Genetic Basis and Evolutionary Forces of Sexually Dimorphic Color Variation in a Toad-Headed Agamid Lizard.

In the animal kingdom, sexually dimorphic color variation is a widespread phenomenon that significantly influences survival and reproductive success. However, the genetic underpinnings of this variation remain inadequately understood. Our investigation into sexually dimorphic color variation in the desert-dwelling Guinan population of the toad-headed agamid lizard (Phrynocephalus putjatai) utilized a multidisciplinary approach, encompassing phenotypic, ultrastructural, biochemical, genomic analyses, and behavioral experiments. Our findings unveil the association between distinct skin colorations and varying levels of carotenoid and pteridine pigments. The red coloration in males is determined by a genomic region on chromosome 14, housing four pigmentation genes: BCO2 and three 6-pyruvoyltetrahydropterin synthases. A Guinan population-specific nonsynonymous single nucleotide polymorphism in BCO2 is predicted to alter the electrostatic potential within the binding domain of the BCO2-ß-carotene complex, influencing their interaction. Additionally, the gene MAP7 on chromosome 2 emerges as a potential contributor to the blue coloration in subadults and adult females. Sex-specific expression patterns point to steroid hormone-associated genes (SULT2B1 and SRD5A2) as potential upstream regulators influencing sexually dimorphic coloration. Visual modeling and field experiments support the potential selective advantages of vibrant coloration in desert environments. This implies that natural selection, potentially coupled with assortative mating, might have played a role in fixing color alleles, contributing to prevalence in the local desert habitat. This study provides novel insights into the genetic basis of carotenoid and pteridine-based color variation, shedding light on the evolution of sexually dimorphic coloration in animals. Moreover, it advances our understanding of the driving forces behind such intricate coloration patterns.

Elevational variation in metabolic rate, feeding capacity and their associations in the Asiatic toad Bufo gargarizans.

Foraging behavior is known to place demands on the metabolic characteristics of anurans. Active foragers feeding on sedentary prey typically have high aerobic capacity and low anaerobic capacity, whereas sit-and-wait foragers feeding on active and mobile prey have the opposite pattern. Thus, the energetic demands of foraging may influence their metabolic adaptations to harsh environments, such as high elevations. Anurans that engage in active foraging have been found to increase maximum metabolic rate (MMR) and aerobic scope (AS, the difference between MMR and resting metabolic rate, RMR) at high elevations. However, data are lacking in amphibian ambush foragers. In this study, we examined the RMR, MMR, AS, and feeding capacity of a sit-and-wait forager ─the Asiatic toad (Bufo gargarizans), from two populations that are in close geographic proximity but differ by 1350 m in elevation. Our results show that there is no elevational variation in RMR and feeding capacity in either males or females. However, there are sex-specific variations in MMR and AS along an elevational gradient; females from high elevations have lower MMR and smaller net AS than their counterparts from low elevations while males maintain similar MMR and net AS across elevations. Furthermore, aerobic performances do not appear to be associated with feeding capacity at either the individual or population level. Our results support the hypothesis that sit-and-wait foragers may not increase their aerobic capacity as a strategy in hypoxic and low food availability environments and the role of sex in these adaptive adjustments should not be overlooked.

Impacts of Quaternary glaciation, geological history and geography on animal species history in continental East Asia: A phylogeographic review.

Continental East Asia has a mild Pleistocene climate and a complex recent geological history. Phylogeographic studies of animals over the last 30 years have produced several distinctive patterns. Glaciation refugia are numerous and are not restricted to any particular regions. Most of them are localized and species-specific, although several large refugia, for example the mountains of SW China, are shared by multiple species and have refugia-within-refugia. Furthermore, postglaciation range expansion events vary greatly in time, scale and direction. Large-scale south-to-north post-LGM expansions are few and mostly occurred in the northern regions. Additionally, several unique geographic features, including the three-step terrain of China and the northern arid belt, have significant impacts on many species histories. Overall, the impacts of Pleistocene glaciations, particularly the LGM, on species history vary drastically from nondetectable to significant. The impacts are the least for species from the southwestern region and are most dominant for species from the north. Geological events play a more significant role in shaping species history than Pleistocene climatic changes. Phylogeographic patterns among animals species are highly consistent with those of plants. Future phylogeographic endeavour in East Asia should be hypothesis-driven and seek processes that underlie common patterns. The wide use of genomic data allow accurate estimates of historical population processes and exploration of older history beyond the Pleistocene.

