Adding nuclear rhodopsin data where mitochondrial COI indicates discrepancies – can this marker help to explain conflicts in cyprinids?

DNA barcoding is a fast and reliable tool for species identification, and has been successfully applied to a wide range of freshwater fishes. The limitations reported were mainly attributed to effects of geographic scale, taxon-sampling, incomplete lineage sorting, or mitochondrial introgression. However, the metrics for the success of assigning unknown samples to species or genera also depend on a suited taxonomic framework. A simultaneous use of the mitochondrial COI and the nuclear RHO gene turned out to be advantageous for the barcode efficiency in a few previous studies. Here, we examine 14 cyprinid fish genera, with a total of 74 species, where standard DNA barcoding failed to identify closely related species unambiguously. Eight of the genera (Acanthobrama, Alburnus, Chondrostoma, Gobio, Mirogrex, Phoxinus, Scardinius, and Squalius) contain species that exhibit very low interspecific divergence, Research Article Open Access © 2015 S. Behrens-Chapuis et al. licensee De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. S. Behrens-Chapuis*, F. Herder, H. R. Esmaeili, J. Freyhof, N. A. Hamidan, M. Özuluğ, R. Šanda, M. F. Geiger Adding nuclear rhodopsin data where mitochondrial COI indicates discrepancies – can this marker help to explain conflicts in cyprinids? *Corresponding author: S. Behrens-Chapuis, Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute for Animal Biodiversity, Adenauerallee 160, 53113 Bonn, Germany, E-mail: sichapuis@t-online.de F. Herder, M. F. Geiger, Zoologisches Forschungsmuseum Alexander Koenig, Leibniz Institute for Animal Biodiversity, Adenauerallee 160, 53113 Bonn, Germany H. R. Esmaeili, Ichthyology Research Laboratory, Department of Biology, College of Sciences, Shiraz University, Shiraz, Iran J. Freyhof, German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Deutscher Platz 5a, 04103 Leipzig, Germany N. A. Hamidan, The Royal Society for the Conservation of Nature – Conservation Division, P.O. Box 1215, Jubaiha 11941, Jordan and Centre for Conservation Ecology and Environmental Science, School of Applied Sciences, Bournemouth University, Poole, BH12 5BB, U.K. M. Özuluğ, Istanbul University, Science Faculty, Department of Biology, 34134 Vezneciler, Istanbul, Turkey R. Šanda, National Museum, Václavské nám. 68, 115 79 Prague, Czech Republic Unauthenticated Download Date | 7/20/18 5:52 AM 188 S. Behrens-Chapuis, et al. has been considered that the mental disc in labeonine cyprinids has evolved and been reduced independently in several lineages [29]. This new hypothesis was supported by phylogenetic analyses of nuclear and mitochondrial genes in labeonines by Yang et al. [30]. Likewise, mtCOI-based findings in Hamidan et al. [31] and Geiger et al. [10] showed the same pattern with different labeonine genera (defined by the mental disc apparatus) to be nested within Garra, which could be explained by past hybridization events. This pheneticmitochondrial incongruence in the Garra species complex represents an ideal case to be examined by an additional genetic marker with different evolutionary rate and genomic position. Several studies have tried to overcome uncertainties in species identification derived from analysing exclusively the mtCOI region by supplementing this marker with nuclear sequences [32-35]. The nuclear rhodopsin gene (ncRHO) is an intronless gene [36], which has been recommended for teleost fishes by Sevilla et al. [37], and been applied in different phylogenetic analyses [30,38,39]. A first comparison of mtCOI to ncRHO for species identification in billfishes revealed a discrepancy between both markers in the species-complex of white and striped marlin [40]. A direct comparison of individuals where sequences for both loci were available revealed that those, which are distinguishable by mtCOI lack a clear separation by the nuclear marker and vice versa [40]. In French chubs mtCOI clearly separated the common chub S. cephalus from the Catalan chub S. laietanus by revealing two divergent haplotype clusters, whereas the topology based on nuclear rhodopsin was uncertain, showing