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Article

Molecular Phylogeny and Evolutionary History of the Genus Cyprinus (Teleostei: Cypriniformes)

by
Yanyan Chen
1,2,†,
Heng Xiao
3,
Zhaoping Yue
4,
Xiaoyun Wu
5,
Ruiguang Zan
2,*,† and
Shanyuan Chen
3,*
1
College of Life Science and Engineering, Henan University of Urban Construction, Pingdingshan 467036, China
2
School of Life Sciences, Yunnan University, Kunming 650091, China
3
School of Ecology and Environmental Science, Yunnan University, Kunming 650091, China
4
Reproductive Center, Xiangtan Maternal and Child Health Care Hospital, Xiangtan 411100, China
5
Key Laboratory of Puer Tea Science of Ministry of Education, Yunnan Agricultural University, Kunming 650201, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fishes 2025, 10(3), 121; https://doi.org/10.3390/fishes10030121
Submission received: 20 January 2025 / Revised: 3 March 2025 / Accepted: 6 March 2025 / Published: 10 March 2025
(This article belongs to the Section Taxonomy, Evolution, and Biogeography)
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Abstract

The genus Cyprinus encompasses economically vital freshwater fish species; yet the phylogenetic relationships and evolutionary history of many taxa within this genus remain unresolved. To address this knowledge gap, we reconstructed the molecular phylogenetic and estimated divergence times using complete mitochondrial cytochrome b (CYTB) sequences of 76 Cyprinidae specimens, within Cyprinidae, including 4 outgroup species. Phylogenetic trees were reconstructed using maximum likelihood (ML) and Bayesian inference (BI) methods, while divergence times were estimated using a Bayesian relaxed molecular clock approach. The results confirmed the monophyly of the genus Cyprinus. The relationships among C. (Cyprinus) multitaeniata, C. (C.) pellegrini, C. (C.) acutidorsalis, and three Erhai Lake species (C. (C.) longipectoralis, C. (C.) barbatus, and C. (C.) chilia) were resolved with strong support. Cyprinus (C.) multitaeniata is basal. The species in Erhai Lake form a monophyletic group, and C. (C.) acutidorsalis is at the top of the phylogenetic tree. The taxonomic delineation within the genus Cyprinus remains controversial, particularly regarding the proposed division into two subgenera (Cyprinus and Mesocyprinus), which has been historically constrained by limited specimen availability for Mesocyprinus. Our comprehensive phylogenetic analysis reveals significant evolutionary divergence patterns: The genus Cyprinus diverged from Carassius during the 56.9 Mya. Notably, the Erhai Lake radiation species (C. (C.) longipectoralis, C. (C.) barbatus, and C. (C.) chilia) originated during 2.03 Mya, while the Lake Biwa endemic C. (C.) haematopterus demonstrates 8.7 Mya. We identified a late Pleistocene speciation event (0.75 Mya) in C. (C.) acutidorsalis, coinciding with its adaptation to brackish water ecosystems. The native C. (C.) pellegrini of Xingyun Lake and Chilu Lake may have originated 4.8 Mya, when the ancient lake that its ancestral population inhabited became isolated. These findings provide robust molecular evidence supporting the recognition of two evolutionary distinct subgenera within Cyprinus.
Keywords:
Cyprinus species; phylogenetics; divergence time; CYTB
Key Contribution: Our study examines the molecular phylogeny of the economically important freshwater fish genus Cyprinus. We found that the genus Cyprinus is monophyletic. Relationships between C. multitaeniata, C. pellegrini, C. acutidorsalis, and three Erhai Lake species were resolved with strong support. Our dating estimates suggest that Cyprinus diverged from Carassius 56.8 Mya, and that the three Erhai Lake species originated 2.03 Mya. These results resolve long-standing issues related to the systematics and evolutionary history of Cyprinus.

