last Glacial Maximum, which is consistent with the expanded suitable range of the Last Glacial Maximum. Pre-Last Glacial Maximum growth of population is presented for most lineages of E. argus. The Glacial Maximum contraction model and the previous multiple glacial refugia hypotheses are rejected. This may be due to an increase in the amount of climatically favorable habitats in Northeast Asia. Furthermore, E. argus barbouri most likely represents an invalid taxon. The present study is the first to report a range-wide phylogeography of reptiles over such a large region in Northeast Asia. Our results make a significant contribution towards understanding the biogeography of the entire Northeast Asia.

A complete mitogenome of the Przewalski's Wonder Gecko (Teratoscincus przewalskii) from the Junggar Basin in Northwest China with its phylogenetic implications.

A complete mitogenome of the Przewalski's Wonder Gecko (Teratoscincus przewalskii) from the Junggar Basin in Northwest China was determined by using polymerase chain reaction and directly sequenced with the primer walking method. The total length was 17,184 bp, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs) and a control region (CR). The order and structure of the genes were identical to those of congeners. The 13 PCGs contain four start codons (ATG, GTG, ATA, and ATC), three complete stop codons (TAA, TAG, and AGG), and two incomplete stop codons (T-, TA-). The concatenated PCGs were used to perform Bayesian phylogenetic analyses together with mitogenome data of the family Sphaerodactylidae and related representative taxa available in GenBank. The resulting tree recovered the monophyly of Sphaerodactylidae, and confirmed the sister relationship between T. przewalskii and T. roborowskii with strong support. The newly determined mitogenome will provide fundamental data for understanding the population genetic structure of T. pzrewalskii in particular, and the mitochondrial DNA evolution in Sphaerodactylidae in general.

Sex-Linked Loci on the W Chromosome in the Multi-Ocellated Racerunner (Eremias multiocellata) Confirm Genetic Sex-Determination Stability in Lacertid Lizards.

The multi-ocellated racerunner, Eremias multiocellata, was considered to have temperature-dependent sex determination (TSD), as its sex ratio can be influenced at different temperatures. However, such an observation contrasts with recent findings that suggest TSD is less common than previously thought. Here, a genotyping-by-sequencing (GBS) approach was employed to identify sex-linked markers in the E. multiocellata, for which the mechanism choice of TSD or GSD is still controversial. We preliminarily identified 119 sex-linked markers based on sex-associated sex-specific sequences, 97% of which indicated female heterogamety. After eliminating the false positives, 38 sex-linked markers were recognized, all of which showed the ZW/ZZ system. Then, eight of the novel markers were verified by PCR amplification from 15 populations of E. multiocellata, which support the GSD in E. multiocellata without geographic variation. To test the conservation of sex chromosome in Eremias, the eight markers were further cross-tested by PCR amplification in 10 individuals of the Mongolian racerunner (Eremias argus), two of which exhibited cross-utility. The novel sex-linked markers could be mapped on the W chromosome of the sand lizard (Lacerta agilis). Our finding that the sex-linked markers are shared in closely related species, along with a conserved synteny of the W chromosome, further supports the homology and conservation of sex chromosomes in the lacertid lizards.

Phylogeography and Ecological Niche Modeling of the Alashan Pit Viper (Gloydius cognatus; Reptilia, Viperidae) in Northwest China and Adjacent Areas.

The joint impacts of historical geological events and Quaternary climatic oscillations in Northwest China on species evolution have been examined extensively in plant under a phylogeographic perspective. However, animal phylogeographic analyses in this region are still limited. The Alashan pit viper, Gloydius cognatus, occurs primarily in arid Northwest China and adjacent areas. Based on variation at two mtDNA genes (ND4 and Cytb) in 27 individuals representing 24 populations, the spatial genetic structure and demographic history of G. cognatus were examined across its geographic range. Phylogenetic analyses revealed two well-supported allopatric clades (each with two distinct subclades/lineages), distributed across the southern (Qaidam Basin, Lanzhou Basin, and Zoige Basin [S1]; Loess Plateau [S2]) and northern (Ily Basin [N1]; Junggar Basin and Mongolian Plateau [N2]) regions. AMOVA analysis demonstrated that over 76% of the observed genetic variation was related to these lineage splits, indicating substantial genetic differentiation among the four lineages. A strong pattern of isolation-by-distance across the sampling populations suggested that geographic distance principally shaped the genetic structure. The four lineages diverged by 0.9-2.2% for the concatenated data, which were estimated to have coalesced ~1.17 million years ago (Mya), suggesting that the expansions of the Badain Jaran, Tengger, and Mu Us deserts during the Xixiabangma glaciation likely interrupted gene flow and triggered the observed divergence in the southern and northern regions. Subsequently, the early Pleistocene integration of the Yellow River and associated deserts expansion promoted the differentiation of S1 and S2 lineages (~0.9 Mya). Both mitochondrial evidence and ecological niche modeling (ENM) reject the signature of demographic and range contractions during the LGM for G. cognatus. In addition, ENM predicts that the suitable habitat of G. cognatus will contract in the future. As such, the conservation and management of ESUs should be a priority. Our findings provide the first insights on the lineage diversification and population dynamics of the Alashan pit viper in relation to geological history and Pleistocene climatic oscillations in arid Northwest China.

Accommodating heterogenous rates of evolution in molecular divergence dating methods: an example using intercontinental dispersal of Plestiodon (Eumeces) lizards.

