Close relatedness between mitochondrial DNA from seven Anser goose species

The phylogenetic relationships of seven goose species and two of the subspecies representing the genus Anser were studied by approximately 1180 bp of mitochondrial DNA tRNAglu, control region and tRNAphe sequences. Despite obvious morphological and behavioural affinities among the species, their evolutionary relationships have not been studied previously. The small amount of genetic differentiation observed in the mitochondrial DNA indicates an extremely close evolutionary relationship between the Anser species. The sequence divergences between the species (0.9–5.5%) are among the lowest reported for avian species with speciation events of Anser geese dating to late Pliocene and Pleistocene. The species grouped into four mtDNA lineages: (1) snow and Ross’ goose, (2) greylag goose, (3) white‐fronted goose, and (4) bean, pink‐footed and lesser white‐fronted goose. The phylogenetic relationships of the most closely related species, bean, pink‐footed and lesser white‐fronted goose, indicate a period of rapid cladogenesis. The poor agreement between morphological relationships and the phylogenetic relationships indicated by mtDNA sequences implies that either ancestral polymorphism and lineage sorting, hybridization and introgression or convergent evolution has been involved.     

Parallel plumage colour evolution and introgressive hybridization in wheatears

Genetic and phenotypic mosaics, in which various phenotypes and different genomic regions show discordant patterns of species or population divergence, offer unique opportunities to study the role of ancestral and introgressed genetic variation in phenotypic evolution. Here, we investigated the evolution of discordant phenotypic and genetic divergence in a monophyletic clade of four songbird taxa—pied wheatear (O. pleschanka), Cyprus wheatear (Oenanthe cypriaca), and western and eastern subspecies of black‐eared wheatear (O. h. hispanica and O. h. melanoleuca). Phenotypically, black back and neck sides distinguish pied and Cyprus wheatears from the white‐backed/necked black‐eared wheatears. Meanwhile, mitochondrial variation only distinguishes western black‐eared wheatear. In the absence of nuclear genetic data, and given frequent hybridization among eastern black‐eared and pied wheatear, it remains unclear whether introgression is responsible for discordance between mitochondrial divergence patterns and phenotypic similarities, or whether plumage coloration evolved in parallel. Multispecies coalescent analyses of about 20,000 SNPs obtained from RAD data mapped to a draft genome assembly resolve the species tree, provide evidence for the parallel evolution of colour phenotypes and establish western and eastern black‐eared wheatears as independent taxa that should be recognized as full species. The presence of the entire admixture spectrum in the Iranian hybrid zone and the detection of footprints of introgression from pied into eastern black‐eared wheatear beyond the hybrid zone despite strong geographic structure of ancestry proportions furthermore suggest a potential role for introgression in parallel plumage colour evolution. Our results support the importance of standing heterospecific and/or ancestral variation in phenotypic evolution.     

Rapid diversification associated with ecological specialization in Neotropical Adelpha butterflies

Rapid diversification is often associated with morphological or ecological adaptations that allow organisms to radiate into novel niches. Neotropical Adelpha butterflies, which comprise over 200 species and subspecies, are characterized by extraordinary breadth in host plant use and wing colour patterns compared to their closest relatives. To examine the relationship between phenotypic and species diversification, we reconstructed the phylogenetic history of Adelpha and its temperate sister genus Limenitis using genomewide restriction‐site‐associated DNA (RAD) sequencing. Despite a declining fraction of shared markers with increasing evolutionary distance, the RAD‐Seq data consistently generated well‐supported trees using a variety of phylogenetic methods. These well‐resolved phylogenies allow the identification of an ecologically important relationship with a toxic host plant family, as well as the confirmation of widespread, convergent wing pattern mimicry throughout the genus. Taken together, our results support the hypothesis that evolutionary innovations in both larvae and adults have permitted the colonization of novel host plants and fuelled adaptive diversification within this large butterfly radiation.     

