Mitogenomic Characterization and Phylogenetic Placement of African Hind, Cephalopholis taeniops: Shedding Light on the Evolution of Groupers (Serranidae: Epinephelinae).

The global exploration of evolutionary trends in groupers, based on mitogenomes, is currently underway. This research extensively investigates the structure of and variations in Cephalopholis species mitogenomes, along with their phylogenetic relationships, focusing specifically on Cephalopholis taeniops from the Eastern Atlantic Ocean. The generated mitogenome spans 16,572 base pairs and exhibits a gene order analogous to that of the ancestral teleost's, featuring 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and an AT-rich control region. The mitogenome of C. taeniops displays an AT bias (54.99%), aligning with related species. The majority of PCGs in the mitogenome initiate with the start codon ATG, with the exceptions being COI (GTG) and atp6 (TTG). The relative synonymous codon usage analysis revealed the maximum abundance of leucine, proline, serine, and threonine. The nonsynonymous/synonymous ratios were <1, which indicates a strong negative selection among all PCGs of the Cephalopholis species. In C. taeniops, the prevalent transfer RNAs display conventional cloverleaf secondary structures, except for tRNA-serine (GCT), which lacks a dihydrouracil (DHU) stem. A comparative examination of conserved domains and sequence blocks across various Cephalopholis species indicates noteworthy variations in length and nucleotide diversity. Maximum likelihood, neighbor-joining, and Bayesian phylogenetic analyses, employing the concatenated PCGs and a combination of PCGs + rRNAs, distinctly separate all Cephalopholis species, including C. taeniops. Overall, these findings deepen our understanding of evolutionary relationships among serranid groupers, emphasizing the significance of structural considerations in mitogenomic analyses.

Insights into the Mitochondrial Genetic Makeup and Miocene Colonization of Primitive Flatfishes (Pleuronectiformes: Psettodidae) in the East Atlantic and Indo-West Pacific Ocean.

The mitogenomic evolution of the Psettodes flatfishes is still poorly known from their range distribution in eastern Atlantic and Indo-West Pacific Oceans. The study delves into the matrilineal evolutionary pathway of these primitive flatfishes, with a specific focus on the complete mitogenome of the Psettodes belcheri species, as determined through next-generation sequencing. The mitogenome in question spans a length of 16,747 base pairs and comprises a total of 37 genes, including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a control region. Notably, the mitogenome of P. belcheri exhibits a bias towards AT base pairs, with a composition of 54.15%, mirroring a similar bias observed in its close relative, Psettodes erumei, which showcases percentages of 53.07% and 53.61%. Most of the protein-coding genes commence with an ATG initiation codon, except for Cytochrome c oxidase I (COI), which initiates with a GTG codon. Additionally, four protein-coding genes commence with a TAA termination codon, while seven others exhibit incomplete termination codons. Furthermore, two protein-coding genes, namely NAD1 and NAD6, terminate with AGG and TAG stop codons, respectively. In the mitogenome of P. belcheri, the majority of transfer RNAs demonstrate the classical cloverleaf secondary structures, except for tRNA-serine, which lacks a DHU stem. Comparative analysis of conserved blocks within the control regions of two Psettodidae species unveiled that the CSB-II block extended to a length of 51 base pairs, surpassing the other blocks and encompassing highly variable sites. A comprehensive phylogenetic analysis using mitochondrial genomes (13 concatenated PCGs) categorized various Pleuronectiformes species, highlighting the basal position of the Psettodidae family and showed monophyletic clustering of Psettodes species. The approximate divergence time (35-10 MYA) between P. belcheri and P. erumei was estimated, providing insights into their separation and colonization during the early Miocene. The TimeTree analysis also estimated the divergence of two suborders, Psettodoidei and Pleuronectoidei, during the late Paleocene to early Eocene (56.87 MYA). The distribution patterns of Psettodes flatfishes were influenced by ocean currents and environmental conditions, contributing to their ecological speciation. In the face of climate change and anthropogenic activities, the conservation implications of Psettodes flatfishes are emphasized, underscoring the need for regulated harvesting and adaptive management strategies to ensure their survival in changing marine ecosystems. Overall, this study contributes to understanding the evolutionary history, genetic diversity, and conservation needs of Psettodes flatfishes globally. However, the multifaceted exploration of mitogenome and larger-scale genomic data of Psettodes flatfish will provide invaluable insights into their genetic characterization, evolutionary history, environmental adaptation, and conservation in the eastern Atlantic and Indo-West Pacific Oceans.

