First report of Perkinsus marinus occurrence associated with wild Pacific oysters Crassostrea gigas from the west coast of Korea.

This study reports the occurrence of Perkinsus marinus associated with wild Pacific oyster (Crassostrea gigas) specimens collected along the west coast of Korea. Confirmation of P. marinus presence was achieved by conventional PCR using World Organization of Animal Health (WOAH)-recommended primers that specifically targeted regions of the rDNA locus (ITS1, 5.8S, and ITS2). Sequencing of 10 samples revealed two distinct sequences differing by a single base pair, indicating potential haplotype variability. One sequence closely resembled the P. marinus strain found in Maryland, USA, whereas the other exhibited divergence, indicative of species diversity in the Korean strain, as was evident from the haplotype network analysis. Further validation involved the Ray's Fluid Thioglycollate Medium (RFTM) assay, which initially yielded inconclusive results, possibly due to low infection intensity. Subsequently, RFTM and 2 M NaOH assays conducted on the isolates in the present study, cultured P. marinus cells in standard DMEM/F12 medium, and a positive P. marinus strain (ATCC 50509), revealed characteristic hypnospores of P. marinus upon Lugol's iodine staining. These comprehensive investigations underscore the conclusive confirmation of P. marinus in Korean waters and mark a significant milestone in our understanding of the distribution and characteristics of this parasite in previously unreported regions.

Phylogeography of the Korean endemic Coreoleuciscus (Cypriniformes: Gobionidae): the genetic evidence of colonization through Eurasian continent to the Korean Peninsula during Late Plio-Pleistocene.

BACKGROUND: Freshwater endemism is thought to have been formed through the vicariance of connected water systems or the process by which ancestral populations colonized specific areas. The Korean Peninsula is well recognized for its high level of freshwater endemism with about 40% of freshwater fish species being endemic. OBJECTIVE: In this study, we attempted to reconstruct the process of speciation and phylogenetic dispersal of Coreoleuciscus species, which is endemic in the Korean Peninsula. METHODS: We used fossil-calibrated divergence time estimation and ancestral distributional reconstruction to infer phylogeographic reconstruction of Coreoleuciscus based on mitochondrial cytochrome c oxidate subunit I (COI) sequences (1551 bp). RESULTS: Our phylogeographic analysis based on a total of 626 individuals revealed that the two Coreoleuciscus species have originated from the independent colonization of different lineages in the ancestral populations, probably during the Late Plio-Pleistocene. The full-scale expansion of Coreoleuciscus populations appears to have taken place after major river structures were completed on the Korean Peninsula. We also provided evidence that the common ancestors of Coreoleuciscus was distributed in Eastern Eurasian continent and subsequently dispersed into the tip of East Asia. High genetic diversity was mainly concentrated in large drainage populations, while small populations showed an monomorphism, which could give important implications for planning the conservation and management of Coreoleuciscus. CONCLUSIONS: The phylogenetic background of the rheophilic Coreoleuciscus species can be explained by the colonizer hypothesis that the endemic freshwater fish originated from the common ancestor in continental region.

Neutral and adaptive drivers of genomic change in introduced brook trout (Salvelinus fontinalis) populations revealed by pooled sequencing.

Understanding the drivers of successful species invasions is important for conserving native biodiversity and for mitigating the economic impacts of introduced species. However, whole-genome resolution investigations of the underlying contributions of neutral and adaptive genetic variation in successful introductions are rare. Increased propagule pressure should result in greater neutral genetic variation, while environmental differences should elicit selective pressures on introduced populations, leading to adaptive differentiation. We investigated neutral and adaptive variation among nine introduced brook trout (Salvelinus fontinalis) populations using whole-genome pooled sequencing. The populations inhabit isolated alpine lakes in western Canada and descend from a common source, with an average of ~19 (range of 7-41) generations since introduction. We found some evidence of bottlenecks without recovery, no strong evidence of purifying selection, and little support that varying propagule pressure or differences in local environments shaped observed neutral genetic variation differences. Putative adaptive loci analysis revealed nonconvergent patterns of adaptive differentiation among lakes with minimal putatively adaptive loci (0.001%-0.15%) that did not correspond with tested environmental variables. Our results suggest that (i) introduction success is not always strongly influenced by genetic load; (ii) observed differentiation among introduced populations can be idiosyncratic, population-specific, or stochastic; and (iii) conservatively, in some introduced species, colonization barriers may be overcome by support through one aspect of propagule pressure or benign environmental conditions.