1. Introduction

The genus Cyprinus Linnaeus 1758 has been recognized as the most widely distributed taxon within the tribe Cyprinini sensu stricto of the subfamily Cyprininae sensu stricto, following the taxonomic framework established by Cavender and Coburn 1922 [1]. The current taxonomic classification proposed by Luo and Yue (2000) [2] delineates two subgenera: Cyprinus (Cyprinus), comprising 11 species, and Cyprinus (Mesocyprinus), encompassing 5 species. Of these 16 recognized species, 15 are indigenous to China, with 11 species (68.8%) exhibiting restricted ranges in Yunnan Province. Nevertheless, there is still a lack of complete consensus regarding the relationships within this genus, the validity of its subspecies (especially those within Cyprinus carpio Linnaeus 1758), and the appropriate taxonomic level of these subspecies [2,3,4,5,6,7].
Historically, research on Cyprinus fish has a long-standing and rich background. In the early stages, morphological studies were the primary approach. Scholars meticulously observed and compared external morphological characteristics of Cyprinus fish, such as body shape, the number of scales, and the quantity and shape of fin rays, in an attempt to establish taxonomic relationships among species [8,9]. Nevertheless, this morphology-based research method has certain limitations. The similarities and variabilities of morphological characteristics among different species make it difficult to precisely define taxonomic boundaries, leading to significant taxonomic controversies, especially for species or subspecies with similar morphologies [10].
With the development of molecular biology techniques, numerous molecular analyses have been applied to the study of Cyprinus fish in the past few decades. However, the majority of these studies have almost solely focused on Cyprinus carpio. For instance, within the genus, Cyprinus carpio has the largest distribution in Eurasia and has been extensively domesticated; there has been considerable research on its intraspecific relationships at the level of populations, strains, breeds, etc., including the origin of the domestic strain and the validity of the subspecies [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27]. Additionally, the genetic similarity of four Cyprinus species in Erhai Lake (Dali, China) has been analyzed to confirm their sympatric speciation [28,29]. Despite the abundance of research on Cyprinus carpio, its taxonomy remains incompletely resolved due to the lack of research on the phylogenetic relationships between Cyprinus carpio and other species within the same subgenus.
Despite the abundance of research on C. carpio, its taxonomy remains incompletely resolved due to the lack of research on the phylogenetic relationships between C. carpio and other species within the same subgenus. For example, in the analysis of C. carpio rubrofuscus Lacepède 1803 (i.e., C. c. viridiviolaceus), the inclusion of too few samples makes it difficult to determine whether it is a valid subspecies [19]. In another study, five individuals from the Amur River were used in an intraspecies mtDNA phylogenetic analysis; and the results suggested that C. c. carpio was not a valid subspecies [15]. However, there are doubts about whether all these individuals were wild [16]. Therefore, the literature is currently unclear about whether this species is monophyletic.
Chen and Yang carried out a cladistic analysis specifically on the subgenus C. (Mesocyprinus). However, a morphological approach has yet to be utilized for studying the phylogeny of the genus Cyprinus or subgenus C. (Cyprinus) [7]. In the past two decades, although there have been many molecular analyses of the genus Cyprinus, almost all have centered on the species Cyprinus carpio. Moreover, there have been no molecular phylogenetic analyses to date that encompass the entire genus and its subgenera. As a result, there is a lack of clear molecular information regarding the interspecific relationships and evolutionary history of this genus.
In the present study, we conduct a molecular phylogenetic analysis to resolve the phylogeny of the genus Cyprinus, its subgenus C. (Cyprinus), and particularly C. carpio. To achieve this, we have collected samples over an extended period. Our sample collection includes specimens of the subspecies C. c. haematopterus from three different river systems, including the Songhua River in the Amur River system; five samples of the subgenus C. (Cyprinus), the only brackish-water species; four lacustrine native species; and one species within the subgenus C. (Mesocyprinus). Nine other species in this genus, eight of which are lacustrine, may already be extinct. Although the number of available species is relatively small, we believe that the diversity and distribution of these samples are sufficient to uncover the inter-relationships and evolutionary histories within this group. Our aim was to clarify the phylogeny, origin, and pattern of speciation of C. carpio.
We address two questions arising from the comparison of two phylogenetic studies of cyprinids (mainly in Cyprininae sensu Cavender and Coburn 1992). The first study is Wang et al. which used the CYTB gene as a marker [30], and the second is by Yang et al. [31], in which five mtDNA genes were used as markers. The questions are as follows. First, if a sufficient number of taxa are included in the analysis and the correct outgroup is used, and if the three groups—V (Procypris), VI (Sinocyclocheilus), and VIII (Cyprinus, Carassius, Carassioides, and Puntius semifasciolatus Günther, 1868)—from Wang et al. are considered as a new ingroup, can this ingroup be resolved as monophyletic using only CYTB as the marker [30], and will this ingroup correspond to clade 5 of Yang et al. [31]? Second, can P. semifasciolatus be resolved as a member of this monophyletic group along with Carassius, Carassioides, and Cyprinus [31]?
For reconstructing accurate phylogenies, sampling more taxa is more effective than increasing the number of characters [32]. Moreover, appropriate outgroup selection can lead to correct conclusions about the monophyly of the ingroup, and the most appropriate outgroup is the sister group of the ingroup [13]. The present study is designed based on these facts.
Here, we used Procypris, Cyprinus, Carassius, Carassioides, Sinocyclocheilus, and P. semifasciolatus as the ingroup and sampled as extensively as possible. All members of this ingroup, except P. semifasciolatus, corresponded at the generic level to clade 5 of Yang et al. [31]. In Yang et al., clades 4 and 5 were resolved to have a sister relationship with strong support. Clade 4 of Yang et al. corresponds to clade IX of Wang et al. [30]; we select some representative species from this clade as the out-group.
We used the CYTB gene, which has been widely applied as a marker in cyprinid phylogenetic studies [30,33,34,35,36,37,38,39,40,41]. It has been demonstrated to be suitable for resolving cyprinid phylogeny and can enable maximum incorporation of related taxa into the analysis. To better understand the origin and evolutionary histories of the related taxa and their possible relationships to known geological events, we also estimate the divergence times.
The objectives of this analysis are: (1) to reveal the phylogenetic relationships among species in the genus Cyprinus, especially in the subgenus C. (Cyprinus), and to understand the origins, origin times, and speciation patterns of these species and groups; (2) to reconstruct the intraspecific phylogeny of C. (C.) carpio, to comment on the remaining controversial issues, and to understand how its characteristics relate to our results. Our secondary aim is to evaluate the performance of CYTB as a marker in resolving phylogenies within this group. Furthermore, we seek to verify the composition of clade 5 of Yang et al. [31]. Finally, we aimed to answer the questions raised earlier, which originated from the study by Wang et al. [31].