Whole-genome resequencing reveals complex effects of geographical-palaeoclimatic interactions on diversification of moustache toads in East Asia.

Geographical features and palaeoclimatic fluctuations are two classical evolutionary forces that shape genetic diversification within species. Fine-grained analysis of the mechanisms involved through population demographic processes, however, remains limited. Taking advantage of two recently published reference genomes, we resequenced the genomes and examined the evolutionary history of the moustache toads, a group endemic to East Asia where complex topography and fluctuating palaeoclimate are known to have had profound impacts on organisms. Moustache toads probably originated in southeast Yunnan, China, and diversified towards the northwestern of Yunnan, as well as central and eastern China. Further exploration based on three widespread species (Leptobrachium ailaonicum, L. boringii and L. liui) using demographic modelling and species distribution models revealed that mountains and river valleys in East Asia not only functioned as geographical barriers, but also provided dispersal corridors and facilitated continuous migration or post-glacial secondary contact among moustache toad populations. Furthermore, periodic oscillation of effective population sizes accompanying fluctuations of historical temperature and population contraction at the Last Glacial Maximum support the widespread impact of climatic changes of the Pleistocene on species diversification in East Asia. This impact was moderate for populations of L. ailaonicum and L. boringii in the southwestern mountains but severe for populations of L. liui in the eastern lowland regions of continental East Asia, which is supported by different degrees of change of their effective population sizes. Our findings reveal mechanisms underlying genetic diversification among moustache toads, and highlight the power of genomic data and demographic modelling for examining complex historical population-level processes and for understanding how geographical and palaeoclimatic factors interactively shape current intraspecific diversity.

Keep numbers in view: red-eared sliders (Trachemys scripta elegans) learn to discriminate relative quantities.

The ability to discriminate relative quantities, one of the numerical competences, is considered an adaptive trait in uncertain environments. Besides humans, previous studies have reported this capacity in several non-human primates and birds. Here, we test whether red-eared sliders (Trachemys scripta elegans) can discriminate different relative quantities. Subjects were first trained to distinguish different stimuli with food reward. Then, they were tested with novel stimulus pairs to demonstrate how they distinguished the stimuli. The results show that most subjects can complete the initial training and use relative quantity rather than absolute quantity to make choices during the testing phase. This study provides behavioural evidence of relative quantity discrimination in a reptile species and suggests that such capacity may be widespread among vertebrates.

Modified Metabolism and Response to UV Radiation: Gene Expression Variations Along an Elevational Gradient in the Asiatic Toad (Bufo gargarizans).

High-elevation adaptation provides an excellent system for examining adaptive evolution, and adaptive variations may manifest at gene expression or any other phenotypic levels. We examined gene expression profiles of Asiatic toads (Bufo gargarizans) along an elevational gradient from both wild and common-garden acclimated populations. Asiatic toads originated from high altitudes have distinctive gene expression patterns. We identified 18 fixed differentially expressed genes (DEGs), which are different in both wild and acclimated samples, and 1217 plastic DEGs, which are different among wild samples. The expression levels of most genes were linearly correlated with altitude gradient and down-regulated in high-altitude populations. Expression variations of several genes associated with metabolic process are fixed, and we also identified a co-expression module that is significantly different between acclimated populations and has functions related to DNA repair. The differential expression of the vast majority genes, however, are due to phenotypic plasticity, revealing the highly plastic nature of gene expression variations. Expression modification of some specific genes related to metabolism and response to UV radiation play crucial role in adaptation to high altitude for Asiatic toads. Common-garden experiments are essential for evaluating adaptive evolution of natural populations.

A Re-Assessment of Positive Selection on Mitochondrial Genomes of High-Elevation Phrynocephalus Lizards.

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.

Isolation and reconnection: Demographic history and multiple contact zones of the green odorous frog (Odorrana margaretae) around the Sichuan Basin.

