DNA barcoding the ichthyofauna of the Beibu Gulf: Implications for fisheries management in a seafood market hub.

The Beibu Gulf in China is situated in the tropics, in the western Pacific Ocean. It is an emblematic region combining proximity to a marine biodiversity hotspot and a major seafood hub. Intensification of marine fishing and ocean warming led to a drastic decline in fish populations in the Beibu Gulf during the last decades. This situation urges the development of molecular resources of the Beibu Gulf fish fauna in order to enable automated molecular identifications at the species level for next-generation monitoring. With this objective, we present the results of a large-scale campaign to DNA barcode fishes of the Beibu Gulf. We successfully generated 789 new DNA barcodes corresponding to 263 species which, together with 291 sequences mined from Genbank and BOLD, resulted in a reference library of 1080 sequences from 285 species. Based on the use of four DNA-based species delimitation methods (BIN, ASAP, mPTP, mGMYC), a total of 285 Molecular Operational Taxonomical Units (MOTUs). A single case of cryptic diversity was detected in Scomberomorus guttatus and a single species pair was not captured by delimitation methods. Intraspecific K2P genetic distances averaged 0.36% among sequences within species, whereas K2P genetic distances among species within genera averaged 6.96%. The most speciose families in open water trawling differ from those at fish market, and discrepancies with historical data are discussed in the light of recently documented stock collapses.

Multilocus Phylogeography and Population Genetic Analyses of Opsariichthys hainanensis Reveal Pleistocene Isolation Followed by High Gene Flow around the Gulf of Tonkin.

The ichthyofauna of continental islands is characterized by immigration through a land bridge due to fluctuating sea levels. Hainan Island is adjacent to the southern margin of mainland China and provides opportunities for understanding the origin and diversification of freshwater fishes. The aim of our study was to evaluate the level of genetic variation and phylogeographic structure of Opsariichthys hainanensis on Hainan Island and mainland China, using mtDNA cyt b gene (1140 bp) and D-loop (926 bp), nuclear RAG1 gene (1506 bp), and 12 microsatellite loci. Mitochondrial phylogenetic analysis identified five major lineages according to the geographical distribution from different populations. We suggested that two dispersal events occurred: the population in the Changhua River migrated to the Red River (Lineage B), and the populations in the South Hainan region moved northwards to the North Hainan region. However, populations in Northwest Hainan Island dispersed to the populations around the Gulf of Tonkin (Lineage A1) and populations in Northeast Hainan Island dispersed to the populations in mainland China (Lineage A2). Our results indicated that the populations of O. hainanensis suffered a bottleneck event followed by a recent population expansion supported by the ABC analysis. We suggest that O. hainanensis populations were found mostly in the lowlands and a lack of suitable freshwater habitat in southern mainland China and Hainan during the Last Interglacial period, and then expansion occurred during the Last Glacial Maximum.

Genetic pattern and demographic history of cutlassfish (Trichiurus nanhaiensis) in South China Sea by the influence of Pleistocene climatic oscillations.

Trichiurus nanhaiensis is one of the most important commercial fish species in the South China Sea. This study aimed to investigate the level of genetic variation and population genetic structure of T. nanhaiensis in the South China Sea for the first time, using 281 individuals collected from seven locations along the coast of mainland China, Taiwan, and Hainan Island. A high level of haplotype diversity and low nucleotide diversity were detected in the mitochondrial DNA cyt b gene and nuDNA RYR 3 gene. The overall expected heterozygosity (He = 0.693) among the seven populations ranged from 0.681 to 0.706 in microsatellite DNA data, which revealed high levels of genetic diversity. Significant genetic differentiation was found in Taidong populations in Taiwan, revealing the prevention of gene flow caused by the Kuroshio Current. Two major lineages based on the cyt b gene suggested that the Taiwan Strait acted as a geographic barrier for T. nanhaiensis during the glacier periods in the late Pleistocene. The Bayesian skyline plot also revealed that population demographic expansion of T. nanhaiensis was estimated to have occurred in 0.1 Mya. Our results indicated that all populations of T. nanhaiensis had experienced a recent genetic bottleneck following recent expansion based on ABC analysis.

Population genetic diversity and structure of Rhinogobius candidianus (Gobiidae) in Taiwan: Translocation and release.

