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 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 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

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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 and 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 , , 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, , Complete mitochondrial genome sequences were generated Glaridoglanis and 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 the mtDNA, we chose four nuclear genes partitionings. Optimum models (see Table S3) were iden- [rhodopsin (RH), glycosyltransferase (Gylt), myosin tified with MODELTEST 3.7 [33]. (myh6), heavy polypeptide 6; si:ch211-105n9.1-like pro- Phylogenetic relationships were inferred using maxi- tein gene (Ptr, a potential homolog of Ptchd4)] for mum likelihood (ML) and Bayesian interference (BI) sequencing due to their successful performance in previous methods based on the above partition strategies. Maximum phylogenetic studies [23, 24]. PCR conditions used for likelihood trees were obtained from PHYML [34] with 200 amplification of the nuclear genes were identical to those in bootstrap replicates and from RAxML [35, 36] with 1000 Li et al. [23], with primer sequences listed in Table S2. bootstraps. Bayesian analysis was conducted using Purified and cloned products were sequenced at a com- MrBayes 3.1.2 [37] with four independent Metropolis- mercial facility (BGI, Beijing, China) with the same pri- coupled Markov chain Monte Carlo (MCMC) chains run- mers used for PCR. Additional sequencing primers were ning simultaneously (temperature = 0.5, three heated and designed for the middle of sequences when entire ampli- one cold to enhance mixing) for 107 generations and fication products could not be sequenced using just forward sampling one tree per 100 generations. The first 50,000 and reverse primers. trees were discarded as part of the burn-in procedure, and model parameters were set according to the MODELTEST 2.3 Sequence analysis results (Table S3). Bayesian analysis was repeated starting from different random trees twice. In addition, maximum DNA sequences were analyzed using Lasergene version parsimony analyses were performed on mitochondrial 7.0 (DNASTAR). Contig assembly was performed using protein-coding genes using PAUP* [38] (heuristic search, SeqMan. Multiple sequence alignment was performed tree bisection and reconnection, TBR). Weighted analyses using MegAlign. All protein-coding, rRNA and tRNA were performed in which the third codon positions were genes were identified by comparison with known sequen- underweighted, in order to minimize the effect of transi- ces from other catfish taxa, such as Ictalurus punctatus tions that may accumulate at a high frequency at the third [25], Cranoglanis bouderius [26], Pseudobagrus tokiensis codon. The weightings of the first and second codon [27] and Pangasianodon gigas [28]. All tRNA genes were positions have been progressively increased over the third confirmed with tRNAscan-SE Search Server available codon positions using two differential weighting strategies online (http://lowelab.ucsc.edu/tRNAscan-SE/). Multiple of the three codon positions (2:2:1, 2:4:1) given the fact alignments were conducted for the 12 protein-coding (ND6 that transitions are more frequent than transversions [39]. was excluded due to its unusual substitution pattern Similar analyses were justified and performed by Hillis [7, 29–31], 22 tRNAs and two rRNA genes using CLUS- et al. [40] and Barker and Lanyon [41]. A single weighting TAL W. Ambiguous regions (including highly variable scheme was used for downweighting transitions regions of tRNA loops and rRNA and noncoding control (transversions were weighted two or three times over regions) were excluded from analysis. Nuclear gene transitions). To explore possible reasons for differences in sequences were submitted to a BLAST search of GenBank phylogenetic topologies generated by the four nuclear and translated into amino acids to ensure that the target genes, the above methods were also used to construct the sequences had been amplified and that the sequences were phylogenetic relationships using data for each of the three intact. different codon positions for the four nuclear genes. In comparisons of different phylogenetic hypotheses 2.4 Phylogenetic analyses generated depicting relationships of sisorid species, site- wise log-likelihoods were calculated using CONSEL [42] For phylogenetic analysis, we used the following partition with topologies generated by PAUP* [38], and the p value strategies: (1) four partitions, with the 12 mitochondrial for each hypothesis was calculated using the approximately protein-coding genes, 22 tRNAs genes, two rRNA genes unbiased (AU) [43] and the Shimodaira–Hasegawa (SH) and nuclear genes being separate partitions; (2) no parti- [44] tests. tions, with the 12 mitochondrial protein-coding genes, 22 The compositional heterogeneity test implemented in tRNAs genes, two rRNA genes and nuclear genes treated Statio (http://homes.bio.psu.edu/people/faculty/Nei/ as one group; (3) 12 partitions, with each of the 12 software.htm), using the method of Rzhetsky and Nei 123 Sci. Bull.