Identifying and dating historical biological events is a fundamental goal of evolutionary biology, and recent analytical advances permit the modeling of factors known to affect both the accuracy and the precision of molecular date estimates. As the use of multilocus data sets becomes increasingly routine, it becomes more important to evaluate the potentially confounding effects of rate heterogeneity both within (e.g., codon positions) and among loci when estimating divergence times. Here, using Plestiodon lizards as a test case, we examine the effects of accommodating rate heterogeneity among data partitions on divergence time estimation. Plestiodon inhabits both East Asia and North America, yet both the geographic origin of the genus and timing of dispersal between the continents have been debated. For each of the eight independently evolving loci and a combined data set, we conduct single model and partitioned analyses. We found that extreme saturation has obscured the underlying rate of evolution in the mitochondrial DNA (mtDNA), resulting in severe underestimation of the rate in this locus. As a result, the age of the crown Plestiodon clade was overestimated by 15-17 Myr by the unpartitioned analysis of the combined loci data. However, the application of partition-specific models to the combined data resulted in ages that were fully congruent with those inferred by the individual nuclear loci. Although partitioning improved divergence date estimates of the mtDNA-only analysis, the ages were nonetheless overestimated, thus indicating an inadequacy of our current models to capture the complex nature of mtDNA evolution in over large time scales. Finally, the statistically incongruent age distributions inferred by the partitioned and unpartitioned analyses of the combined data support mutually exclusive hypotheses of the timing of intercontinental dispersal of Plestiodon from Asia to North America. Analyses that best capture the rate of evolution in the combined data set infer that this exchange occurred via Beringia ∼18.0-30 Ma.

Complete Mitochondrial Genomes of Five Racerunners (Lacertidae: Eremias) and Comparison with Other Lacertids: Insights into the Structure and Evolution of the Control Region.

Comparative studies on mitochondrial genomes (mitogenomes) as well as the structure and evolution of the mitochondrial control region are few in the Lacertidae family. Here, the complete mitogenomes of five individuals of Eremias scripta (2 individuals), Eremias nikolskii, Eremias szczerbaki, and Eremias yarkandensis were determined using next-generation sequencing and were compared with other lacertids available in GenBank. The circular mitogenomes comprised the standard set of 13 protein-coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and a long non-coding control region (CR). The extent of purifying selection was less pronounced for the COIII and ND2 genes in comparison with the rest of the PCGs. The codons encoding Leucine (CUN), Threonine, and Isolecucine were the three most frequently present. The secondary structure of rRNA of Lacertidae (herein, E. scripta KZL15 as an example) comprised four domains and 28 helices for 12S rRNA, with six domains and 50 helices for 16S rRNA. Five types and twenty-one subtypes of CR in Lacertidae were described by following the criteria of the presence and position of tandem repeats (TR), termination-associated sequence 1 (TAS1), termination-associated sequence 2 (TAS2), conserved sequence block 1 (CBS1), conserved sequence block 2 (CSB2), and conserved sequence block 3 (CSB3). The compositions of conserved structural elements in four genera, Acanthodactylus, Darevskia, Eremias, and Takydromus, were further explored in detail. The base composition of TAS2 - TATACATTAT in Lacertidae was updated. In addition, the motif "TAGCGGCTTTTTTG" of tandem repeats in Eremias and the motif "GCGGCTT" in Takydromus were presented. Nucleotide lengths between CSB2 and CSB3 remained 35 bp in Eremias and Darevskia. The phylogenetic analyses of Lacertidae recovered the higher-level relationships among the three subfamilies and corroborated a hard polytomy in the Lacertinae phylogeny. The phylogenetic position of E. nikolskii challenged the monophyly of the subgenus Pareremias within Eremias. Some mismatches between the types of CR and their phylogeny demonstrated the complicated evolutionary signals of CR such as convergent evolution. These findings will promote research on the structure and evolution of the CR and highlight the need for more mitogenomes in Lacertidae.

Complete mitochondrial genome of the Kyrghyz racerunner (Eremias nikolskii Bedriaga, 1905) from Kyrgyzstan.

The complete mitochondrial genome (mitogenome) of the Kyrghyz racerunner (Eremias nikoskii Bedriaga, 1905) from Kyrgyzstan was determined for the first time by next-generation sequencing. The mitogenome was 20,840 bp in length and comprised the standard set of 13 protein-coding genes (PCGs), 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. The 13 concatenated PCGs were used to implement Bayesian phylogenetic analyses together with some congeners and three representative lacertids retrieved from GenBank. The monophyly of both Eremias and its viviparous group was recovered in the Bayesian phylogenetic tree, while the subgenus Pareremias was paraphyletic with respect to E. nikoskii. The mitogenome of E. nikoskii will faciliate the research on species delimitation, molecular evolution, and phylogenetic inference in the racerunner lizards.

Comparative Analysis of Mitochondrial Genomes in Two Subspecies of the Sunwatcher Toad-Headed Agama (Phrynocephalus helioscopus): Prevalent Intraspecific Gene Rearrangements in Phrynocephalus.

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.

The complete mitochondrial genome of Metidiocerus impressifrons (Kirschbaum, 1868) (Hemiptera: Cicadellidae: Idiocerinae).

We determined the complete mitochondrial genome (mitogenome) of the leafhopper Metidiocerus impressifrons by next-generation sequencing. The mitogenome sequence was 16,426 bp in length and consists of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. Moreover, the nucleotide composition biases toward A and T, which together made up 78.2% of the entirety. The complete mitochondrial genomes of Metidiocerus impressifrons and other 27 species were used for phylogenetic analysis using the Bayesian method. The above results would facilitate our understanding of the evolution of Idiocerinae mitochondrial genome.