Rhinogobius candidianus is a freshwater goby distributed in north, northwest, west, and south Taiwan, but this species has been introduced to east Taiwan and became dominant. To investigate its native population genetic diversity and structure and evaluate the sources and diversity of translocated populations, the mitochondrial DNA control region and cytochrome b gene (1981 bp) from 220 specimens were analyzed. These results indicated that (1) the east populations originated from two sources in west Taiwan; (2) translocated populations exist in east Taiwan and south Taiwan; (3) many populations have likely been moved secondarily by human intervention; (4) the effective size of the populations had declined greatly; (5) within the native populations, the ancestral populations colonized Taiwan during the land bridge phase in the Pleistocene through north Taiwan; (6) the landform changes in Taiwan shaped the population structure; and (7) the landforms of the coastline during glaciation also shaped the native range. The low-level genetic diversity, high population differentiation, and population decline greatly suggest the need for resource management and conservation interventions. Four clades (α-δ) should be managed as four distinct evolutionarily significant units, while the translocated populations should be managed as separate management units. Moreover, the translocated populations in east Taiwan should be evaluated and monitored carefully.

Complete mitogenomes of four Trichiurus species: A taxonomic review of the T.lepturus species complex.

Four Trichiurus species, T.japonicus, T.lepturus, T.nanhaiensis, and T.brevis, from the coasts of the China Seas, have been identified and their entire mitochondrial genomes (mitogenomes) have been sequenced by next-generation sequencing technology. A comparative analysis of five mitogenomes was conducted, including the mitogenome of T.gangeticus. The mitogenomes contained 16.568-16.840 bp and encoded 36 typical mitochondrial genes (13 protein-coding, 2 ribosomal RNA-coding, and 21 transfer RNA-coding genes) and two typical noncoding control regions. Although tRNAPro is absent from Trichiurus mitogenomes, when compared with the 22 tRNAs reported in other vertebrates, the gene arrangements in the mitogenomes of the studied species are consistent with those in most teleost mitogenomes. The full-length sequences and protein-coding genes (PCGs) in the mitogenomes of the five species had obvious AT biases and negative GC skew values. Our study indicate that the specimens in the Indian Ocean are neither T.lepturus nor T.nanhaiensis but they are T.gangeticus; the Trichiurus species composition in the Indian Ocean is totally different from that in Pacific and Atlantic oceans; there are at least two Trichiurus species in Indian Ocean; and the worldwide systematics and diversity of the genus Trichiurus need to be reviewed.

Phylogeographic structure of the dwarf snakehead (Channa gachua) around Gulf of Tonkin: Historical biogeography and pronounced effects of sea-level changes.

Geological events, landscape features, and climate fluctuations have shaped the distribution of genetic diversity and evolutionary history in freshwater fish, but little attention has been paid to that around the Gulf of Tonkin; therefore, we investigated the phylogeographic structure of the dwarf snakehead (Channa gachua) on Hainan Island and mainland China, as well as two populations in Vietnam. We attempted to elucidate the origins of freshwater fish in South Hainan by incorporating genetic data from DNA markers on both the mitochondrial cytochrome b gene (cyt b) and the nuclear recombination-activating gene 1 (RAG-1). Mitochondrial phylogenetic analysis identified two major lineages (lineages A and B), which may represent separate species. Divergence data suggested that C. gachua populations diverged between 0.516 and 2.376 myr. The divergence of the two cryptic species is congruent with sea-level rise, which subsequently isolated Hainan from the mainland. During the Pleistocene glaciations, the entire region of the Gulf of Tonkin and the Qiongzhou Strait became part of the coastal plain of the Asian continent, which might have resulted in the current distribution patterns and dispersal routes of C. gachua populations. The formation of three sublineages in lineage A indicated that the Gulf of Tonkin was a geographical barrier between Hainan Island and mainland China but not between Vietnam and Hainan Island. The results of this study may help to elucidate the origins of freshwater fish in South Hainan and the phylogeographic structure of C. gachua.

Amphidromous but endemic: Population connectivity of Rhinogobius gigas (Teleostei: Gobioidei).

Rhinogobius gigas is an amphidromous fish endemic to eastern Taiwan. Fishes with the diadromous behavior are expected to have a broader distribution range and higher genetic homogeneity despite that some amphidromous fishes with limited distribution are observed and R. gigas is an additional exception with a limited distribution range. Rhinogobius gigas has been documented to be retained inshore near the river plume with a short pelagic larval duration of 30-40 days, which may account for the endemism of this species. The short marine larval stage of R. gigas may imply a population genetic structure and the aim of the present study is to test whether the population genetic structure is present in R. gigas. To test the population genetic structure, fragments of mitochondrial displacement loop and cytochrome c oxidase subunit I were sequenced to provide molecular inference for genetic structure among populations. Sixty-nine haplotypes were identified among 191 R. gigas from 10 populations of eastern Taiwan and the mean haplotype and nucleotide diversities for all samples were 0.956 and 0.0024, respectively, implying a bottleneck followed by a recent population expansion further supported by Fu's Fs (-26.6; p < 0.001) and Tajima's D (-1.5; p = 0.037) values. The phylogenetic analysis revealed lack of genetic structure and the bush-like median joining network without commonly shared haplotypes supports the same scenario. The genetic homogeneity is probably due to the amphidromous life history providing the opportunity for passive larval transportation among the rivers through coastal currents in eastern Taiwan. The endemism to eastern Taiwan may be a consequence of complicated interactions among short pelagic larval duration, interspecific competition and coastal currents.

