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Article

Historical Landscape Evolution Shaped the Phylogeography and Population History of the Cyprinid Fishes of Acrossocheilus (Cypriniformes: Cyprinidae) According to Mitochondrial DNA in Zhejiang Province, China

by
Mu-Yang Zhou
1,†,
Jun-Jie Wang
2,†,
Jian-Feng Ren
1,
Fan Li
3,
Jin-Xian Wu
2,
Jia-Jun Zhou
4,
Jia-Le Li
1,
Jin-Quan Yang
1,* and
Hung-Du Lin
5,*
1
Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, Shanghai Ocean University, Shanghai 201306, China
2
Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring, School of Life Science, South China Normal University, Guangzhou 510631, China
3
Shanghai Natural History Museum, Branch of Shanghai Science and Technology Museum, Shanghai 201306, China
4
Zhejiang Forest Resource Monitoring Center, Hangzhou 310020, China
5
The Affiliated School of National Tainan First Senior High School, Tainan 701, Taiwan
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Diversity 2023, 15(3), 425; https://doi.org/10.3390/d15030425
Submission received: 29 January 2023 / Revised: 5 March 2023 / Accepted: 10 March 2023 / Published: 14 March 2023
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Abstract

:
Geological events and landscape features, as well as changes in the climate during the Pliocene period, have shaped the distribution of genetic diversity and demographic history of freshwater fish in mainland China. In this study, we investigated the phylogeny and population genetic structure of Acrossocheilus species (A. fasciatus, A. kreyenbergii and A. wenchowensis) in the Zhejiang region by the mitochondrial cytochrome b (cyt-b) and control (D-loop) region s. Mitochondrial phylogenetic analysis revealed three major lineages (lineages A, B and C), which may represent A. fasciatus, A. wenchowensis and A. kreyenbergii, respectively. Our results revealed that A. fasciatus and A. wenchowensis diverged from A. kreyenbergii in the Zhejiang region. The uplift of the Wuyi Mountains and the Xianxia Mountains served as an important geographic barrier in the diversification of the three Acrossocheilus species. The most recent common ancestors (TMRCAs) of the three lineages and lineages A + B were dated to 1.859 and 1.614 myr, respectively. Our results indicate that the effective population sizes of A. fasciatus and A. wenchowensis remained constant from the past to the present, as supported by ABC analysis. For conservation and protection, a strategy is required because of their genetic uniqueness, and we suggest that the two regions divided by the Xianxia Mountains be regarded as different management units (Mus), conforming to the major zoological regions of the Zhejiang region.

1. Introduction

There are 1323 freshwater fish species in mainland China, accounting for 9.6% of global fish species, indicating high fish diversity in this region [1]. Limited dispersal and low connectivity due to geographic barriers drive fine-scale population structure and reflect changes in the river network and biogeographic history. Therefore, freshwater fish are faring worse than terrestrial vertebrates, and the decline in freshwater fish populations is globally much higher (81%) than that observed in terrestrial (38%) environments [2]. Freshwater ecosystems have been subjected to the most severe impacts of multiple environmental stressors due to rapid economic development and fast urbanization over the last 30 years in mainland China, such as flow regulation (e.g., damming and hydropower), water pollution, overharvesting, illegal fishing and the introduction of invasive species. Understanding the genetic diversity patterns and biological processes of freshwater fish would help to design comprehensive conservation schemes.
The history of Zhejiang’s hydrological network is associated with complex geographical barriers driven by the development of mountainous topography and Pliocene climate fluctuation [3]. According to fundamental geohistory events and the ichthyofauna’s structure, the Wuyi-Xianxialing Mountains act as an important geographic barrier between the Yangtze River basin and independent coastal rivers of the Zhejiang region, including the Qiantang River, Oujiang River and Jiaojiang River. During the glacial period, the mouths of streams may have become confluent, and connections between hydrogeographic basins may have occurred because the continental shelf of the East China Sea was largely above water [4]. These geographic events would have left an imprint on the fish fauna’s composition and some fishes’ genetic structure within these drainages. Therefore, phylogeographic studies of fish in freshwater are important for understanding the history of the reorganization of rivers among drainages. The growing number of freshwater fish phylogeographic studies in southern mainland China has enabled us to assess biogeographic history and genetic diversity for conservation. Previous studies have mainly focused on the relationship between the Yangtze River and Qiantang River in the northern Zhejiang region (e.g., Huigobio chenhsienensis [4]; Squalidus argentatus [5]; Sinibrama macrops [6]; Sarcocheilichthys sinensis [7]). Presently, little is known about the phylogeographic structure and demographic history of freshwater fish populations in Zhejiang’s hydrological network, especially in the southern Zhejiang region, and the genetic variability of the species has not been assessed to date.
The genus Acrossocheilus Oshima, 1919, is composed of small to medium-sized barbine species and comprises 26 valid species distributed in Southeast Asia [8]. Landscape evolution can influence the evolution of freshwater fish species [9]. The Yangtze River is the northernmost limit of the distribution of this genus. According to previous studies, the Nanling and Wuyi Mountains played a key role as geographical barriers to the dispersal of Acrossocheilus species on either slope in southeast mainland China. There are three species distributed in the Yangtze River and northern region of the Wuyi Mountains (Zhejiang Province), namely Acrossocheilus wenchowensis, A. fasciatus and A. kreyenbergii. According to previous phylogenetic analyses based on whole-mitogenomic data, A. wenchowensis, A. fasciatus and A. kreyenbergii were monophyletic, while A. wenchowensis was sister to A. fasciatus and clustered with A. kreyenbergii [10]. Acrossocheilus kreyenbergii was distributed in the Yangtze-Pearl Rivers. The distributions of A. fasciatus and A. wenchowensis were divided by the Xianxia Mountains on the northern edge of the Wuyi Mountain chains as geographical barriers. Acrossocheilus fasciatus was mainly distributed in the northern Zhejiang region, including the Qiantang River, Yongjiang River and Jiaojiang River, while A. wenchowensis was mainly only distributed in the southern Zhejiang region, mainly in the Ou River and its adjacent water systems. However, escaped non-native fish had already changed the original fish community, and this trend was further intensified by artificial fish propagation and release in mainland China [11]. Based on field investigations by the authors, some individuals of A. fasciatus have been found in the wild in the southern Zhejiang basin, probably as a result of anthropogenic release by fish keepers.
Over the past 20 years, rapid urbanization and infrastructure has developed in the Zhejiang region, threatening the biodiversity of freshwater fish. For local governments and fishery farmers, the release of hatchery-reared individuals can be a viable approach to increasing the productivity of a fishery. However, artificial propagations and release can also lead to a loss of genetic diversity and a change in population structure. Before the implementation of conservation strategies, it is highly recommended that a clear picture be drawn of the genetic diversity of the population and the structure of the introduced population.
In this study, mitochondrial cytochrome b gene (cyt b) and control region (D-loop) sequences were obtained from three Acrossocheilus species in the Zhejiang region covering the distribution drainages of A. fasciatus and A. wenchowensis. The purpose of this study was to address three main questions as follows: (1) What is the phylogeny and population genetic structure of Acrossocheilus species in the Zhejiang region; (2) How did Acrossocheilus species colonize the rivers of different geographical basins on the Yangtze River by the Zhejiang region under the influences of paleoclimatic events and Pleistocene sea-level changes?; and (3) What competing scenarios of demographic and phylogenetic population history, tested using the approximate Bayesian computation (ABC) procedure, have potentially contributed to the modern distribution of A. fasciatus, A. kreyenbergii and A. wenchowensis?

