Genome assembly and population genomic data of a pulmonate snail Ellobium chinense.

Ellobium chinense is an airbreathing, pulmonate gastropod species that inhabits saltmarshes in estuaries of the northwestern Pacific. Due to a rapid population decline and their unique ecological niche in estuarine ecosystems, this species has attracted special attention regarding their conservation and the genomic basis of adaptation to frequently changing environments. Here we report a draft genome assembly of E. chinense with a total size of 949.470 Mb and a scaffold N50 of 1.465 Mb. Comparative genomic analysis revealed that the GO terms enriched among four gastropod species are related to signal transduction involved in maintaining electrochemical gradients across the cell membrane. Population genomic analysis using the MSMC model for 14 re-sequenced individuals revealed a drastic decline in Korean and Japanese populations during the last glacial period, while the southern Chinese population retained a much larger effective population size (Ne). These contrasting demographic changes might be attributed to multiple environmental factors during the glacial-interglacial cycles. This study provides valuable genomic resources for understanding adaptation and historical demographic responses to climate change.

Morphological and molecular analysis of the tropical hermit crab Calcinus vachoni (Decapoda, Diogenidae) and its potential association with colonial anemone.

Calcinus is the colorful hermit crab genus belonging to the family Diogenidae and is often found in coral reefs of the tropical Indo-West Pacific region, including southern Honshu, Japan, which is the northern limit of their occurrence. In the present study, we found C. vachoni for the first time in the intertidal zone of Jeju Island-the southernmost island of South Korea. We examined their morphology and provided a diagnosis of their morphological details with illustrations. In addition, the Korean Calcinus population was genetically characterized using mtDNA cox1 sequences and by placing them into three previously reported regional haplogroups. The phylogenetic tree from maximum likelihood analysis revealed that Korean C. vachoni is assigned to the C. vachoni haplogroups exclusively, one of the three well-supported mitochondrial haplogroups with distinct geographic ranges (i.e., C. vachoni, C. aff. vachoni Cooks, and C. aff. vachoni Mascarenes). This result provides new information on the species distribution of C.vachoni, extending their geographic range further north into the southern coast of Korea. In this study, we also first report the potential association of C. vachoni with their co-occurring colonial anemone species Palythoa aff. mutuki and dead coral head of Pocillopora species based on our on-site observation and a public coral collection database of Calcinus species. However, their ecological association with co-occurring coral species is putatively assumed for now and therefore has to be validated by compelling evidence from further field observation and experimental studies (i.e., whether the presence/absence of colonial anemones affects the behavior and survival of the hermit crabs).

Complete mitochondrial genome of Conuslischkeanus Weinkauff, 1875 (Neogastropoda, Conidae) and phylogenetic implications of the evolutionary diversification of dietary types of Conus species.

The family Conidae, commonly known as cone snails, is one of the most intriguing gastropod groups owing to their diverse array of feeding behaviors (diets) and toxin peptides (conotoxins). Conuslischkeanus Weinkauff, 1875 is a worm-hunting species widely distributed from Africa to the Northwest Pacific. In this study, we report the mitochondrial genome sequence of C.lischkeanus and inferred its phylogenetic relationship with other Conus species. Its mitochondrial genome is a circular DNA molecule (16,120 bp in size) composed of 37 genes: 13 protein-coding genes (PCGs), 22 transfer RNA genes, and two ribosomal RNA genes. Phylogenetic analyses of concatenated nucleotide sequences of 13 PCGs and two ribosomal RNA genes showed that C.lischkeanus belongs to the subgenus Lividoconus group, which is grouped with species of the subgenus Virgiconus, and a member of the largest assemblage of worm-hunting (vermivorous) species at the most basal position in this group. Mitochondrial genome phylogeny supports the previous hypothesis that the ancestral diet of cone snails was worm-hunting, and that other dietary types (molluscivous or piscivorous) have secondarily evolved multiple times from different origins. This new, complete mitochondrial genome information provides valuable insights into the mitochondrial genome diversity and molecular phylogeny of Conus species.

Morphological and molecular analyses of parasitic barnacles (Crustacea: Cirripedia: Rhizocephala) in Korea: preliminary data for the taxonomy and host ranges of Korean species.