Genetic diversity of Rhinogobius delicatus (Perciformes: Gobiidae): origins of the freshwater fish in East Taiwan.

Mitochondrial DNA cytochrome b and d-loop sequences (1,984 bp) from 92 specimens of the freshwater goby Rhinogobius delicatus from seven drainages in East Taiwan were identified as two major lineages exhibiting a southern or northern distribution. The existence of low genetic diversity, a pattern of population decline and high population differentiation (FST=0.711) support the need for the development of management strategies for the conservation of localized populations. The results of a statistical dispersal-vicariance analysis suggested that the ancestral populations of R. delicatus were widely distributed in East Taiwan. Compared with the phylogeographic patterns of the other endemic eastern Taiwan freshwater fishes, Onychostoma alticorpus, Aphyocypris kikuckii and Hemimyzon taitungensis, our study suggests that the freshwater fishes colonized East Taiwan through northeastern and southwestern Taiwan, although the ancestral populations colonized the island before it reached its present shape.

Genetic diversity and structure of the Chinese lake gudgeon (Sarcocheilichthys sinensis).

Mitochondrial DNA cytochrome b and d-loop sequences (2,137 bp) in 65 specimens of Sarcocheilichthys sinensis from five populations were identified as two lineages (I and II). The pairwise genetic distance between lineages I and II was 1.94%. SAMOVA analyses suggested that the best grouping occurred at three groups, Yangtze, Qiantang and Minjiang Rivers. High haplotype diversity (0.949) and low nucleotide diversity (θπ = 1.067%) were detected. The results of the neutrality tests, mismatch distribution and approximate Bayesian computation (ABC) did not support demographic expansions. The results of phylogenetic analysis, statistical dispersal-vicariance analysis (S-DIVA), ABC, MIGRATE-N and the time to the most recent common ancestor (TMRCA) indicated two colonization routes. First, before the Wuyi Mountains lifted, S. sinensis dispersed from the Yangtze River to the Minjiang River. Second, during glaciation, the continental shelf was exposed, which contributed to the dispersion of populations from the Yangtze River.

Spatial genetic structure of Opsariichthys hainanensis in South China.

South China presents an excellent opportunity to build a phylogeographic paradigm for complex geological history, including mountain lifting, climate change, and river capture/reversal events. The phylogeography of cyprinids, particularly Opsariichthys hainanensis, an endemic species restricted to South China, was examined to explore the relationship between the populations in Red River, Hainan Island and its adjacent mainland China. A total of 37 haplotypes were genotyped for the mitochondrial cytochrome b (Cyt b) gene in 115 specimens from 11 river systems. Relatively high levels of haplotype diversity (h = 0.946) and low levels of nucleotide diversity (π = 0.014) were detected in O. hainanensis. Four major phylogenetic haplotype groups revealed a relationship between phylogeny and geography. Our results found that (i) the ancestral populations of O. hainanensis were distributed south of the Wuzhishan and Yinggeling mountains, including the Changhua River on Hainan Island, and then spread to the surrounding areas, (ii) the admixtures within lineages occurred between the Red River in North Vietnam and the Changhua River in western Hainan Island and (iii) indicated that the exposure of straits and shelves under water retreat, provides opportunities for population dispersion during glaciations.

Complete mitochondrial genome of the freshwater fish Onychostoma lepturum (Teleostei, Cyprinidae): genome characterization and phylogenetic analysis.

The cyprinid genus Onychostoma Günther, 1896 consists of 24 valid species distributed in Southeast Asia, including Taiwan, Hainan, mainland China and the Indochina region. In the present study, we determined the complete mitochondrial genome of O. lepturum, which is 16,598 bp in length, containing 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a typical control region (D-loop). To verify the molecular phylogeny of the subfamily Acrossocheilinae, we provide new insights to better understand the taxonomic status of Acrossocheilus, Onychostoma and Folifer brevifilis. The phylogenetic trees presented three major clades based on the 13 protein-coding genes from 28 Acrossocheilinae species. Clades I and II represent the Onychostoma and Acrossocheilus groups, respectively. Species of Acrossocheilus, Onychostoma and F. brevifilis are included in Clade III, which is considered as an ancestral group. This work provides genomic variation information and improves our understanding of the Acrossocheilinae mitogenome, which will be most valuable in providing new insights for phylogenetic analysis and population genetics research.

