Description of three new species of Gyrodactylus von Nordmann, 1832 (Monogenea: Gyrodactylidae) on bitterling fishes (Acheilognathinae) from China.

Three new species of Gyrodactylus are described from three species of bitterling in Donghu Lake, China: Gyrodactylus ocellorhodei n. sp. from Rhodeus ocellatus; G. sinenorhodei n. sp. from Rhodeus sinensis; and G. acheilorhodei n. sp. from Acheilognathus macropterus. All the three new species showed similar opisthaptor morphology, especially the marginal hooks: all had a slender and perpendicular sickle shaft, and flat sickle base with distinct heel and inner arch which was different from the G. rhodei-group species parasitic on bitterling. Multivariate analyses based on hamulus and marginal hooks suggested that these three new species cannot be completely distinguished, despite some morphology divergence observed in certain less reliable morphometric features, such as hamulus root length, ventral bar total length and process shape. These three new species shared an identical 18S ribosomal RNA gene sequence, while the variation in the Internal Transcribed Spacers (ITS1-ITS2) sequence among them (8.4-11.2%, K2P) far exceeded the 1% ITS sequence difference that had been suggested as a threshold for species delimitation of Gyrodactylus. Phylogenetic analysis based on ITS1-ITS2 showed that all these sequenced Gyrodactylus spp. parasitic on the subfamily Acheilognathinae host formed a monophyletic group. However, a clear differentiation (18.9-20.9%, K2P of ITS1-ITS2) could be found between the subgroup from China (G. ocellorhodei n. sp., G. sinenorhodei n. sp. and G. acheilorhodei n. sp.) and that from Europe (G. rhodei).

Novel insights into adaptive evolution based on the unusual AT-skew in Acheilognathus gracilis mitogenome and phylogenetic relationships of bitterling.

Acheilognathus gracilis, a bitterling species, distribute in lower reaches of Yangtze River. They are identified as the top-priority bitterling species for conservation as having high evolutionary distinctiveness and are at risk of extinction. In present study, we first sequenced the complete mitogenome of A. gracilis and analyzed its phylogenetic position using 13 PCGs. The A. gracilis mitogenome is 16,774 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, a control region and the origin of the light strand replication. The overall base composition of A. gracilis in descending order is T 27.9 %, A 27.7 %, C 26.1 % and G 18.3 %, shows a unusual AT-skew with slightly negative. Further investigation revealed A. gracilis uses excess T over A in NADH dehydrogenase 5 (nd5), whereas the most of other bitterlings are biased toward to use A not T, implying there is likely to be unique strategy of adaptive evolution in A. gracilis. We also compared 13 PCGs of 30 bitterling mitogenomes and the results exhibit highly conservative. Phylogenetic trees constructed by 13 PCGs strongly support the monophyly of Acheilognathus and the paraphyly of Rhodeus and Tanakia. Current results will provide valuable information for follow-up research on conservation of species facing with serious population decline and can provide novel insights into the phylogenetic analysis and evolutionary biology research.

The complete mitogenome of Paratanakia chii (Cypriniformes; Cyprinidae).

Paratanakia chii is a bitterling fish of the genus Paratanakia, subfamily Acheilognathinae and family Cyprinidae. The mitochondrial DNA sequence of P. chii is reported in this paper. The complete mitochondrial genome of P. chii is 16,575 bp in length, including 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and 1 displacement loop (D-loop). The genome sequence is consistent with those of most other carp. The majority of PCGs have AT- (Met) start codons and TA- end codons. The A + T contents of the genome, PCGs, transfer RNAs (tRNAs), and ribosomal RNAs (rRNAs) are 56.92%, 58.07%, 56.34%, and 54.21%, respectively. Phylogenetic analysis showed that P. chii is most closely related to Tanankia himantegus. These data will benefit relative ecological and phylogenetic studies.

Reproductive ecology and adaptive host choice correlated with body size in an autumn-spawning bitterling Acheilognathus typus.

