In this study, a total of 24 species were reported, including two new species: a cryptic species in the T. minxianensis population and a cryptic species in the T. robusta population. The morphological and molecular data were consistent in 14 of the 22 species identified. The results show that there are two cryptic species that can be described in the biodiversity hotspot area, which reinforces the general view that there is still a large amount of unrecorded diversity in the plateau loach. There is only one haplotype in the clade of T. bleekeri and T. orientalis. It is necessary to collect more specimens and add sequences, but we do not rule out the possibility of identifying more cryptic species.
Different numbers of MOTUs were identified in the four DNA barcode analysis methods: 17 different MOTUs were identified using the PTP and GMYC models and 19 MOTUs were identified using the ABGD and BOLD methods. T. shiyangensis and T. leptosoma cannot be distinguished by the PTP or GMYC models, but the ABGD and BOLD methods allow different MOTUs to be assigned to each species (Figure 3). The inconsistent results of the four methods may be due to differences in the methods used to distinguish species, The ABGD and BOLD methods are based on the genetic distance between species and distinguish species by the difference of intraspecific and interspecific genetic distances. The BOLD method defaults to a genetic distance threshold of 2.2% and ABGD of 2.8%, resulting in the same number of MOUTs defined by the two methods. Although it has been pointed out that the RESL in the BOLD system has a stronger taxonomic performance than that in the ABGD system, showing better species identification and MOTU assignment results (Ratnasingham and Hebert, 2013), the two methods in this study achieved the same results, which may be related to the identified species. A key aspect implicit in DNA barcoding analysis is the genetic distance threshold values used to define the MOTUs. COI genetic distance values from 1% (Hubert et al., 2008) to 2% (Keskin et al., 2013) have been considered the threshold values for fish DNA barcoding analysis. However, these values are derived from comparative analyses of species diversity in different aquatic ecosystems. For example, 2% is used to represent the DNA barcodes for the community of fish in certain rivers (Pereira et al., 2013). However, when DNA barcoding analysis was used for a group of closely related species (e.g., the same genus), a lower genetic distance value has been reported (Carvalho et al., 2011; Pereira et al., 2011, 2013). In particular, a low threshold value of 0.92% is needed to distinguish MOTUs in the genus Laemolyta (Anostomidae) (Ramirez and Galetti, 2015). Although most of the values obtained in this paper are above 1.47% (14 out of 18 MOTUs, Table 2), the maximum threshold value of related species detected between the obtained MOTUs is 0.40%, and some species have shared haplotypes. The existence of haplotype sharing among different species of plateau fishes may be related to complex species differentiation mechanism or convergent evolution of local adaptation (Shen et al., 2019; Chen et al., 2020). A lower threshold of genetic distance may be obtained when the genetic relationships between different species within a genus are analysed. This approach based on genetic distance analysis is easy to run, but it lacks phylogenetic content, the boundaries used to distinguish species are artificial and lacks the objectivity of species evolution (Ortiz and Francke, 2016). GMYC and PTP are species defining methods based on evolutionary trees, GMYC is based on the ultrametric trees to define species (Fujisawa and Barraclough, 2013), and PTP is based on substitution calibrated trees to define species, which avoids the potential pitfalls of constructing time-calibrated species phylogeny. (Zhang et al., 2013). We believe that tree-based techniques are effective in identifying individual species because identifying a particular branch representing a particular species requires a threshold to represent the clade length and/or the pair distance used to distinguish differences between individuals.( Gustafsson, et al., 2009; Vieites et al., 2009; Lim and Meier, 2011).Such thresholds are also required when DNA barcoding data is analysed using clustering methods and based on distance methods. A technical problem with clustering is that pair distances of three or more sequences need not be equal, so strict thresholds are usually impossible to apply (Meier et al. 2006). Both methods defined 17 MOUTs in this study. Clearly, the accuracy of DNA barcoding methods depends largely on the target species being analysed (Pentinsaari et al., 2017)
The difference in the number of MOTUs detected by the different analysis methods was mainly seen in two pairs of MOTUs: the genetic distance between T. shiyangensis and T. stoliczkae was relatively low (2.65%), as was the genetic distance between T. leptosoma and T. papilloso-labiatus (1.47%). These relatively low genetic distance values may be related to the late differentiation of these MOTUs. Notably, the MOTUs of relatively recent origin had less time than species of distant origin to accumulate genetic differences, which hindered their correct identification, even though the species differ greatly in their morphological characteristics. T. papilloso-labiatus has obvious swim bladder, while T. leptosoma does not (Zhao, 1984). The characteristics of the genetic diversity of these species are the same: there is a relatively high level of haplotype diversity (> 0.5) and relatively low levels of nucleotide diversity (< 0.5%) (Table 1). This indicates that after the differentiation of these species, influenced by the founder effect and environmental heterogeneity caused by water system changes, the population rapidly accumulated variation, resulting in a high haplotype diversity index. The accumulation time of the nucleotide diversity index was much longer than that of the haplotype diversity index. In terms of geographical distribution, these two species are mainly distributed in the Shulehe River and Heihe River. The possibility of sympatric speciation exists, but this needs to be confirmed by further analysis.