The green odorous frog (Odorrana margaretae) displays a circular distribution around the Sichuan Basin of western China and possesses multiple replicate hybrid zones between lineages with high levels of divergence. To gain an understanding of the speciation process, we obtained 1540 SNPs from 29 populations and 227 individuals using ddRAD sequencing. Population structure analysis revealed three groups within the species: the West, North & South, and East groups. Demographic inference showed that they were initially isolated at ~2 million years ago, and subsequent post-glacial expansion produced the current circular distribution with four secondary contact zones. Hybridization in those zones involved lineages with various levels of divergence and produced greatly different patterns of introgression. Contact zones between the East and North & South groups (E-S and E-N) had contrast admixture levels but both showed a general lack of potential barrier loci. Meanwhile, the reconnection of the West and North & South groups produced two contact zones along the rim of the Basin. The S-W zone had extensive admixture while the N-W zone had limited admixture within a narrow geographic distance. Both showed substantial barrier effects, and a large number of potential barrier loci were shared. We also detected strong coupling among these loci. The N-W hybrid zone involved two highly-diverged lineages (FST = 0.704) and many loci have reached fixation around the hybrid zone. This study system offers a unique opportunity to understand the dynamics of introgression in contact zones and the architecture of reproductive isolation at different stages of speciation.

UCE Phylogenomics, detection of a putative hybrid population, and one older mitogenomic node age of Batrachuperus salamanders.

The prevalence of incomplete lineage sorting complicates the examination of hybridization and species-level paraphyly with gene trees of a small number of loci. In Asian mountain salamanders of the genus Batrachuperus, possible hybridization and species paraphyly had been identified by utilizing mitochondrial genealogy and fixed allozyme differences. Here we sampled 2909 UCEs in 44 local populations from all six Batrachuperus species, inferred gene and species trees, compared them with mitochondrial and allozyme results, and examined the potential hybridization and species paraphyly. The clustering pattern of single-locus trees, increased proportion of heterozygous SNPs, allele frequency-based migration edge estimation, and intrapopulation long branches (as expected from an increase of genetic lineage and nucleotide diversity) support that an eastern B. karlschmidti population has experienced admixture with B. tibetanus. On the 2909-UCE concatenated and species trees, lower nodal supports were observed when similar proportions of loci agreed with alternative topologies, i.e., a reciprocal monophyly between a Pengxian lineage and the remainder of B. pinchonii (0.379) or a paraphyly of the latter with respect to Pengxian (0.362). The UCE phylogenomics agreed with the relatively recent groupings in the allozyme dendrogram. Despite incomplete lineage sorting, the mitochondrial trees were similar to the UCE trees for deeper relationships of the genus. However, one significant branch-length level discordance was identified. The branch between the common ancestor of B. daochengensis and B. yenyuanensis and common ancestor of the genus was approximately three times shorter on the mitochondrial tree than on the UCE tree, suggesting that the split of the mitochondrial lineages was likely a few million years earlier than the split of species. This finding supports considering possible ancestral polymorphism when interpreting different divergence dates estimated from mitochondrial and genome-wide data.

Molecular convergent and parallel evolution among four high-elevation anuran species from the Tibetan region.

BACKGROUND: To date, evidence for the relative prevalence or rarity of molecular convergent and parallel evolution is conflicting, and understanding of how these processes contribute to adaptation is limited. We compared four high-elevation anuran species (Bufo tibetanus, Nanorana parkeri, Rana kukunoris and Scutiger boulengeri) from the Tibetan region, and examined convergent and parallel amino acid substitutions between them and how they may have contributed to high-elevation adaptation. RESULTS: Genomic data of the four high-elevation species and eight of their low-elevation close relatives were gathered. A total of 1098 orthologs shared by all species were identified. We first conducted pairwise comparisons using Zhang and Kumar's test. Then, the Rconv index was calculated and convergence/divergence correlation plotting was conducted. Furthermore, genes under positive selection and with elevated evolutionary rate were examined. We detected a large number of amino acid sites with convergent or parallel substitutions. Several pairs of high-elevation species, in particular, R. kukunoris vs N. parkeri and B. tibetanus vs S. boulengeri, had excessive amounts of convergent substitutions compared to neutral expectation. Nevertheless, these sites were mostly concentrated in a small number of genes (3-32), and no genome-wide convergence was detected. Furthermore, the majority of these convergent genes were neither under detectable positive selection nor had elevated evolutionary rates, although functional prediction analysis suggested some of the convergent genes could potentially contribute to high-elevation adaptation. CONCLUSIONS: There is a substantial amount of convergent evolution at the amino-acid level among high-elevation amphibians, although these sites are concentrated in a few genes, not widespread across the genomes. This may attribute to the fact that all the target species are from the same environment. The relative prevalence of convergent substitutions among high-elevation amphibians provides an excellent opportunity for further study of molecular convergent evolution.

Revisiting the evolution of the North American tetraploid treefrog (Hyla versicolor).