The life history and reproductive ecology of an autumn-spawning bitterling Acheilognathus typus were studied under natural and experimental conditions. In the study pond, the embryos of A. typus emerged from mussels in May and grew rapidly until August, whereas overwintered age-1 fish grew slowly. Adult A. typus in the pond was smaller (32-47 mm in standard length) than they were in other habitats and mainly spawned in smaller mussels. The number of A. typus embryos in mussels was negatively correlated with the shell length of the mussel, and a lower number of embryos were observed in larger mussels (over 110 mm in shell length). In the mussel size-choice experiment conducted in an enclosure, smaller A. typus selected smaller mussels, and larger A. typus selected larger mussels for spawning. In some cases, smaller A. typus spawned in larger mussels and the number of spawned eggs ejected increased by over four times compared with other cases. These results of the enclosure experiment explained the lower number of embryos in larger mussels in the study pond. In addition, reproductive traits such as ovipositor length and the number of ovulated eggs of female A. typus, which are considered to contribute to their size-dependent host utilization, were positively correlated with their standard length. Because A. typus is geologically or seasonally isolated from other bitterling species, this size-dependent host utilization contributes to a reduction in intraspecies rather than interspecies competition.

Complete mitochondrial genome of Rhodeuscyanorostris (Teleostei, Cyprinidae): characterization and phylogenetic analysis.

Rhodeuscyanorostris Li, Liao & Arai, 2020 is a freshwater fish that is endemic to China and restricted to Chengdu City in Sichuan Province. This study is the first to sequence and characterize the complete mitochondrial genome of R.cyanorostris. The mitogenome of R.cyanorostris is 16580 bp in length, including 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and a control region (D-loop). The base composition of the sequence is 28.5% A, 27.6% C, 26.4% T, and 17.5% G, with a bias toward A+T. The genome structure, nucleotide composition, and codon usage of the mitogenome of R.cyanorostris are consistent with those of other species of Rhodeus. To verify the molecular phylogeny of the genus Rhodeus, we provide new insights to better understand the taxonomic status of R.cyanorostris. The phylogenetic trees present four major clades based on 19 mitogenomic sequences from 16 Rhodeus species. Rhodeuscyanorostris exhibits the closest phylogenetic relationship with R.pseudosericeus, R.amarus, and R.sericeus. This study discloses the complete mitochondrial genome sequence of R.cyanorostris for the first time and provides the most comprehensive phylogenetic reconstruction of the genus Rhodeus based on whole mitochondrial genome sequences. The information obtained in this study will provide new insights for conservation, phylogenetic analysis, and evolutionary biology research.

Intensity-dependent energetic costs in a reciprocal parasitic relationship.

Parasitic infections elicit host defences that pose energetic trade-offs with other fitness-related traits. Bitterling fishes and unionid mussels are involved in a two-way parasitic interaction. Bitterling exploit mussels by ovipositing into their gills. In turn, mussel larvae (glochidia) develop on the epidermis and gills of fish. Hosts have evolved behavioural responses to reduce parasite load, suggesting that glochidia and bitterling parasitism are costly. We examined the energetic cost of parasitism on both sides of this relationship. We used intermittent flow-through respirometry to measure (1) standard metabolic rate (SMR) of individual duck mussels Anodonta anatina (a common bitterling host) before and during infection by embryos of the European bitterling Rhodeus amarus, and (2) SMR and maximum oxygen uptake (MO2max) of individual R. amarus before and during infection with glochidia of the Chinese pond mussel Sinanodonta woodiana (a mussel species that successfully infects bitterling). As predicted, we observed an increase in mussel SMR when infected by bitterling embryos and an increased SMR in glochidia-infected bitterling, though this was significantly mediated by the time post-infection. Contrary to our predictions, glochidia infection did not impair MO2max and the number of glochidia attached to gills positively (rather than negatively) correlated with MO2max. The results suggest that tolerance is the prevailing coping mechanism for both fish and mussels when infected, while resistance mechanisms appear to be confined to the behavioural level.

Infection Status with Clonorchis sinensis Metacercariae in Fish from Yangcheon (Stream) in Sancheong-gun, Gyeongsangnam-do, Korea.