[45], was used to examine possibilities of phylogenetic 2.7 Biogeographic analysis correlations. To examine whether nucleotide substitution saturation affected phylogenetic tree reconstruction, it was To explore the historical processes leading to the extant tested using the index of substitution saturation (Iss) of Xia distributions of the Chinese Sisoridae, we performed a et al. [46] implemented in DAMBE 5.2.13 [47]; results are reconstruction using a likelihood method implemented in shown in Table S4. the program Lagrange20110117 [55]. The rivers containing species of Sisoridae were coded as: A (Tsangpo), B (Brahmaputra), C (Irrawaddy), D [Salween (Nujiang)], E 2.5 Molecular dating [Lancangjiang (Upper River)], F [Jinshajiang (Upper Yangtze)] and G (). The dispersal probabil- The divergence times for the Sisoridae were determined ity between rivers was set to 1.0. Parsimony-based and using the protein-coding genes with the Bayesian molec- Bayesian analyses were performed using S-DIVA (RASP) ular dating program PAML/multidivtime (MULTI- under the default settings [56]. Our Bayesian phylogenetic DISTRIBUTE program), following instructions in the tree (Fig. 1), with outgroups removed, was used for all program manual [48–50]. Temporal constraints were from biogeographic investigations. the fossil age of (Miocene to Pliocene, 5.3–1.8 Mya, node d in Fig. 1;[51]) and the results from Peng et al. [7] for the root of glyptosternoids (node g, 3 Results Fig. 1; 10 Mya) and Guo et al. [6] for the root of the phylogeny (node 1, Fig. 1; 19–24 Mya) for its limited fossil 3.1 Sequence characteristics record in this lineage and the different approaches in dating this linage [6, 7]. Divergence time estimates were deter- Our final combined DNA alignment for the 15 taxa con- mined using a relaxed molecular clock approach from tains 17,007 nucleotide sites for mitochondrial and nuclear MCMCTree [52], with the ages and taxa identified above genes, and is summarized in Table S1. The data include [5–7]. Time estimates were conducted under the indepen- 10,174 bp of mitochondrial protein-coding sequence, dent rates model, with the likelihood function calculated 2259 bp of ribosomal RNA sequence (12S rRNA and 16S (usedata = 1 seed =-1), or not (usedata = 0). Diver- rRNA), 1472 bp of tRNA, 819 bp of glycosyltransferase gence time was sampled once every 2 iterations, after an (Gylt), 732 bp of myosin heavy polypeptide 6 (myh6), initial burnin of 2000 iterations, until 20,000 samples were 720 bp of Ptr and 831 bp of RH. Of the aligned bases, gathered. Convergence of the chains to a stationary dis- 10,455 bp were invariant with 6543 bp being variable, of tribution was examined using TRACER V1.4 [53], and the which 4697 were parsimony informative. All sequences are simulation was repeated once (usedata = 1, and the seed deposited in GenBank, and their accession numbers are for second run was the result of the first analysis) to con- listed in Tables S1 and S6. firm that the results were very similar between runs. 3.2 The phylogeny of the Chinese sisorids Results are shown in Table S5. and the monophyly of glyptosternoids