An example of incompletely separated species was also found. T. minxianensis, T. robusta, T. pappenheimi and T. siluroides are not sufficiently differentiated by COI gene differences, and there are also shared haplotypes among the four species (Figure 5). These phenomena can be explained as frequent Mitochondrial DNA introgression events before species differentiation (Feng et al., 2018) or phenotypic plasticity in fish (Robinson and Parsons, 2002; Thibert-Plante and Hendry, 2011). The morphological characteristics of T. hsutschouensis, which was identified as an independent species isolated from T. robusta, include bare and scaleless bodies and a relatively low ratio of body length to body height (Wang, 1991). T. robusta only has residual scales in specific parts of its body. The Jinghe River population of T. robusta has scales along the lateral line from the caudal fin to the front of the dorsal fin. Moreover, the Jinghe River population and other populations of T. robusta were clustered into two clades (Figure 3), and the genetic distance between the populations reached 7.9% (Table 2). These phenomena suggested the existence of cryptic species of T. robusta. There was no difference between T. minxianensis and T. minxianensis sp1 in the degradation of the swim bladder, whether the end of the pelvic fin reached the anus, the starting point of the dorsal fin and the pelvic fin relative to each other or the morphological measurement data. But the scales of T. minxianensis sp1 were only found in the caudal peduncle and this is quite different from T. minxianensis, in which all the body parts except the head have obvious round scales. The genetic distance between the two populations was 7.4% (Table 2), which indicated that there were cryptic species in T. minxianensis. Similar to this example of incomplete species separation, Wang (1991) argued that the plateau loach groups without scales (T. hsutschouensis) come from scaly groups (T. minxianensis) following the degeneration of scales. The groups with remnant body scales (T. robusta) are the intermediate species between the two types. The presence or absence of scales marks a leap in the evolution of plateau loach populations. The cryptic species found in this study provide more evidence for this speculation.
The morphological characteristics and molecular characteristics were inconsistent in T. pseudoscleroptera and T. scleroptera. The two species have similar appearances but different internal anatomical structure. The anterior and posterior segments of the swim bladder of T. pseudoscleroptera were the same size, with a long pouch or oblong oval shape and no pyloric caecum. The posterior chamber of the swim bladder of T. scleroptera is developed, the anterior segment is thin and the posterior segment is enlarged into a long pouch (Zhu et al., 1981). Without the comparison of internal anatomical structure, these species are easy to misidentify and morphological identification may be incorrect (He et al., 2008). However, due to the low interspecific distance between the two species (0.40%), the two MOTUs cannot be correctly distinguished. This inconsistency was also found between T. dalaica and T. stoliczkai. The posterior chamber of T. dalaica's swim bladder was oval, while the posterior chamber of T. stoliczkai 's swim bladder was degraded; this feature can be used to accurately distinguish the two species.
As shown by the two cases reported here, the DNA barcoding did not show enough difference to distinguish similar species because the lineages were not completely divided into different clades. The reason for this phenomenon is the process of incomplete lineage sorting. Due to the extremely short time of species differentiation, ancestral traits are randomly fixed in the differentiated species (Fontenot et al., 2011; Leavitt et al., 2017). Similar phenomena have been found in Psorophora (Chan-Chable et al., 2016), Syngnathidae (Zhang et al., 2017) and Laemolyta (Ramirez et al., 2015), and mixed lineage cases are particularly common in plateau fish (Shen et al., 2018). In this sense, to find evidence of reproductive isolation, it is important to combine nuclear genetic and ecological data for further research (Mardulyn et al., 2011; Versteirt et al., 2015; Beebe, 2018).
It is easy to identify species with morphological characteristics that are not significantly different as a single species. For example, T. bleekeri and T. polyfasciata have very similar morphological characteristics, there is no significant difference in the quantitative traits in different proportions of their bodies, and they have been identified as the same nominal species. Ding et al. (1996) believed that they should be divided into two different species based on molecular data and pointed out that the main distinguishing feature was that there were 10-12 wide, dark brown horizontal stripes on the side of the body. However, even among T. bleekeri individuals collected from the same site, the horizontal stripes on the side of its body can range from 0-10. Of the specimens collected from Wenchuanhe River in Sichuan Province, most had 5-7 horizontal stripes, and almost none had more than 10. It was concluded that the validity of T. polyfasciata was still questionable (He et al., 2008). In this study, the numbers of these two species of plateau loach collected were relatively small, with 10 T. bleekeri and 5 T. polyfasciata, and 7-9 horizontal stripes were observed on the sides of the fish bodies. The division into two different species was also not supported by morphology, but the genetic distance between the two species reached 8.57%, far exceeding the threshold of genetic distance within the species of 2% (Pereira et al., 2013). Therefore, it is speculated that these two species have undergone genetic differentiation in terms of genetic material, but due to the small size of the individual (the length of the collected sample is 5-8 cm), the morphological difference is not obvious, so they have historically been regarded as one species. Obviously, the body colour or body markings of the plateau loach may not be an effective classification feature for the identification of species and cannot be used as the main basis for identification.