Hybridization between two parapatric ranid frog species in the northern Sierra Nevada,California, USA

Contact zones between species provide a unique opportunity to test whether taxa can hybridize or not. Cross‐breeding or hybridization between closely related taxa can promote gene flow (introgression) between species, adaptation, or even speciation. Though hybridization events may be short‐lived and difficult to detect in the field, genetic data can provide information about the level of introgression between closely related taxa. Hybridization can promote introgression between species, which may be an important evolutionary mechanism for either homogenization (reversing initial divergence between species) or reproductive isolation (potentially leading to speciation). Here, we used thousands of genetic markers from nuclear DNA to detect hybridization between two parapatric frog species (Rana boylii and Rana sierrae) in the Sierra Nevada of California. Based on principal components analysis, admixture, and analysis of heterozygosity at species diagnostic SNPs, we detected two F1 hybrid individuals in the Feather River basin, as well as a weak signal of introgression and gene flow between the frog species compared with frog populations from two other adjacent watersheds. This study provides the first documentation of hybridization and introgression between these two species, which are of conservation concern.     

The role of mitochondrial introgression in illuminating the evolutionary history of Nearctic treefrogs

Inferring the evolutionary history of lineages often becomes difficult when gene histories are in conflict with each other. Introgression, for example, can cause DNA sequences from one species to be more similar to sequences of a different species and lead to incongruence amongst gene trees. However, incorporating congruent and incongruent locus‐specific phylogenetic estimates with the geographical distribution of lineages may provide valuable insight into evolutionary processes important to speciation. In this study, we investigated mitochondrial introgression within the Hyla eximia group to better understand its role in illuminating the evolutionary history and phylogeography of these treefrogs. We reconstructed and compared the matrilineal history of the Hyla eximia group with estimates of evolutionary history inferred from nuclear genes. We tested for introgression within the mitochondrial and nuclear genes using a posterior predictive checking approach. Reconstructions of the species tree based on the mitochondrial DNA (mtDNA) and nuclear DNA data were strongly discordant. Introgression between lineages was widespread in the mtDNA data set (145 occurrences amongst 11 of the 16 lineages), but uncommon in the nuclear genes (12 occurrences amongst four of the 16 lineages). Nonetheless, the geographical structuring of mtDNA within species provides valuable information on biogeographical areas, ancient areas of hybridization, and unique histories of lineages within the H. eximia group. These results suggest that the combination of nuclear, mitochondrial, and spatial information can provide a more complete picture of ‘how evolutionary history played out’, particularly in cases where mitochondrial introgression is known to occur. © 2014 The Linnean Society of London     

Maintenance of species boundaries in three sympatric Ligularia (Senecioneae,Asteraceae) species

The key process in speciation concerns the formation and maintenance of reproductive isolating barriers between diverging lineages. Although species boundaries are frequently investigated between two species across many taxa, reproductive isolating barriers among multiple species (>2) that would represent the most common phenomenon in nature, remain to be clarified. Here, we use double digest restriction‐site associated DNA (ddRAD) sequencing to examine patterns of hybridization at a sympatric site where three Ligularia species grow together and verify whether those patterns contribute to the maintenance of boundaries among species. The results based on the RAD SNP datasets indicated hybridization Ligularia cyathiceps × L. duciformis and L. duciformis × L. yunnanensis were both restricted to F₁s plus a few first‐generation backcrosses and no gene introgression were identified, giving rise to strong reproductive isolation among hybridizing species. Moreover, hybrid swarm simulation, using HYBRIDLAB, indicated the RAD SNP datasets had sufficient discriminatory power for accurate hybrid detection. We conclude that parental species show strong reproductive isolation and they still maintain species boundaries, which may be the key mechanism to maintain species diversity of Ligularia in the eastern Qinghai‐Tibetan Plateau and adjacent areas. Moreover, this study highlights the effectiveness of RAD sequencing in hybridization studies.     

