SCIENCE CHINA Life Sciences • RESEARCH PAPER • September 2012 Vol.55 No.9: 761–773 doi: 10.1007/s11427-012-4366-z Cyprinid phylogeny based on Bayesian and maximum likelihood analyses of partitioned data: implications for Cyprinidae systematics WANG XuZhen1, GAN XiaoNi1, LI JunBing1, MAYDEN Richard L.2 & HE ShunPing1* 1Key Laboratory of Aquatic Biodiversity and Conservation of Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China; 2Department of Biology, Saint Louis University, Saint Louis, Missouri 63103-2010, USA Received April 25, 2012; accepted July 16, 2012 Cyprinidae is the biggest family of freshwater fish, but the phylogenetic relationships among its higher-level taxa are not yet fully resolved. In this study, we used the nuclear recombination activating gene 2 and the mitochondrial 16S ribosomal RNA and cytochrome b genes to reconstruct cyprinid phylogeny. Our aims were to (i) demonstrate the effects of partitioned phylo- genetic analyses on phylogeny reconstruction of cyprinid fishes; (ii) provide new insights into the phylogeny of cyprinids. Our study indicated that unpartitioned strategy was optimal for our analyses; partitioned analyses did not provide better-resolved or -supported estimates of cyprinid phylogeny. Bayesian analyses support the following relationships among the major mono- phyletic groups within Cyprinidae: (Cyprininae, Labeoninae), ((Acheilognathinae, ((Leuciscinae, Tincinae), Gobioninae)), Xenocyprininae). The placement of Danioninae was poorly resolved. Estimates of divergence dates within the family showed that radiation of the major cyprinid groups occurred during the Late Oligocene through the Late Miocene. Our phylogenetic analyses improved our understanding of the evolutionary history of this important fish family. Cyprinidae, phylogeny, molecular dating, partitioned analyses Citation: Wang X Z, Gan X N, Li J B, et al. Cyprinid phylogeny based on Bayesian and maximum likelihood analyses of partitioned data: implications for Cyprinidae systematics. Sci China Life Sci, 2012, 55: 761–773, doi: 10.1007/s11427-012-4366-z The family Cyprinidae is the largest freshwater fish family subfamilies are vaguely defined or supported by few mor- and includes an estimated 2420 species in about 220 genera phological characteristics [2]. [1]. The large number of species, wide geographic distribu- Cyprinidae has been conventionally divided into two tion, and considerable morphological diversity make the major lineages, the cyprinine (barbine) and the leuciscine cyprinid fishes taxonomically difficult [2] and a challenge groups. Overall, morphology has provided few insights into for cladistic analysis. The history of Cyprinidae classifica- cyprinid relationships below the family level and failed to tion was well documented by Hensel [3], and numerous reach agreement on the number and the monophyly of sub- efforts have been made to partition cyprinids into subfami- families within Cyprinidae. Chen et al. [8] published the lies using morphological or anatomical characteristics cladistic evaluation of cyprinid subfamily relationships and [2,48]. However, the systematic relationships among many the additional morphological studies by Cavender and Co- cyprinid subfamilies are poorly understood, because the burn [9] and Howes [2] attempted a coherent classification of all cyprinid groups, including the monotypic genus Tinca. In these previous studies, conflicting arrangements of the *Corresponding author (email: [email protected]) © The Author(s) 2012. This article is published with open access at Springerlink.com life.scichina.com www.springer.com/scp 762 Wang X Z, et al. Sci China Life Sci September (2012) Vol.55 No.9 subfamilies Tincinae, Rasborinae, and Gobioninae were partitioned phylogenetic analyses have become increasingly proposed. popular in recent years. Partitioned phylogenetic analyses Recently, molecular phylogenetic analyses have been use separate nucleotide substitution models (and associated performed on Cyprinidae. In general, most of the molecular parameters) for subsets of the data, to better explore parti- studies of European cyprinids [1016] have been phyloge- tion-specific models of evolution and to reduce systematic netically congruent. For example, all of these studies sup- error, thus yielding more accurate phylogenies. Generally, ported the nesting of Alburninae [2] within a paraphyletic partitioned phylogenetic analyses are undertaken in a Leuciscinae, but not the usual dichotomy between barbelled Bayesian framework [21], but recently, mixed-model search cyprinines (subfamilies Cyprininae, Gobioninae, and Ras- methods using maximum likelihood (ML) have become borinae) and leuciscines lacking or sporadically possessing available [22]. Furthermore, an