Stout et al. BMC Evolutionary Biology (2016) 16:244 DOI 10.1186/s12862-016-0819-5 RESEARCH ARTICLE Open Access Resolving Cypriniformes relationships using an anchored enrichment approach Carla C. Stout1*†, Milton Tan1†, Alan R. Lemmon2, Emily Moriarty Lemmon3 and Jonathan W. Armbruster1 Abstract Background: Cypriniformes (minnows, carps, loaches, and suckers) is the largest group of freshwater fishes in the world (~4300 described species). Despite much attention, previous attempts to elucidate relationships using molecular and morphological characters have been incongruent. In this study we present the first phylogenomic analysis using anchored hybrid enrichment for 172 taxa to represent the order (plus three out-group taxa), which is the largest dataset for the order to date (219 loci, 315,288 bp, average locus length of 1011 bp). Results: Concatenation analysis establishes a robust tree with 97 % of nodes at 100 % bootstrap support. Species tree analysis was highly congruent with the concatenation analysis with only two major differences: monophyly of Cobitoidei and placement of Danionidae. Conclusions: Most major clades obtained in prior molecular studies were validated as monophyletic, and we provide robust resolution for the relationships among these clades for the first time. These relationships can be used as a framework for addressing a variety of evolutionary questions (e.g. phylogeography, polyploidization, diversification, trait evolution, comparative genomics) for which Cypriniformes is ideally suited. Keywords: Fish, High-throughput sequencing, Phylogenetics, Ostariophysi, Cyprinidae Background carp), and many popular aquarium species (rasboras and Cypriniformes (minnows, carps, loaches, and suckers) is barbs). the largest group of freshwater fishes in the world. Di- For taxonomic clarity, this study follows the propos- versity ranges from some of the smallest vertebrates in ition by Mayden and Chen [1] that elevates subfamilies the world (Paedocypris, 7.9 mm in standard length) to within Cyprinidae to the family level based on consistent members of Tor (almost 3 m SL) [1]. The number of support of major clades. Superfamilies are elevated to valid species is currently estimated at around 4300 [2] the suborder level to be consistent with the recognition with as many as 2500 still awaiting description [3]. To of suborders as the taxonomic level above family and place the Cypriniformes into perspective, about one below order in the classification of bony fishes [5, 6]. third of freshwater fish species is a cypriniform and Other taxonomic assignments follow designations estab- about 6 % of all vertebrate species is a cyprinform [2]. lished by Tang et al. [7], Kottelat [8], van der Laan et al. Species of Cypriniformes are distributed in freshwater [9] and Yang et al. [10]. Because of the great diversity habitats across Asia, Europe, Africa, and North America within Cypriniformes, most phylogenetic studies have [4]. Example representatives include the zebrafish (Danio focused on smaller groups within the order (for example rerio), a model organism used in genomic and develop- [11–14]). Approaches used to resolve relationships at mental biology, important aquaculture species like the these levels have typically included standard methods common carp (Cyprinus carpio), major invasive species using PCR to amplify targeted mitochondrial and/or nu- to North America such as Hypophthalmichthys (silver clear genes [11–19]. These approaches have had varied success at elucidating relationships at these taxonomic * Correspondence: [email protected] levels, but deeper, all-inclusive studies have resulted in †Equal contributors conflicting phylogenies. These major differences in find- 1 Department of Biological Sciences, Auburn University, 101 Rouse Life ings even include two publications in the same volume Sciences Building, Auburn, AL 36849, USA Full list of author information is available at the end of the article [1, 19] whose results are incongruent. Morphological © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Stout et al. BMC Evolutionary Biology (2016) 16:244 Page 2 of 13 studies have also been at odds with the molecular hy- relationships are resolved can researchers begin to take potheses, particularly concerning placement of the advantage of the size, diversity, and distribution of paedomorphic taxa (Danionella, Paedocypris, and Sun- Cypriniformes to gain insight into various biological dadanio) [1, 20–22]. The results of analyses