Geographical and temporal origins of Neocaridina species (Decapoda: Caridea: Atyidae) in Taiwan.

BACKGROUND: The freshwater species on Taiwan Island have been documented to have originated from mainland China and the Japanese islands from multiple events and by multiple colonization routes. Moreover, the sequences from the mitochondrial DNA cytochrome c oxidase subunit I (COI) have been used for DNA barcoding to identify the species. This study used the COI sequences to identify Neocaridina species in Taiwan and to examine their geographical and temporal origins. RESULTS: In total, 479 specimens were collected from 35 localities, which covered almost all rivers in Taiwan. In addition, some sequences were downloaded from GenBank. The maximum likelihood (ML) tree displayed that all sequences were sorted into 13 taxa (clades), and all sequences in Taiwan were sorted into four clades. The Bayesian skyline plots revealed that these four Neocaridina species have declined recently in Taiwan. CONCLUSIONS: All results support that (1) there are four Neocaridina species in Taiwan, which are N. davidi, N. saccam, N. ketagalan and an undescribed Neocaridina species (N. sp.); (2) these four species colonized Taiwan Island in four colonization events; (3) N. sp. colonized Taiwan first; (4) after the island reached its shape, N. ketagalan and N. saccam colonized Taiwan from the Japanese islands and mainland China, respectively; (5) N. davidi colonized northern Taiwan last; and (6) the cyclic glacial and landform changes in East Asia shaped the colonization events and population structures of the Neocaridina species.

Population structure of Aphyocypris normalis: phylogeography and systematics.

Aphyocypris normalis (Teleostei: Cyprinidae) is an endemic species in South China, but little is known about its genetic structure. This study examined the population structure of A. normalis using sequences of the mitochondrial DNA control region and cytochrome b gene (2,086 bp). In total, 107 specimens were collected from nine populations. All 105 mtDNA haplotypes were identified as belonging to two allopatric phylogroups. The results of a statistical dispersal-vicariance analysis (S-DIVA) suggested that the ancestral populations of A. normalis were distributed widely on Hainan Island and east of the Leizhou Peninsula. A comparison of the fixation indices NST (0.532) and GST (0.004) revealed that the phylogeny and geography had a significant relationship. Our study found that (1) the Wuzhishan and Yinggeling Mountain Range was an important barrier limiting gene exchange between populations on both sides; (2) cyclic climate changes may have shaped migrations and population differentiations; and (3) different colonization times caused different population diversities between codistributed species. In addition, the inter- and intraspecific diversities of the genus Aphyocypris were estimated.

The complete mitochondrial genome of Pseudorasbora interrupta and phylogeny of Pseudorasbora.

We sequenced the mitochondrial genome of Pseudorasbora interrupta using Illumina technology and additional Sanger sequencing. The assembled 16601 bp mitogenome had a GC content of 40.98% and consisted of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one non-coding control region (CR), with a gene order identical to the fishes. In addition, we downloaded the mitogenome of the closely related species Topmouth Gudgeon P. parva. The mitogenomes of P. interrupta and P. parva showed a sequence identity of 99.2% with the previously published P. parva mitogenome. The phylogenetic reconstruction based on 14 Gobioninae mitogenomes supported P. interrupta and its sister species P. parva as a monophyletic group. However, Pseudorasbora was proved to be a polyphyletic group which means amendments will be needed for the taxonomy of this genus.

Characterization of the complete mitochondrial genome of the coral reef fish, Hemigymnus melapterus (Pisces: Labridae) and its phylogenetic implications.

Hemigymnus melapterus belongs to the family Labridae, which inhabit in coastal and continental shelf waters. The entire mitochondrial genome of H. melapterus is 16,527 base pairs (bp) in length and contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. The overall base composition is 27.56% A, 25.58% T, 30.02% C, and 16.85% G, showing AT-rich feature (53.14%). Phylogenetic analysis based on 13 protein-coding genes shows the H. melapterus has the closest evolutionary relationship with Stethojulis strigiventer. This work provides valuable genome variation information, which will be useful for phylogenetic analysis and population genetics research.

Mitochondrial Genetic Diversity of the Freshwater Snail Melanoides tuberculata.