2. Materials and Methods

2.1. Sample Collection

A total of 80 specimens were examined in this study, among them 37 specimens of the subfamily Cyprininae were collected in this study (Table 1), 31 from the subgenus C. (Cyprinus), 2 from the subgenus C (Mesocyprinus), and 4 specimens from Carassius, and previously published mtDNA sequences of other 39 individuals and 4 outgroup species were downloaded from GenBank (Table 2) and included in our analyses. Tissue samples were preserved in 100% ethanol, and voucher specimens were deposited in the Zoological Museum of Yunnan University. Additionally, Schizothorax argentatus Kessler 1874, Barbus barbus Linnaeus 1758, Spinibarbus denticulatus Chu et Cui 1989, and Gyrinocheilus aymonieri Tirant 1883 were included as outgroup taxa. The distribution of the genus Cyprinus in China is showed in Figure 1.

2.2. DNA Preparation, PCR Amplification, and Sequencing

Genomic DNA was extracted from muscle tissues using a standard phenol-chloroform protocol. Complete sequences of CYTB were amplified by PCR, using the primers L14724, L14737, and H15915 [42,43]. PCR amplifications were carried out in 50 μL reaction mixtures consisting of 5 μL 10 × PCR buffer (TaKaRa Biotechnology Co. Ltd., Dalian, China) 0.2 mM dNTPs, 0.2 μM of each primer, 1.5 U Taq DNA polymerase (TaKaRa Biotechnology Co. Ltd., Dalian, China), and approximately 50 ng genomic DNA, filled to 50 μL with sterilized water. Amplification was implemented as follows: denaturing at 94 °C for 5 min; 35 cycles of denaturation at 94 °C for 1 min; annealing at 52 °C for 1 min; and extension at 72 °C for 1 min. PCR products were electrophoresed in a 1.5% agarose gel, and successful amplifications were sequenced in an ABI Prism 3730 (Applied Biosystems Inc., ABI) automatic sequencer. Sequencing used both PCR primers and internal primers (L15138, L15286, L15519, H15374, and H15560) [42,43].