Differential patterns of diversity at neutral and adaptive loci in endangered Rhodeus pseudosericeus populations.

Given the fact that threatened species are often composed of isolated small populations, spatial continuity or demography of the populations may be major factors that have shaped the species' genetic diversity. Thus, neutral loci have been the most commonly-used markers in conservation genetics. However, the populations under the influence of different environmental factors may have evolved in response to different selective pressures, which cannot be fully reflected in neutral genetic variation. Rhodeus pseudosericeus, a bitterling species (Acheilognathidae; Cypriniformes) endemic to the Korean Peninsula, are only found in some limited areas of three rivers, Daecheon, Han and Muhan, that flow into the west coast. Here, we genotyped 24 microsatellite loci and two loci (DAB1 and DAB3) of MHC class II peptide-binding ß1 domain for 222 individuals collected from seven populations. Our microsatellite analysis revealed distinctive differentiation between the populations of Daecheon and Muhan Rivers and the Han River populations, and populations were structured into two subgroups within the Han River. Apparent positive selection signatures were found in the peptide-binding residues (PBRs) of the MHC loci. The allelic distribution of MHC showed a degree of differentiation between the populations of Daecheon and Muhan Rivers and the Han River populations, partially similar to the results obtained for microsatellites, however showed rather complex patterns among populations in the Han River. Considering the apparent differences in the distribution of supertypes obtained based on the physicochemical differences induced by the polymorphisms of these PBRs, the differentiation in DAB1 between the two regional groups may result in the differences in immune function. No differentiation between these two regions was observed in the supertyping of DAB3, probably indicating that only DAB1 was associated with the response to locally specialized antigenic peptides.

Diversification and colonization processes in Gobioidei predicted based on mitochondrial 12S rRNA with focusing on Oxudercidae.

Gobioidei is one of the largest vertebrate taxa with over 2000 species observed around the globe. The largest group in Gobioidei is gobies that had been classified as one family, Gobiidae, based on morphological features. Molecular phylogenetic studies revealed that gobies consisted of two monophyletic families, Gobiidae and Oxudercidae, in which 19 lineages have been proposed, despite some claims arisen about the relationship among these lineages or species. We analyzed 58 Gobioidei species, including 45 East Asian oxudercids, based on 12S rRNA sequences to reconstruct the spatiotemporal diversification history of gobies. Our analysis yielded the results compatible with the previous reports in a large framework. The common ancestor of Gobiidae and Oxudercidae were estimated to appear at 38.66 Mya. Genus-level splits occurred in Gobiidae and Oxudercidae predominantly at Miocene and late Miocene to early Pleistocene, respectively. Gobies have likely originated in many parts of the northern and western Pacific Ocean, of which a large proportion of Oxudercidae have adapted to various environments in the North Pacific.

Early Life History of Coreoperca herzi in Han River, Korea.

This study aimed to investigate early life of Coreoperca herzi living in Han River and compare morphological differences between different groups during the development in order to provide basic data for relevant taxonomic research. On average, one female individual spawned 541 to 861 eggs (average 701) at once. After 259 hours, the tail broke out of the egg membrane and hatching began. Immediately after hatching, the larvae were average 7.81±0.10 mm (n=5) in total length. 60 days the juvenile was average 35.9±1.30 mm (n=5) in total length. The white spots spread to the rest of the body, rending the same pattern as that on the body of their broodstock fish.

