Sci. Bull. www.scibull.com DOI 10.1007/s11434-016-1104-0 www.springer.com/scp Article Life & Medical Sciences Diversification of Sisorid catfishes (Teleostei: Siluriformes) in relation to the orogeny of the Himalayan Plateau Chuanjiang Zhou • Xuzhen Wang • Xiaoni Gan • Yaping Zhang • David M. Irwin • Richard L. Mayden • Shunping He Received: 17 December 2015 / Revised: 15 March 2016 / Accepted: 31 March 2016 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2016 Abstract Abstract Sisorid catfishes are primarily limited traits that were presumably in response to environmental in distribution to rivers of the Himalayan region and pressure involving the rapid flowing river system that were Tibetan Plateau. These species have external morphologies generated during the uplift of the Tibetan Plateau (UTP). that are adapted for extremely fast-flowing riverine sys- Molecular dating indicates that the Chinese sisorids and the tems. Given the diversity of the group and the above glyptosternoids originated at the later Miocene (*10.9–9.8 qualities of these catfishes, this lineage serves as an ideal Mya), and with further biogeographic analyses indicates group for inferring the geological history of this region that the species of Sisoridae likely originated from a widely based on their phylogenetic relationships reflecting evolu- distributed ancestor. Moreover, the divergence of the tionary history. We sequenced the complete mitochondrial Sisoridae in China can be divided into two phases consis- genome and four nuclear genes of representative sisorids tent with the UTP. All of these results indicate that the distributed across river systems in China. Phylogenetic diversification and dispersal events in this lineage occurred analyses strongly support the monophyly of the Sisoridae as a result of drainage systems formed during and after the and the glyptosternoids. An analysis of the reconstructed UTP in the late Miocene and Quaternary periods. ancestral states derived from inferred genealogical rela- tionships suggests that the evolution of this lineage was Keywords Phylogeny Á Diversification and dispersal Á accompanied by convergent evolution in morphological Biogeography Á Divergence time estimation Á Tibetan Database linking All the data used in this manuscript are archived in Plateau Á Sisoridae Á Catfish GenBank (Table S1). Electronic supplementary material The online version of this article (doi:10.1007/s11434-016-1104-0) contains supplementary material, which is available to authorized users. C. Zhou Á X. Wang Á X. Gan Á S. He (&) C. Zhou Key Laboratory of Aquatic Biodiversity and Conservation of College of Fisheries, Henan Normal University, Chinese Academy of Sciences, Institute of Hydrobiology, Xinxiang 453007, China Chinese Academy of Sciences, Wuhan 430072, China e-mail: [email protected] D. M. Irwin Department of Laboratory Medicine and Pathobiology, C. Zhou University of Toronto, Toronto, ON, Canada Key Laboratory of Freshwater Fish Reproduction and Development (Southwest University), Ministry of Education, D. M. Irwin School of Life Science, Southwest University, Banting and Best Diabetes Centre, University of Toronto, Chongqing 400715, China Toronto, ON, Canada C. Zhou Á Y. Zhang Á D. M. Irwin R. L. Mayden State Key Laboratory of Genetic Resources and Evolution, Department of Biology, 3507 Laclede Ave., Saint Louis Kunming Institute of Zoology, Chinese Academy of Sciences, University, St. Louis, MO 63103, USA Kunming 650223, China 123 Sci. Bull. 1 Introduction China, especially the relationships within the glyptoster- noids, to serve as an evolutionary backdrop for two other It is believed that an organism’s habitat plays a crucial role objectives. These included the reconstruction of the in both adaptation and diversification, and that changes in ancestral states of morphological characters in this lineage habitats most frequently associated by species ultimately and test hypotheses concerning the morphological spe- impact their distributions and diversification of the biota in cialization and speciation relative to the uplift of the a particular region [1]. In turn, information on the phylo- Himalayas. Molecular clock approaches were used to infer genetic relationships of species within the context of his- divergence dates for this molecular phylogeny to test torical biogeography of a lineage reflects a history of a whether the diversification and evolution of the sisorids region where a species, or lineage, occurs [2]. The conti- were associated with the UTP and, if so, to examine the nental collision between India and Asia resulted in the landscape of adaptive evolution for this lineage in response uplift of the Tibetan Plateau (UTP) in the late Eocene to the uplift events. [3, 4]. Since this estimated time of impact, the continuing UTP has impacted much of the fauna of the region (e.g., fish [5–7], frogs [8] and pika [9]), as well as the climate 2 Materials and methods [10] and river flows and their patterns of connectivity in this region [11]. 