2. Materials and Methods

2.1. Sample Collection and Sequencing

A total of 160 individuals of Acrossocheilus were collected from 12 populations of 11 rivers in the Zhejiang region and the Yangtze River, including one population of A. kreyenbergii, four populations of A. wenchowensis and seven populations of A. fasciatus, during 2018–2020 (Table 1, Figure 1). All specimens from the field were collected with seines, anesthesia was administered by immersion into MS 222 (Sigma, St Louis, MO, USA), and the specimens were preserved in 90% ethanol. The samples were deposited in the laboratory of Jin-Quan Yang, Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, and all procedures were carried out in accordance with the guidelines and approval of the Animal Research and Ethics Committee of Shanghai Ocean University (permissions, SHOU-DW-2018-021).
Total genomic DNA was extracted from the tissue of the fins using the DNeasy Blood and Tissue Kit (QIAGEN, Valencia, CA, USA). The complete mitochondrial cyt b gene (1140 bp) and control region (D-loop) region (949 bp) were amplified by polymerase chain reaction (PCR) using the primers CBW-F (5′-AGAAGCGACGGCGATTAG-3′) plus CBW-R (5′-GAGCCAGTGGTGGGAGTTA-3′) and LOOP-F (5′-AAGCATCGGTCTTGTAATCC-3′) plus LOOP-R (5′-CCATCTTGGCATCTTCAGTG-3′), respectively. PCRs were performed in 25 μL total volume containing genomic DNA 3 μL, Taq mix 12.5 μL, 1 µL of each primer and ddH2O 7.5 μL. PCR was performed in an Eppendorf Mastercycler under the following conditions: 94 °C for 4 min for initial denaturation, followed by 35 cycles of 94 °C for 30 s, 55 °C (cyt b); and 53 °C (D-loop) for 40 s, and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. All PCR products were purified, and the cycle sequencing reactions were run on an ABI PRISM 3730XL sequencer (Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator kit (Applied Biosystems). The chromatograms were analyzed using CHROMA software, and sequence alignment was performed manually in BioEdit version 7.2.2.5 [12]. Sequences were submitted to GenBank under the accession numbers OP556253–OP556315 for cyt b and OP556361–OP556476 for the D-loop.

