Population Morphometry of Conger myriaster (Anguilliformes: Congridae) along the Coast of China: Implications for Population Structure and Fishery Management.

In this study, the traditional morphometry method was applied to analyze the standardized measurements together with the meristic counts so as to provide supplementary information for fishery biology, population assessment, and fishery resources protection of C. myriaster. The results of one-way analysis of variance (ANOVA) showed that the greatest divergence was observed between the Dalian and Qingdao populations, whereas the smallest difference was found between the Lianyungang and Zhoushan populations. Statistical difference in tail length (TAL) was detected between all populations. The morphological traits with high C.D values were mostly related to body weight (BW), confirming greater potential variations of these weight-related traits. Principal component analysis (PCA) extracted 7 principal components (PCs) with eigenvalues greater than 1, and the cumulative contribution rate was 72.790%. The results of cluster analysis, together with the PCA and DFA, supported separating the populations into three groups linked with their geographic distribution and their specific environment localization. Considering the particularity of the natural environment of the Bohai Sea and the sophisticated oceanic circulations of the Shandong Peninsula, the relationships of C. myriaster populations in the northwest Pacific Ocean along the China coast were closely related to their geographical distributions and oceanic circulations.

The First Genome-Wide Survey of Shortbelly Eel (Dysomma anguillare Barnard, 1923) to Provide Genomic Characteristics, Microsatellite Markers and Complete Mitogenome Information.

Dysomma anguillare is a demersal eel widespread distributing in tropical waters of the Indo-West Pacific and Atlantic. As an important component of the coastal fishery and marine ecosystem, the lack of genomic information for this species severely restricts the progress of relevant researches. In this study, the abecedarian genome-wide characteristics and phylogenetic relationships analyses were carried out based on next-generation sequencing (NGS) platform. The revised genome size was approximately 1 310 Mb, with the largest scaffold length reaching 23 878 bp through K-mer (K = 17) method. The heterozygosity, repetitive rate and average GC content were about 0.94%, 51.93% and 42.23%, respectively. A total of 1 160 104 microsatellite motifs were identified from the de novo assembled genome of D. anguillare, in which dinucleotide repeats accounted for the largest proportion (592 234, 51.05%), the highest occurrence frequency (14.58%) as well as the largest relative abundance (379.27/Mb). The high-polymorphic and moderate-polymorphic loci composed around 73% of the total single sequence repeats (SSRs), showing a latent capacity for subsequent population genetic structure and genetic diversity appraisal researches. Another byproduct of whole-genome sequencing, the double-stranded and circular mitogenome (16 690 bp) was assembled to investigate the evolutionary relationships of D. anguillare. The phylogenic tree constructed with maximum likelihood (ML) method showed that D. anguillare was closely related to Synaphobranchidae species, and the molecular systematic results further supported classical taxonomy status of D. anguillare.

[Mitochondrial genome sequence characteristics and phylogenetic analysis of Schizothorax argentatus].

Schizothorax argentatus that only distributes in the Ili River basin in Xinjiang is one of the rare and endangered species of schizothorax in China, thus has high scientific and economic values. In this study, the complete mitochondrial genome sequence of S. argenteus with a length of 16 580 bp was obtained by high-throughput sequencing. The gene compositions and arrangement were similar to those of typical vertebrates. It contained 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a non-coding region (D-loop). The nucleotide compositions were A (30.25%), G (17.28%), C (27.20%), and T (25.27%), respectively, showing obvious AT bias and anti-G bias. Among the tRNA genes, only tRNA-Ser(GCU) could not form a typical cloverleaf structure due to the lack of dihydrouracil arm. The AT-skew and GC-skew values of the ND6 gene were fluctuating the most, suggesting that the gene may experience different selection and mutation pressures from other genes. The mitochondrial control region of S. argenteus contained three different domains, i.e., termination sequence region (ETAS), central conserved region (CSB-F, CSB-E, CSB-D, and CSB-B), and conserved sequence region (CSB1, CSB2, and CSB3). The conserved sequence fragment TT (AT) nGTG, which was ubiquitous in Cypriniformes, was identified at about 50 bp downstream CSB3. Phylogenetic relationships based on the complete mitochondrial genome sequence of 28 Schizothorax species showed that S. argenteus had differentiated earlier and had a distant relationship with other species, which may be closely related to the geographical location and the hydrological environment where it lives.