2.6 Ancestral morphological state reconstruction Results of the partitioned and unpartitioned Bayesian and ML nucleotide analyses (PHYML and partitioned RAxML) To examine the evolution of the external morphology and of the mitochondrial and mitochondrial ? nuclear feeding habits of sisorid species, we reconstructed ancestral sequence datasets showed a marked consistency in topo- states for 12 morphological characters of the Chinese logical congruence, differing only in the support values for Sisoridae. These included the following labial fold state 1 some nodes (Fig. 1). MP (weighting positions first codon/ (presence or absence of a labial fold); 2 (continuous or second codon/third codon = 2:2:1, Fig. S1) based on the discontinuous posterior labial fold); 3 (tooth type); 4 (tooth mitochondrial protein-coding genes yielded similar results, band shape on the upper jaw); 5 (tooth band shape on the while relationships from the MP-based analysis using lower jaw); 6 (tooth band extended or not on the upper weightings of the three codon positions (weighting posi- jaw); 7 (whether branchiostegal membranes are absolute); tions first codon:second codon:third codon = 2:4:1, 8 (whether body type is depressed); 9 (presence of adherent Fig. S2) differed only slightly. These results are consistent apparatus on the chest); 10 (number of pectoral fin rays); with the phylogeny generated with morphological data 11 (width of gill opening) and 12 (feeding habit). The based on the osteological characteristics by He [14], but ancestral states for these characteristics were estimated by differed from the results derived from analyses of only maximum likelihood and maximum parsimony using nuclear gene sequences, either with or without partitioning Mesquite [54]. (Figs. S3, S4). Support values, however, for many of the

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Liobagrus anguillicauda

a Ictalurus punetaus b Cranoglanis bouderius

Pseudechenneis sulcatus

Bagarius yarrelli

c d Gagata dolichonema e trilineatus Glyptosternoids f Glyptosternum maculatum

Exostoma labiatum g Glaridoglanis andersonii h kamengensis i j Euchiloglanis kishinouyei k macropterus 0.1 substitutions per site l Pareuchiloglanis gracilicaudata m Pseudexostoma yunnanensis

a b c d e f g h i j k l m

PHYML 195/200 200/200 - 200/200 200/200 119/200 200/200 98/200 200/200 200/200 200/200200/200 200/200 RAxML 100 100 - 100 100 94 98 - 99 100 100 100 100

BPA - 100 100 100 100 100 100 82 100 100 100 100 100

UBPA - 100 100 100 100 100 100 97 100 100 100 100 100

Fig. 1 Phylogenetic relationships of species of Sisoridae sampled from the wide region of the Tibetan Plateau and Himalayan regions in China. Relationships were inferred from an analysis of mitochondrial genomes and four nuclear genes sequences [RH: rhodopsin, Gylt: glycosyltransferase, myh6: myosin heavy polypeptide 6; Ptr (si:ch211-105n9.1-like protein gene, a potential homolog of Ptchd4)]. Letters identify branches that have branch support values from PHYML, RAxML, partitioned Bayesian and unpartitioned Bayesian (UBA) methods, shown in the table below the phylogeny. The table shows branches that were unresolved. The scale bar represents 0.1 substitutions per site. Image of Ictalurus punctatus is from the Internet (http://www.fao.org/fishery/culturedspecies/Ictalurus_punctatus/zh), and other images are from Fauna Sinica nodes on the nuclear gene phylogeny were lower than those variations in effective population sizes that would impact derived from trees derived from analyses of the mito- nuclear gene phylogenies greater than phylogenies gener- chondrial data. We believe that the inferences generated ated from mitochondrial sequences [58]. Furthermore, from nuclear gene sequence data are likely misleading for phylogenies generated from the mitochondrial sequences, the following reasons: (1) There is significant base com- in contrast to those of nuclear sequences, consistently position heterogeneity between the four nuclear genes (e.g., support the monophyly of Sisoridae and glyptosternoids, as third position of codons of the four genes in the combined found by previous researchers [6, 7, 13, 14]. Our CONSEL data, Table S4), which can generate incorrect tree topolo- results also suggested that the results based on the mito- gies [57]; (2) Trees based on synonymous and nonsyn- chondrial or the mitochondrial ? nuclear dataset are more onymous sites were not congruent, suggesting the acceptable and yield topologies with higher support values possibility of convergent evolution (results not shown). (Table S7) [43, 44]. We therefore used the phylogeny Synonymous sites were not saturated (Table S4); and (3) generated from the mitochondrial or mitochondrial ? nu- incomplete lineage sorting may have occurred due to clear sequence datasets for the remainder of our analyses.