2.2. Sequence Variations, Genetic Structure, and Phylogenetic Analyses

Sequences of the entire cyt b and D-loop regions for all 160 samples were aligned with Clustal X v2.1 [13]. The genetic diversity of each population was determined, such as the number of haplotypes (Nh), haplotype diversity (h) and nucleotide diversity (θπ and θω) [14], using DnaSP v5.0 software [15]. To detect any phylogeographic structure, we calculated two genetic differentiation indices (GST and NST) following the method of Pons and Petit [16] using the software DnaSP v5.0 [15]. Pairwise FST between all pairs of populations and analysis of molecular variance (AMOVA) were used to examine the spatial partitioning of genetic variation among populations in Arlequin version 3.5 [17], with tests of statistical significance performed with 10,000 simulation steps for each comparison. For the hierarchical analysis, populations based on geographical barriers were grouped together under four scenarios as follows: (1) Scenario I, three species groups were primarily divided into A. fasciatus, A. wenchowensis and A. kreyenbergii; (2) Scenario II, two geographical groups were primarily divided by the Xianxia Mountains in A. fasciatus; (3) Scenario III, two geographical groups were primarily divided by the Qiantang and Tiaoxi Rivers and others in A. fasciatus; and (4) Scenario IV, two geographical groups were primarily divided by the Feiyun River and others in A. wenchowensis.
Phylogenetic trees were constructed using neighbor-joining (NJ), Bayesian inference (BI) and maximum likelihood (ML) approaches with MEGA-X [18], MrBayes v3.1.2 [19] and PhyML 3.0 [20], respectively. The best-fitting evolutionary substitution model was determined for the mitochondrial sequences (cyt b + D-loop) by SMS (Smart Model Selection) in PhyML with the Akaike information criterion (AIC) [21]. We constructed the phylogenetic tree and estimated divergence dates using the Bayesian strict-clock model approach implemented in BEAST 1.8.2 [22] with 107 MCMC steps and the first 10% taken as burn-in. Mutation rates for the mitochondrial cyt b and D-loop regions were regarded as 3.6% and 0.76% per million years, respectively [23]. Convergence and adequate effective sample sizes were checked with Tracer v1.6 [24], ensuring that the effective sample size (ESS) values were higher than 200 for all parameters. The maximum clade credibility (MCC) tree was generated using TreeAnnotator v.2.2.1 [25] in the BEAST package to summarize trees based on mean height, with the first 10% of the trees discarded as burn-in, and the results were visualized in FigTree 1.4.3 [26]. To present interrelationships among haplotypes for the mitochondrial cyt b and D-loop sequences, a network with information on frequency and type-sharing among populations was created using the minimum spanning network method (MINSPNET algorithm in Arlequin 3.5) [26]. Historical demography and biogeographic analysis were used to test for possible historical population expansion, a statistical analysis of the neutrality test was carried out (Tajima’s D test [27] and Fu’s Fs test [28]) and the distribution of mismatches was determined using DnaSP v5.0 [15]. In addition, Bayesian skyline plot (BSP) analyses were performed using BEAST v1.8.2 [22] for A. fasciatus and A. wenchowensis to estimate the change in population size over time. Mutation rates were as described in the text, and plots were generated using 20,000 iterations after a burn-in of 10,000 iterations. Convergence of the MCMC runs (ESSs > 200) was analyzed with Tracer v1.6 [24], and the results were used to construct a maximum clade credibility tree with 20% burn-in for each discarded chain. We used the statistical Bayesian binary MCMC (BBM) method implemented in RASP 3.2 [29] with default parameters to reconstruct the ancestral state of the area. The distributions of populations were divided into three biogeographic units based on the sampling and distribution range of Acrossocheilus as follows: (1) the Yangtze River region (AKSZ); (2) the northern Zhejiang region (AFHZ, AFNB, AFKH, AFXJ and AFWL); and (3) the southern Zhejiang region (AWLS, AWQY, AWTS and AWFD).

2.3. ABC Analyses Using DIYABC

To better understand the evolutionary and demographic histories of A. fasciatus, A. wenchowensis and A. kreyenbergii, we used coalescence-based approximate Bayesian computation (ABC) implemented in DIY-ABC v.2.0 [30] for the mtDNA cyt b + D-loop region, which was then used to discriminate the most probable scenario among a set of alternative historical and demographic scenarios. First, we compared three possible evolutionary scenarios for A. fasciatus, A. wenchowensis and A. kreyenbergii in ABC 1 analyses (Figure 2A). These were as follows: Scenario 1: two species (A. fasciatus and A. wenchowensis) diverged simultaneously at t1 and diverged from an ancestral species (A. kreyenbergii) at t2; Scenario 2: two species (A. fasciatus and A. kreyenbergii) diverged simultaneously at t1 and diverged from an ancestral species (A. wenchowensis) at t2; and Scenario 3: two species (A. kreyenbergii and A. wenchowensis) diverged simultaneously at t1 and diverged from an ancestral species (A. fasciatus) at t2. Second, based on the complex history, the five possible demographic histories of A. fasciatus and A. wenchowensis following the recommendations proposed by Cabrera and Palsboll [31] were tested in ABC 2 and 3 analyses, respectively (Figure 2B). In scenario A (CON model), the populations of A. fasciatus and A. wenchowensis remained constant in size over time. In scenario B (DEC model), populations of A. fasciatus and A. wenchowensis experienced a bottleneck event. In scenario C (INC model), populations of A. fasciatus and A. wenchowensis recently expanded. In scenario D (INCDEC model), populations of A. fasciatus and A. wenchowensis experienced ancient expansion, followed by shrinkage. In scenario E (DECINC model), populations of A. fasciatus and A. wenchowensis experienced old shrinkage, followed by expansion. For mitochondrial data, we assumed an HKY mutation model. Default settings were used for all other parameters. For each scenario, the reference table was built with 3,000,000 simulated datasets using all statistics. The determination of the best-supported scenario was used to compare the posterior distribution probability with logistic regression, that is, the assumption that the best-supported scenario had the highest posterior probability. The posterior probability of each model based on 1% of the simulated datasets for each scenario was assessed using both direct and logistic approaches.

3. Results

3.1. Genetic Diversity of A. fasciatus, A. wenchowensis and A. kreyenbergii

The mitochondrial cyt b + D-loop region from Acrossocheilus comprised 2057 base pairs (bps) (cyt b: 1140 bp; D-loop: 917 bp), which consisted of 14.1% guanine, 30.9% adenine, 29.2% thymine and 25.9% cytosine (40.0% GC content). A total of 537 variable sites were observed, of which 405 were parsimony informative. From 160 specimens of the 12 Acrossocheilus populations, we identified 124 haplotypes of which 110 were characteristic of A. fasciatus and 88 of A. wenchowensis (Table 1). There were no shared haplotypes between populations of A. fasciatus and A. wenchowensis. The average haplotype diversity within A. fasciatus was high (0.994), ranging from 0.643 (AFHZ) to 1.000 (AFWL), and the average nucleotide diversity (θπ) for this species was low (0.018), ranging from 0.003 (AFHZ) to 0.0142 (AFNB) (Table 1). Within A. wenchowensis, the average haplotype diversity was high (0.975), ranging from 0.000 (AWTS) to 0.982 (AWFD), and the average nucleotide diversity (θπ) was low (0.013), ranging from 0.000 (AWTS) to 0.011 (AWFD) (Table 1). The average haplotype diversity was high (0.800), and the nucleotide diversity (θπ) was low (0.016) in A. kreyenbergii. Estimates of current (θπ) and historical (θω) genetic diversity (a higher θω than θπ) in the three Acrossocheilus species revealed that the population of Acrossocheilus declined while expanding locally (Table 1). NST being much larger than GST revealed the presence of a phylogeographic structure in A. fasciatus and A. wenchowensis (A. fasciatus: 0.524 and 0.028, respectively; A. wenchowensis: 0.552 and 0.064, respectively) [16].