Mitogenomic Characterization of Cameroonian Endemic Coptodon camerunensis (Cichliformes: Cichlidae) and Matrilineal Phylogeny of Old-World Cichlids.

The mitogenomic evolution of old-world cichlids is still largely incomplete in Western Africa. In this present study, the complete mitogenome of the Cameroon endemic cichlid, Coptodon camerunensis, was determined by next-generation sequencing. The mitogenome was 16,557 bp long and encoded with 37 genes (13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a control region). The C. camerunensis mitogenome is AT-biased (52.63%), as exhibited in its congener, Coptodon zillii (52.76% and 53.04%). The majority of PCGs start with an ATG initiation codon, except COI, which starts with a GTG codon and five PCGs and ends with the TAA termination codon and except seven PCGs with an incomplete termination codon. In C. camerunensis mitogenome, most tRNAs showed classical cloverleaf secondary structures, except tRNA-serine with a lack of DHU stem. Comparative analyses of the conserved blocks of two Coptodonini species control regions revealed that the CSB-II block was longer than other blocks and contained highly variable sites. Using 13 concatenated PCGs, the mitogenome-based Bayesian phylogeny easily distinguished all the examined old-world cichlids. Except for Oreochromini and Coptodinini tribe members, the majority of the taxa exhibited monophyletic clustering within their respective lineages. C. camerunensis clustered closely with Heterotilapia buttikoferi (tribe Heterotilapiini) and had paraphyletic clustering with its congener, C. zillii. The Oreochromini species also displayed paraphyletic grouping, and the genus Oreochromis showed a close relationship with Coptodinini and Heterotilapiini species. In addition, illustrating the known distribution patterns of old-world cichlids, the present study is congruent with the previous hypothesis and proclaims that prehistoric geological evolution plays a key role in the hydroclimate of the African continent during Mesozoic, which simultaneously disperses and/or colonizes cichlids in different ichthyological provinces and Rift Lake systems in Africa. The present study suggests that further mitogenomes of cichlid species are required, especially from western Africa, to understand their unique evolution and adaptation.

Mitochondrial DNA Corroborates the Genetic Variability of Clarias Catfishes (Siluriformes, Clariidae) from Cameroon.

The airbreathing walking catfish (Clariidae: Clarias) comprises 32 species that are endemic to African freshwater systems. The species-level identification of this group is challenging due to their complex taxonomy and polymorphism. Prior to this study, the biological and ecological studies were restricted to a single species, Clarias gariepinus, resulting in a biased view of their genetic diversity in African waters. Here, we generated the 63-mitochondrial Cytochrome c oxidase subunit 1 (COI) gene sequences of Clarias camerunensis and Clarias gariepinus from the Nyong River in Cameroon. Both C. camerunensis and C. gariepinus species maintained adequate intra-species (2.7% and 2.31%) and inter-species (6.9% to 16.8% and 11.4% to 15.1%) genetic distances with other Clarias congeners distributed in African and Asian/Southeast Asian drainages. The mtCOI sequences revealed 13 and 20 unique haplotypes of C. camerunensis and C. gariepinus, respectively. The TCS networks revealed distinct haplotypes of C. camerunensis and shared haplotypes of C. gariepinus in African waters. The multiple species delimitation approaches (ABGD and PTP) revealed a total of 20 and 22 molecular operational taxonomic units (MOTUs), respectively. Among the two Clarias species examined, we found more than one MOTU in C. camerunensis, which is consistent with population structure and tree topology results. The phylogeny generated through Bayesian Inference analysis clearly separated C. camerunensis and C. gariepinus from other Clarias species with high posterior probability supports. The present study elucidates the occurrence of possible cryptic diversity and allopatric speciation of C. camerunensis in African drainages. Further, the present study confirms the reduced genetic diversity of C. gariepinus across its native and introduced range, which might have been induced by unscientific aquaculture practices. The study recommends a similar approach to the same and related species from different river basins to illuminate the true diversity of Clarias species in Africa and other countries.