Morphological and molecular analyses of Korean rhizocephalan barnacle species were performed to examine their host ranges and taxonomy. Morphological examination and molecular analysis of mtDNA cox1, 16S, and nuclear 18S rRNA sequences revealed nine rhizocephalan species from three genera of the two families, Sacculinidae and Polyascidae. Phylogenetic analysis of molecular sequences revealed two new species candidates in the genus Parasacculina, and three Sacculina species (S. pilosella, S. pinnotherae, and S. imberbis) were transferred to the genus Parasacculina. Examination of host ranges revealed higher host specificity and lower infestation rates in Korean rhizocephalan species than rhizocephalans from other geographic regions. This is the first report of the taxonomy, species diversity, and host ranges of Korean parasitic rhizocephalan barnacles based on their morphological and molecular analyses. More information from extensive sampling of parasitic barnacles from a wide range of crustacean host species is necessary to fully understand their taxonomy, prevalence on decapod hosts, and phylogenetic relationships among major rhizocephalan taxa.

Phylogeography of Mytilisepta virgata (Mytilidae: Bivalvia) in the northwestern Pacific: Cryptic mitochondrial lineages and mito-nuclear discordance.

The purplish bifurcate mussel Mytilisepta virgata is widely distributed and represents one of the major components of the intertidal community in the northwestern Pacific (NWP). Here, we characterized population genetic structure of NWP populations throughout nearly their whole distribution range using both mitochondrial (mtDNA cox1) and nuclear (ITS1) markers. Population genetic analyses for mtDNA cox 1 sequences revealed two monophyletic lineages (i.e., southern and northern lineages) geographically distributed according to the two different surface water temperature zones in the NWP. The timing of the lineage split is estimated at the Pliocene- mid-Pleistocene (5.49-1.61 Mya), which is consistent with the timing of the historical isolation of the East Sea/Sea of Japan from the South and East China Seas due to sea level decline during glacial cycles. Historical sea level fluctuation during the Pliocene-Pleistocene and subsequent adaptation of mussels to different surface water temperature zones may have contributed to shaping the contemporary genetic diversity and deep divergence of the two mitochondrial lineages. In contrast to mtDNA sequences, a clear lineage split between the two mitochondrial lineages was not found in ITS1 sequences, which showed a star-like structure composed of a mixture of southern and northern mitochondrial lineages. Possible reasons for this type of mito-nuclear discordance include stochastic divergence in the coalescent processes of the two molecular markers, or balancing selection under different marine environments. Cryptic speciation cannot be ruled out from these results, and future work using genomic analyses is required to address whether the thermal physiology of these mussels corresponds to the deep divergence of their mitochondrial genes and to test for the existence of morphologically indistinguishable but genetically separate cryptic species.

Life-history features and oceanography drive phylogeographic patterns of the chiton Acanthochitona cf. rubrolineata (Lischke, 1873) in the northwestern Pacific.

Chitons are a group of marine mollusks (class Polyplacophora) characterized by having eight articulating shell plates on their dorsal body surface. They represent suitable materials for studying the spatiotemporal processes that underlie population differentiation and speciation in ocean environments. Here we performed population genetic analyses on the northwestern Pacific chiton Acanthochitona cf. rubrolineata (Lischke, 1873) using two mitochondrial gene fragments (COI and 16S) from 180 individuals sampled from 11 populations among the coastal waters of Korea, Japan, and China. The phylogenetic network uncovered a reticulated relationship with several sub-haplogroups for all A. cf. rubrolineata haplotypes. SAMOVA analyses suggested the best grouping occurred at three groups (ΦCT = 0.151, P < 0.0001), which geographically corresponds to hydrographic discontinuity among the coastal regions of Korea, Japan, and China. The assumed limited dispersal ability of A. cf. rubrolineata, coupled with northeasterly flowing, trifurcate warm currents, might have contributed to the genetic differentiation among the three groups. Meanwhile, a high level of within-group genetic homogeneity was detected, indicating extensive coastal currents might facilitate gene flow among the populations within each group. Bayesian skyline plots demonstrated significant population expansion after the Last Glacial Period (110-25 thousand years ago) for all studied populations except the Japan group. Together these results suggest that the present-day phylogeographic patterns of A. cf. rubrolineata are strongly affected by the interplay of historical and/or contemporary oceanography and species-specific life-history features.