3.2. Genetic Structure and Phylogenetic Reconstruction

Pairwise estimates of FST between populations based on the cyt b + D-loop region ranged from 0.098 (between the AFXJ and AFWL populations) to 0.900 (between the AFHZ and AFWL populations) (mean value 0.588) in A. fasciatus, and ranged from 0.500 (between the AWFD and AWLS populations) to 0.948 (between the AWTS and AWQY populations) (mean value 0.688) in A. wenchowensis, indicating very high levels of genetic differentiation among all populations (Table 2). Hierarchical analysis of molecular variance (AMOVA) based on the three Acrossocheilus species revealed significant spatial genetic structuring among groups, with 63.31% of the variation occurring at this level (FCT = 0.633, p < 0.000), but only 16.02% (FSC = 0.436, p < 0.000) occurred among populations within groups and 20.66% (FST = 0.793, p < 0.000) within populations (Table 3). Based on two geographical groups (Scenarios II and III) in A. fasciatus, AMOVA revealed that 27.03% and 10.15% of the variation occurred among groups, 32.96% and 34.32% of the variation occurred among populations within groups, and 64.68% and 55.53% of the variation occurred within populations (Table 3). Based on two geographical groups in A. wenchowensis, AMOVA showed that 25.03% of the variation was present among groups, 40.30% of the variation was present among populations within groups and 34.67% of the variation was present within populations (Table 3).
Phylogenetic analyses based on the NJ, ML and BI approaches recovered the same tree topology. Three major lineages (A, B and C) were identified according to the haplotypes’ distribution pattern from different populations based on the mtDNA cyt b + D-loop region. Lineage A, belonging to A. fasciatus, was divided into three sublineages (A1, A2 and A3). Lineage A1 was distributed among populations AFKH, AFWC, AFXJ, AFLS and AFHZ, and lineages A2 and A3 were composed of individuals from populations AFNB and AFXJ + AFWL, respectively (Figure 3). Lineage B, belonging to A. wenchowensis, included specimens from populations AWQY, AWFD, AWTS and AWLS in the southern Zhejiang region (Figure 3). Lineage C, belonging to A. kreyenbergii, was only composed of one population (SZ) and was found in the Yangtze River.
The spanning network confirmed the presence of the three major lineages of mtDNA (A, B and C), with lineage A being interior and the others (lineages B and C) being located at the tip (Figure 4). Lineage A formed three major sublineages (A1, A2 and A3), with sublineage A2 being located in the interior and the others being located at the tip (Figure 4). The most recent common ancestors (TMRCAs) of all lineages, estimated in BEAST analyses, occurred 1.859 (95% HPD: 1.647–2.095) myr. Molecular dating estimated that lineage A (A. fasciatus) and lineage B (A. wenchowensis) lived 1.614 (95% HPD: 1.394–1.836) myr. Dating estimates for lineage A (A. fasciatus) and lineage B (A. wenchowensis) coalesced to a TMRCA 1.059 (95% HPD: 0.885–1.238) myr ago and 0.555 (95% HPD: 0.452–0.671) myr ago, respectively. Lineage A could be subdivided into three sublineages in A. fasciatus, namely A1, A2 and A3, which diverged from each other at 0.668, 0.838 and 0.837 myr, respectively.

3.3. Historical Demography and Biogeographic Analysis

Multimodal distribution was observed in the mismatch distribution analysis of A. fasciatus and A. wenchowensis. According to the neutrality test statistics, the values of Tajima’s D tests were negative but nonsignificant in A. fasciatus and A. wenchowensis (Tajima’s D = −1.72153, 0.10 > p > 0.05 and Tajima’s D = −1.32297, p > 0.10, respectively). However, the values of Fu’s Fs tests were negative and significant in A. fasciatus and A. wenchowensis (Fu’s Fs = −31.277, p < 0.000 and Fu’s Fs = −1.670, p < 0.075, respectively). Fu’s Fs test is more sensitive to recent population events [28]. In addition, comparing current and historical genetic diversity in A. fasciatus and A. wenchowensis (θω (0.03767) > θπ (0.01835) and θω (0.02072) > θπ (0.01346), respectively), the populations of A. fasciatus and A. wenchowensis showed a pattern of decline [32]. The Bayesian skyline plots (BSPs) also indicated a decline in effective population size 20,000 years ago (20 Kya) in A. fasciatus and A. wenchowensis (Figure 5). However, the neutrality test (Fu’s Fs test) results were consistent with a model of demographic growth, while the mismatch distribution analysis and Bayesian skyline plot (BSP) results did not support population expansion in A. fasciatus and A. wenchowensis.