Comparative Mitogenomic Analysis of Two Snake Eels Reveals Irregular Gene Rearrangement and Phylogenetic Implications of Ophichthidae.

The family Ophichthidae has the largest number and the most various species (about 359 valid species) in the order Anguilliformes worldwide. Both morphological and molecular characteristics have been used to assess their taxonomic status. However, due to the ambiguous morphological features, molecular data such as mitochondrial DNA sequences have been implemented for the correct identification and classification of these fishes. In this study, the gene arrangement and structure characteristics of two Ophichthidae mitochondrial genomes were investigated for the first time. The total mitogenome lengths of O. evermanni and O. erabo were 17,759 bp and 17,856 bp, respectively. Comparing with the ancestral mitochondrial gene order, the irregular gene rearrangement happened between ND6 and tRNA-Pro (P) genes with another similar control region emerging between tRNA-Thr (T) and ND6 genes, which could be explained by the tandem duplication and random loss (TDRL) model appropriately. ML phylogenetic tree demonstrated that the family Ophichthidae was monophyletic origin, but genus Ophichthus might be polyphyletic because of the confused cluster relationships among different species.

Genomic microsatellite characteristics analysis of Dysommaanguillare (Anguilliformes, Dysommidae), based on high-throughput sequencing technology.

Microsatellite loci were screened from the genomic data of Dysommaanguillare and their composition and distribution were analysed by bioinformatics for the first time. The results showed that 4,060,742 scaffolds with a total length of 1,562 Mb were obtained by high-throughput sequencing and 1,160,104 microsatellite loci were obtained by MISA screening, which were distributed on 770,294 scaffolds. The occurrence frequency and relative abundance were 28.57% and 743/Mb, respectively. Amongst the six complete microsatellite types, dinucleotide repeats accounted for the largest proportion (592,234, 51.05%), the highest occurrence frequency (14.58%) and the largest relative abundance (379.27/Mb). A total of 1488 microsatellite repeats were detected in the genome of D.anguillare, amongst which the hexanucleotide repeat motifs were the most abundant (608), followed by pentanucleotide repeat motifs (574), tetranucleotide repeat motifs (232), trinucleotide repeat motifs (59), dinucleotide repeat motifs (11) and mononucleotide repeat motifs (4). The abundance of microsatellites of the same repeat type decreased with the increase of copy numbers. Amongst the six types of nucleotide repeats, the preponderance of repeated motifs are A (191,390, 43.77%), CA (150,240, 25.37%), AAT (13,168, 14.05%), CACG (2,649, 8.14%), TAATG (119, 19.16%) and CCCTAA (190, 19.16%, 7.65%), respectively. The data of the number, distribution and abundance of different types of microsatellites in the genome of D.anguillare were obtained in this study, which would lay a foundation for the development of high-quality microsatellite markers of D.anguillare in the future.

Single-molecule Real-time (SMRT) Sequencing Facilitates Transcriptome Research and Genome Annotation of the Fish Sillago sinica.

As a newly described Sillaginidae species, Chinese sillago (Sillago sinica) needs a better understanding of gene annotation information. In this study, we reported the first full-length transcriptome data of S. sinica using the PacBio isoform sequencing Iso-seq and a description of transcriptome structure analysis. A total of 454,979 high-quality full-length transcripts were obtained by single-molecule real-time (SMRT) sequencing, which was corrected by Illumina sequencing data. After that, 66,948 non-redundant full-length transcripts were generated after mapping to the reference genome of S. sinica, including 49 fusion isoforms and 9,250 novel isoforms. 63,459 isoforms were successfully annotated by one of the Nr, Nt, SwissProt, Pfam, KOG, GO, and KEGG databases. Additionally, 30,987 alternative polyadenylation (APA) sites, 451,867 alternative splicing (AS) events, 21,928 long non-coding RNAs (lncRNAs) and 12,911 transcription factors (TFs) were identified. The full-length transcripts of S. sinica would provide a precious resource for characterizing the transcriptome of S. sinica and for the further study of gene function and regulatory mechanism of this species.

Genome-wide survey and genetic characteristics of Ophichthus evermanni based on Illumina sequencing platform.