2.3. Data Analyses

All sequences were checked and aligned using SeqMan and EditSeq implemented in DNASTAR v. 7.1 (DNASTAR Inc., Madison, WI, USA). The newly obtained sequences in this study, together with previously published sequences, were used for subsequent analyses. CYTB sequences were aligned using ClustalW1.8.3 implemented in MEGA v. 12 [44]. The aligned sequence data were then used to calculate nucleotide composition and transitional/transversional ratios in MEGA v. 12 [44]. Nucleotide substitution saturation was analyzed using DAMBE 5.3.48 [45].
We performed phylogenetic analyses using Bayesian inference (BI) in MrBayes v. 3.2. [46] and maximum likelihood (ML) using RAxML-NG v. 1.2.0 [47]. The best-fitting nucleotide substitution model of CYTB sequences was detected by jModeltest 2.17, using the likelihood ratio test [48]. GTRCAT_GAMMAI was selected as the best-fitting nucleotide substitution model for the ML and BI analysis. For the ML analyses, nodal support was assessed using nonparametric bootstrapping of 1000 pseudo-replicates [49]. For BI analyses, random starting trees were used, and four independent Markov chain Monte Carlo chains (one cold and three heated) were run for 1 × 107 generations, sampling the Markov chain at intervals of 100 generations, for a total size of 1 × 105 generations. The samples prior to reaching stationarity (25,000 trees) were discarded as burn-in. The remaining sample trees were then used to generate a majority rule consensus tree that was used to determine the Bayesian posterior probabilities of the clades.

2.4. Divergence Time Estimation Analyses

The divergence times within the genus Cyprinus were estimated by the Bayesian relaxed clock method using the MCMCtree program within PAML v. 4.9 [50]. To include reliable fossils of Cypriniformes, we used sequences from Yang et al. [31], excluding their clade 5. We also used the sequences produced in our study. To achieve computational efficiency, we used a normal approximation to the maximum likelihood estimates of branch lengths. The substitution rate was estimated using the BASEML program of PAML v. 4.9 [50]: the shape parameter (α), the scale parameter (β) for the gamma prior of the root rate parameter, and the rate drift parameters were determined by the suggested procedures. The time unit was 100 Mya (million years).
The root age (basal Cypriniformes) was assumed to be 94.9 Mya [51], based on Near et al. Seven time-points were used to calibrate clock dates. (1) A date of 94.9 Mya (78.9–113.3 Mya) was used for estimating the divergence of Cyprinidae and Catostomidae [51]. (2) The fossil of Amyzon (49.4 Mya) was used for estimating the divergence of Ictiobus and Hypentelium [51]. (3) The earliest fossil record of Parabarbus is from the early medium Eocene (48.6 Mya) [52]; we have therefore assumed that the split between the clade of Probarbus jullieni Sauvage 1880 and Catlocarpio siamensis Boulenger 1898, and the remaining clades of Cyprininae sensu lato, was at least 48.6 Mya. (4) The earliest fossil of Eoprocypris maomingensis Dunn 1942 was recorded from the late Eocene (37.2–33.9 Mya) [53]; we therefore assumed that the split between the clade of the Procypris and Cyprinus/Carassius occurred at least 18 Mya. (5) The earliest fossil of Huashancyprinus robustispinus was from the Oligocene (33.9–23.03 Mya) [54]; we therefore assumed that the split between the clade of the Cyprinus and Carassius was at least 23.03 Mya. (6) Barbus bohemicus and Barbus sp. were reported from the Czech Republic and dated from 18–19 Mya [52]; we therefore assumed that the split between the clade of the Barbus and its sister group (Luciobarbus and Capoeta) was at least 18 Mya. (7) The opening of the Gibraltar Strait occurred about 5.33 Mya; we used this as the time when the North African barbs separated from the Iberian barbs [55]. The Markov Chain was run for 1.0 × 108 generations and sampled every 100 generations after an initial burn-in of 1 × 106 generations. The chain was then sampled every 1000 generations.

3. Results

3.1. Sequences Characters

We obtained complete CYTB sequences (1140 bp) of 45 specimens of the genus Cyprinus. None of the protein-coding sequences had premature stop codons or ambiguous nucleotides in translation, indicating that these sequences were for functional genes [56]. The CYTB sequences exhibited 152 variable sites. On the basis of all sequences, the mean nucleotide base composition of CYTB was 29.9%, 29.9%, 14.0%, and 26.2% for A, C, G, and T, respectively. The ratio of transitional/transversional pairs was 8.02.