According to geological history, Peninsular Malaysia and Borneo formed at different times and were once connected during Quaternary glaciations. To determine how this history has influenced phylogeography, our study examined the population genetic structure of the tropical freshwater gastropod Melanoides tuberculata across Peninsular Malaysia and Borneo using the sequences from mitochondrial DNA 16S rRNA and cytochrome oxidase subunit I genes (1168 bp). In total, 104 specimens were collected from seventeen populations. All mtDNA haplotypes were identified as belonging to two highly divergent lineages, and these lineages were almost allopatric in their distributions. Our study found that the freshwater fauna in Malaysia might be divided into four regions: northeast Peninsular Malaysia, northwest Peninsular Malaysia, south Peninsular Malaysia, and Borneo. The phylogeography of M. tuberculata in Malaysia was shaped by the landforms of Peninsular Malaysia and by the paleo-river systems in the Sunda continental shelf. In addition, our study found that these two lineages in Malaysia have invaded the globe. These results suggest that Malaysia is located in important shipping lanes throughout the world, and the populations of M. tuberculate might be widely distributed throughout the world by shipping.

Mitochondrial diversity and phylogeography of Acrossocheilus paradoxus (Teleostei: Cyprinidae).

Mitochondrial DNA cytochrome b sequences (1141 bp) in 229 specimens of Acrossocheilus paradoxus from 26 populations were identified as four lineages. The pairwise genetic distances among these four lineages ranged from 1.57 to 2.37% (mean= 2.00%). Statistical dispersal-vicariance analysis suggests that the ancestral populations were distributed over mainland China and Northern and Western Taiwan. Approximate Bayesian computation approaches show that the three lineages in Taiwan originated from the lineage in mainland China through three colonization routes during two glaciations. The results indicated that during the glaciation and inter-glacial periods, the Taiwan Strait was exposed and sank, which contributed to the dispersion and differentiation of populations. Furthermore, the populations of A. paradoxus colonized Taiwan through a land bridge to the north of the Formosa Bank, and the Miaoli Plateau in Taiwan was an important barrier that limited gene exchange between populations on both the sides.

New insights on geographical/ecological populations within Coilia nasus (Clupeiformes: Engraulidae) based on mitochondrial DNA and microsatellites.

The Coilia nasus is an important fish species, which is of commercial values in China. In order to manage the fisheries resources and the development of sustainable fishing strategies to protect this species, 11 microsatellite loci polymorphisms and mtDNA COI sequences were used to examine the genetic diversity of C. nasus in Japan and China. In total, the 40 COI haplotypes and 93 microsatellite alleles were detected. The mtDNA phylogeny did not support population grouping, but the distribution patterns of mtDNA haplotypes and the results of STRUCTURE analysis based on microsatellite indicated a degree of genetic isolation in this species. Our study suggested that the lack of a population genetic structure might result in its amphidromous life cycle, and the geographical distance and habitat fragments might cause isolated populations. Thus, the sampling populations of C. nasus in Japan and China could be divided as four geographical/ecological populations.

Complete mitochondrial genome and the phylogenetic position of the pelagic octopus Tremoctopus violaceus (Mollusca: Tremoctopodidae).

Tremoctopus violaceus is a small species of pelagic octopods which inhabit in subtropical and tropical open oceans. The mitogenome of T. violaceus is 16,015 base pairs (bp) in length and contained 13 protein-coding genes, two rRNA genes, and 22 tRNA genes. Sequence analysis showed that the overall base composition is 31.5% for A, 40.47% for T, 7.79% for C, and 20.24% for G. The newly characterized complete mitochondrial genome of T. violaceus will provide essential data for further studies of this endangered species.

Phylogeography and genetic population structure of the spadenose shark (Scoliodon macrorhynchos) from the Chinese coast.

The population structure of Scoliodon macrorhynchos from the Chinese coast was investigated using the mitochondrial control region. All 19 mtDNA haplotypes from 219 sequences were identified. Relatively high average haplotype diversity (0.797) and relatively low average nucleotide diversity (0.0013) were found together with a recent and sudden population expansion. Analysis of the mismatch distributions, neutrality tests and Bayesian skyline plot showed a pattern consistent with a recent population expansion event that may have taken place during the last glacial maximum (LGM). The analysis of molecular variance (AMOVA) showed the low genetic differentiation between the populations, which may be a general feature of sharks living in coastal areas. The phylogenetic and cluster analysis of the mtDNA indicates that two putative groups (K = 2) existed in S. macrorhynchos, showing that the Taiwan Strait acted as a biogeographic barrier during major drops in the sea level in the late Pliocene epoch.