Mitochondriomics of Clarias Fishes (Siluriformes: Clariidae) with a New Assembly of Clarias camerunensis: Insights into the Genetic Characterization and Diversification.

The mitogenome of an endemic catfish Clarias camerunensis was determined from the Cameroon water. This circular mitogenome was 16,511 bp in length and comprised 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a single AT-rich control region. The heavy strand accommodates 28 genes, whereas the light strand is constituted by ND6 and eight transfer RNA (tRNA) genes. The C. camerunensis mitochondrial genome is AT biased (56.89%), as showcased in other Clarias species. The comparative analyses revealed that most of the Clarias species have 6 overlapping and 11 intergenic spacer regions. Most of the PCGs were initiated and terminated with the ATG start codon and TAA stop codon, respectively. The tRNAs of C. camerunensis folded into the distinctive cloverleaf secondary structure, except trnS1. The placement of the conserved domains in the control region was similar in all the Clarias species with highly variable nucleotides in CSB-I. Both maximum likelihood and Bayesian-based matrilineal phylogenies distinctly separated all Clarias species into five clades on the basis of their known distributions (South China, Sundaland, Indochina, India, and Africa). The TimeTree analysis revealed that the two major clades (Indo-Africa and Asia) of Clarias species might have diverged during the Paleogene (≈28.66 MYA). Our findings revealed the separation of Indian species (C. dussumieri) and African species (C. camerunensis and Clarias gariepinus) took place during the Paleogene, as well as the South Chinese species (Clarias fuscus) and Sundaland species (Clarias batrachus) splits from the Indochinese species (Clarias macrocephalus) during the Neogene through independent colonization. This pattern of biotic relationships highlights the influence of topography and geological events in determining the evolutionary history of Clarias species. The enrichment of mitogenomic data and multiple nuclear loci from their native range or type locality will confirm the true diversification of Clarias species in African and Asian countries.

Molecular Diet Analysis of Adélie Penguins (Pygoscelis adeliae) in the Ross Sea Using Fecal DNA.

The diet of Adélie penguins, Pygoscelis adeliae, is a useful indicator in understanding the ecological conditions of their habitats. The diets of Adélie penguins were studied using metabarcoding and quantitative PCR (qPCR) analyses of fecal DNA from seven habitats along the Ross Sea region. Using metabarcoding analysis with dual universal primers (18Sv9 and miniFish), the overall diet composition and detailed information about piscine prey were clearly elucidated. It was found that two krill species (Euphausia superba and Euphausia crystallorophias) and notothenioid fish were the most abundant in the diets of Adélie penguins. Among the notothenioid prey, Pleuragramma antarctica (56.50%) and Pagothenia borchgrevinki (18.21%) were the two most abundant species. qPCR analysis showed a significant geographic difference in the composition of main prey. Penguins inhabiting outbound parts of the Ross Sea (Capes Adare (CA) and Duke of York Island (DY)) mainly preyed on E. superba, without any significant changes in prey composition. By contrast, those inhabiting the inbound parts of the Ross Sea (Edmonson Point (EP) and Inexpressible Island (II)) preyed on E. crystallorophias and notothenioid fish rather than E. superba. Compared with the outbound habitats, prey compositions for penguins inhabiting the inbound regions were significantly different year to year, which was presumably due to the food availability based on the annual environmental and meteorological conditions of the coastal region along with the inbound parts of the Ross Sea.

The complete mitochondrial genome of the Korean endemic species Cobitis hankugensis (Kim, Park, Son & Nalbant, 2003).

As one of efforts to conserve a genetic resource of the endemic cobitid species in the Korean peninsula, the complete mitogenome of Cobitis hankugensis (Kim, Park, Son & Nalbant, 2003) was determined using Illumina MiSeq system. The circular mitogenome was 16,557 bp length and encoded 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 tRNA genes, and a control region. Only the COX1 gene was identified with an aberrant initiation codon GTG, and an incomplete termination codon (T-/TA-) was identified in six PCGs including COX2, COX3, ND2, ND3, ND4, and Cytb genes. Phylogenetic analysis using 30 mitochondrial genomes belonging to Cobitidae, Botiidae, and Gyrinocheilidae showed that the highest identity (92.38%) with Kichulchoia brevifasciata (NC_027166). The complete mitogenome of C. hankugensis, an endemic species in Korea, will provide fundamental data on the evolutionary relationship of Cobitidae species.