Hyla chrysoscelis and H. versicolor are common treefrogs in eastern North America and are a cryptic diploid-tetraploid species pair. They are morphologically identical but H. versicolor is a tetraploid. They can be identified acoustically by the male's advertisement mating call, which has a pulse repetition rate that has twice as many pulses per second in the diploid species, H. chrysoscelis. We used isozymes, microsatellite DNA alleles, and mitochondrial cytochrome b sequences to test the hypothesis that gene exchange occurs between the diploid and tetraploid species in sympatric populations. Each method provided results that are best explained by occasional hybridization of female H. versicolor and male H. chrysoscelis. We propose that H. versicolor first arose from an autotriploid H. chrysoscelis female that produced unreduced triploid eggs. After H. versicolor became established, genes could be passed from H. chrysoscelis to H. versicolor in sympatric populations when these species hybridize. Their F1 female progeny produce unreduced triploid eggs that are fertilized by haploid H. chrysoscelis sperm to reconstitute H. versicolor. Genes can be passed from diploid H. chrysoscelis to tetraploid H. versicolor in sympatric populations.

Evolutionary melting pots and reproductive isolation: A ring-shaped diversification of an odorous frog (Odorrana margaratea) around the Sichuan Basin.

Quaternary climatic oscillations and geography are of primary importance in shaping intraspecific genetic diversity. We examined the diversification patterns and inferred processes for the green odorous frog (Odorrana margaretae) of western China. Species distribution modelling showed that the species has a continuous circular distribution around the Sichuan Basin while the basin itself is largely uninhabitable. Population genetic and phylogenetic analyses revealed that the species has a ring-shaped divergent pattern. While the chain of populations around the Basin maintains a mostly gradual and continuous genetic variation, populations between the north and west showed little gene exchange. Two processes, glacial refugial history and geography, likely contributed to the observed patterns. Our genetic clustering analysis revealed two clusters, suggesting two refugial groups among the populations, one from the west and the other from the east. Postglacial expansion may have created two contact zones. One at the south had extensive population admixture and produced a gradual transition between the western and eastern populations. Consequently, this region has the highest genetic diversity and represents an evolutionary "melting pot." In contrast, the second contact zone at the northwestern side of the Basin has limited admixture, suggesting partial reproductive isolation between the northern and western populations. Furthermore, an isolation-by-distance analysis revealed a strong correlation (Mantel r = 0.745) between the genetic and geographic distances, when we assumed that populations were connected following the circular distribution without gene flow across the NW contact zone. We also explored alternative explanations, such as a one-refugium scenario. With its micro-ring, the green odorous frog is poised to make an excellent model system for understanding the process of speciation.

Testing domain general learning in an Australian lizard.

A key question in cognition is whether animals that are proficient in a specific cognitive domain (domain specific hypothesis), such as spatial learning, are also proficient in other domains (domain general hypothesis) or whether there is a trade-off. Studies testing among these hypotheses are biased towards mammals and birds. To understand constraints on the evolution of cognition more generally, we need broader taxonomic and phylogenetic coverage. We used Australian eastern water skinks (Eulamprus quoyii) with known spatial learning ability in three additional tasks: an instrumental and two discrimination tasks. Under domain specific learning we predicted that lizards that were good at spatial learning would perform less well in the discrimination tasks. Conversely, we predicted that lizards that did not meet our criterion for spatial learning would likewise perform better in discrimination tasks. Lizards with domain general learning should perform approximately equally well (or poorly) in these tasks. Lizards classified as spatial learners performed no differently to non-spatial learners in both the instrumental and discrimination learning tasks. Nevertheless, lizards were proficient in all tasks. Our results reveal two patterns: domain general learning in spatial learners and domain specific learning in non-spatial learners. We suggest that delineating learning into domain general and domain specific may be overly simplistic and we need to instead focus on individual variation in learning ability, which ultimately, is likely to play a key role in fitness. These results, in combination with previously published work on this species, suggests that this species has behavioral flexibility because they are competent across multiple cognitive domains and are capable of reversal learning.

Molecular Convergent Evolution of the MYBPC2 Gene Among Three High-Elevation Amphibian Species.

We report a strong pattern of molecular-level convergent/parallel evolution of the MYBPC2 gene. Three high-elevation amphibian species, Bufo gargarizans minshanicus, Nanorana pleskei, Rana kukunoris, revealed remarkable numbers of convergent and parallel amino acid substitutions. On the MYBPC2 gene tree of eleven anurans, the three distantly related species formed a strongly supported clade that was away from their respective relatives. Furthermore, we generated both model-based and empirical data-based null distributions for neutral convergent evolution. All three pairwise comparisons among the three species showed significantly more convergent and parallel substitutions than the null distributions. This study adds to the very small roster of clear cases of non-neutral molecular convergent evolution (e.g. prestin, rhodopsin). Molecular convergent evolution has significant implications in biology and detailed case studies will likely provide more insight into its genetic mechanisms.