3.4. Approximate Bayesian Computation (ABC) Scenarios

We used ABC analysis to determine the possible evolutionary and demographic histories of A. fasciatus, A. wenchowensis and A. kreyenbergii. In the ABC 1 model, the highest posterior probability after evaluation using the possible evolutionary history of A. fasciatus, A. wenchowensis and A. kreyenbergii was Scenario 1 (posterior probability = 1.0000 [1.0000, 1.0000]). This result reveals that A. fasciatus and A. wenchowensis diverged from A. kreyenbergii in the Zhejiang region. In the ABC 2 and 3 models, the possible demographic histories of A. fasciatus and A. wenchowensis were identified. In the ABC 2 and 3 analyses, the “CON model” was highly favored (posterior probability = 0.9939 [0.9735,1.0000]; 0.9907 [0.9831,0.9984]) over the other models in A. fasciatus and A. wenchowensis, respectively. This result reveals that the effective population sizes of A. fasciatus and A. wenchowensis were constant from the past to the present.

4. Discussion

4.1. Genetic Diversity

In the last decade, the biodiversity of freshwater ecosystems has declined more rapidly than that of terrestrial or marine ecosystems in mainland China (e.g., overfishing and anthropogenic disturbance) [33]. Genetic diversity plays a significant role in promoting the survival of species and their adaptation, as well as restraining species’ ability to adapt to changes in the environment. In our study, analyses of the mitochondrial cyt b + D-loop regions in Acrossocheilus populations revealed high haplotype diversity and low nucleotide diversity, which was higher than that estimated in previous studies for other freshwater species in the Zhejiang region (e.g., H. chenhsienensis [4]; S. macrops [6]). Compared to other Acrossocheilus species, A. fasciatus still exhibited a higher level of nucleotide diversity (e.g., A. longipinnis [34]; A. yunnanensis [35]; A. paradoxus [36]). The populations of A. fasciatus also exhibited higher haplotype diversity and higher levels of nucleotide diversity than those of A. wenchowensis in the Zhejiang region. The distribution of A. fasciatus mainly included the northern Zhejiang region, especially the Qiantang River, the largest river in the Zhejiang region. We suggest that A. fasciatus, with a wider distribution range, shows higher overall genetic diversity than A. wenchowensis and other species with more geographically confined distributions. In A. fasciatus, lower nucleotide diversity was found in smaller rivers (e.g., Tiaoxi River and Jinqing River), showing a positive relationship between the area of the drainage basin and genetic diversity. Lower genetic diversity is likely the result of habitat fragmentation, inbreeding and genetic drift following a bottleneck or founder effect. The genetic diversity of freshwater fish in montane streams has shown high levels of genetic diversity among populations and low levels within populations [37]. High levels of genetic diversity with many unique haplotypes and no shared haplotypes among populations indicated limited gene flow in A. fasciatus and A. wenchowensis.

4.2. Population Structure and Demographic History

The population analyses of Acrossocheilus showed a strong relationship between geography and phylogeny in A. fasciatus and A. wenchowensis (A. fasciatus: 0.524 (NST) and 0.028 (GST), respectively; A. wenchowensis: 0.552 (NST) and 0.064 (GST), respectively), indicating phylogeographic structure. Pairwise FST values suggested high genetic differentiation among populations, except among the AFLS, AFWC and AFKH populations. We suggest a possible increase in genetic differentiation with an increase in stream fragmentation due to increased genetic drift and decreased gene flow [38]. According to the network, we suggest that low genetic differentiation in AFLS and AFWC was consistent with them being translocated populations from the Qiantang River (AFKH). The average pairwise FST values were 0.588 and 0.688 in A. fasciatus and A. wenchowensis, respectively. The higher genetic differentiation of Acrossocheilus populations in comparison to those of other sympatric freshwater fishes in the Zhejiang region is due to the sympatric species being headwater species (e.g., S. macrops [6]; S. argentatus [5]). The results of AMOVA revealed that 63.31% of the total genetic variation occurred among the three Acrossocheilus species. However, 27.03% of the total genetic variation occurred between the two geographical groups primarily divided by the Jinqing River and others in A. fasciatus. Barriers to fish migration in a stream reduces genetic exchange between populations, resulting in high levels of divergence that are usually typical in freshwater species [39].
Previous studies have revealed that sea-level variations have influenced the demographic history of freshwater fishes in the Zhejiang region during glacial cycles, such as H. chenhsienensis [4], S. argentatus [5], S. sinensis [7] and Rhynchocypris oxycephalus [37]. Our data indicate that populations of A. fasciatus and A. wenchowensis carried the genetic signal of similar historical demographic processes after they split. Populations with high haplotype diversity and low nucleotide diversity were observed, and populations with a star-shaped haplotype network, indicating population expansion after a period of small effective population size, occurred in A. fasciatus and A. wenchowensis [40]. In addition, a neutrality test (Fu’s Fs test) indicated that range expansion occurred in A. fasciatus and A. wenchowensis. However, other analyses of demographic history (mismatch distribution analysis and Bayesian skyline plot (BSP) analysis) did not support population expansion for A. fasciatus and A. wenchowensis. Although several demographic analyses displayed different results, the results of ABC analysis supported population constancy. We suggest that A. fasciatus and A. wenchowensis experienced population range expansion and differentiation but population size contraction. Previous studies have revealed a similar scenario, such as in S. sinensis [7]. However, other freshwater fish studies in the Zhejiang region revealed population expansion of some species, such as S. macrops [6], S. argentatus [5] and H. chenhsienensis [4]. These results revealed that the effects of population expansion are more significant for downstream fishes than for upstream fishes in the Zhejiang region. During the glacial cycle leading to changes in sea level, the habitat area did not increase for A. fasciatus and A. wenchowensis due to their more restricted habitat in the headwaters of the drainage system.