The mitochondrial genome of Acrobeloides varius (Cephalobomorpha) confirms non-monophyly of Tylenchina (Nematoda).

The infraorder Cephalobomorpha is a diverse and ecologically important nematode group found in almost all terrestrial environments. In a recent nematode classification system based on SSU rDNA, Cephalobomorpha was classified within the suborder Tylenchina with Panagrolaimomorpha, Tylenchomorpha and Drilonematomorpha. However, phylogenetic relationships among species within Tylenchina are not always consistent, and the phylogenetic position of Cephalobomorpha is still uncertain. In this study, in order to examine phylogenetic relationships of Cephalobomorpha with other nematode groups, we determined the complete mitochondrial genome sequence of Acrobeloides varius, the first sequenced representative of Cephalobomorpha, and used this sequence for phylogenetic analyses along with 101 other nematode species. Phylogenetic analyses using amino acid and nucleotide sequence data of 12 protein-coding genes strongly support a sister relationship between the two cephalobomorpha species A. varius and Acrobeles complexus (represented by a partial mt genome sequence). In this mitochondrial genome phylogeny, Cephalobomorpha was sister to all chromadorean species (excluding Plectus acuminatus of Plectida) and separated from Panagrolaimomorpha and Tylenchomorpha, rendering Tylenchina non-monophyletic. Mitochondrial gene order among Tylenchina species is not conserved, and gene clusters shared between A. varius and A. complexus are very limited. Results from phylogenetic analysis and gene order comparison confirms Tylenchina is not monophyletic. To better understand phylogenetic relationships among Tylenchina members, additional mitochondrial genome information is needed from underrepresented taxa representing Panagrolaimomorpha and Cephalobomorpha.

A mitochondrial genome phylogeny of Mytilidae (Bivalvia: Mytilida).

The family Mytilidae is a family of bivalve mussels that are distributed worldwide in diverse marine habitats. Within the family, classification systems and phylogenetic relationships among subfamilies remain not yet fully resolved. In this study, we newly determined 9 mitochondrial genome sequences from 7 subfamilies: Bathymodiolus thermophilus (Bathymodiolinae), Modiolus nipponicus (Modiolinae), Lithophaga curta (the first representative of Lithophaginae), Brachidontes mutabilis (Brachidontinae), Mytilisepta virgata (Brachidontinae), Mytilisepta keenae (Brachidontinae), Crenomytilus grayanus (Mytilinae), Gregariella coralliophaga (Crenellinae), and Septifer bilocularis (the first representative of Septiferinae). Phylogenetic trees using maximum likelihood and Bayesian inference methods for 28 mitochondrial genomes (including 19 previously published sequences) showed two major clades with high support values: Clade 1 ((Bathymodiolinae + Modiolinae) + (Lithophaginae + Limnoperninae)) and Clade 2 (((Mytilinae + Crenellinae) + Septiferinae) + Brachidontinae). The position of the genus Lithophaga (representing Lithophaginae) differed from a previously published molecular phylogeny. Divergence time analysis with a molecular clock indicated that lineage splitting among the major subfamilies of Mytilidae (including the habitat transition from marine to freshwater environments by ancestral Limnoperninae) occurred in the Mesozoic period, coinciding with high diversification rates of marine fauna during that time. This is the first mitochondrial genome-based phylogenetic study of the Mytilidae that covers nearly all subfamily members, excluding the subfamily Dacrydiinae.

Impacts of Salt Stress on Locomotor and Transcriptomic Responses in the Intertidal Gastropod Batillaria attramentaria.