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Phylogenies generated by the mitochondrial and the 3.4 Molecular dating mitochondrial ? nuclear datasets reveal the monophyly for Chinese Sisoridae and glyptosternoid catfishes with very Little difference was found for molecular dating between high support (PP = 1.00 and BP = 100, Fig. 1). In these the two likelihood values obtained from baseml analyses, Pseudecheneis sulcatus forms the sister group to (ln L =-67,727.334885) and estbranches analysis other Chinese sisorids, and glyptosternoid and non-glyp- (ln L =-67,871.346773) [50]. Using the MULTI- tosternoid fishes form sister clades with high support values DIVTIME and a relaxed clock analysis, Chinese Sisoridae (Fig. 1). Glyptosternum maculatum forms the basal sister were estimated to have originated in the late Miocene group to all other glyptosternoids. The placement of Ex- (&11.9 Mya), the glyptosternoids in late Miocene (*9.8 ostoma labiatum has long been controversial [6, 7]; herein, Mya), and the specialized glyptosternoids, such as Cre- the forms the basal sister group to remaining glyp- teuchiloglanis kamengensis, Euchiloglanis kishinouyei, tosternoids, at the apex of the glyptosternoid lineage Par- Oreoglanis macropterus, Pareuchiloglanis gracilicaudata, euchiloglanis gracilicaudata and Pseudexostoma and Pseudexostoma yunnanensis, between the Pleistocene yunnanensis, and this clade was sister to Oreoglanis mac- and Holocene (Fig. 3; Table S4). Results from MCMCTree ropterus. Pareuchiloglanis is not monophyletic; rather, were similar to the results of MULTIDIVTIME (Table S4), independent lineages of this artificial genus are sister but revealed a wider 95 % confidence interval. The larger groups to other unrelated clades. One is the most closely confidence interval may be due to the differences in the related to a clade formed by Euchiloglanis kishinouyei, estimations by MCMCTree, which uses soft bounds [52] Oreoglanis macropterus and Pseudexostoma yunnanensis, and user guide for PAML, compared to MULTIDIVTIME, while the other is sister to Pseudexostoma yunnanensis. which uses hard bounds. Since calibration information is These results are consistent with those of He et al. [13]. set using a wide range and only one is available, the results of these analyses must have wider 95 % confidence inter- 3.3 Ancestral state reconstruction vals. As such, it is our opinion that results from the MULTIDIVTIME estimations are likely more accurate. A total of 12 morphological and biological traits were Below, to be concise, discussions are based on results from successfully evaluated in reconstructed ancestral states MULTIDIVTIME. (Fig. 2). Among these characters, the states of the posterior labial fold vary from absent to a discontinuous posterior 3.5 Biogeographic analysis labial fold, and a continuous posterior labial fold (Fig. 2, the posterior labial fold states a, b and c). The upper tooth Likelihood, parsimony and Bayesian methods were used to band varies along a continuum from not being separated to reconstruct the historical ranges of the hypothetical separate. The numbers of pectoral fin rays range from 7 to ancestors of these fishes, all of which produced similar 9 in outgroups and 9 to 20 in ingroups, and the width of the results. Ancestral lineages of species of Sisoridae and gill opening decreases. Tooth type did not reveal any glyptosternoids examined in this study occurred over a consistent direction of change, although most of the species large region (Fig. 4, node 23 and node 22). More details are have pointed teeth; however, the specialized provided in Table S8 and Fig. S5. species(Glyptosternum maculatum, Creteuchiloglanis kamengensis (the former Pareuchiloglanis kamengensi, [15]), Euchiloglanis kishinouyei, Pareuchiloglanis gracil- 3.6 Origin and expansion of Sisoridae icaudata, Glaridoglanis andersonii, Oreoglanis macro- and glyptosternoids pterus, Pseudexostoma yunnanensis) possess different types of teeth. These include Exostoma-like teeth (pointed Three different explanations have been suggested for the teeth and spade-like teeth in the upper and lower jaws: extant distribution pattern of the species of Sisoridae in Glyptosternum maculatum, Creteuchiloglanis kamengen- China [12, 13, 60]. Phylogenetic resolution of clades sis, Euchiloglanis kishinouyei, Pareuchiloglanis gracili- herein, together with biogeographic results, tends to cor- caudata), incisiform teeth (Glaridoglanis andersonii), roborate He’s [13, 60] explanation (Fig. 1) and the distri- pointed teeth in the upper jaw and spade-like teeth in the butions of the species. Our analysis suggests that the lower jaw (Oreoglanis macropterus), or pointed teeth in species Sisoridae and the glyptosternoids originated from a the medial area with spade-like teeth in the later area of the widely distributed ancestor, which were then isolated into upper jaw and lower jaw (Pseudexostoma yunnanensis). the different river basins during the UTP. Following these We hypothesize these are correlated with their feeding events, species or clades then spread to downstream por- habits (pointed-form: fish and arthropod; shovel-form: tions of these rivers (Mekong, current Yangtze, Yuanjiang, algae; Exostoma-like tooth: both algae and arthropod) [59]. Pearl River and Salween rivers; Figs. 3, S5). 123 Sci. Bull.