Historical introgression among the American live oaks and the comparative nature of tests for introgression

Introgressive hybridization challenges the concepts we use to define species and infer phylogenetic relationships. Methods for inferring historical introgression from the genomes of extant species, such as ABBA‐BABA tests, are widely used, however, their results can be easily misinterpreted. Because these tests are inherently comparative, they are sensitive to the effects of missing data (unsampled species) and nonindependence (hierarchical relationships among species). We demonstrate this using genomic RADseq data sampled from all extant species in the American live oaks (Quercus series Virentes), a group notorious for hybridization. By considering all species and their phylogenetic relationships, we were able to distinguish true hybridizing lineages from those that falsely appear admixed. Six of seven species show evidence of admixture, often with multiple other species, but which is explained by introgression among a few related lineages occurring in close proximity. We identify the Cuban oak as the most admixed lineage and test alternative scenarios for its origin. The live oaks form a continuous ring‐like distribution around the Gulf of Mexico, connected in Cuba, across which they could effectively exchange alleles. However, introgression appears highly localized, suggesting that oak species boundaries and their geographic ranges have remained relatively stable over evolutionary time.     

Is RAD‐seq suitable for phylogenetic inference? An in silico assessment and optimization

Inferring phylogenetic relationships between closely related taxa can be hindered by three factors: (1) the lack of informative molecular variation at short evolutionary timescale; (2) the lack of established markers in poorly studied taxa; and (3) the potential phylogenetic conflicts among different genomic regions due to incomplete lineage sorting or introgression. In this context, Restriction site Associated DNA sequencing (RAD‐seq) seems promising as this technique can generate sequence data from numerous DNA fragments scattered throughout the genome, from a large number of samples, and without preliminary knowledge on the taxa under study. However, divergence beyond the within‐species level will necessarily reduce the number of conserved and non‐duplicated restriction sites, and therefore the number of loci usable for phylogenetic inference. Here, we assess the suitability of RAD‐seq for phylogeny using a simulated experiment on the 12 Drosophila genomes, with divergence times ranging from 5 to 63 million years. These simulations show that RAD‐seq allows the recovery of the known Drosophila phylogeny with strong statistical support, even for relatively ancient nodes. Notably, this conclusion is robust to the potentially confounding effects of sequencing errors, heterozygosity, and low coverage. We further show that clustering RAD‐seq data using the BLASTN and SiLiX programs significantly improves the recovery of orthologous RAD loci compared with previously proposed approaches, especially for distantly related species. This study therefore validates the view that RAD sequencing is a powerful tool for phylogenetic inference.     

THE INTERFACE BETWEEN PHYLOGENETICS AND POPULATION GENETICS: INVESTIGATING GENE TREES,SPECIES TREES,AND POPULATION DYNAMICS IN THE PHYLLOPHAGA FRATERNA SPECIES GROUP

This study uses traditional and contemporary phylogenetic and population genetic analyses to assess the causes of discordance (i.e., lineage sorting and introgression) among mitochondrial and nuclear gene trees for a clade of eastern North American scarab beetles (fraterna species group, genus Phyllophaga). I estimated gene trees using individual and combined analysis of one mitochondrial and two nuclear loci in MrBayes , and inferred a species tree using a hierarchical coalescent approach based on all loci in the program Best . Because hybridization violates the assumptions of Best , I tested for introgression by comparing species monophyly between the mitochondrial and nuclear gene trees based on the prediction that cytoplasmic genomes introgress more readily than nuclear genomes. Haplotype exclusivity was identified using Bayesian tests of monophyly and the genealogical sorting index. I used the results of the phylogenetic analyses and monophyly tests to develop an explicit hypothesis of introgression that could be tested in the program IMa. Results from these analyses provided evidence for introgression across clades within the fraterna group. The tiered analytical approach used in this study demonstrated how the use of multiple methods can identify when assumptions are violated and methods are prone to yield misleading results.     