appropriately-partitioned barbels (subfamilies Acheilognathinae, Leuciscinae, Cultri- data-set should be well modeled but not over-partitioned, nae, and Alburninae) [2]. Because cyprinids are most di- because the over-parameterization (including over-parti- verse in Asiatic waters [17], phylogenetic studies that in- tioning) could result in parameter nonidentifiability, in- clude Asian species would greatly advance cyprinid sys- creased variance, improper posterior distributions, and un- tematics [1820]. Cunha et al. [18] identified an Asian due influence of the priors [23]. Alternatives to Bayes fac- group consisting of cultrins+acheilognathins+gobionins+ tors for phylogenetic model selection that use explicit pa- xenocyprinins within the Cyprinidae using cytochrome b rameterization penalties are now available for partitioned (Cytb) gene sequences. Other molecular phylogenies of East analyses [23]. Asian cyprinids indicated two principal lineages within Cy- We performed ML and Bayesian analyses of partitioned prinidae and provided phylogenetic evidence for the mon- data to reconstruct the phylogeny of cyprinid fishes, and ophyly of cultrins-xenocyprins and affiliated groups [19,20]. also used relaxed molecular clock approaches to estimate However, these molecular analyses were heavily based on the dates of cladogenetic events within the family. Our main partial mtDNA sequences, and resulted in phylogenetic aims were (i) to demonstrate how partitioning concatenated trees with limited resolution and little discrimination among data affected phylogenetic reconstruction of cyprinid fishes; alternative phylogenetic hypotheses. (ii) to test the monophyly of the currently-recognized sub- The current cyprinid classification developed in the ab- families within Cyprinidae; and (iii) to discuss the taxo- sence of a strong phylogenetic framework and is largely onomic implications of the recovered clades. morphology based; few revisions have resulted from recent molecular evidence, due to the limited taxon sampling in 1 Materials and methods those studies. Some critical areas of Cyprinidae phylogeny and systematics remain unresolved. First, a majority of des- 1.1 Taxon sampling and total DNA isolation ignated cyprinid subfamilies have not been tested for mon- ophyly with either molecular or morphological data, and Our samples include 103 cyprinid species representing all molecular data [1820] has failed to support the monophyly major morphological groups and all 12 subfamilies within of many morphologically-defined subfamilies, e.g., Ras- Cyprinidae [4]. Outgroup taxa were selected based on the borinae [8] and Leuciscinae [2,4]. Second, previous anal- consensus that Cypriniformes is a monophyletic group yses have not agreed on the phylogenetic positions of Ras- [24,25]. Therefore, six cypriniform fishes outside Cyprini- borinae, Tincinae, and Acheilognathinae. In recent molecu- dae were included in our analyses (Catostomidae, Balitori- lar phylogenies, relationships among these subgroups re- dae, Cobitidae, and Gyrinocheilidae) (Table 1). mained unclear, because corresponding nodes were gener- Field-collected fish muscle or fin tissues were fixed in ally not statistically supported. Third, although the leucis- 95% ethanol and kept at 20°C in the laboratory until DNA cine and cyprinine subdivisions of Cyprinidae are widely extraction. Total genomic DNA was isolated from muscle accepted, the higher-level taxonomic relationships within or fin tissues using the phenol/chloroform extraction proce- these clades remain unresolved. dure [26]. Molecular phylogenetic analyses of East Asian cyprinid resulted in substantial disagreement on the classification of 1.2 DNA sequences collection and alignment subfamilies compared with the traditional taxonomy [19,20]. Therefore, extensive sampling of Asian cyprinids would The nuclear RAG2 gene and mitochondrial genes were am- provide further insights into the phylogenetic systematics of plified from total DNA extracts via polymerase chain reac- this family. The present paper used extensive taxon sam- tion (PCR) using published and/or optimized primers pling and concatenated sequence data for the nuclear re- [2729]. Reaction mixtures contained approximately 100 ng combination activating gene 2 (RAG2) and the mitochon- of DNA template, 5 µL of 10× reaction buffer, 2 µL dNTPs drial 16S ribosomal RNA (16S rRNA) and Cytb genes to (each 2.5 mmol L1), 2.0 U Taq polymerase, and 1 µL of reconstruct the phylogeny of cyprinids. each oligonucleotide primer (10 µmol L1 each ), in a final To analyze DNA sequence data-sets with multiple genes, volume 50 µL. The PCR amplification profile included an Wang X Z, et al. Sci China Life Sci September (2012) Vol.55 No.9 763 Table 1 Cyprinid