4.3. Phylogeography of Acrossocheilus

The history of Zhejiang’s hydrological network is associated with complex geographical barriers driven by the development of mountainous topography and the history of basin connections due to the reduced sea levels that led to drastic changes in the biogeography and evolution of freshwater fishes. Previous phylogeographic studies have revealed that the populations of freshwater fish in the middle and lower reaches of the Yangtze River are more closely related to those in the river of the Zhejiang region (e.g., Qiantang River) than to those in the river of the Fujian region (e.g., Minjiang River) (e.g., S. argentatus, [5]; S. sinensis, [7]). According to our phylogenetic analysis, A. kreyenbergii occupied the basal position and A. wenchowensis was sister to A. fasciatus. The results of the BBM analysis based on the results of historical distribution estimation indicated that possible ancestral populations of A. kreyenbergii were distributed in the Yangtze River. Previous studies have interpreted the genetic structure of freshwater fish in the Zhejiang region to have mainly been caused by dispersal and vicariance (e.g., H. chenhsienensis, [4]; S. sinensis, [7]). For example, the uplift of the Wuyi Mountains caused genetic differences in fishes between the Yangtze River (A. kreyenbergii) and the northern Zhejiang region (A. fasciatus); the uplift of the Xianxia Mountains caused genetic differences in fishes between the northern Zhejiang region (A. fasciatus) and the southern Zhejiang region (A. wenchowensis). These phenomena suggest that the geographical origin of freshwater fish in the Zhejiang region is the Yangtze River. These results are also supported by DIY ABC simulation, which showed that A. kreyenbergii was the ancestral species in the Yangtze River. During Pleistocene glaciations, the East China Sea was exposed due to the decrease in sea level, prompting historical colonization from the lower reaches of the Yangtze River to the Qiantang River and Ou River. In our study, lineages A and B of A. fasciatus were separated from each other over geographic space (Tiantai Mountain), and the two lineages were linked by 48 mutational steps, demonstrating deep genetic differentiation. Previous biogeographic studies revealed that A. fasciatus was not distributed in the southern Zhejiang region [41]. However, in our study, we found that A. fasciatus had been introduced to the Oujiang River and the Feiyun River. Two populations (AFLS and AFWC) were defined as translocated populations because they were not distributed in the native area and all haplotypes of the populations (AFLS and AFWC) belonged to lineage A1. It is possible that the populations AFLS and AFWC were introduced from the Qiantang River. The topological relationships of A. wenchowensis inferred from the phylogenetic analysis support the formation of four sublineages in lineage B, recovered according to the distribution pattern from different populations (Figure 3). The high genetic differentiation in A. wenchowensis stems largely from the fact that freshwater habitats are strongly fragmented and isolated, owing to the lack of interconnecting rivers. BEAST analysis estimated an approximate age of 1.614 (1.394–1.836) myr for A. fasciatus and A. wenchowensis in the Zhejiang region. This high level of divergence likely reflects long-term isolation between A. fasciatus and A. wenchowensis. A scenario involving similar events that shaped the distributions and genetic population structures via geographical divergence supports a shared history for these codistributed freshwater fishes (e.g., H. chenhsienensis [4]; S. parvus [3]).

5. Conclusions

In conclusion, our results suggest that the Quaternary climatic oscillations in the Zhejiang region had no significant effects on current patterns of A. fasciatus and A. wenchowensis. Due to the uplifts of the Wuyi Mountains and Xianxia Mountains, ancestral populations were fragmented among A. kreyenbergii, A. fasciatus and A. wenchowensis, resulting in a lack of association between phylogenetic structure and drainage system. After this divergence, Acrossocheilus populations were constricted by geographic barriers and thus experienced long, independent evolutionary histories with no shared haplotypes between populations. Analysis of demographic history based on ABC revealed that A. fasciatus and A. wenchowensis maintained stable population sizes over time during climatic oscillations. However, this study is the only one that investigates the phylogeographic histories of Acrossocheilus species in the Zhejiang region and provides a more detailed understanding of factors that influence the phylogeographic history of this region’s fish fauna.
The identification of conservation units (CUs), such as evolutionarily significant units (ESUs) and management units (MUs), is an important concept in conservation genetics [42]. Evolutionarily significant units (ESUs) show substantial reproductive isolation from other conspecific populations and have been geographically isolated for a long time, with genetic uniqueness and independence [42]. Management units (MUs) are basic units for management and are considered essential for maintaining long-term persistence. In our studies, the three reciprocally monophyletic lineages in A. fasciatus and one monophyletic lineage in A. wenchowensis should be considered ESUs due to the presence of genetically differentiated lineages with geographical isolation. For conservation and protection, a strategy is required because of their genetic uniqueness. We suggest that the two regions divided by the Xianxia Mountains be regarded as different MUs, conforming to the major zoological regions of the Zhejiang region.

Author Contributions

Conceptualization, J.-Q.Y., J.-L.L. and H.-D.L.; methodology, M.-Y.Z., J.-J.W., F.L. and J.-F.R.; software, M.-Y.Z., J.-X.W. and J.-J.W.; validation, J.-J.Z., H.-D.L. and J.-Q.Y.; formal analysis, M.-Y.Z., J.-J.Z. and H.-D.L.; writing—original draft preparation, J.-J.W., F.L., J.-Q.Y. and H.-D.L.; writing—review and editing, H.-D.L. All authors have read and agreed to the published version of the manuscript.

Funding

The research was supported by the National Natural Science Foundation of China (No. 31872207), National Key Research and Development Program of China (No. 2022YFD2400102) and the China-ASEAN Maritime Cooperation Fund (CAMC-2018F).