Ophichthidae fishes limit to continental shelf of all tropical and subtropical oceans and contain more than 350 species, representing the greatest specialization diversity in the order Anguiliformes. In the present study, we conducted a genome survey sequencing (GSS) analysis of Ophichthus evermanni by Illumina sequencing platform to briefly reveal its genomic characteristics and phylogenetic relationship. The first de novo assembled 1.97 Gb draft genome of O. evermanni was predicted based on K-mer analysis without obvious nucleotide bias. The heterozygosity ratio was 0.70%, and the sequence repeat ratio was calculated to be 43.30%. A total of 9016 putative coding genes were successfully predicted, in which 3587 unigenes were identified by gene ontology (GO) analysis and 4375 unigenes were classified into cluster of orthologous groups for enkaryotic complete genomes (KOG) functional categories. About 2,812,813 microsatellite motifs including mono-, di-, tri-, tetra-, penta- and hexanucleotide motifs were identified, with an occurrence frequency of 23.32%. The most abundant type was dinucleotide repeat motifs, accounting for 49.19% of the total repeat types. The mitochondrial genome, as a byproduct of GSS, was assembled to investigate the evolutionary relationships between O. evermanni and its relatives. Bayesian inference (BI) phylogenetic tree inferring from concatenated 12 protein-coding genes (PCGs) showed complicated relationships among Ophichthidae species, indicating a polyphyletic origin of the family. The results would achieve more thorough genetic information of snake eels and provide a theoretical basis and reference for further genome-wide analysis of O. evermanni.

Comprehensive Draft Genome Analyses of Three Rockfishes (Scorpaeniformes, Sebastiscus) via Genome Survey Sequencing.

Sebastiscus species, marine rockfishes, are of essential economic value. However, the genomic data of this genus is lacking and incomplete. Here, whole genome sequencing of all species of Sebastiscus was conducted to provide fundamental genomic information. The genome sizes were estimated to be 802.49 Mb (S. albofasciatus), 786.79 Mb (S. tertius), and 776.00 Mb (S. marmoratus) by using k-mer analyses. The draft genome sequences were initially assembled, and genome-wide microsatellite motifs were identified. The heterozygosity, repeat ratios, and numbers of microsatellite motifs all suggested possibly that S. tertius is more closely related to S. albofasciatus than S. marmoratus at the genetic level. Moreover, the complete mitochondrial genome sequences were assembled from the whole genome data and the phylogenetic analyses genetically supported the validation of Sebastiscus species. This study provides an important genome resource for further studies of Sebastiscus species.

Characterization and phylogenetic analyses of the complete mitochondrial genome of Sillaginopsis panijus (Perciformes: Sillaginidae).

The complete mitochondrial genome of Sillaginopsis panijus has been determined for the first time using Sanger Dideoxy DNA sequencing. The mitogenome is a circular molecule of 16,529 bp in length. It contains 37 mitochondrial genes (13 protein-coding genes, two ribosomal RNA, and 22 transfer RNA) and a control region as other bony fishes. In the phylogenetic analysis using 12H-strand protein-coding genes, monotypic S. panijus is situated separately from the genus Sillago. The present phylogeny supports its taxonomic position according to morphology and will be helpful for evolutionary analysis.

Genome-Wide Survey Reveals the Microsatellite Characteristics and Phylogenetic Relationships of Harpadon nehereus.

Harpadon nehereus forms one of the most important commercial fisheries along the Bay of Bengal and the southeast coast of China. In this study, the genome-wide survey dataset first produced using next-generation sequencing (NGS) was used to provide general information on the genome size, heterozygosity and repeat sequence ratio of H. nehereus. About 68.74 GB of high-quality sequence data were obtained in total and the genome size was estimated to be 1315 Mb with the 17-mer frequency distribution. The sequence repeat ratio and heterozygosity were calculated to be 52.49% and 0.67%, respectively. A total of 1,027,651 microsatellite motifs were identified and dinucleotide repeat was the most dominant simple sequence repeat (SSR) motif with a frequency of 54.35%. As a by-product of whole genome sequencing, the mitochondrial genome is a powerful tool to investigate the evolutionary relationships between H. nehereus and its relatives. The maximum likelihood (ML) phylogenetic tree was constructed according to the concatenated matrix of amino acids translated from the 13 protein-coding genes (PCGs). Monophyly of two species of the genus Harpadon was revealed in the present study and they formed a monophyletic clade with Saurida with a high bootstrap value of 100%. The results would help to push back the frontiers of genomics and open the doors of molecular diversity as well as conservation genetics studies on this species.

Comparative analysis of the complete mitochondrial genomes of three rockfishes (Scorpaeniformes, Sebastiscus) and insights into the phylogenetic relationships of Sebastidae.