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yangcheon (a branch of Gyeongho-gang), which is located in Sancheong-gun, Gyeongsangnam-do, the Republic of Korea. Total 2,201 fishes in 26 species were examined by the artificial digestion method through 7 years. CsMc were detected in 1,171 (53.2%) fishes in 21 spp. (80.8%) and their density was 85 per fish infected. Total 532 (99.6%) out of 534 Pungtungia herzi (index fish) examined were infected with 147 CsMc per fish infected. Metacercarial densities in this fish were highest in 2015 (179 CsMc), followed by 2012 (168), 2013 (152), 2016 (145), 2014 (114), and 2017 (89) respectively. In the gobioninid fish group, i.e., P. herzi, Sarcocheilichthys spp., Squalidus spp., Pseudogobio esocinus, Hemibarbus longirostris, and Hemibarbus labeo, 841 (92.7%) fishes were infected with 117 CsMc per fish infected. Total 250 (54.7%) acheilognathinid fish (bitterlings), Acheilognathus spp. and Acanthorhodeus spp. were infected with 5.8 CsMc. In the rasborinid fish (chubs) group, i.e., Zacco platypus, Zacco temminckii, and Zacco koreanus, 77 (13.7%) out of 563 fish examined were infected with 2.4 CsMc in average. The susceptibility indices of CsMc were 49.09 in the overall positive fish group, 104.15 in the gobioninid group, 3.17 in the acheilognathinid group and 0.35 in the rasborinid fish group respectively. Only 1 CsMc was detected in 3 fish species, Coreoperca herzi, Channa argus, and Lepomis macrochirus, respectively. Conclusively, it was confirmed that CsMc are moderately prevalent in fishes from Yangcheon in Sancheon-gun, Gyeongsangnam-do, Korea.

Unusual AT-skew of Sinorhodeus microlepis mitogenome provides new insights into mitogenome features and phylogenetic implications of bitterling fishes.

Sinorhodeus microlepis (S. microlepis) is recently described as a new species and represents a new genus Sinorhodeu of the subfamily Acheilognathinae. In this study, we first sequenced the complete mitogenome of S. microlepis and compared with the other 29 bitterling mitogenomes. The S. microlepis mitogenome is 16,591 bp in length and contains 37 genes. Gene distribution pattern is identical among 30 bitterling mitogenomes. A significant linear correlation between A+T% and AT-skew were found among 29 bitterling mitogenomes, except S. microlepis shows unusual AT-skew with slightly negative in tRNAs and PCGs. Bitterling mitogenomes exhibit highly conserved usage bias of start codon, relative synonymous codons and amino acids, overlaps and non-coding intergenic spacers. Phylogenetic trees constructed by 13 PCGs strongly support the polyphyly of the genus Acheilognathus and the paraphyly of Rhodeus and Tanakia. Together with the unusual characters of S. microlepis mitogenomes and phylogenetic trees, S. microlepis should be a sister species to the genus Rhodeu that might diverge about 13.69 Ma (95% HPD: 12.96-14.48 Ma).

Infection Status with Clonorchis sinensis Metacercariae in Fish from Yangcheon (Stream) in Sancheong-gun, Gyeongsangnam-do, Korea

The infection status with Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fishes from Yangcheon (a branch of Gyeongho-gang), which is located in Sancheong-gun, Gyeongsangnam-do, the Republic of Korea. Total 2,201 fishes in 26 species were examined by the artificial digestion method through 7 years. CsMc were detected in 1,171 (53.2%) fishes in 21 spp. (80.8%) and their density was 85 per fish infected. Total 532 (99.6%) out of 534 Pungtungia herzi (index fish) examined were infected with 147 CsMc per fish infected. Metacercarial densities in this fish were highest in 2015 (179 CsMc), followed by 2012 (168), 2013 (152), 2016 (145), 2014 (114), and 2017 (89) respectively. In the gobioninid fish group, i.e., P. herzi, Sarcocheilichthys spp., Squalidus spp., Pseudogobio esocinus, Hemibarbus longirostris, and Hemibarbus labeo, 841 (92.7%) fishes were infected with 117 CsMc per fish infected. Total 250 (54.7%) acheilognathinid fish (bitterlings), Acheilognathus spp. and Acanthorhodeus spp. were infected with 5.8 CsMc. In the rasborinid fish (chubs) group, i.e., Zacco platypus, Zacco temminckii, and Zacco koreanus, 77 (13.7%) out of 563 fish examined were infected with 2.4 CsMc in average. The susceptibility indices of CsMc were 49.09 in the overall positive fish group, 104.15 in the gobioninid group, 3.17 in the acheilognathinid group and 0.35 in the rasborinid fish group respectively. Only 1 CsMc was detected in 3 fish species, Coreoperca herzi, Channa argus, and Lepomis macrochirus, respectively. Conclusively, it was confirmed that CsMc are moderately prevalent in fishes from Yangcheon in Sancheon-gun, Gyeongsangnam-do, Korea.