Radiation and reticulation: extensive introgressive hybridization in the carabid beetles Ohomopterus inferred from mitochondrial gene genealogy

 In the evolutionary process of an animal lineage, interactions in secondary contacts of differentiated populations and introgressive hybridization may play an important role. In the Japanese islands, the carabid subgenus Ohomopterus (genus Carabus) exhibits a marked differentiation in body size and genital morphology. Although geographical differentiation is apparent, two or three species usually coexist at many localities. Their reproductive isolation relies on body size differences, chemical cues for mate recognition, and a species-specific genital lock-and-key system. However, these isolation mechanisms are not always effective enough to prevent interspecific hybridization. An initial assessment of the species-level phylogeny with mitochondrial gene sequences revealed that the gene genealogy is highly inconsistent with the morphology-based taxonomy. A comparison of mitochondrial and nuclear gene genealogies showed that these are strongly incongruent with each other, while the nuclear gene genealogy is more consistent with traditional taxonomy, indicating the repeated occurrence of introgression of mitochondria across species. Here, two different cases of mitochondrial introgression among Ohomopterus species are described in detail, one for parapatric species and the other for sympatric species. First, mitochondrial haplotypes and sequences were studied in Carabus insulicola and three taxa parapatric with C. insulicola, at least two of which hybridize with C. insulicola naturally. Among the four species studied, directional introgressions of mitochondria across boundary zones were detected. Second, in the Mt. Kongo area in central Honshu, which harbors five species, introgression of mitochondria among four out of the five species was detected, despite the apparent absence of on-going natural hybridization. These inferred cases of mitochondrial introgression indicate that species interactions through hybridization could have played an important role at various stages in the evolution of Ohomopterus. Received: April 12, 2002 / Accepted: October 17, 2002 Acknowledgments I am grateful to Alfried P. Vogler for a long-lasting collaboration in the molecular phylogenetic study of Ohomopterus. R. Ishikawa, K. Kubota, M. Ujiie, Y. Takami, and F. Kusumoto have also collaborated at various stages of this study. Thanks are also due to K. Miyashita, T. Funakoshi, H. Fujimoto, T. Dejima, Y. Nagahata, T. Miyagawa, K. Yodoe, H. Kadowaki, S. Nakamine, Y. Oka, H. Tanaka, T. Tanabe, K. Kusakari, and T. Okumura for their care of specimens. Supported by grants-in-aid from the Japan Society for the Promotion of Science (Nos. 09640748, 11304056).     

Widespread decoupling of mtDNA variation and species integrity in Grammia tiger moths (Lepidoptera: Noctuidae)

Abstract. We investigate the diversity of the North American tiger moth genus Grammia Rambur (Lepidoptera: Noctuidae) by comparing mitochondrial DNA (mtDNA) ‘barcode’ fragments of cytochrome oxidase I with non‐molecular characters such as morphology, ecology, behaviour and distribution. Mitochondrial DNA genealogy is strikingly at odds with morpho‐species taxonomy for most of the 28 sampled species, as haplotypic polyphyly not only is taxonomically widespread, but involves multiple shared haplotypes among two to four species. Morpho‐ecological traits show that those species sharing haplotypes are often not closely related. Furthermore, high mtDNA divergences occur within species. Haplotypic variation is highly discordant with species taxonomy, but variation at a continental scale reveals significant geographic structuring of haplogroups, transcending morpho‐species boundaries. A nested clade analysis and comparison of non‐molecular with mtDNA data indicate that most discordance between mtDNA and taxonomy in Grammia is explained best by taxonomically and geographically widespread ongoing hybridization events resulting in mtDNA introgression. We hypothesize that broad areas of sympatry, interspecifically compatible genitalic structure, and species overlap in pheromone components facilitate hybridization, with disparate interspecies abundances promoting mitochondrial introgression. The molecular evolution of Grammia challenges the view that interspecific gene exchange occurs rarely and is restricted to recently diverged species. These results show the value of mtDNA in detecting cryptic hybridization, while highlighting the inherent dangers of drawing taxonomic conclusions based solely on mtDNA.     