3.2. Phylogenetic Analyses

Eighty complete CYTB sequences (1140 bp) were used for constructing phylogenetic trees (Table 1 and Table 2). The phylogenetic trees recovered using BI and ML methods had identical topologies for the main clades; we therefore present only the Bayesian tree (Figure 2). The monophyly of Cyprinus was strongly supported (BP = 92%, Bayesian posterior probability (BPP) = 100%) in all cases. Five major clades were identified: Sinocyclocheilus (Clade I), Procypris (Clade II), Puntius (Clade III), Carassius/Carassioides (Clade IV), and Cyprinus (Clade V), respectively (Figure 2).
Clade I (BP = 92%, BPP = 100%), which was basal to the tree, contained all the species of Sinocyclocheilus used in the present study, and the topological structure was similar to that in previous studies [42,57]. Clade II (BP = 100%, BPP = 100%) comprised two Procypris species, P. mera and P. rabaudi. Clade III (BP = 100%, BPP = 100%) comprised two Puntius semifasciolatus (Puntius semifasciolatus has been reclassified as Barbodes semifasciolatus). Clade IV (BP = 90%; BPP = 100%) comprised all specimens of Carassioides and Carassius; these genera were resolved as sister groups. Clade V (BP = 99%; BPP = 100%) comprised all specimens of the genus Cyprinus. Within this clade, C. (C.) multitaeniata Pellegrin and Chevey 1936 was basal and was sister to all other species of the subgenus C. (Cyprinus). Four lacustrine native species and one brackish water species were resolved with strong support: C. (C.) pellegrini Tchang 1933 was basal to C. (Cyprinus), C. (C.) acutidorsalis Wang 1979 was at the top of the tree, and the three Erhai Lake species formed a monophyletic group. Cyprinus carpio was polyphyletic; nevertheless, the LBW strain was clustered as the basal group within C. (Cyprinus), with strong support. C. c. haematopterus from the Volga River was clustered as a clade along with C. c. haematopterus from northeastern China.

3.3. Divergence Time Estimation

Dating estimates suggest that C. (Cyprinus) diverged from Carassius about 56.9 Mya (Figure 3). In Cyprinus, the divergence of C. (C.) multitaeniata was the earliest divergence within the group, at 32.49 Mya (41.65–71.65 Mya). Cyprinus c. haematopterus of Lake Biwa originated about 8.73 Mya. The three species from Erhai Lake clustered as a monophyletic group that originated about 2.03 Mya. The species Cyprinus (C.) pellegrini, which is native to Xingyun Lake and Chilu Lake, originated about 4.8 Mya, and C. (C.) acutidorsalis possibly originated about 0.75 Mya, as an adaptation to the brackish water environment, and C. (C.) multitaeniata originated about 32.5 Mya. Therefore, this study provides evidence that C. (C.) acutidorsalis is not the most primitive species of C. (Cyprinus). Cyprinus (C.) pellegrini, which is native to Xingyun Lake and Chilu Lake, might have originated at about 4.8 Mya, when the ancient lake that its ancestral population inhabited was isolated.
The Erhai Lake region was the center of distribution of the subfamily Cyprininaesensu stricto [41]; previous studies have shown that the Erhai basin was formed during the intersection of the Pliocene and Pleistocene (2.588 Mya); Erhai Lake, a faulted lake, was formed in the early Pleistocene (2.588–1.806 Mya) [58,59,60]. According to our estimates, the three species from Erhai Lake (C. (C.) longipectoralis Chen and Huang 1977, C. (C.) barbatus, and C. (C.) chilia Wu, Yang, and Huang 1963 might have originated in the early Pleistocene (2.03 Mya), which is consistent with the date when Erhai Lake formed. According to our estimation, the divergence time of the three C. c. haematopterus samples of Lake Biwa (Japan) was 8.7 Mya, which is earlier than the formation of Lake Biwa; this is inconsistent with the view that the water systems of Japan were connected to those of the continent 5.332 Mya [61]. Our estimate, however, is consistent with the formation of the ancient Sea of Japan. This implies that the geological isolation of these water systems from the continental water system might have occurred at the start of the Miocene. This suggests paths for further geological research.