Institutional Review Board Statement

The samples were deposited in the laboratory of Jin-Quan Yang, Shanghai Universities Key Laboratory of Marine Animal Taxonomy and Evolution, and all procedures were carried out in accordance with the guidelines and approval of the Animal Re-search and Ethics Committee of Shanghai Ocean University (permissions, SHOU-DW-2018-021).

Data Availability Statement

GenBank under the accession numbers OP556253–OP556315 for cyt b and OP556361–OP556476 for the D-loop.

Acknowledgments

In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Map showing the 12 sampling localities of the 3 Acrossocheilus species examined in this study. Collection sites (triangles, squares, star) correspond to locations given in the text and Table 1.
Figure 1. Map showing the 12 sampling localities of the 3 Acrossocheilus species examined in this study. Collection sites (triangles, squares, star) correspond to locations given in the text and Table 1.
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Figure 2. (A) Schematic representation of three evolutionary scenarios for A. fasciatus, A. wenchowensis and A. kreyenbergii tested by approximate Bayesian computation (ABC 1). Time and effective population size are not to scale. (B) Schematic representation of five demographic scenarios for A. fasciatus and A. wenchowensis tested by approximate Bayesian computation (ABC 2 and 3). Time (T1–T3) and effective population size (N–N5) are not to scale.
Figure 2. (A) Schematic representation of three evolutionary scenarios for A. fasciatus, A. wenchowensis and A. kreyenbergii tested by approximate Bayesian computation (ABC 1). Time and effective population size are not to scale. (B) Schematic representation of five demographic scenarios for A. fasciatus and A. wenchowensis tested by approximate Bayesian computation (ABC 2 and 3). Time (T1–T3) and effective population size (N–N5) are not to scale.
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Figure 3. BI tree of genetic relationships based on the mitochondrial cyt b + D-loop region among 12 populations of three Acrossocheilus species using 123 haplotypes. The values above the branches are the posterior probabilities for bootstrap values for the NJ, ML and Bayesian analyses. Spinibarbus hollandi is shown as an outgroup (NCBI accession No. AP012063.1). The results are also presented based on the statistical Bayesian binary MCMC (BBM) method implemented in RASP 3.2. ● indicates vicariance events; ✩ indicates dispersal events. A: Northern Zhejiang region; B: Southern Zhejiang region; C: Yangtze River region.
Figure 3. BI tree of genetic relationships based on the mitochondrial cyt b + D-loop region among 12 populations of three Acrossocheilus species using 123 haplotypes. The values above the branches are the posterior probabilities for bootstrap values for the NJ, ML and Bayesian analyses. Spinibarbus hollandi is shown as an outgroup (NCBI accession No. AP012063.1). The results are also presented based on the statistical Bayesian binary MCMC (BBM) method implemented in RASP 3.2. ● indicates vicariance events; ✩ indicates dispersal events. A: Northern Zhejiang region; B: Southern Zhejiang region; C: Yangtze River region.
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Figure 4. Minimum spanning network (MSN) based on mutations between haplotypes observed in populations of three Acrossocheilus species. Haplotype designations (Table 1) are indicated next to each circle. Locality designations (see Figure 1) for specimens possessing each haplotype are indicated inside the circles. The sizes of the circles are proportional to the number of individuals represented. The length of the lines between circles is roughly proportional to the estimated number of mutational steps between the haplotypes. Clades (A)–(C) is shown on the map with corresponding colors for each lineage.
Figure 4. Minimum spanning network (MSN) based on mutations between haplotypes observed in populations of three Acrossocheilus species. Haplotype designations (Table 1) are indicated next to each circle. Locality designations (see Figure 1) for specimens possessing each haplotype are indicated inside the circles. The sizes of the circles are proportional to the number of individuals represented. The length of the lines between circles is roughly proportional to the estimated number of mutational steps between the haplotypes. Clades (A)–(C) is shown on the map with corresponding colors for each lineage.
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Figure 5. Bayesian skyline plot of effective population sizes over time for (A) A. fasciatus and (B) A. wenchowensis. The X-axis is the time scale in millions of years, and the Y-axis is the estimated effective population size in units of Nes, the product of effective population size and generation length in years (log transformed). The solid line indicates the median estimate, whereas the thinner lines indicate the 95% credibility intervals.
Figure 5. Bayesian skyline plot of effective population sizes over time for (A) A. fasciatus and (B) A. wenchowensis. The X-axis is the time scale in millions of years, and the Y-axis is the estimated effective population size in units of Nes, the product of effective population size and generation length in years (log transformed). The solid line indicates the median estimate, whereas the thinner lines indicate the 95% credibility intervals.