Phylogeography of Endangered Bitterling Acheilognathus melanogaster Endemic to Eastern Japan.

The bitterling Acheilognathus melanogaster is a critically endangered primary freshwater fish endemic to the Pacific side of eastern Japan. To elucidate A. melanogaster genetic structure, we investigated phylogeography in nine populations, using gene sequences of mitochondrial Cytochrome b (Cytb), as well as nuclear Rhodopsin (Rho) and glycosyltransferase (Glyt). We found four Cytb-based geographical clusters unevenly divided between the northern and southern regions, with smaller groups in the south. Of the nuclear genes, Glyt did not show geographical differentiation, whereas Rho formed two clusters: one widely occurring and another restricted to central regions. Genetic diversity was generally higher in southern than in northern populations. Our results suggest that conservation of southern local populations is particularly important in maintaining the genetic diversity of this endangered fish.

Prevalence and Intensity of Clonorchis sinensis Metacercariae in Freshwater Fish from Wicheon Stream in Gunwi-gun, Gyeongsangbuk-do, Korea.

The infection status of Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fish from a highly prevalent site, Wicheon (a branch of Nakdong-gang), which is located in Gunwi-gun, Gyeongsangbuk-do, the Republic of Korea. Total 1,162 fish in 32 species were examined by the artificial digestion method through 6 years. CsMc were detected in 720 (67.5%) out of 1,067 fish (26 spp.) and their density was 610 per fish infected. In the susceptible gobioninid fish group, i.e., Pungtungia herzi, Squalidus gracilis majimae, Squalidus japonicus coreanus, Sarcocheilichthys variegatus wakiyae and Pseudorasbora parva, all of 323 fish were infected with an average of 1,310 CsMc. Total 23 (95.8%) gobioninid fish, i.e., Pseudogobio esocinus, Abbottina springeri, Hemibarbus longirostris, Microphysogobio koreensis, and Microphysogobio jeoni, were infected with 127 CsMc in average. In the acheilognathinid fish (bitterlings) group, the prevalence was 77.0%, and the density was 50 CsMc per fish infected. In the rasborinid fish (chubs) group, i.e., Zacco platypus, Zacco temminckii, Zacco koreanus, and Opsariichthys uncirostris amurensis, 147 (36.5%) out of 403 fish examined were infected with 15 CsMc in average. The susceptibility indices of CsMc were 412 in the overall positive fish group, 1,310 in the gobioninid group-1, 122 in the gobioninid group-2, 38.5 in the acheilognathinid group, and 5.5 in the rasborinid fish group. Conclusively, it was confirmed that CsMc are highly prevalent in fish from Wicheon, and their infection tendency varied according to the subfamily groups in Cyprinidae fish hosts.

Prevalence and Intensity of Clonorchis sinensis Metacercariae in Freshwater Fish from Wicheon Stream in Gunwi-gun, Gyeongsangbuk-do, Korea