Gene expression variations in high-altitude adaptation: a case study of the Asiatic toad (Bufo gargarizans).

BACKGROUND: Genome-wide investigation of molecular mechanisms for high-altitude adaptation has attracted great attention in the last few years. In order to understand the contribution of gene expression level variations to high-altitude adaptation in Asiatic toads (Bufo gargarizans), we implemented a reciprocal transplant experiment between low- and high-altitude sites and sequenced 12 transcriptomes from brain, heart, and liver tissues. RESULTS: A large number of genes with expression differences (DEGs) between high- and low-altitude individuals (193 fixed and 844 plastic) were identified, and the majority of them were tissue specific. Heart displayed the largest number of DEGs, both plastic and fixed. Fixed DEGs were particularly concentrated in functions associated with muscle contraction, and the majority of them were down-regulated in high-altitude individuals. Plastic DEGs were highly concentrated in several energy metabolism related functional categories, and the majority of them were also down-regulated at high-altitude environments. In liver samples, genes associated with nutrient metabolism experienced a broad-scale expression down-regulation in high-altitude toads. CONCLUSIONS: These broadly suppressed expression patterns at high altitudes are in strong contrast to those of endothermic homeotherms, suggesting poikilothermic vertebrates may have adopted different strategies at high altitudes. Our results strongly support that both genotypic specialization and phenotypic plasticity play crucial role in adaptation to high altitude for Asiatic toads. Poikilothermic vertebrates are among the most hypoxia-tolerant animals known, and many molecular mechanisms remain elusive. We hope that our results will provide useful directions for future research.

Genetic signals of high-altitude adaptation in amphibians: a comparative transcriptome analysis.

BACKGROUND: High-altitude adaptation provides an excellent system for studying how organisms cope with multiple environmental stressors and interacting genetic modifications. To explore the genetic basis of high-altitude adaptation in poikilothermic animals, we acquired transcriptome sequences from a high-altitude population and a low-altitude population of the Asiatic toad (Bufo gargarizans). Transcriptome data from another high-altitude amphibian, Rana kukunoris and its low-altitude relative R. chensiensis, which are from a previous study, were also incorporated into our comparative analysis. RESULTS: More than 40,000 transcripts were obtained from each transcriptome, and 5107 one-to-one orthologs were identified among the four taxa for comparative analysis. A total of 29 (Bufo) and 33 (Rana) putative positively selected genes were identified for the two high-altitude species, which were mainly concentrated in nutrient metabolism related functions. Using SNP-tagging and FST outlier analysis, we further tested 89 other nutrient metabolism related genes for signatures of natural selection, and found that two genes, CAPN2 and ITPR1, were likely under balancing selection. We did not detect any positively selected genes associated with response to hypoxia. CONCLUSIONS: Amphibians clearly employ different genetic mechanisms for high-altitude adaptation compared to endotherms. Modifications of genes associated with nutrient metabolism feature prominently while genes related to hypoxia tolerance appear to be insignificant. Poikilotherms represent the majority of animal diversity, and we hope that our results will provide useful directions for future studies of amphibians as well as other poikilotherms.

Rates and patterns of molecular evolution in freshwater versus terrestrial insects.

Insect lineages have crossed between terrestrial and aquatic habitats many times, for both immature and adult life stages. We explore patterns in molecular evolutionary rates between 42 sister pairs of related terrestrial and freshwater insect clades using publicly available protein-coding DNA sequence data from the orders Coleoptera, Diptera, Lepidoptera, Hemiptera, Mecoptera, Trichoptera, and Neuroptera. We furthermore test for habitat-associated convergent molecular evolution in the cytochrome c oxidase subunit I (COI) gene in general and at a particular amino acid site previously reported to exhibit habitat-linked convergence within an aquatic beetle group. While ratios of nonsynonymous-to-synonymous substitutions across available loci were higher in terrestrial than freshwater-associated taxa in 26 of 42 lineage pairs, a stronger trend was observed (20 of 31, pbinomial = 0.15, pWilcoxon = 0.017) when examining only terrestrial-aquatic pairs including fully aquatic taxa. We did not observe any widespread changes at particular amino acid sites in COI associated with habitat shifts, although there may be general differences in selection regime linked to habitat.