Salinity is one of the most crucial environmental factors that structures biogeographic boundaries of aquatic organisms, affecting distribution, abundance, and behavior. However, the association between behavior and gene regulation underlying acclimation to changes in salinity remains poorly understood. In this study, we investigated the effects of salinity stress on behavior (movement distance) and patterns of gene expression (using RNA sequencing) of the intertidal gastropod Batillaria attramentaria. We examined responses to short-term (1-hour) and long-term (30-day) acclimation to a range of salinities (43, 33 [control], 23, 13, and 3 psu). We found that the intertidal B. attramentaria is able to tolerate a broad range of salinity from 13 to 43 psu but not the acute low salinity of 3 psu. Behavioral experiments showed that salt stress significantly influenced snails' movement, with lower salinity resulting in shorter movement distance. Transcriptomic analyses revealed critical metabolic pathways and genes potentially involved in acclimation to salinity stress, including ionic and osmotic regulation, signal and hormonal transduction pathways, water exchange, cell protection, and gene regulation or epigenetic modification. In general, our study presents a robust, integrative laboratory-based approach to investigate the effects of salt stress on a nonmodel gastropod facing detrimental consequences of environmental change. The current genetic results provide a wealth of reference data for further research on mechanisms of ionic and osmotic regulation and adaptive evolution of this coastal gastropod.

Evidence that alternative transcriptional initiation is largely nonadaptive.

Alternative transcriptional initiation (ATI) refers to the frequent observation that one gene has multiple transcription start sites (TSSs). Although this phenomenon is thought to be adaptive, the specific advantage is rarely known. Here, we propose that each gene has one optimal TSS and that ATI arises primarily from imprecise transcriptional initiation that could be deleterious. This error hypothesis predicts that (i) the TSS diversity of a gene reduces with its expression level; (ii) the fractional use of the major TSS increases, but that of each minor TSS decreases, with the gene expression level; and (iii) cis-elements for major TSSs are selectively constrained, while those for minor TSSs are not. By contrast, the adaptive hypothesis does not make these predictions a priori. Our analysis of human and mouse transcriptomes confirms each of the three predictions. These and other findings strongly suggest that ATI predominantly results from molecular errors, requiring a major revision of our understanding of the precision and regulation of transcription.

Phylogeography of the highly invasive sugar beet nematode, Heterodera schachtii (Schmidt, 1871), based on microsatellites.

Plant-parasitic nematodes (PPNs) threaten crop production worldwide. Yet few studies have examined their intraspecific genetic diversity or patterns of invasion, critical data for managing the spread of these cryptic pests. The sugar beet nematode Heterodera schachtii, a global invader that parasitizes over 200 plant species, represents a model for addressing important questions about the invasion genetics of PPNs. Here, a phylogeographic study using 15 microsatellite markers was conducted on 231 H. schachtii individuals sampled from four continents, and invasion history was reconstructed through an approximate Bayesian computation approach, with emphasis on the origin of newly discovered populations in Korea. Multiple analyses confirmed the existence of cryptic lineages within this species, with the Korean populations comprising one group (group 1) and the populations from Europe, Australia, North America, and western Asia comprising another (group 2). No multilocus genotypes were shared between the two groups, and large genetic distance was inferred between them. Population subdivision was also revealed among the populations of group 2 in both population comparison and STRUCTURE analyses, mostly due to different divergent times between invasive and source populations. The Korean populations showed substantial genetic homogeneity and likely originated from a single invasion event. However, none of the other studied populations were implicated as the source. Further studies with additional populations are needed to better describe the distribution of the potential source population for the East Asian lineage.

A revision of the genus Littorina (Mollusca: Gastropoda) in Korea.

Littorina Férussac, 1822 is an abundant genus of small gastropods found in the upper littoral zone of rocky seashores worldwide. Although ecologically important, shell-based species identification in this genus is challenging due to phenotypic variation in shell morphology and lack of diagnostic characters among morphologically similar species. In this study, we revised the taxonomy of Korean Littorina species using morphological characters (shell and radula) and cox1 mitochondrial DNA sequences for three Korean species: L. brevicula, L. sitkana, and L. horikawai. Results suggest that L. sitkana was erroneously reported as L. kasatka in a previous study. A new record for Littorina horikawai (Matsubayashi & Habe in Habe, 1979), previously unknown from Korea, is described, which can be distinguished from L. sitkana by the presence of alternating white and brown spiral ribs on each whorl. Comparison of the mtDNA cox1 gene sequences shows very low intraspecific variation even between geographically distant populations. A phylogenetic tree supports a close relationship between L. horikawai and L. sitkana, consistent with earlier phylogenetic studies.