Mitochondrial genome is a powerful molecule marker to provide information for phylogenetic relationships and revealing molecular evolution in ichthyological studies. Sebastiscus species, a marine rockfish, are of essential economic value. However, the taxonomic status and phylogenetic relationships of Sebastidae have been controversial so far. Here, the mitochondrial genomes (mitogenomes) of three species, S. tertius, S. albofasciatus, and S. marmoratus, were systemically investigated. The lengths of the mitogenomes' sequences of S. tertius, S. albofasciatus, and S. marmoratus were 16910, 17056, and 17580 bp, respectively. It contained 13 protein-coding genes (PCGs), two ribosomal RNAs (rRNAs), 22 transfer RNA (tRNA) genes, and one identical control region (D-loop) among the three species. The genetic distance and Ka/Ks ratio analyses indicated 13 PCGs were suffering purifying selection and the selection pressures were different from certain deep-sea fishes, which were most likely due to the difference in their living environment. The phylogenetic tree was constructed by Bayesian Inference (BI) and Maximum Likelihood (ML). Most interestingly, the results indicated that Sebastidae and Scorpaenidae were grouped into a separate branch, so the taxonomic status of Sebastidae should be classified into subfamily Sebastinae. Our results may lead to a taxonomic revision of Scorpaenoidei.

Population Genomic Analysis of Two Endemic Schizothoracins Reveals Their Genetic Differences and Underlying Selection Associated with Altitude and Temperature.

Schizothoracins are a group of cyprinid fishes distributed throughout the Qinghai-Tibet Plateau, which can be classified in three grades: primitive, specialised and highly specialised according to adaptation ability to plateau environments. As the only specialised schizothoracins in Xinjiang, China, Diptychus maculates and Gymnodiptychus dybowskii are ideal materials for adaptive evolution research. Based on single-nucleotide polymorphism (SNP) loci detected by specific-locus amplified fragment (SLAF) technology, the genome-wide genetic diversities of these two species from nine sites in Xinjiang were evaluated. D.maculates in the Muzat River (BM) and G. dybowskii in the Kaidu River (LKG) presented the lowest genetic diversity levels, whereas D. maculates in the Kumarik River (BK) and G.dybowskii in the Kashi River (LK) were just the opposite. Cluster and principal component analysis demonstrated a distant genetic affinity between D. maculates in the Tashkurgan River (BT) and other populations. Outlier SNP loci were discovered both in D. maculates and G. dybowskii. The coalescent Bayenv and latent factor mixed model (LFMM) methods showed that a total of thirteen and eighteen SNPs in D. maculates were associated with altitude and temperature gradient, respectively. No intersection was revealed in G. dybowskii. The results indicated that D. maculates was subject to much greater divergent selection pressure. A strong signal of isolation-by-distance (IBD) was detected across D. maculates (Mantel test, rs = 0.65; p = 0.05), indicating an evident geographical isolation in the Tarim River. Isolation-by-environment (IBE) analysis implied that temperature and altitude selections were more intensive in D. maculates, with greater environmental variation resulting in weak gene flow.

Levels and patterns of genetic variation in Japanese whiting (Sillago japonica) based on mitochondrial DNA control region.

Japanese whiting (Sillago japonica) is a commercially important demersal fish distributed along the coasts of East Asia and becomes recently an aquaculture species. Despite its commercial importance, the levels and patterns of population genetic variation remain poorly understood. In this study, 346 specimens were collected from 14 localities along the coastal waters of China, Korea and Japan and their genetic variation was analyzed with mtDNA control region (D-loop) sequences. A total of 131 polymorphic sites were detected which determined 294 haplotypes. A pattern with high levels of haplotype diversity (h = 0.999 ± 0.001) and nucleotide diversity (л = 0.030 ± 0.015) was detected in the examined range. Analyses of analysis of molecular variance (AMOVA) and Fst showed that no significant genetic differentiation existed among China, Korea and Japan populations, excepting for the populations between Ise Bay (IBP) sample and the other ones. Minimum spanning tree (MST), neutrality tests, mismatch distribution and Bayesian skyline analyses indicated that the species along coastline of China, Korea and Japan have experienced population expansions originated in its most recent history at about 106-423 kya during the late Pleistocene glaciations and deglaciations periods.

Transcriptome analysis for identification of candidate genes related to sex determination and growth in Charybdis japonica.