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold‐resistant leaf beetles, Gonioctena quinquepunctata and G. intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD‐seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species‐specific SNPs (displaying a fixed difference between species) a priori and second by using model‐based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.     

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.     

Discordance between morphological and molecular species boundaries among Caribbean species of the reef sponge Callyspongia

Sponges are among the most species‐rich and ecologically important taxa on coral reefs, yet documenting their diversity is difficult due to the simplicity and plasticity of their morphological characters. Genetic attempts to identify species are hampered by the slow rate of mitochondrial sequence evolution characteristic of sponges and some other basal metazoans. Here we determine species boundaries of the Caribbean coral reef sponge genus Callyspongia using a multilocus, model‐based approach. Based on sequence data from one mitochondrial (COI), one ribosomal (28S), and two single‐copy nuclear protein‐coding genes, we found evolutionarily distinct lineages were not concordant with current species designations in Callyspongia. While C. fallax, C. tenerrima, and C. plicifera were reciprocally monophyletic, four taxa with different morphologies (C. armigera, C. longissima, C. eschrichtii, and C. vaginalis) formed a monophyletic group and genetic distances among these taxa overlapped distances within them. A model‐based method of species delimitation supported collapsing these four into a single evolutionary lineage. Variation in spicule size among these four taxa was partitioned geographically, not by current species designations, indicating that in Callyspongia, these key taxonomic characters are poor indicators of genetic differentiation. Taken together, our results suggest a complex relationship between morphology and species boundaries in sponges.     

Phylogeny of Dendroctonus bark beetles (Coleoptera: Curculionidae: Scolytinae) inferred from morphological and molecular data

Bark beetles in the genus Dendroctonus may attack and kill several species of coniferous trees, some of them causing major economic losses in temperate forests throughout North and Central America. For this reason, they have been widely studied. However, various aspects of the taxonomy and evolutionary history of the group remain contentious. The genus has been subdivided in species groups according to morphological, biological, karyological or molecular attributes, but the evolutionary affinities among species and species groups within the genus remain uncertain. In this study, phylogenetic relationships among Dendroctonus species were reassessed through parsimony‐based cladistic analysis of morphological and DNA sequence data. Phylogenetic inference was based on 36 morphological characters and on mitochondrial DNA sequences of the cytochrome oxidase I (COI) gene. Analyses were carried out for each dataset, as well as for the combined data analysed simultaneously, under equal and implied weights. According to the combined analysis, the genus Dendroctonus is a monophyletic group defined by at least three synapomorphic characters and there are four main lineages of varied composition and diversity within the genus. Within these lineages, several monophyletic groups match, to some extent, species groups defined by previous authors, but certain groups proposed by those authors are polyphyletic or paraphyletic.     

Molecular phylogenetic analyses of nuclear and plastid DNA sequences support dysploid and polyploid chromosome number changes and reticulate evolution in the diversification of Melampodium (Millerieae, Asteraceae)

Chromosome evolution (including polyploidy, dysploidy, and structural changes) as well as hybridization and introgression are recognized as important aspects in plant speciation. A suitable group for investigating the evolutionary role of chromosome number changes and reticulation is the medium-sized genus Melampodium (Millerieae, Asteraceae), which contains several chromosome base numbers (x = 9, 10, 11, 12, 14) and a number of polyploid species, including putative allopolyploids. A molecular phylogenetic analysis employing both nuclear (ITS) and plastid (matK) DNA sequences, and including all species of the genus, suggests that chromosome base numbers are predictive of evolutionary lineages within Melampodium. Dysploidy, therefore, has clearly been important during evolution of the group. Reticulate evolution is evident with allopolyploids, which prevail over autopolyploids and several of which are confirmed here for the first time, and also (but less often) on the diploid level. Within sect. Melampodium, the complex pattern of bifurcating phylogenetic structure among diploid taxa overlain by reticulate relationships from allopolyploids has non-trivial implications for intrasectional classification.     