The infection status of Clonorchis sinensis metacercariae (CsMc) was examined in freshwater fish from a highly prevalent site, Wicheon (a branch of Nakdong-gang), which is located in Gunwi-gun, Gyeongsangbuk-do, the Republic of Korea. Total 1,162 fish in 32 species were examined by the artificial digestion method through 6 years. CsMc were detected in 720 (67.5%) out of 1,067 fish (26 spp.) and their density was 610 per fish infected. In the susceptible gobioninid fish group, i.e., Pungtungia herzi, Squalidus gracilis majimae, Squalidus japonicus coreanus, Sarcocheilichthys variegatus wakiyae and Pseudorasbora parva, all of 323 fish were infected with an average of 1,310 CsMc. Total 23 (95.8%) gobioninid fish, i.e., Pseudogobio esocinus, Abbottina springeri, Hemibarbus longirostris, Microphysogobio koreensis, and Microphysogobio jeoni, were infected with 127 CsMc in average. In the acheilognathinid fish (bitterlings) group, the prevalence was 77.0%, and the density was 50 CsMc per fish infected. In the rasborinid fish (chubs) group, i.e., Zacco platypus, Zacco temminckii, Zacco koreanus, and Opsariichthys uncirostris amurensis, 147 (36.5%) out of 403 fish examined were infected with 15 CsMc in average. The susceptibility indices of CsMc were 412 in the overall positive fish group, 1,310 in the gobioninid group-1, 122 in the gobioninid group-2, 38.5 in the acheilognathinid group, and 5.5 in the rasborinid fish group. Conclusively, it was confirmed that CsMc are highly prevalent in fish from Wicheon, and their infection tendency varied according to the subfamily groups in Cyprinidae fish hosts.

Mitochondrial genome of Paracheilognathus imberbis (Cypriniformes: Cyprinidae: Acheilognathinae).

In this study, the complete mitochondrial genome (mitogenome) sequence of Paracheilognathus imberbis (Cypriniformes: Cyprinidae: Acheilognathinae) was determined by long PCR and primer-walking methods. The complete mitochondrial genome is 16,819 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A(29.73%), T(27.25%), C(26.63%) and G(17.40%), respectively. The mitogenome of P. imberbis displayed novel gene order arrangement compared with published Rhodeus sinensis to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Acheilognathinae.

Complete mitochondrial genome of Rhodeus lighti (Cypriniformes: Cyprinidae).

In this study, the complete mitochondrial genome (mitogenome) sequence of Rhodeus lighti (Cypriniformes: Cyprinidae) was determined by long PCR and primer walking methods. The complete mitochondrial genome is 16,677 bp in length and contains 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes as well as a displacement loop (D-loop). The overall base composition of the genome is A (28.87%), T (27.22%), C (26.53%) and G (17.38%). The mitogenome of R. lighti displayed novel gene order arrangement compared with published Rhodeus sinensis to date. The mitogenome would contribute to resolving phylogenetic position and interrelationships of Acheilognathinae.

Complete mitochondrial genome of endangered Rhodeus pseudosericeus and its implications for the reconstruction of phylogenetic relationship among Acheilognathinae species.

Rhodeus pseudosericeus is a native bitterling to the Korean Peninsula and found in very limited areas with small census size. Here, its complete mitochondrial genome was analyzed to provide novel data for the reconstruction of phylogenetic relationship among Acheilognathinae species. The genome was a 16,574 bp long consisting of 1 putative control region, 2 rRNA genes, 22 tRNA and 13 protein-coding genes. The gene arrangement was completely identical to those observed in other Acheilognathinae species as well as in other cyprinid species. In our phylogenetic analyses, three major genera of Acheilognathinae indepedently formed monophyletic groups in the tree reconstructed based on the whole genome sequences, whereas Rhodeus was not recovered as a single monophyly when solely considering protein-coding genes, indicating that the taxonomic reevaluation is still required in this subfamily.

Complete mitochondrial genome of the Chinese bitterling Rhodeus sinensis (Cypriniformes: Cyprinidae).

The mitochondrial genome of the Chinese bitterling Rhodeus sinesis is a circular molecule of 16,677 bp in length, containing 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and two main non-coding regions (the control region and the origin of the light strand replication). The gene composition and order of which were similar to most other vertebrates. All protein-coding genes are initiated with ATG except for COX 1, which begin with GTG instead. However, the termination codons of 13 protein-coding genes are varied with TAA, TA, T or TAG. The molecular data we presented here could provide useful information for the studies on species identification, evolutionary relationships and population genetics of the Acheilognathinae.

Phylogenetic relationships of Acheilognathidae (Cypriniformes: Cyprinoidea) as revealed from evidence of both nuclear and mitochondrial gene sequence variation: evidence for necessary taxonomic revision in the family and the identification of cryptic species.