4. Discussion

4.1. The Phylogeny and Geography Distribution of Cyprinus

The species within C. (Cyprinus) that we analyzed were resolved as a monophyletic group (BPP = 100%, BP = 100%) and as a sister group to C. (C.) multitaeniata (BPP = 100%, BP = 98%). Most Chinese ichthyologists, including the authors of the present study, consider the species we studied to belong to C. (Cyprinus); those in the sister group C. (C.) multitaeniata, however, are widely considered to belong to a different subgenus, C. (Mesocyprinus) [3,7]. Although the results of the present study suggest that Cyprinus can be divided into two subgenera, they are not conclusive because we included only one species of the subgenus C. (Mesocyprinus) in our analysis. The disagreements over this question [2,3,4,5,7] therefore remain unsettled.
Surprisingly, within the monophyletic C. (Cyprinus) group, most of the species are nested within the polyphyletic clade of C. (C.) carpio. Groups that were nested outside of C. (C.) carpio include Cyprinus (C.) acutidorsalis (which inhabits brackish water), C. (C.) pellegrini (which occurs only in Chilu Lake and Xingyun Lake), and the monophyletic group comprising C. (C.) longipectoralis, C. (C.) barbatus, and C. (C.) chilia from Erhai Lake. Furthermore, different populations of the same species, in this case the population of C. (C.) chilia from Erhai Lake, were not monophyletic, as is consistent with previous studies [28]. C. (C.) chilia exhibits high genetic diversity [62], potentially due to hybridization with other cyprinid species in Erhai Lake. Hybridization may result in offspring inheriting genetic material from divergent parental lineages, thereby generating phylogenetic relationships with other cyprinid species that appear ambiguous in cladistic analyses [29]. Furthermore, Ren et al. demonstrated that gene flow is a key driver of phylogenetic incongruence among cyprinids, and C. (C.) chilia may have experienced genetic introgression from related cyprinid species [63]. These factors may be the reasons for C. (C.) chilia not being a monophyletic group. Samples from different river systems are clustered in different clades according to the rivers; except for three species from Erhai Lake, our results were consistent with the study of Zheng et al. that the genetic divergence of sympatric speciation was lower than in allopatric speciation [29]. These results indicate that different species of C. (Cyprinus) have a clear geographical structure, and this may imply that geographical isolation was the main factor leading to genetic differentiation. Interspecific relationships such as this are rare within Cyprinidae. The implications of these findings are that all the species within C. (Cyprinus) that we analyzed, including the two populations of C. (C.) chilia, are derived from different ancestral populations within the C. (C.) carpio clade.
The subspecies division of Cyprinus carpio remains controversial. Chen and Huang (1977) considered C. (C.) carpio to have four subspecies: C. (C.) carpio, C. (C.) chilia, C. c. rubrofuscus, and C. c. haematopterus [3]. In contrast, Wang (1979) considered C. (C.) chilia to be a separate species and merged the other three into one species together with other subspecies. Our study does not support either of these views, as neither C. (C.) chilia nor C. c. rubrofuscus formed monophyletic clades.

4.2. Divergence Times in Cyprinus (Cyprinus)

The origin and evolutionary history of Cyprinus (Cyprinus) remain a subject of active debate among ichthyologists [20,64]. Previous studies, often limited by the inclusion of only a small number of taxa, have left the divergence times within this genus poorly resolved. In the present study, we analyzed five species from Cyprinus (Cyprinus), one species from C. (Mesocyprinus), and C. carpio to clarify the divergence times of these groups. Earlier studies, such as those by Bermingham et al. and Bown et al., proposed that a genetic distance of 0.02 based on CYTB analysis corresponds to 1 million years of divergence in Osteichthyes [65,66]. However, these estimates are now considered outdated, as more recent molecular clock calibrations and genomic analyses have refined our understanding of divergence times in teleost fishes. For instance, Yao et al. suggested that the C. (C.) acutidorsalis diverged from C. carpio around 0.65 Mya, prior to the origin of C. (C.) multitaeniata [67]. In contrast, our findings indicate that C. (C.) acutidorsalis likely originated approximately 0.75 Mya, potentially as an adaptation to the brackish water environment, while C. (C.) multitaeniata diverged much earlier, around 32.5 Mya. These results challenge the notion that C. (C.) acutidorsalis is not the most primitive species within C. (Cyprinus). Additionally, we estimate that C. (C.) pellegrini, endemic to Xingyun Lake and Chilu Lake, originated approximately 4.8 Mya, coinciding with the isolation of the ancient lake inhabited by its ancestral population.
The Erhai Lake region is recognized as a center of diversification for the subfamily Cyprininae sensu stricto. Geological evidence indicates that the Erhai basin formed during the transition from the Pliocene to the Pleistocene (2.588 Mya); with Erhai Lake, itself, emerging as a faulted lake in the early Pleistocene (2.588–1.806 Mya) [58,59,60]. Our estimates suggest that the three species endemic to Erhai Lake (C. (C.) longipectoralis, C. (C.) barbatus, and C. (C.) chilia diverged around 2.03 Mya, consistent with the timing of the lake’s formation. In contrast, our analysis places the divergence time of the three C. c. haematopterus samples from Lake Biwa (Japan) at approximately 8.7 Mya, predating the formation of Lake Biwa itself. This finding challenges the hypothesis that the water systems of Japan were connected to those of the Asian mainland around 5.332 Mya [61]. Instead, our estimate aligns more closely with the formation of the ancient Sea of Japan, suggesting that the geological isolation of these water systems from the continental water network may have occurred as early as the Miocene. This discrepancy highlights the need for further geological and phylogeographic research to reconcile these timelines.