Genome‐wide sequence information reveals recurrent hybridization among diploid wheat wild relatives

Many conflicting hypotheses regarding the relationships among crops and wild species closely related to wheat (the genera Aegilops, Amblyopyrum, and Triticum) have been postulated. The contribution of hybridization to the evolution of these taxa is intensely discussed. To determine possible causes for this, and provide a phylogeny of the diploid taxa based on genome‐wide sequence information, independent data were obtained from genotyping‐by‐sequencing and a target‐enrichment experiment that returned 244 low‐copy nuclear loci. The data were analyzed using Bayesian, likelihood and coalescent‐based methods. D statistics were used to test if incomplete lineage sorting alone or together with hybridization is the source for incongruent gene trees. Here we present the phylogeny of all diploid species of the wheat wild relatives. We hypothesize that most of the wheat‐group species were shaped by a primordial homoploid hybrid speciation event involving the ancestral Triticum and Am. muticum lineages to form all other species except Ae. speltoides. This hybridization event was followed by multiple introgressions affecting all taxa except Triticum. Mostly progenitors of the extant species were involved in these processes, while recent interspecific gene flow seems insignificant. The composite nature of many genomes of wheat‐group taxa results in complicated patterns of diploid contributions when these lineages are involved in polyploid formation, which is, for example, the case for tetraploid and hexaploid wheats. Our analysis provides phylogenetic relationships and a testable hypothesis for the genome compositions in the basic evolutionary units within the wheat group of Triticeae.     

From hybridization to introgression between two closely related sympatric ant species

Interspecific hybridization is becoming more frequent worldwide due to increasing global changes and translocations of organisms. For individual organisms, the most significant negative consequences are sterility or inviability of hybrid offspring. However, hybridization sometimes leads to fertile offspring, promoting introgression from one species into another. In such situations, hybridization can play a key role in evolution and speciation. Combining hypervariable DNA (microsatellites) and mitochondrial DNA markers with the use of several modeling methods allow an efficient detection of hybridization processes. The present study therefore investigates hybridization between two ant species, Tetramorium immigrans and T. caespitum, using multiple methods, and systematically comparing results with simulated data to ensure accurate identification of hybrids. Introgression was revealed both by backcross detection based on 14 nuclear microsatellite loci and by mitochondrial‐nuclear discordance based on comparison with mitochondrial DNA (cytochrome c oxidase subunit I). Results were spatially consistent, with hybrids located at latitudes where parental species are sympatric. The causes and consequences of hybridization and introgression between T. caespitum and T. immigrans remain to be further investigated, especially because T. immigrans could be an invasive species in France.     

Genetic diversity and evolutionary history of the Schizothorax species complex in the Lancang River (upper Mekong)

The genus Schizothorax (Cyprinidae), one of the most diverse genera of ichthyofauna of the Qinghai‐Tibetan Plateau (QTP), is a good candidate for investigating patterns of genetic variation and evolutionary mechanisms. In this study, sequences from the mitochondrial control region, the cytochrome b gene, and two nuclear genes were used to re‐examine the genetic diversity and investigate the evolutionary history of the Schizothorax species complex inhabiting the Lancang River. Three maternal clades were detected in the Schizothorax species complex, but frequent nuclear allele sharing also occurred among the three maternal clades. A discrepancy between topologies of mitochondrial and nuclear loci might result from introgression or/and incomplete lineage sorting. The divergence of the clades of the Schizothorax species complex was closely related to the Late Pliocene and Early Pleistocene orogenesis of the QTP and Southwest Mountains of China. Demographic analyses indicated that the species complex subsequently persisted in situ with stable populations during Pleistocene glacial cycling, which suggested that Pleistocene climate changes did not exert a remarkable influence on the species complex. Our study provides a comprehensive analysis of the genetic diversity and evolutionary history of the Schizothorax species complex in the Lancang River.