Evidence of an ancient connectivity and biogeodispersal of a bitterling species, Rhodeus notatus, across the Korean Peninsula.

The modern-day distribution of freshwater fishes throughout multiple rivers is likely the result of past migration during times when currently separate drainages were once connected. Here, we used mitochondrial and microsatellite analyses for 248 individuals of Rhodeus notatus collected from seven different rivers to obtain better understand historical gene flow of freshwater fish on the Korean Peninsula. Based on our phylogenetic analyses, this Korean species originated through the paleo-Yellow River from China and first colonized near the west coast. These genetic data also provided evidence of estuary coalescences among the rivers flowing to the west and southwest coast on well-developed continental shelf. In addition, the pattern of population structure revealed the biogeodispersal route from the west coast to the south coast. It could be inferred that massive migration was not involved in the formation of southern populations, since the signature of historical genetic drift was clearly observed. Our study is the first genetic attempt to confirm hypotheses describing the migration of freshwater species towards the end of East Asia, which have previously been developed using only geological reasoning.

Polymorphism of MHC class IIB in an acheilognathid species, Rhodeus sinensis shaped by historical selection and recombination.

BACKGROUND: Rhodeus sinensis is a bitterling species occurring throughout the numerous freshwater systems on the East Asia. Here, we analyzed the diversity of the MHC class IIB (DAB) genes from this species, which may offer meaningful insights into evolutionary processes in this species as well as other bitterlings. RESULTS: Using cDNA and gDNA samples from 50 individuals, we discovered classical 140 allelic sequences that could be allocated into either DAB1 (Rhsi-DAB1) or DAB3 (Rhsi-DAB3). DAB sequences completely lacking the intron, but identical or similar to Rhsi-DAB1, were also discovered from our gDNA samples, and this intron loss likely originated from the retrotransposition events of processed mDNA. The ß1 domain was the most polymorphic in both Rhsi-DAB1 and -DAB3. Putative peptide biding residues (PBRs) in Rhsi-DAB1, but not in Rhsi-DAB3, exhibited a significant dN/dS, presumably indicating that different selection pressures have acted on those two DABs. Recombination between different alleles seemed to have contributed to the increase of diversity in Rhsi-DABs. Upon phylogenetic analysis, Rhsi-DAB1 and -DAB3 formed independent clusters. Several alleles from other species of Cypriniformes were embedded in the clade of Rhsi-DAB1, whereas Rhsi-DAB3 clustered with alleles from the wider range of taxa (Cyprinodontiformes), indicating that these two Rhsi-DABs have taken different historical paths. CONCLUSIONS: A great deal of MHC class IIB allelic diversity was found in R. sinensis, and gene duplication, selection and recombination may have contributed to this diversity. Based on our data, it is presumed that such historical processes have commonly or differently acted on the polymorphism of Rhsi-DAB1 and -DAB3.

Mass mortality in Korean bay scallop (Argopecten irradians) associated with Ostreid Herpesvirus-1 µVar.

Since November 2017, mass mortalities of larvae of bay scallop (Argopecten irradians) were reported in hatcheries located at the southern area of Republic of Korea. Over 90% of larvae aged 5-10 days sank to the bottom of the tank and died. The hatcheries could not produce spat, and thus artificial seed production industry incurred huge losses. We identified Ostreid Herpesvirus-1 µVar (OsHV-1 µVar) associated with mass mortality by PCR, sequencing and transmission electron microscopy (TEM). All the samples were positive for OsHV-1 µVar with 99% sequence identity to previously reported OsHV-1 µVar sequences. Partial sequence of ORF-4 of OsHV-1 detected in this study was more closely related to sequences isolated from Europe. This is the first report to confirm the mortality caused by an OsHV-1 infection in the bay scallop.

The genetic structure of Squalidus multimaculatus revealing the historical pattern of serial colonization on the tip of East Asian continent.