Pectoral fin ray number Pseudechenneis sulcatus 10–12

Bagarius yarrelli 11 99/97/99/99/94 Gagata dolichonema 11 99/98/99/99/99 94/96/99/98/94 Glyptothorax trilineatus 9–11

Glyptosternum maculatum 11

95/91/99/90/ Exostoma labiatum 10–11

99/70/99/73/ Glaridoglanis andersonii 10–11

99/90/99/74/ Creteuchiloglanis kamengensis 14–16

99/92/93/71/94 Euchiloglanis kishinouyei 12–14

99/83/50/68/99 Oreoglanis macropterus 18–20

98/57/94/96/99 Pareuchiloglanis gracilicaudata 13–15 99/54/99/99/99 Pseudexostoma yunnanensis 16–18

(a) (b) (c) (d) (e) (f)

No posterior labial fold Discontiguous posterior labial fold Separated tooth band in the upper jaw

Undivided tooth band in the upper jaw Have posterior labial fold Contiguous posterior labial fold

Large gill-opening Moderate gill-opening Small gill-opening

Fig. 2 Ancestral state reconstructions for five traits found in Chinese species of Sisoridae. Ancestral state reconstruction was generated using phylogeny from Fig. 1 (outgroups removed). The five morphological traits include: a presence or absence of posterior labial folds (open triangle = no posterior labial fold; red = have posterior labial fold), b continuous or discontinuous posterior labial folds (orange moon = discontinuous posterior labial folds; blue moon = continuous posterior labial folds), c Tooth band in upper jaw (red five-pointed star = separated tooth band in the upper jaw; open five-pointed star = undivided tooth band in the upper jaw), d Extent of gill opening (blue circle = large gill opening; orange circle = moderate gill opening; cyan circle = small gill opening), and e Pectoral fin ray number (numbers indicated after species name). The numbers to the right of the internodes are the proportional of likelihoods for the five traits listed in the same order. The last proportional likelihoods have different colors to indicate different states [black = pectoral fin ray number (PFRN) \ 10; red = PFRN 10–12; blue = PFRN [ 12]

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Pseudechenneis sulcatus Outgroup Bagarius yarrelli 11.9(10.1,13.2) 8.4(6.5,9.8) Gagata dolichonema 3.3(1.4,4.6)