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Table
Table 1. Sample information and genetic diversity of the cyt b + D-loop region in the genus Acrossocheilus.
Table 1. Sample information and genetic diversity of the cyt b + D-loop region in the genus Acrossocheilus.
SpeciesRiver SystemLocation (Abb.)HaplotypeLatitude and LongitudeSample Size (n)Haplotype Number (Nh)Haplotype Diversity (h)Nucleotide Diversity (θπ)Nucleotide Diversity (θw)
A. fasciatusTiaoxi RiverHuzhou (AFHZ)Hap1–430°38′ N, 119°40′ E840.6430.002940.00454
Yongjiang RiverNingbo (AFNB)Hap56–6829°49′ N, 121°31′ E15130.9710.014270.02380
Qiantang RiverKaihua (AFKH)Hap5–4429°08′ N, 118°25′ E49400.9900.013330.01831
Jiaojiang RiverXianju (AFXJ)Hap77–8828°51′ N, 120°44′ E16120.9420.014130.01940
Jinqing RiverWenling (AFWL)Hap75–7628°22′ N, 121°23′ E221.0000.004900.00490
Oujiang RiveLishui (AFLS)Hap45–5528°28′ N, 119°55′ E14110.9340.009430.00895
Feiyun RiverWencheng (AFWC)Hap69–7427°47′ N, 120°05′ E661.0000.005920.00580
110880.9940.018350.03767
A. wenchowensisOujiang RiverLishui (AWLS)Hap108–11728°28′ N, 119°55′ E14100.9230.007120.00877
Jiaoxi RiverQingyuan (AWQY)Hap118–12227°37′ N, 119°04′ E650.9330.001240.00150
Feiyun RiverTaishun (AWTS)Hap12327°34′ N, 119°45′ E41000
Longshanxi RiverFuding (AWFD)Hap98–10727°19′ N, 120°13′ E11100.9820.011080.01423
35260.9750.013460.02072
A. kreyenbergiiYangtze RiverSuzhuang (AKSZ)Hap89–9729°10′ N, 118°07′ E1590.8000.016220.01415
Total 1601230.9940.035260.05524
Table
Table 2. Matrix of pairwise FST values among twelve populations in three Acrossocheilus species based on the mitochondrial cyt b + D-loop region (below diagonal) and p values (above diagonal).
Table 2. Matrix of pairwise FST values among twelve populations in three Acrossocheilus species based on the mitochondrial cyt b + D-loop region (below diagonal) and p values (above diagonal).
AFHZAFNBAFKHAFXJAFWLAFLSAFWCAWLSAWQYAWTSAWFDAKSZ
AFHZ 0.0000.0000.0000.0630.0000.0000.0000.0000.0000.0000.000
AFNB0.473 0.0000.0000.0090.0000.0000.0000.0000.0000.0000.000
AFKH 0.3770.186 0.0000.0090.0000.0810.0000.0000.0000.0000.000
AFXJ0.7370.5620.580 0.5130.0000.0000.0000.0000.0000.0000.000
AFWL0.9000.7350.7510.098 0.0000.0450.0090.0540.0720.0000.009
AFLS0.3860.2400.1310.6300.802 0.0090.0000.0000.0000.0000.000
AFWC0.5540.2580.1190.6570.8390.198 0.0000.0000.0000.0000.000
AWLS 0.9050.7960.8000.8170.9040.8380.865 0.0000.0000.0000.000
AWQY0.9600.8460.8520.8630.9520.8910.9220.5380.0090.0000.000
AWTS0.9710.8510.8570.8690.9620.8990.9320.7710.948 0.0000.000
AWFD0.8810.7780.7860.7930.8780.8190.8450.5000.6140.749 0.000
AKSZ0.8220.7340.7330.7360.8210.7650.7900.8200.8580.8590.804
Table
Table 3. Analysis of molecular variance (AMOVA) for Acrossocheilus based on the mtDNA cyt b + D-loop region.
Table 3. Analysis of molecular variance (AMOVA) for Acrossocheilus based on the mtDNA cyt b + D-loop region.
Scheme Category Description% Var.Statisticp
Scenario I: three species groups (A. fasciatus, A. wenchowensis and A. kreyenbergii)
Among groups63.31FCT = 0.6330.000
Among populations within groups16.02FSC = 0.4360.000
Within populations20.66FST = 0.7930.000
Scenario II: two geographical groups primarily divided by the Xianxia Mountains in A. fasciatus (HZ, KH, NB, XJ) (WL)
Among groups27.03FCT = −0.2700.402
Among populations within groups32.96FSC = 0.4510.000
Within populations40.02FST = 0.5990.000
Scenario III: two geographical groups primarily divided by the Qiantang and Tiaoxi Rivers and others in A. fasciatus (HZ, KH, LS, WC, WL) (NB, XJ)
Among groups10.15FCT = 0.1010.189
Among populations within groups34.32FSC = 0.3810.000
Within populations55.53FST = 0.4440.000
Scenario IV: two geographical groups primarily divided by the Feiyun River and others in A. wenchowensis (TS) (LS, FD, QY)
Among groups25.03FCT = 0.2500.245
Among populations within groups40.30FSC = 0.5370.000
Within populations34.67FST = 0.6530.000
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Zhou, M.-Y.; Wang, J.-J.; Ren, J.-F.; Li, F.; Wu, J.-X.; Zhou, J.-J.; Li, J.-L.; Yang, J.-Q.; Lin, H.-D. Historical Landscape Evolution Shaped the Phylogeography and Population History of the Cyprinid Fishes of Acrossocheilus (Cypriniformes: Cyprinidae) According to Mitochondrial DNA in Zhejiang Province, China. Diversity 2023, 15, 425. https://doi.org/10.3390/d15030425

AMA Style

Zhou M-Y, Wang J-J, Ren J-F, Li F, Wu J-X, Zhou J-J, Li J-L, Yang J-Q, Lin H-D. Historical Landscape Evolution Shaped the Phylogeography and Population History of the Cyprinid Fishes of Acrossocheilus (Cypriniformes: Cyprinidae) According to Mitochondrial DNA in Zhejiang Province, China. Diversity. 2023; 15(3):425. https://doi.org/10.3390/d15030425

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Zhou, Mu-Yang, Jun-Jie Wang, Jian-Feng Ren, Fan Li, Jin-Xian Wu, Jia-Jun Zhou, Jia-Le Li, Jin-Quan Yang, and Hung-Du Lin. 2023. "Historical Landscape Evolution Shaped the Phylogeography and Population History of the Cyprinid Fishes of Acrossocheilus (Cypriniformes: Cyprinidae) According to Mitochondrial DNA in Zhejiang Province, China" Diversity 15, no. 3: 425. https://doi.org/10.3390/d15030425

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