The bipartite mitochondrial genome of Ruizia karukerae (Rhigonematomorpha, Nematoda).

Mitochondrial genes and whole mitochondrial genome sequences are widely used as molecular markers in studying population genetics and resolving both deep and shallow nodes in phylogenetics. In animals the mitochondrial genome is generally composed of a single chromosome, but mystifying exceptions sometimes occur. We determined the complete mitochondrial genome of the millipede-parasitic nematode Ruizia karukerae and found its mitochondrial genome consists of two circular chromosomes, which is highly unusual in bilateral animals. Chromosome I is 7,659 bp and includes six protein-coding genes, two rRNA genes and nine tRNA genes. Chromosome II comprises 7,647 bp, with seven protein-coding genes and 16 tRNA genes. Interestingly, both chromosomes share a 1,010 bp sequence containing duplicate copies of cox2 and three tRNA genes (trnD, trnG and trnH), and the nucleotide sequences between the duplicated homologous gene copies are nearly identical, suggesting a possible recent genesis for this bipartite mitochondrial genome. Given that little is known about the formation, maintenance or evolution of abnormal mitochondrial genome structures, R. karukerae mtDNA may provide an important early glimpse into this process.

Population Genomic Analysis Reveals Contrasting Demographic Changes of Two Closely Related Dolphin Species in the Last Glacial.

Population genomic data can be used to infer historical effective population sizes (Ne), which help study the impact of past climate changes on biodiversity. Previous genome sequencing of one individual of the common bottlenose dolphin Tursiops truncatus revealed an unusual, sharp rise in Ne during the last glacial, raising questions about the reliability, generality, underlying cause, and biological implication of this finding. Here we first verify this result by additional sampling of T. truncatus. We then sequence and analyze the genomes of its close relative, the Indo-Pacific bottlenose dolphin T. aduncus. The two species exhibit contrasting demographic changes in the last glacial, likely through actual changes in population size and/or alterations in the level of gene flow among populations. Our findings suggest that even closely related species can have drastically different responses to climatic changes, making predicting the fate of individual species in the ongoing global warming a serious challenge.

The developmental transcriptome atlas of the spoon worm Urechis unicinctus (Echiurida: Annelida).

Background: Echiurida is one of the most intriguing major subgroups of annelida because, unlike most other annelids, echiurids lack metameric body segmentation as adults. For this reason, transcriptome analyses from various developmental stages of echiurid species can be of substantial value for understanding precise expression levels and the complex regulatory networks during early and larval development. Results: A total of 914 million raw RNA-Seq reads were produced from 14 developmental stages of Urechis unicinctus and were de novo assembled into contigs spanning 63,928,225 bp with an N50 length of 2700 bp. The resulting comprehensive transcriptome database of the early developmental stages of U. unicinctus consists of 20,305 representative functional protein-coding transcripts. Approximately 66% of unigenes were assigned to superphylum-level taxa, including Lophotrochozoa (40%). The completeness of the transcriptome assembly was assessed using benchmarking universal single-copy orthologs; 75.7% of the single-copy orthologs were presented in our transcriptome database. We observed 3 distinct patterns of global transcriptome profiles from 14 developmental stages and identified 12,705 genes that showed dynamic regulation patterns during the differentiation and maturation of U. unicinctus cells. Conclusions: We present the first large-scale developmental transcriptome dataset of U. unicinctus and provide a general overview of the dynamics of global gene expression changes during its early developmental stages. The analysis of time-course gene expression data is a first step toward understanding the complex developmental gene regulatory networks in U. unicinctus and will furnish a valuable resource for analyzing the functions of gene repertoires in various developmental phases.

Description of Pseudacrobeles (Pseudacrobeles) curvatus sp. n. (Cephalobidae: Rhabditida) in South Korea.

Pseudacrobeles (Pseudacrobeles) curvatus sp. n. was collected from potato fields in Gyeongsangnam-do, South Korea. The new species shares morphological characters typical of the genus Pseudacrobeles, including three lateral incisures that fade posteriorly near the phasmid openings. The new species differs from other Pseudacrobeles species by its smaller body size and a comparatively shorter corpus relative to the isthmus length. In this study, we provide a comparison of morphometrics and diagnostic features of Pseudacrobeles species and molecular sequence data from the D2-D3 regions of the 28S ribosomal DNA (rDNA) and ITS1-5.8S-ITS2 region of rDNA from the new species, which can be used as molecular barcode sequences.