10.9(9.3,11.9) Glyptothorax trilineatus

Glyptosternum maculatum

9.8(8.2,11.0) Exostoma labiatum

9.4(7.8,10.6) Glaridoglanis andersonii

7.9(6.3, 9.2) Creteuchiloglanis kamengensis

1.6(1.2,2.1) Euchiloglanis kishinouyei

0.9(0.6,1.2) Oreoglanis macropterus 0.6(0.4,0.9) 0.1 Pareuchiloglanis gracilicaudata 0.5(0.3,0.8) Pseudexostoma yunnanensis 14 13 12 11 109 8 7 6 5 43210 mya A B C D A: Late Miocene B: Pliocene C: Pleistocene D: Holocene

Fig. 3 (Color online) Time tree for Chinese Sisoridae. The tree topology derived from MULTIDIVTIME is consistent with the Bayesian inference shown in Fig. 1. Branch lengths are proportional to divergence times. Numbers to the right of nodes are the estimates of the mean divergence times (in Mya). Numbers in parentheses represent 95 % confidence intervals

4 Discussion in only the Yangtze River [59]. In previous studies, Euchiloglanis kishinouyei was considered to be closely Different analytical strategies using a dataset of complete related to Glyptosternum maculatum, because of shared mitochondrial genomes or mitochondrial ? nuclear external characters such as having large gill openings, tooth sequences resulted in robust and consistent phylogenetic bands extending on both sides of the upper jaw, and the low relationships, all with high support values, for the species number of ramified pectoral fins [61]. Given the likely high of Sisoridae sampled in China (Fig. 1). These inferred degree of selection on external morphological features in relationships do, however, differ from that of Peng et al. [7] the extreme high gradient of Himalayan rivers, the conflict at some nodes. However, estimates of sister-group rela- between the hypotheses derived from the above morpho- tionships elucidated by Peng et al. [7] were derived only logical comparisons and the current genomic findings may from mitochondrial Cyt b sequences [7]. Our results, be the result of convergent evolution. however, are consistent with those of He [13], based on 60 Our analysis of 12 morphological characters in com- osteological characters and those of Guo et al. [6] (based parison to phylogenetic results of species of Sisoridae on Cyt b and 16S rRNA). As found previous in studies resolved herein reveal extensive homoplasy of external [6, 7], Pseudecheneis sulcatus forms the sister group of the features, suggesting independent ecological adaptations, glyptosternoid lineage (Fig. 1). Glyptosternum maculatum high gradient and fast-water habitats of the UTP. Most is the sister group to the remaining glyptosternoid taxa, and species of Sisoridae from China and all glyptosternoids phylogenies based on molecular data and the interior possess adaptive features for decreased water resistance anatomical morphological characters are in agreement, and increased adherence to smooth stones, including with the exception of Euchiloglanis kishinouyei distributed depressed heads and bodies and protruding pectoral fins