Population Genetic Structure and Species Status of Asiatic Toads (Bufo gargarizans) in Western China.

We investigated the population genetic structure of Asiatic toads (Bufo gargarizans) from the mountains of western China to determine their species status, using genotypic data of ten microsatellite DNA loci and DNA sequences from one mitochondrial gene. A total of 197 samples from eight sites were examined, which cover a large range of elevations (559-3457 m), as well as all three traditionally defined species (or subspecies). AMOVA did not reveal any particularly large among-groups structure, whether the sites were grouped by drainage, elevation, region, or species (subspecies). Individual assignment tests placed all samples into two genetic clusters, which largely corresponded to their geographic locations. An isolation-by-distance pattern was also detected when an outlier population (site 3) was excluded. Furthermore, a mitochondrial gene tree revealed deep divergence among haplotypes, sometimes within the same site. The clade patterns were partially associated with geographic distribution but had no resemblance to the traditional 2- or 3-species classification. Overall, these toad populations harbor a large amount of genetic diversity and have very high population differentiation, but taken together the evidence suggests that all populations belong to a single species. Our results are consistent with most previous molecular studies, and we recommend using Bufo gargarizans to represent all Asiatic toad populations from western China without subspecies division.

Hybridization and mitochondrial genome introgression between Rana chensinensis and R. kukunoris.

Mitochondrial genome (mito-genome) introgression among metazoans is commonplace, and several biological processes may promote such introgression. We examined two proposed processes for the mito-genome introgression between Rana chensinensis and R. kukunoris: natural hybridization and sex-biased dispersal. We sampled 477 individuals from 28 sites in the potential hybrid zone in the western Tsinling Mountains. Mitochondrial gene (cyt-b) trees were used to examine the introgression events. Microsatellite DNA loci, cyt-b and morphological data were used to identify hybrids and to examine the extent of natural hybridization. We detected rampant bidirectional introgressions, both ancient and recent, between the two species. Furthermore, we found a wide hybrid zone, and frequent and asymmetric hybridization. The hybrid zone cline analysis revealed a clear mitochondrial-nuclear discordance; while most nuclear markers displayed similar and steep clines, cyt-b had a displaced cline centre and a more gradual and wider cline. We also detected strong and asymmetric historical maternal gene flow across the hybrid zone. This widespread hybridization and detected low mito-nuclear conflicts may, at least partially, explain the high frequency of introgression. Lastly, microsatellite data and population genetic methods were used to assess sex-biased dispersal. A weak pattern of female-biased dispersal was detected in both species, suggesting it may not play an important role in the observed introgression. Our data are consistent with the hybridization hypothesis, but support for the sex-biased dispersal hypothesis is weak. We further suggest that selective advantages of the R. kukunoris-type mito-genome in thermal adaptation may also contribute to the introgression between the two species.

A phylogeographic evaluation of the Amolops mantzorum species group: cryptic species and plateau uplift.

Phylogeographic analysis brings organism phylogeny, regional geological/climatic history, and population demography together, and provides insights into species history and speciation. Using DNA sequence data from a mitochondrial marker (cytochrome b) and a nuclear marker (cmyc intron 2), we examined phylogeography of the Amolops mantzorum species group. We first constructed Bayesian and maximum-likelihood gene trees and medium-joint networks for the recovered haplotypes, and estimated divergence time for each major lineage. Species boundaries were then delineated using the general mixed Yule-coalescent model (GMYC) and a Bayesian species-delimitation method (BP&P). Potential gene flow between putative species was also estimated using the isolation-with-migration model. Furthermore, species-distribution modeling was used to probe linkage between climatic changes and speciation. Lastly, extended Bayesian skyline plotting was employed to reveal historical demography for each putative species. Our analyses clearly delimit nine species in the group, including four well-recognized species and five putative species, of which three are often listed as synonyms of A. mantzorum. The nominal species A. mantzorum may in fact include two cryptic species. Divergence-time estimates align the speciation events with the recent intense uplift of the Tibetan Plateau in the last 3.6 million years. The species-distribution modeling also reveals different habitat preferences among species that are potentially linked to climatic changes associated with the uplift. Furthermore, three species sustained continuous population growth through the last glaciation, while others expanded only after the last glaciation. The eastern escarpment of Tibet is a biodiversity hotspot; its rich species diversity, unique topography, and well-established geological history provide an excellent system for evolutionary studies.