Bitterlings are relatively small cypriniform species and extremely interesting evolutionarily due to their unusual reproductive behaviors and their coevolutionary relationships with freshwater mussels. As a group, they have attracted a great deal of attention in biological studies. Understanding the origin and evolution of their mating system demands a well-corroborated hypothesis of their evolutionary relationships. In this study, we provide the most comprehensive phylogenetic reconstruction of species relationships of the group based on partitioned maximum likelihood and Bayesian methods using DNA sequence variation of nuclear and mitochondrial genes on 41 species, several subspecies and three undescribed species. Our findings support the monophyly of the Acheilognathidae. Two of the three currently recognized genera are not monophyletic and the family can be subdivided into six clades. These clades are further regarded as genera based on both their phylogenetic relationships and a reappraisal of morphological characters. We present a revised classification for the Acheilognathidae with five genera/lineages: Rhodeus, Acheilognathus (new constitution), Tanakia (new constitution), Paratanakia gen. nov., and Pseudorhodeus gen. nov. and an unnamed clade containing five species currently referred to as "Acheilognathus". Gene trees of several bitterling species indicate that the taxa are not monophyletic. This result highlights a potentially dramatic underestimation of species diversity in this family. Using our new phylogenetic framework, we discuss the evolution of the Acheilognathidae relative to classification, taxonomy and biogeography.

The function of multiple ejaculations in bitterling.

In some taxa, males perform multiple ejaculations, which may function in sperm competition or in maintaining a baseline density of spermatozoa in the female reproductive tract to ensure fertilization, a process that has been termed 'topping up'. We investigated the function of multiple ejaculations in two species of bitterling, the European bitterling (Rhodeus amarus) and Chinese rose bitterling (Rhodeus ocellatus). Bitterling oviposit in living freshwater mussels, with fertilization taking place within the mussel gill cavity. Thus, although fertilization is external, the mussel is analogous to the female reproductive tract in an internally fertilizing species. We measured the frequency of ejaculations and mussel inspections by individual males of two bitterling species in 28 replicated mesocosms and examined focal male responses to rival ejaculations and the presence of females in spawning condition. We used a model of ejaculatory behaviour to simulate the temporal abundance of spermatozoa in mussels. Male R. amarus exhibited high rates of ejaculation and inspection of the siphons of mussels and increased their ejaculation rate in response to the presence of females in spawning condition. Rhodeus ocellatus showed lower overall rates of ejaculation, but significantly elevated ejaculation rate in response to rival ejaculations. The ejaculatory strategy of R. amarus is one that maintains a minimum level of spermatozoa in mussels, which is elevated when the probability of oviposition increases. In contrast, R. ocellatus engages more directly in sperm competition with rivals. We discuss these results in the context of the function of multiple ejaculations and male mating tactics.

Molecular phylogeny and conservation priorities of the subfamily Acheilognathinae (Teleostei: Cyprinidae).

It is increasingly accepted that conservation work should consider the evolutionary history of target species. Fishes in the subfamily Acheilognathinae, family Cyprinidae, are, with the exception of three species exclusively distributed in Europe, restricted to Asia and show a distinct spawning behavior in laying their eggs in gill chambers of freshwater mussels. At present, many of the 70 species recognized in this group are facing with serious population decline in China and Japan, and their phylogenetic relationships are not well resolved. In the present study, based on mtDNA cyt b and 12S rRNA gene sequences, we reconstructed a more detailed species-level phylogenetic tree of this group, and assessed species conservation priorities based on their evolutionary distinctiveness. Molecular phylogenetic analyses showed that the Acheilognathinae contains two major clades: Acheilognathus clade and Tanakia-Rhodeus clade. Based on this phylogenetic result, conservation priority analyses were conducted using ED (evolutionary distinctiveness)/HED (heightened evolutionary distinctiveness), and EDGE (evolutionary distinctiveness and global endangeredness)/HEDGE (heightened evolutionary distinctiveness and global endangeredness) methods. The results suggested that T. himantegus, T. lanceolata, A. gracilis, A. imberbis, T. tanago, and A. longipinnis should be ranked as the top-priority species for conservation. According to our results, we also discussed the current conservation efforts of the bitterling fishes and gave suggestions for future work.