2.1 Samples and genomic DNA extraction Glyptosternoid fishes are freshwater catfish species of the family Sisoridae (Order Siluriformes) and form a lin- All experimental protocols were approved by the ethics eage mainly distributed in the incredibly fast-flowing rivers committee of the Institute of Hydrobiology at the Chinese around the Tibetan Plateau and eastern Himalayas [6]. Academy of Sciences. Twelve sisorid species and their Glyptosternoids are extremely well adapted to the rapidly close relatives [6, 7], Liobagrus anguillicauda (Siluri- flowing water environment and show a series of adaptive formes: Amblycipitidae), Cranoglanis bouderius (Siluri- structures to aid in their maintaining position in extreme formes: Cranoglanididae) and Ictalurus punctatus currents [6, 7]. Sisorid species distributed in China are (Ictaluridae), were used in this study (sample information currently classified in 13 genera. All of these genera, provided in Table S1). Total genomic DNA was extracted except Bagarius, Gagata, Pseudecheneis and Glyptotho- from muscle tissue using the standard phenol–chloroform rax, are glyptosternoids [12–15]. While several studies extraction technique with slight modification [22]. have investigated the phylogeny, biogeography and evo- lution of the glyptosternoid species, these reports have 2.2 PCR amplification, cloning and sequencing generated inconsistent phylogenetic results, and the relative positions of Pseudecheneis, Euchiloglanis, Exostoma, Complete mitochondrial genome sequences were generated Glaridoglanis and Glyptosternon remain unclear from overlapping fragments using 22 pairs of amplification [6, 7, 13, 14, 16]. The current conflicting and inconclusive primers and standard PCR methods with genomic DNA. hypotheses of relationships of species are most likely a Amplification primers used were previously described [15] consequence of limited and different taxon and character and were based on conserved portions of the complete sampling, as described by Mayden et al. [17], Chen et al. mitochondrial genome sequences of Corydoras rabauti [18] and Mayden and Chen [19] for inferring the phylo- (GenBank AB054128), Pseudobagrus tokiensis genetic relationships of Cypriniformes, and also docu- (AB054127), Liobagrus obesus (NC_008232), Cra- mented by Hillis [20] and Hillis et al. [21]. Due to the noglanis bouderius (NC_008280), Ictalurus punctatus unique distribution and morphology of fishes of this lin- (AF482987) and Danio rerio (AC024175). Sequences of eage, the relationships between speciation, evolution and primers are listed in Table S2; the 22 primer pairs amplify biogeography of these species, and the UTP has become an overlapping segments, yielding the complete mitochondrial area of intense research [1, 6, 7, 12–14, 16]. genomes of the targeted species. PCR reactions were per- In this study, we sequenced complete mitochondrial formed in 50 lL reaction volumes containing 5 lL109 genomes from 13 species, as well as four nuclear genes PCR buffer (Takara, Dalian, China), 4 lL 2.5 mmol/L from 12 of these 13 species, which represent all of the dNTPs (Takara) 2 lL 10 mmol/L each primer, 0.5 lL5U/ genera of the sisorid fishes in China and their close relative lL Taq polymerase (rTaq, Takara) and 1.0 lL containing Liobagrus anguillicauda, and we also included in our approximately 40 ng of template DNA. PCR cycle condi- analysis the Ictalurus punctatus and Cranoglanis bouderius tions were as follows: pre-denaturation at 94 °C for 3 min, as they are close relatives according to Guo [6] and Peng followed by 35 cycles of denaturation at 94 °C for 30 s, [7]. The objective of this study was to infer a robust phy- annealing at 54–56 °C for 30 s, extension at 72 °C for logenetic framework of relationships for sisorid catfishes of 2 min and a final extension at 72 °C for 10 min. PCR 123 Sci. Bull. products were purified after separation by electrophoresis mitochondrial protein-coding genes as separate partitions; in a 1.5 % agarose gel using a purification kit (Promega, (4) no partitions of the 12 mitochondrial protein-coding Beijing, China). Purified DNA fragments were used for genes; (5) no partitions of the four nuclear genes and (6) direct sequencing, and fragments that did not generate four partitions, with each of the four nuclear genes having a reliable sequence (due to repeats of G or T) were subcloned separate partition. PartitionFinder [32] was also used to into a PMD18-T vector (Takara) and then sequenced. In select the best schemes and comparisons with the above addition to