Mitochondrial DNA and Distribution Modelling Evidenced the Lost Genetic Diversity and Wild-Residence of Star Tortoise, Geochelone elegans (Testudines: Testudinidae) in India.

The Indian star tortoise (Geochelone elegans) is a massively traded animal in South Asia. To mitigate this risk, the conservation agencies recommended guidelines to safeguard this charismatic species in nature. We adopted mitochondrial DNA-based investigation and performed species distribution modeling of G. elegans throughout its distribution range in the Indian subcontinent. The genetic analyses revealed weak genetic landscape shape interpolations, low intraspecific distances (0% to 1.5%) with mixed haplotype diversity, and a single molecular operational taxonomic unit (MOTU) in the cytochrome b gene dataset. The star tortoise, G. elegans, and its sister species Geochelone platynota showed a monophyletic clustering in the Bayesian (BA) phylogeny. We also attempt to understand the habitat suitability and quality of G. elegans in its distribution range. Our results suggest that, out of the extant area, only 56,495 km2 (9.90%) is suitable for this species, with regions of highest suitability in Sri Lanka. Comparative habitat quality estimation suggests the patch shape complexity and habitat fragmentation are greater in the western and southern ranges of India, which have been greatly influenced by an increased level of urbanization and agriculture practices. We have also provided a retrospect on the potential threat to G. elegans related to the wildlife trade on the regional and international spectrum. Our results detected multiple trading hubs and junctions overlying within the suitable ranges which need special attention in the vicinity. The present study calls for a proper conservation strategy to combat the fragmented distribution and explicitly recommends intensive genetic screening of founder individuals or isolated adult colonies, implementing scientific breeding, and subsequent wild release to restore the lost genetic diversity of star tortoises.

Characterization of the complete mitochondrial genome of the Northern Mud Gudgeon, Ophiocara porocephala (Perciformes: Eleotridae) with phylogenetic implications.

The first mitochondrial genome of Ophiocara porocephala was determined by the combination of next-generation sequencing (NGS) and Sanger sequencing methods. A complete circular mitogenome of O. porocephala (16,529 bp) consisted of 13 protein-coding genes, 22 transfer RNAs, two ribosomal RNAs, and two non-coding regions, including a control region (D-loop) and a light strand origin of replication (OL). Two start codons (ATG and GTG) and four stop codons (TAG, TAA, TA-, and T-) were used in all the PCGs. Except for ND6 and eight transfer RNAs (tRNAs), all the other genes were encoded in the heavy strand. Based on phylogenetic analysis, O. porocephala formed a clade with three other species in the subfamily Butinae, while the other 10 made a subfamily Eleotrinae clade.

Development of the cephalopod-specific universal primer set and its application for the metabarcoding analysis of planktonic cephalopods in Korean waters.

Although spatiotemporal analysis of the cephalopod larvae provides the useful information for the effective management of their resources, it has been difficult mainly due to their low numbers in the mixed zooplankton net samples and difficulty in morphological identification. In order to analyze the planktonic cephalopods using next-generation sequencing (NGS), we have designed a cephalopod-specific universal (CPD) primer set targeting a region covering mitochondrial cytochrome b and ND6 genes based on the currently identified 36 complete cephalopod mitochondrial genome sequences in the GenBank database. The expected amplicon sizes by CPD primers were between 465 and 471 bp, which was applicable to the MiSeq system (Illumina, San Diego, CA, USA). NGS results of pooled DNAs from 8 months (including 739 zooplankton net samples) collected from Korean waters in 2016 showed the exclusive cephalopod sequences with little contaminant sequences supporting the specificity of CPD primer set. Total 47 representative cephalopod haplotypes (seven families and 10 genera) were obtained from 1,439,414 merged reads. Among the total analyzed haplotypes, Watasenia scintillans, Todarodes pacificus, and Sepiola birostrata were the most abundant species in Korean waters. Two "unidentified" clades in order Oegopsida were identified, which was showed less than 90% sequence identity but closely related to Enoploteuthidae and Idiosepiidae, respectively. Monthly changes in proportions of each haplotype were also identified, which may reflect its reproduction and spawning period. The larvae of W. scintillans was dominant from February to June, while high proportions of other cephalopod taxa were also identified from August to November. Only single haplotype was dominant in W. scintillans (Type 2) throughout the year, while two distinct haplotypes showed seasonal differences in T. pacificus.