to date facets, such as biogeography, timing of diversifications, mean that this radiation of organisms that is nearly the morphological and ecological evolution, and compara- size of the Mammalia and that is the predominant fresh- tive genomics. water order of fishes has an unsettled taxonomy and phylogeny despite the fact that it has been very highly Methods studied. With the vertebrate developmental model (zeb- Taxon selection and tissue preparation rafish) being part of the Cypriniformes, we are currently The 172 taxa selected for this study (Additional file 1: lacking a basic understanding of the evolutionary con- Table S1) represent almost all families within the order. text of its characteristics, and it is clear that new ap- Families not represented in this study are: Psilorhynchi- proaches to the phylogenetics of this very important dae (26 species), Barbuccidae (two species), Tincidae (13 group of fishes must be employed. species), Serpenticobitidae (three species), Ellopostomi- To date, the only nuclear genomic scale study [23] dae (two species) and Leptobarbidae (five species). Spe- consisted of 100 genes and was limited to only thirteen cies were chosen based on tissue availability and because individuals, most of which belong to Xenocyprididae of their incorporation in recent studies that will allow within Cyprinoidei. The large number of taxa in Cyprini- for direct comparisons [11, 13, 14, 26, 30, 38, 39]. Type formes has forced researches to either focus on a small genera for each of the families were included if available. subset of representatives with an increasing number of Exceptions include Botiidae, Balitoridae, Gastromyzonti- molecular loci, or focus on large taxonomic representa- dae, and Xenocyprididae, but in these cases other repre- tion with relatively fewer numbers of markers. sentatives were chosen based on their supported Evaluating tree topologies from previous large-scale inclusion within their respected families according to studies has led to moderate consensus supporting mono- previous studies [8, 40]. Three outgroup taxa were phyly for some clades within the order, including fam- chosen to represent the three other ostariophysan or- ilies of loaches (e.g. Botiidae, Cobitidae, Balitoridae, ders: Siluriformes, Gymnotiformes, and Characiformes. Nemacheilidae), Catostomidae (suckers), Cyprinidae, Whole genomic DNA was prepared using the Omega- Xenocyprididae, Gobionidae, Leuciscidae, and Acheilog- biotek E.Z.N.A. animal tissue extraction kit (product nathidae [1, 19, 24–33]. Despite support for monophyly #D3396-02) and verified for quality and quantity using of many families, clear establishment of the relationships gel electrophoresis and nanodrop, respectively. among them still remains elusive. Other families, most notably Danionidae, have been more problematic, with Locus selection and probe design paedomorphic genera like Paedocypris and Sundadanio Although the Anchored Hybrid Enrichment kit devel- changing placement across trees employing both mor- oped for vertebrates by Lemmon et al. [36] contains a phological and varying molecular data [1, 19–21, 31, 34]. fish reference (Danio) and has been utilized in teleosts If analyses result in incongruent relationships due to with moderate success [41], we desired an enrichment conflict or weak phylogenetic signal among individual tool more efficient and appropriate for phylogenomics in genes, the next approach to establishing robust reso- teleosts. Because of the complex nature of teleost gen- lution would be to incorporate high-throughput sequen- ome evolution, which involved multiple whole-genome cing data that can increase the signal to noise ratio and duplications and lineage-specific gene losses [42], it is reduce stochastic error. New methods have been estab- impractical to identify a set of loci that are truly single- lished that have been specifically tailored for use in sys- copy across all of Teleostei. Previous studies claiming to tematics [35–37] and that address problems typical of have identified single-copy loci in teleosts (e.g. [43]) transcriptome approaches for phylogenomics. These likely only identified loci that were single-copy in the problems include tissue preservation, orthology assess- species they considered; evaluation of those loci in add- ment, missing data, and resolution capabilities across itional teleost lineages suggests that these loci are not various taxonomic levels [35–37]. All of these factors universally single-copy (see