Wang et al. BMC Evol Biol (2020) 20:151 https://doi.org/10.1186/s12862-020-01718-0 RESEARCH ARTICLE Open Access DNA barcoding reveals cryptic diversity in the underestimated genus Triplophysa (Cypriniformes: Cobitidae, Nemacheilinae) from the northeastern Qinghai-Tibet Plateau Tai Wang1,2, Yan‑ping Zhang2, Zhuo‑yu Yang2, Zhe Liu1* and Yan‑yan Du2* Abstract Background: The northeastern part of the Qinghai‑Tibet Plateau (QTP) presents a high number of plateau loach species. As one of the three major groups of fshes distributed on the QTP, plateau loach has high ecological value. However, the taxonomy and systematics of these fsh are still controversial, and a large number of new species have been reported. The reason for this phenomenon is that the degree of morphological variation is low, the phylogenetic information provided by morphological and anatomical features used for species identifcation is relatively poor, and many cryptic species are observed. Based on the high‑density sampling points from the biodiversity hotspots sur‑ veyed, this study aims to evaluate the biodiversity of plateau loach in the northeastern part of the QTP and reveal the hidden diversity by comparing morphological species with molecular operational taxonomic units (MOTUs). Results: After careful identifcation and comparison of the morphology and DNA barcoding of 1630 specimens, 22 species were identifed, with 20 considered valid local species and two identifed as new species that had not been previously described. Based on the combination of morphological and molecular methods, a total of 24 native spe‑ cies were found, two of which were cryptic species: Triplophysa robusta sp1 and Triplophysa minxianensis sp1. Fourteen of the 24 species form clusters of barcodes that allow them to be reliably identifed. The remaining cases involved 10 closely related species, including rapidly diferentiated species and species that seemed to have experienced incom‑ plete lineage sorting or showed introgressions. Conclusions: The results highlight the need to combine traditional taxonomies with molecular methods to correctly identify species, especially closely related species, such as the plateau loach. This study provides a basis for protecting the biodiversity of plateau loach. Keywords: DNA barcode, Qinghai‑Tibet, Tibetan loach, Cryptic species Background With problems such as global climate change and issues related to populations and the ecological environment, energy and food production is becoming increasingly *Correspondence: [email protected]; [email protected] serious; moreover, achieving sustainable anthropogenic 1 College of Animal Science and Technology, Gansu Agricultural development and understanding and meeting the require- University, Lanzhou, China ments of biodiversity are becoming urgent research issues 2 Gansu Key Laboratory of Cold Water Fishes Germplasm Resources and Genetics Breeding, Gansu Fisheries Research Institute, Lanzhou, [9, 36, 44]. Tere is a major global demand for accurate China and rapid identifcation of species for the protection and © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Wang et al. BMC Evol Biol (2020) 20:151 Page 2 of 15 sustainable use of biodiversity resources. Species identi- are identifed is caused mainly by the existence of cryptic fcation and classifcation is a basic requirement for bio- species or the lack of a careful classifcation review. Te logical research. Based on morphological characteristics, simple body structure and relatively conservative mor- classical taxonomy has made great contributions to spe- phological evolution of the plateau loach fsh coupled cies classifcation, however, due to morphological plastic- with their weak migration ability due to the restrictions ity, traditional taxonomy cannot accurately distinguish all of the water system have led to limited gene exchange species, particularly for similar and related species [54, between diferent populations. Although morphologi- 61]. Terefore, new methods of supporting species iden- cally imperceptible, the process of species diferentiation, tifcation with classical taxonomy methods are needed. including genetic structural diferentiation and reproduc- Tautz et al. [67] frst suggested using DNA sequencing, tive isolation, may have occurred over time; thus, many namely, DNA taxonomy, as the main platform for bio- hidden taxa may have been ignored. Terefore, the genus logical classifcation. Professor Paul Hebert from the Triplophysa should be considered in the study of cryptic University of Guelph in Canada then introduced the diversity. concept of DNA barcoding, highlighting its signifcance Classical morphological classifcation has always to the feld of biological taxonomy and species identifca- played a dominant role in species identifcation, although tion [28, 60] and suggesting the use of the mitochondrial it has limitations. In particular, for fsh of the genus Tri- cytochrome C oxidase subunit I (COI) gene as the basis plophysa, the phenotype is easily afected by biological for animal DNA barcoding. Te applicability of DNA factors and the external environment, and morphologi- barcoding to the identifcation of marine and freshwater cal plasticity is observed; therefore, morphological dif- fsh species has been shown by using a short fragment ferences are not easily detected [27]. Moreover, some of approximately 650 bp from the mitochondrial COI species were named many years ago and were assigned gene to identify species based on sequence diferences relatively simple morphological descriptions. All these [7, 30, 37, 69, 75, 79, 80] . A growing number of studies factors have led to difculties in the subsequent identi- have shown that DNA barcodes are widely used in animal fcation of species and taxonomic research. Due to the species identifcation and classifcation, cryptic species difculty in obtaining detailed data for comparisons, the detection, phylogenetic research, etc. (Smith et al. 2008; distribution of some species may be artifcially expanded Swartz et al. 2008; Rock et al. 2008) [1]. Moreover, DNA and mistakenly divided into diferent geographical popu- barcodes have been used to construct barcode databases, lations [17, 55]. Tis long confusing classifcation his- such as the Barcode of Life Data Systems (BOLD) (https tory is particularly obvious in the species classifcation ://www.bolds ystem s.org), which barcoded approximately of Triplophysa. Fish biodiversity must be identifed and 96,425 fsh specimens belonging to 10,267 species. DNA recorded to achieve efective species conservation and barcoding can complement traditional species identif- predict biodiversity responses to climate change [4, 46, cation as a method of automating and standardizing the 53]. Although DNA barcode technology has certain limi- process of specimen identifcation, thereby reducing the tations, such as improper software models or parameters, dependence on the experience of taxonomists [8, 63]. which lead to excessive division or clustering of species, Te Qinghai-Tibet Plateau (QTP), known as “the DNA barcode itself introgression, pseudogenes, defni- roof of the world”, is rich in biodiversity and a rela- tion of molecule as a result [11, 14, 49, 64], DNA barcod- tively unique area with many endemic species [34]. Te ing has been proposed as an important tool that can help native fsh living in the Qinghai-Tibet region belong to close the large gap in our current understanding of bio- three orders: Salmoniformes, Siluriformes and Cyprini- logical diversity [5, 15, 16, 43]. Some relatively automated formes [76]. Triplophysa, which belongs to the family methods of species division using DNA barcoding data Nemacheilinae (Cypriniformes), is widely distributed on sets have been developed, and they may increase the ef- the QTP and in its adjacent regions [74] and represents ciency and decrease the subjectiveness of species division a special group adapted to the climatic characteristics of [19, 20, 35]. the QTP, such as cool temperatures and oxygen short- To date, studies have not focused on the identifcation ages [76, 84]. In 1992, 33 Triplophysa species were identi- or evaluation of cryptic biodiversity within the genus fed. However, over time, a large number of new species Triplophysa using DNA barcoding in the northeastern have been described, and a total of 140 valid species have QTP. Herein, based on extensive sample collection in this been identifed [27, 40]. Although some synonymous spe- area, DNA barcoding technology was used to evaluate