Characterization of the complete mitochondrial genome of Gangetic ailia, Ailia coila (Siluriformes: Ailiidae).

The first complete mitochondrial genome sequence of Ailia coila from Bangladesh was determined by the bioinformatic assembly of the next generation sequencing (NGS) reads. The constructed circular mitogenome for A. coila was 16,565 bp in length which harbored the canonical 13 protein-coding genes, 22 tRNAs, 2 rRNAs. Two non-coding regions, control region, D-loop (927 bp), and origin of light strand replication, OL (30 bp) were also well conserved in the mitogenome. Among the currently reported mitochondrial genomes in the order Siluriformes, A. coila was most closely related to Eutropiichthys vacha (AB919123) with 85.63% sequence identity.

Characterization of the complete mitochondrial genome of Chionobathyscus dewitti (Perciformes, Channichthyidae).

The complete mitochondrial genome sequence of the Icefish, Chionobathyscus dewitti was determined by the Next Generation Sequencing (NGS) analysis. The complete mitogenome was 17,452 bp in length, which encoded the canonical 13 protein-coding genes, 22 tRNAs, two rRNAs, and two non-coding regions. As shown in the other notothenids, translocation of ND6 and an additional non-coding region were identified, which is different from the typical vertebrate mitochondrial genomes. The C. dewitti was clustered distinctly from the those in the Chinodraco and Chaenocephalus, which supported the idea that this species should be classified in the different genus, Chionobathyscus in the family Channichthyidae.

Characterization of complete mitochondrial genome of Pogonophryne albipinna (Perciformes: Artedidraconidae).

The complete mitochondrial genome of Pogonophryne albipinna was determined by the MiSeq platform, which was the first report in the family Artedidraconidae. The circular form of its mitochondrial genome was 17,086 bp, which contained the canonical eukaryotic 37 genes. The gene orders of P. albipinna was identical to the other icefish species, in which there was additional non-coding region and translocation of ND6 gene. Except for ATP6 gene and COI (GTG), 11 genes begin with the typical start codon, while incomplete stop codons (T- -) were identified in COII, ND4, and CytB. Phylogenetic tree with the currently known mitogenomes in suborder Notothenioidei showed that P. albipinna was located distinctly from those in Bathydraconidae and Nototheniidae forming a unique cluster as Artedidraconidae. The first complete mitochondrial genome of P. albipinna would be the fundamental data to understand the evolutional relationship of icefish species in the Antarctic Oceans.

Metabarcoding analysis of the stomach contents of the Antarctic Toothfish (Dissostichus mawsoni) collected in the Antarctic Ocean.

Stomach contents of the Antarctic toothfish, Dissostichus mawsoni, collected from subareas 58.4 and 88.3, were analyzed using next generation sequencing (NGS) technology. After processing the raw reads generated by the MiSeq platform, a total of 131,233 contigs (130 operational taxonomic units [OTUs]) were obtained from 163 individuals in subarea 58.4, and 75,961 contigs (105 OTUs) from 164 fish in subarea 88.3. At 98% sequence identity, species names were assigned to most OTUs in this study, indicating the quality of the DNA barcode database for the Antarctic Ocean was sufficient for molecular analysis, especially for fish species. A total of 19 species was identified from the stomach of D. mawsoni in this study, which included 14 fish species and five mollusks. More than 90% of contigs belonged to fish species, supporting the postulate that the major prey of D. mawsoni are fish. Two fish species, Macrourus whitsoni and Chionobathyscus dewitti, were the most important prey items (a finding similar to that of previous studies). We also obtained genotypes of prey items by NGS analysis, identifying an additional 17 representative haplotypes in this study. Comparison with three previous morphological studies and the NGS-based molecular identification in this study extended our knowledge regarding the prey of D. mawsoni, which previously was not possible. These results suggested that NGS-based diet studies are possible, if several current technical limitations, including the quality of the barcode database or the development of precise molecular quantification techniques to link them with morphological values, are overcome. To achieve this, additional studies should be conducted on various marine organisms.