Separated river systems could create confluences via two geological processes, estuary coalescence in response to decreasing sea levels and headwater capture, allowing primary freshwater species to disperse across rivers. Squalidus multimaculatus, is an endemic and primary freshwater species restricted to the southeast coast of the Korean Peninsula. The distribution of this species is unique, given that other congeneric species, including its closely related S. gracilis majimae, as well as other cyprind species are observed throughout the peninsula except for the east coast. Phylogeographic analyses were conducted using three mitochondrial loci to identify the origin of S. multimaculatus and the historical pathways of dispersal. A strong phylogenetic affinity between S. multimaculatus and S. g. majimae and the genetic structure among populations indicated that S. multimaculatus originated from the eastward colonization of the common ancestor between S. g. majimae and S. multimaculatus via headwater capture through fault zones within successive mountain range. Following colonization, the ancestral S. multimaculatus likely migrated towards north via estuary coalescence along a well-developed continental shelf. Our study was the first empirical attempt providing insights into how freshwater organisms dispersed to the southernmost tip of East Asia, despite the potential loss of such historical imprints with anthropogenic interference.

Taxonomic characterization of Tanakia species (Acheilognathidae) using DNA barcoding analyses.

Tanakia is a bitterling genus with six species found in Far East Asia. Our aim was to construct the standard DNA barcode database available for the identification of six Tanakia species by comparing the range of intra- and inter-specific genetic distances, identifying the phylogenetic placement of each Tanakia species and providing the unique barcode characteristics that are specific to each species, using cytochrome oxidase I (COI) and cytochrome b (cyt b). Both loci failed to create a perfect barcoding gap between the ranges of inter- and intra-specific genetic distances, though interspecific COI distances were sufficiently greater than intraspecific values with only a few exceptions. In our phylogenetic analyses, T. koreensis and T. signifer did not form a monophyletic cluster of haplotypes in both loci. COI provided clear nucleotide characteristics that distinguish each species, whereas relatively fewer informative sites were found within the range of cyt b. Overall, COI could be regarded as appropriate species identification solution in Tanakia. Our analyses yielded some taxonomic issues that need the further investigation, and are expected to be helpful in the examination for the conservation status of Tanakia species that are on the verge of being endangered.

The complete mitochondrial genomes of three geographical lineages in short ninespine stickleback (Pungitius kaibarae) complex and their phylogenetic implication.

The short ninespine stickleback, Pungitius kaibarae, is a small gasterosteid species complex containing three geographical lineages: ND, NE and SE. Here, complete mitochondrial genomes of these three lineages were analyzed to estimate the genetic differentiation among them and to identify their phylogenetic placement in genus Pungitius. Although the overall genome structure was identical among those three lineages, the genome sizes were slightly different from each other, ranging from 16,489 to 16,500 bp. Upon robust phylogenetic tree inferred by Bayesian algorithm, ND and SE showed relatively higher affinity, and those three lineages formed a monophyletic group with Russian P. tymensis, clearly supporting previous studies.

Complete mitochondrial genome of endangered Rhodeus pseudosericeus and its implications for the reconstruction of phylogenetic relationship among Acheilognathinae species.

Rhodeus pseudosericeus is a native bitterling to the Korean Peninsula and found in very limited areas with small census size. Here, its complete mitochondrial genome was analyzed to provide novel data for the reconstruction of phylogenetic relationship among Acheilognathinae species. The genome was a 16,574 bp long consisting of 1 putative control region, 2 rRNA genes, 22 tRNA and 13 protein-coding genes. The gene arrangement was completely identical to those observed in other Acheilognathinae species as well as in other cyprinid species. In our phylogenetic analyses, three major genera of Acheilognathinae indepedently formed monophyletic groups in the tree reconstructed based on the whole genome sequences, whereas Rhodeus was not recovered as a single monophyly when solely considering protein-coding genes, indicating that the taxonomic reevaluation is still required in this subfamily.