123 Sci. Bull. with pinnate rugae along their rims [59]. The tendency to sampled from China can be divided into two phases. The lack continuous posterior labial folds is thought to be an first phase began during the late Miocene (ca. 10.9 Mya) adaptation in the glyptosternoids for the fast-running and lasted until approximately 8 Mya. Cladogenetic events streams [59] that likely evolved in ancestral lineages during leading to ancestral lineages of the species Pseudecheneis the UTP. The evolution of a labial fold in glyptosternoids is sulcatus, Bagarius yarrelli, Glyptosternum maculatum, hypothesized to provide greater adherence to smooth Exostoma labiatum and Glaridoglanis andersonii origi- stones. It is our hypothesis that the various modifications in nated during this phase. This view is supported by the the characters identified above were evolutionary adapta- results of several previous studies. For example, Harrison tions in this lineage for the torrential waters of this region et al. [63] suggested that the rapid uplifting, and uplifting during drainage captures and the UTP. Thus, many of these of southern Tibet, began approximately 20 Mya and that characters may, as reconstructed herein, represent conver- the area attained its present elevation by 8 Mya. Sun et al. gences. This evolutionary scenario provides an outstanding [64] also suggested that the uplift that occurred between opportunity for detailed studies of convergences in mor- 13.7 and 9 Mya as an important tectonic event, at least phologies, in unrelated species, to similar environments along the northern edge. The rapid increase in the mean [62]. elevation of eastern Tibet was hypothesized from investi- The earliest recorded fossil for a species of Sisoridae is gations by Clark et al. [65] and between 9 and 13 Mya. Bagarius yarrelli from the sub-Himalayan Siwalik Hills, a These views all support results and hypothesis of the cur- formation dating from the Miocene to the Pliocene [51]. rent study. Our molecular dating results indicate that a cladogenetic The second phase of the UTP occurred from the middle event involving Bagarius yarrelli may have occurred dur- Pliocene to 0.5 Mya, especially in the Quaternary period. ing the late Miocene (ca. 8.4 Mya, 95 % CI 6.5–9.8 Mya), Some lineages or specialized species, such as Cre- earlier than suggested by Hora [12] (Pliocene). Similarly, teuchiloglanis kamengensis, Euchiloglanis kishinouyei, most recent common ancestor of glyptosternoid species Oreoglanis macropterus, Pareuchiloglanis gracilicaudata may also have originated at the late Miocene (9.8 Mya, and Pseudexostoma yunnanensis resulted from cladoge- 95 % CI 8.2–11.0 Mya). The dates proposed in this netic events during this phase. These species were highly hypothesis are consistent with those proposed by Peng (ca. adapted to the unique flowing waters in terms of their 6–8 Mya, [7]), but later than those reported by Guo (19–24 external morphologies (e.g., more depressed head and body Mya, [7]). The divergence of the species of Sisoridae and larger numbers of ramified pectoral fins). Furthermore,

(CD) Node 13 Pseudexostoma yunnanensis

Node 14 (E) Pareuchiloglanis gracilicaudata

Node 15 (C) Oreoglanis macropterus

Node 16 (F) Euchiloglanis kishinouyei

Node 17 (ABCDE) Creteuchiloglanis kamengensis

Node 18 (AC) Glaridoglanis andersonii

Node 19 (ABC) Exostoma labiatum

(AB) Glyptosternum maculatum Node 22 (E) Node 20 Gagata dolichonema

Node 21 (CD) Node 23 Glyptothorax trilineatus

(DE) Bagarius yarrelli

(ABCDEG) Pseudechenneis sulcatus

Fig. 4 The known distribution of Chinese Sisoridae. The terminal letters represent the known distribution of Chinese Sisoridae in different rivers: A = Tsangpo, B = Brahmaputra, C = Irrawaddy, D = Salween (Nujiang), E = Lancangjiang (Upper Mekong River), F = Jinshajiang (Upper Yangtze), G = Ganges 123 Sci. Bull. hypotheses formulated herein are also consistent with the Acknowledgments We thank Drs. Xianguang Guo, Xingjian Yue, proposed recent rapid uplift of the plateau, occurring Zuogang Peng, Dengqiang Wang, Jianjun Gao (Yunnan University), Ming Zou, Wenjing Tao, Xiaoping Wang (Yunnan University) and approximately 3.6 Mya. This was accompanied by the Beide Fu and Meiling Yu for assistance in collecting species, pro- formation of the largest glacier in the northern hemisphere, viding tissues, performing experiments, data analysis and helpful rearrangements of river systems prior to that time and the comments on the manuscript. We thank Dr. Peng Zhang (Sun Yat-Sen formation of a particular river system around the Tibetan University) for his constructive suggestions on the manuscript. We thank the two anonymous reviewers for their constructive suggestions Plateau and eastern Himalayas during this period improving the manuscript. This work was supported by the National [11, 66, 67]. Natural Science Foundation of China (31090254, 30770300 and An ancestral Sisoridae species is predicted to have been U1036603), the Chinese Academy of Sciences (KSCX2-EW-Q-12) widely distributed throughout the eastern Himalayas and and the endowment of William S. Barnickle, Saint Louis University, St. Louis, Missouri, USA. the Tibetan areas around the Oligocene–Miocene boundary (19–24 Mya, [6]). These areas include what are now Nepal, Conflict of interest The authors declare that they have no conflict of India and China (Tibet and Yunnan). 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