Molecular characterization of an adiponectin receptor homolog in the white leg shrimp, Litopenaeus vannamei.

Adiponectin (AdipoQ) and its receptors (AdipoRs) are strongly related to growth and development of skeletal muscle, as well as glucose and lipid metabolism in vertebrates. Herein we report the identification of the first full-length cDNA encoding an AdipoR homolog (Liv-AdipoR) from the decapod crustacean Litopenaeus vannamei using a combination of next generation sequencing (NGS) technology and bioinformatics analysis. The full-length Liv-AdipoR (1,245 bp) encoded a protein that exhibited the canonical seven transmembrane domains (7TMs) and the inversed topology that characterize members of the progestin and adipoQ receptor (PAQR) family. Based on the obtained sequence information, only a single orthologous AdipoR gene appears to exist in arthropods, whereas two paralogs, AdipoR1 and AdipoR2, have evolved in vertebrates. Transcriptional analysis suggested that the single Liv-AdipoR gene appears to serve the functions of two mammalian AdipoRs. At 72 h after injection of 50 pmol Liv-AdipoR dsRNA (340 bp) into L. vannamei thoracic muscle and deep abdominal muscle, transcription levels of Liv-AdipoR decreased by 93% and 97%, respectively. This confirmed optimal conditions for RNAi of Liv-AdipoR. Knockdown of Liv-AdipoR resulted in significant changes in the plasma levels of ammonia, 3-methylhistine, and ornithine, but not plasma glucose, suggesting that that Liv-AdipoR is important for maintaining muscle fibers. The chronic effect of Liv-AdipoR dsRNA injection was increased mortality. Transcriptomic analysis showed that 804 contigs were upregulated and 212 contigs were downregulated by the knockdown of Liv-AdipoR in deep abdominal muscle. The significantly upregulated genes were categorized as four main functional groups: RNA-editing and transcriptional regulators, molecular chaperones, metabolic regulators, and channel proteins.

Three cDNAs encoding vitellogenin homologs from Antarctic copepod, Tigriopus kingsejongensis: Cloning and transcriptional analysis in different maturation stages, temperatures, and putative reproductive hormones.

Three full-length cDNAs encoding lipoprotein homologs were identified in Tigriopus kingsejongensis, a newly identified copepod from Antarctica. Structural and transcriptional analyses revealed homology with two vitellogenin-like proteins, Tik-Vg1 and Tik-Vg2, which were 1855 and 1795 amino acids in length, respectively, along with a third protein, Tik-MEP, which produced a 1517-residue protein with similarity to a melanin engaging protein (MEP) in insects Phylogenetic analysis showed that Vgs in Maxillopods including two Tik-Vgs belong to the arthropod vitellogenin-like clade, which includes clottable proteins (CPs) in decapod crustaceans and vitellogenins in insects. Tik-MEP clustered together with insect MEPs, which appear to have evolved before the apoB-like and arthropod Vg-like clades. Interestingly, no genes orthologous to those found in the apoB clade were identified in Maxillopoda, suggesting that functions of large lipid transfer proteins (LLTPs) in reproduction and lipid metabolism may be different from those in insect and decapod crustaceans. As suggested by phylogenetic analyses, the two Tik-Vgs belonging to the arthropod Vg-like clade appear to play major roles in oocyte maturation, while Vgs belonging to the apoB clade function primarily in the reproduction of decapod crustaceans. Transcriptional analysis of Tik-Vg expression revealed a 24-fold increase in mature and ovigerous females compared with immature female, whereas expression of Tik-MEP remained low through all reproductive stages. Acute temperature changes did not affect the transcription of Tik-Vg genes, whereas Tik-MEP appeared to be affected by temperature change. Among the three hormones thought to be involved in molting and reproduction in arthropods, only farnesoic acid (FA) induced transcription of the two Tik-Vg genes. Regardless of developmental stage and hormone treatment, Tik-Vg1 and Tik-Vg2 exhibited a strong positive correlation in expression, suggesting that expression of these genes may be regulated by the same transcriptional machinery.