Herzenstein (1891) identified T. papilloso-labiatus as a subspecies of T. strauchii; this finding was also supported by Zugmeyer (1910). T. strauchii lack a developed mastoid process similar to that of T. papilloso-labiatus. Instead, they have only a strong, naked fold, while the mastoid process on the upper lip of the plateau loach living in the Hexi corridor is obviously a double line, and that on the lower lip is blurred double line. Characteristics such as the mastoid process and strong, naked crease are continuously transitive in a geographical distribution without obvious boundaries. However, the appearance of significant double lines on the mastoid marks discontinuity in the variation, and there are relatively stable differences in a series of other morphological traits. Thus, T. papilloso-labiatus should be regarded as an independent species (Li and Chang, 1974; Zhao, 1984). This is also supported in the phylogenetic tree constructed in this study (Figure 3). T. strauchii and T. papilloso-labiatus are clustered into two different clades and should be independent species.
There is little difference in the morphological characteristics between T. wuweiensis and T. scleroptera. Li and Chang (1974) regarded T. wuweiensis as an independent species based on 7 morphological traits. Zhu and Wu (1975, 1981) believed that there was a certain continuity in the identification characteristics of these two species. However, after collecting specimens of T. scleroptera distributed in the Datonghe River, only one mountain away from the T. wuweiensis specimens, Zhao (1984) believed that there were significant differences between the two species in the number of pectoral fin rays, intestinal shapes and gill rakers, supporting T. wuweiensis as an independent species. In this study, T. wuweiensis and T. scleroptera clustered in different clades, and the two species were greatly differentiated, which also supported the idea that T. wuweiensis is an independent species. The low genetic diversity of T. wuweiensis may be due to the short time since species differentiation and the low haplotype diversity and nucleotide diversity may be caused by the founder effect and the narrow distribution area (the species is only distributed in the east and west Shiyanghe River tributaries).
T. shiyangensis, T. papilloso-labiatus and T. hsutschouensis are distributed in three inland river systems in the Hexi corridor. The maximum intra-species genetic distance of these three species is more than 1%. This may be mainly due to the wide geographic distribution of the three species and the large population differentiation caused by the barriers created by the water systems. This phenomenon also appears in the sympatric distribution of Gymnocypris chilianensis, in which each geographic population is clustered into a single clade, with a large genetic differentiation (Zhao et al., 2011).
The different geographic populations of some widespread species are identified as different species or subspecies due to some more significant morphological differences. For example, T. stoliczkae was divided into 7 subspecies (Herzenstein, 1891) due to the differences in the number of gill rakers, the proportion of quantitative traits and the number of spiral loops of intestinal tubes with changes in altitude or water system. In this study, the samples were collected in three drainage systems (Yellow River, Jialing River and the inland rivers in the Hexi corridor). The maximum genetic distance within the species was greater than 1.2% (Table 2). However, the samples of different water systems have shared haplotypes. This indicates that different geographic populations of T. stoliczkae in the surveyed area are from a common ancestor.
The membranous swim bladder of T. obscura is very developed with a constriction in the middle, and its length accounts for approximately 2/3 of the abdominal cavity. Compared with T. orientalis, its body surface has obvious spines. It is regarded as an independent new species (Li, 2017). In this study, a relatively large number of samples (n=234) were collected in the distribution area. The phylogenetic tree showed that the samples from different water systems were clustered into different clades, the maximum genetic distance within the species was 2%, and the nucleotide diversity and haplotype diversity were relatively high (h=0.887, π = 0.00777). These findings indicate that there is a large differentiation between the two geographically separated populations of T. obscura and the possibility of allopatric speciation. T. obscura and T. orientalis are also divided into two different monophyletic lines in the phylogenetic tree, which is consistent with the results of the analysis of Wu (2017).
Although only 3 specimens of T. sp1 were collected in the Liangdang section of the Jialing River, there are obvious differences in morphological characteristics from other species of plateau loach. It should be identified as a new species that has not been reported, but more specimens should be collected for further confirmation. T. sp2 was collected in the Jialing River, and showed degeneration of the membranous swim bladder, leaving only a small chamber, an anus near the start of the anal fin, the end of the pelvic fin adjacent to the anus, a large spot on the back of the body, a spot on the side of the body and other morphological characteristics which were obviously different from those of the closely related species T. obscura. A detailed description of these newly discovered species is necessary to make it possible to record the relationship between morphology and molecular identification criteria (Versteirt et al., 2015).