Charybdis japonica is an important cultured crab in China and it exhibits sex differences in their growth. Growth is an important economic trait that is controlled by many genes. In order to discover the growth-related regulatory mechanisms, whole-body transcriptomic sequencing and comparative analyses in different genders of C. japonica were conducted based on Illumina RNA-seq technology. In the present study, we obtained 62,879,204 and 60,226,334 clean reads in female and male libraries, respectively. 25,000,000 clean reads of every library were randomly selected and compared with Nt database to examine the possible contamination. Results showed that all clean reads were distributed among C. japonica or other species that were closely relative to this species, indicating no-pollution. De novo assembly was performed and a total of 32,543 and 44,174 unigenes were produced in female and male of C. japonica, respectively. Among all the unigenes, 12,591 and 14,455 unigenes of female and male crabs were annotated based on protein databases. Moreover, a total of 33,926 unigenes were found to contain ORFs and 52,839 SSRs were detected. The contrast between male and female C. japonica identifying 1939 unigenes were significantly differentially expressed. In addition, we specifically discussed some gene functions and pathways potentially associated with sex determination and growth. This is the first systematic report of whole transcriptome in C. japonica. The transcriptome information provides a basic resource for further studies on understanding the molecular basis of biological processes in C. japonica and other crustaceans.

Complete mitochondrial genome of Diptychus maculatus (Cypriniformes: Cyprinidae: Schizothoracinae).

Abstact The complete mitogenome sequence of the mitochondrial genome of Diptychus maculatus was amplified and analyzed in this study. It was 16,895 bp in length and contained 13 protein-coding genes, 2 ribosomal RNA genes, 22 tRNA and 1 control region. Most of the genes were encoded on the heavy strand except for eight tRNA genes and DN6. The overall base composition of the complete mtDNA was 27.80% A, 26.70% T, 19.01% G and 26.49% C. A 312 bp-long sequence containing a tandem repeat array (37 bp) was found between tRNA(Thr) and tRNA(Pro).

The complete mitochondrial DNA genome of Gymnodiptychus dybowskii (Cypriniformes: Cyprinidae: Schizothoracinae).

The complete nucleotide sequence of Gymnodiptychus dybowskii mitogenome (16 677 bp) has been determined, containing 13 protein-coding genes, two ribosomal RNA genes, 22 tRNA genes, and one non-coding control region. The base composition is 28.04% A, 26.92% T, 18.94% G, 26.10% C, with an AT bias of 54.97%. The origin of light-strand replication (OL) is found between tRNAAsn and tRNACys, which has the potential to fold in a step-loop secondary structure with a stem formed by 11 pairs of nucleotides and a loop of 14 nucleotides. The phylogenetic analysis indicates close relationship between genus Gymnodiptychus and Diptychus.

[Phylogeny and divergence time estimation of Schizothoracinae fishes in Xinjiang].

Based on combined data of mitochondrial COI, ND4 and 16S RNA genes, molecular phylogeny of 4 genera, 10 species or subspecies of Schizothoracinae fishes distributed in Xinjiang were analyzed. The molecular clock was calibrated by divergence time of Cyprininae and geological segregation event between the upper Yellow River and Qinghai Lake. Divergence time of Schizothoracinae fishes was calculated, and its relationship with the major geological events and the climate changes in surrounding areas of Tarim Basin was discussed. The results showed that genus Aspiorhynchus did not form an independent clade, but clustered with Schizothorax biddulphi and S. irregularis. Kimura 2-parameter model was used to calculate the genetic distance of COI gene, the genetic distance between genus Aspiorhynchus and Schizothorax did not reach genus level, and Aspiorhynchus laticeps might be a specialized species of genus Schizothorax. Cluster analysis showed a different result with morphological classification method, and it did not support the subgenus division of Schizothorax fishes. Divergence of two groups of primitive Schizothoracinae (8.18Ma) and divergence of Gymnodiptychus dybowskii and Diptychus maculates (7.67Ma) occurred in late Miocene, which might be related with the separation of Kunlun Mountain and north Tianshan Mountain River system that was caused by the uplift of Qinghai-Tibet Plateau and Tianshan Mountain, and the aridification of Tarim Basin. The terrain of Tarim Basin that was affected by Quaternary Himalayan movement was high in west but low in east, as a result, Lop Nor became the center of surrounding mountain rivers in Tarim Basin, which shaped the distribution pattern of genus Schizothorax.