More than meets the eye: The barrier effect of the Yangtze River outflow.

The Yangtze (Changjiang) River enters the East China Sea with huge annual freshwater and sediment deposits. This outflow, known as the Changjiang diluted water (CDW), causes striking ecological gradients that potentially shape coastal species' distribution and differentiation. The CDW has long been rendered as a marine biogeographic boundary separating cold-temperature and warm-water faunas, but it remains unclear whether and to what extent it acts as an intraspecific barrier. Here, we synthesize published phylogeographic studies related to the CDW to address these issues. We find that the influence of the CDW on population differentiation is taxonomically variable, and even congeneric species may respond differently. In studies that claim the CDW is a phylogeographic barrier, the underlying assumptions explaining observed genetic breaks are sometimes incorrect, and some may have misinterpreted results due to conceptual confusion or insufficient geographic sampling. After excluding these studies, the remaining ones generally show shallow genetic divergence but significant population structure for coastal species across the CDW, suggesting that the CDW has not been a historically persistent barrier, but rather has acted as a filter within some species recently, probably after the last glacial maximum.

Draft genome of the sea cucumber Apostichopus japonicus and genetic polymorphism among color variants.

The Japanese sea cucumber (Apostichopus japonicus Selenka 1867) is an economically important species as a source of seafood and ingredient in traditional medicine. It is mainly found off the coasts of northeast Asia. Recently, substantial exploitation and widespread biotic diseases in A. japonicus have generated increasing conservation concern. However, the genomic knowledge base and resources available for researchers to use in managing this natural resource and to establish genetically based breeding systems for sea cucumber aquaculture are still in a nascent stage. A total of 312 Gb of raw sequences were generated using the Illumina HiSeq 2000 platform and assembled to a final size of 0.66 Gb, which is about 80.5% of the estimated genome size (0.82 Gb). We observed nucleotide-level heterozygosity within the assembled genome to be 0.986%. The resulting draft genome assembly comprising 132 607 scaffolds with an N50 value of 10.5 kb contains a total of 21 771 predicted protein-coding genes. We identified 6.6-14.5 million heterozygous single nucleotide polymorphisms in the assembled genome of the three natural color variants (green, red, and black), resulting in an estimated nucleotide diversity of 0.00146. We report the first draft genome of A. japonicus and provide a general overview of the genetic variation in the three major color variants of A. japonicus. These data will help provide a comprehensive view of the genetic, physiological, and evolutionary relationships among color variants in A. japonicus, and will be invaluable resources for sea cucumber genomic research.

Phylogenetic analysis of two Plectus mitochondrial genomes (Nematoda: Plectida) supports a sister group relationship between Plectida and Rhabditida within Chromadorea.

Plectida is an important nematode order with species that occupy many different biological niches. The order includes free-living aquatic and soil-dwelling species, but its phylogenetic position has remained uncertain. We sequenced the complete mitochondrial genomes of two members of this order, Plectus acuminatus and Plectus aquatilis and compared them with those of other major nematode clades. The genome size and base composition of these species are similar to other nematodes; 14,831 and 14,372bp, respectively, with AT contents of 71.0% and 70.1%. Gene content was also similar to other nematodes, but gene order and coding direction of Plectus mtDNAs were dissimilar from other chromadorean species. P. acuminatus and P. aquatilis are the first chromadorean species found to contain a gene inversion. We reconstructed mitochondrial genome phylogenetic trees using nucleotide and amino acid datasets from 87 nematodes that represent major nematode clades, including the Plectus sequences. Trees from phylogenetic analyses using maximum likelihood and Bayesian methods depicted Plectida as the sister group to other sequenced chromadorean nematodes. This finding is consistent with several phylogenetic results based on SSU rDNA, but disagrees with a classification based on morphology. Mitogenomes representing other basal chromadorean groups (Araeolaimida, Monhysterida, Desmodorida, Chromadorida) are needed to confirm their phylogenetic relationships.