nine different haplotypes with neither correspondence to taxonomy, nor to geography [41]. When identifying cyprinids in the aquarium trade, nuclear rhodopsin results were broadly consistent with mtCOI patterns and morphology although ncRHO failed to distinguish closely related species, but proved to be useful to uncover hybridisation between various species of barbs in the ornamental trade sector [42]. Following the three previously mentioned direct comparisons of mtCOI and ncRHO, we first address the question whether the ncRHO marker has the potential to improve species identification where the taxonomic resolution of mtCOI in cyprinid fishes was not sufficient. We include cases where species exhibit mtCOI barcodes with low interspecific divergence, haplotype sharing or potential introgression ([10], Table S2C). The second focus of this study is on labeonine cyprinid genera, where members of Crossocheilus, Hemigrammocapoeta, barcoding proved to be a powerful and reliable tool for discriminating a large range of species, including all major taxa of marine as well as freshwater fishes [e.g. 1,5-12]. The tool has gained additional attention among biologists by having the potential to reveal cryptic diversity, especially given the enormous amount of data collected globally, which can easily be used to compare own sequence data on a regional scale. A high level of intraspecific mtCOI divergence is a likely indicator for cryptic diversity, i.e. putative new, or unrecognized species, pronounced phylogeographic structure, but might also result from introgressive hybridization [1015]. The potential of DNA barcoding appears promising, but has not been without controversy. The latter centred on either the suitability of mtCOI as universal marker, or the most meaningful method to delineate species based on molecular data [16-18]. The possibility to distinguish species unambiguously by DNA barcoding depends on the sequence variation within and between the taxa studied. Freshwater fishes, and in particular members of the speciose family Cyprinidae (minnows, carps), are among the animals known for high rates of hybridization and introgression [19-22]. Hybridization may lead to the transfer of mitochondrial DNA into heterospecific populations (i.e. introgression); the mismatch of nuclear population structure and mitochondrial haplotypes may confound analyses based solely on mitochondrial markers, and lead to misidentifications in barcoding routines [6,23]. Here, we focus on cyprinid species that exhibited low interspecific divergence, or haplotype sharing based on mtCOI data in previous studies [10,12]; these allow to critically assess the potential to resolve taxonomic conflicts as well as the discrimination capacity of the nuclear rhodopsin marker (ncRHO). Cyprinids are the most species-rich family of vertebrates in freshwaters of Eurasia, Africa, and North America [24,25]. Some of the species, like the common carp, the goldfish, or the zebrafish, have substantial commercial cultural and/or scientific importance, whereas the vast majority of the cyprinid species remain known only to a few specialists [26,27]. In order to evaluate the potential of ncRHO to resolve an ongoing taxonomic dispute, we also include labeonine cyprinids (Garra and related genera), characterized by the presence or absence of a conspicuous adhesive organ, the mental disc apparatus. A small or absent disc was interpreted as ancestral character state, whereas a completely developed disc was considered the derived state [28]. More recently, and based on several nuclear genes, it Unauthenticated Download Date | 7/20/18 5:52 AM Adding nuclear rhodopsin data where mitochondrial COI indicates discrepancies ... 189 in a second attempt. The nuclear rhodopsin data were generated using the primers RH 28F [39] and RH 1039R [38]. PCR conditions for both markers using Qiagen Multiplex ® taq were as follows: 15min at 95.0°C; 10 cycles of 35s at 94.0°C, 90s at 52.0-49.0°C (“touch-down”) and 90 s at 72.0°C followed by 25 cycles of 35 s at 94.0°C, 90 s at 55.0°C and 90 s at 72.0°C; 10 min at 72.0°C and hold at 10.0°C. The purified PCR products were sent to Macrogen Europe Laboratories for standard Sanger sequencing in both.