5. Conclusions

In this study, we reconstructed the phylogenetic relationships among species within the genus Cyprinus, with a particular focus on the subgenus C. (Cyprinus). By examining the molecular phylogeny of the economically important freshwater fish genus Cyprinus, we confirmed that Cyprinus is monophyletic. Our analyses resolved the relationships between C. (C.) multitaeniata, C. (C.) pellegrini, C. (C.) acutidorsalis, and three Erhai Lake species with strong support. Divergence time estimates indicate that Cyprinus diverged from Carassius approximately 56.8 Mya, while the three Erhai Lake species originated around 2.03 Mya. These findings provide critical insights into the systematics and evolutionary history of Cyprinus, addressing long-standing questions regarding its phylogenetic relationships and diversification timeline.

Author Contributions

Conceptualization: R.Z., H.X. and S.C.; sample collection: R.Z., Z.Y. and X.W.; methodology: Z.Y., X.W., Y.C., H.X., R.Z. and S.C.; data analysis: Y.C., X.W. and R.Z.; supervision: R.Z., H.X. and S.C.; visualization: Y.C. and R.Z.; writing—original draft preparation: Y.C., X.W. and R.Z.; writing—review and editing: Y.C., H.X., R.Z. and S.C.; funding acquisition: R.Z., H.X. and S.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was partially supported by the National Natural Science Foundation of China (31560111), the Hundred Oversea Talents Recruitment Program of Yunnan Province, the Henan Province Science and Technology Research Project (Grant No. 222102320077).

Institutional Review Board Statement

The use of animals in this study was approved by School of ecology and Environmental Science YNU; Approval date: 13 March 2022.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Figure 1. Distribution of the genus Cyprinus in China. Samples collected from different regions were marked with different symbols.
Figure 1. Distribution of the genus Cyprinus in China. Samples collected from different regions were marked with different symbols.
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Figure 2. Bayesian tree constructed from CYTB sequences for Procypris, Cyprinus, Carassius, Carassioides, Sinocyclocheilus, and Puntius semifasciolatus. Node support values are Bayesian posterior probabilities.
Figure 2. Bayesian tree constructed from CYTB sequences for Procypris, Cyprinus, Carassius, Carassioides, Sinocyclocheilus, and Puntius semifasciolatus. Node support values are Bayesian posterior probabilities.
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Figure 3. Chronogram of the Cyprininae. Divergence times were estimated using the Bayesian relaxed clock method. Time scale is in millions of years ago (Mya). The numbers above the nodes indicate the estimated divergence times. The branches are named according to Yang et al.’s [31] study.
Figure 3. Chronogram of the Cyprininae. Divergence times were estimated using the Bayesian relaxed clock method. Time scale is in millions of years ago (Mya). The numbers above the nodes indicate the estimated divergence times. The branches are named according to Yang et al.’s [31] study.
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Table 1. Species and sampling locations for the species that were collected for this study. N indicates the number of samples.
Table 1. Species and sampling locations for the species that were collected for this study. N indicates the number of samples.
SpeciesLocalityN
Cyprinus pellegriniXingyun Lake, Yunnan, China 5
Cyprinus longipectoralisErhai Lake, Yunnan, China3
Cyprinus barbatusErhai Lake, Yunnan, China2
Cyprinus chiliaErhai Lake, Yunnan, China2
Cyprinus chiliaQiubei Country, Yunnan, China1
Cyprinus carpio rubrofuscusYuanjiang River, Yunnan, China4
Cyprinus carpio haematopterusYuanjiang River, Hunan, China3
Cyprinus carpio haematopterusJinan, Shandong, China3
Cyprinus carpio haematopterusYangtze River, Wuhan, China4
Cyprinus carpio haematopterusSonghuajiang River, Jilin, China3
Cyprinus acutidorsalisQingzhou, Guangxi, China3
Carassius auratus auratusDianchi Lake, Yunnan, China2
Carassius auratus auratusShili Country, Yunnan, China1
Carassius auratus auratusDolsko, Slovenija, Europe1
Table 2. Species, sampling locations, and GenBank accession numbers for species that we did not collect ourselves.
Table 2. Species, sampling locations, and GenBank accession numbers for species that we did not collect ourselves.
SpeciesLocalityAccession No.
Cyprinus carpio carpioVolga River, RussiaAY347294, AY347295
Cyprinus carpio haematopterusLake Biwa, JapanAB158803, AB158804. AB158805
Cyprinus carpio haematopterusYangtze River, Wuhan, ChinaAY347281, AY347291
Cyprinus carpio rubrofuscusYuanjiang River, Yunnan, ChinaAY347280, AY347290
Cyprinus carpio carpioTaiwan, ChinaX61010
Cyprinus multitaeniataQinzhou, Guangxi, ChinaKC696556, KC696557
Carassioides cantonensisQinzhou, Guangxi, ChinaKC696558
Carassius auratus auratus-AB111951
Carassius auratus langsdorfi-AB006953
Carassius carassius-GU135602
Carassius cuvieri-AB045144
Carassius cuvieri-AP011237
Carassius gibelio-GU138989
Procypris meraXijiang, Guangxi, ChinaKC696555
Procypris rabaudiMudong, Chongqing, ChinaNC_011192
Puntius semifasciolatusGuangxi, ChinaAY856116
Puntius semifasciolatusLuoping, Yunnan, ChinaKC696521
Sinocyclocheilus altishoulderusMashan County, GuangxiFJ984568
Sinocyclocheilus anatirostrisLeye County, GuangxiAY854708
Sinocyclocheilus angustiporusLuxi County, YunnanAY854702
Sinocyclocheilus anophthalmusJiuxiang, Yiliang County, YunnanAY854715
Sinocyclocheilus cyphotergousLuodian County, GuizhouAY854711
Sinocyclocheilus grahamiQinglongsi, Kunming, YunnanGQ148557
Sinocyclocheilus guishanensisGuishan, Shilin County, YunnanAY854722
Sinocyclocheilus hyalinusAlugudong, Luxi County, YunnanAY854721
Sinocyclocheilus lateristriatusLuliang County, GuangxiAY854703
Sinocyclocheilus lingyunensisShading, Linyun County, GuangxiAY854691
Sinocyclocheilus macrocephalusHeilongtan, Shilin County, YunnanAY854683
Sinocyclocheilus macrolepisNandan County, GuangxiAY854729
Sinocyclocheilus macrophthalmusXiaao, Duan County, GuangxiHM536792
Sinocyclocheilus malacopterusJinji, Luoping County, YunnanAY854697
Sinocyclocheilus microphthalmusShading, Linyun County, GuangxiAY854690
Sinocyclocheilus purpureusYanshan County, YunnanEU366189
Outgroup
Barbus barbusEurope, FranceAB238965
Gyrinocheilus aymonieriSoutheast AsiaNC008672
Schizothorax argentatusLi River, KazakhstanAY954269
Spinibarbus denticulatusFuxian Lake, Yunnan, ChinaKC696533
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Chen, Y.; Xiao, H.; Yue, Z.; Wu, X.; Zan, R.; Chen, S. Molecular Phylogeny and Evolutionary History of the Genus Cyprinus (Teleostei: Cypriniformes). Fishes 2025, 10, 121. https://doi.org/10.3390/fishes10030121

AMA Style

Chen Y, Xiao H, Yue Z, Wu X, Zan R, Chen S. Molecular Phylogeny and Evolutionary History of the Genus Cyprinus (Teleostei: Cypriniformes). Fishes. 2025; 10(3):121. https://doi.org/10.3390/fishes10030121

Chicago/Turabian Style

Chen, Yanyan, Heng Xiao, Zhaoping Yue, Xiaoyun Wu, Ruiguang Zan, and Shanyuan Chen. 2025. "Molecular Phylogeny and Evolutionary History of the Genus Cyprinus (Teleostei: Cypriniformes)" Fishes 10, no. 3: 121. https://doi.org/10.3390/fishes10030121

APA Style

Chen, Y., Xiao, H., Yue, Z., Wu, X., Zan, R., & Chen, S. (2025). Molecular Phylogeny and Evolutionary History of the Genus Cyprinus (Teleostei: Cypriniformes). Fishes, 10(3), 121. https://doi.org/10.3390/fishes10030121

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