Development of two new sets of PCR primers for eDNA metabarcoding of brittle stars (Echinodermata, Ophiuroidea)

汲んだ水から深海生物の種類を判別 --世界初「クモヒトデメタバーコーティング」技術を開発--. 京都大学プレスリリース.Brittle stars (class Ophiuroidea) are marine invertebrates comprising approximately 2, 100 extant species, and are considered to constitute the most diverse taxon of the phylum Echinodermata. As a non-invasive method for monitoring biodiversity, we developed two new sets of PCR primers for metabarcoding environmental DNA (eDNA) from brittle stars. The new primer sets were designed to amplify 2 short regions of the mitochondrial 16S rRNA gene, comprising a conserved region (111–115 bp, 112 bp on average; named “16SOph1”) and a hyper-variable region (180–195 bp, 185 bp on average; named “16SOph2”) displaying interspecific variation. The performance of the primers was tested using eDNA obtained from two sources: a) rearing water of an 2.5 or 170 L aquarium tanks containing 15 brittle star species and b) from natural seawater collected around Misaki, the Pacific coast of central Japan, at depths ranging from shallow (2 m) to deep (> 200 m) sea. To build a reference library, we obtained 16S rRNA sequences of brittle star specimens collected from around Misaki and from similar depths in Japan, and sequences registered in International Nucleotide Sequence Database Collaboration. As a result of comparison of the obtained eDNA sequences with the reference library 37 (including cryptic species) and 26 brittle star species were detected with certain identities by 16SOph1 and 16SOph2 analyses, respectively. In shallow water, the number of species and reads other than the brittle stars detected with 16SOph1 was less than 10% of the total number. On the other hand, the number of brittle star species and reads detected with 16SOph2 was less than half of the total number, and the number of detected non-brittle star metazoan species ranged from 20 to 46 species across 6 to 8 phyla (only the reads at the “Tank” were less than 0.001%). The number of non-brittle star species and reads at 80 m was less than 10% with both of the primer sets. These findings suggest that 16SOph1 is specific to the brittle star and 16SOph2 is suitable for a variety of marine metazoans. It appears, however, that further optimization of primer sequences would still be necessary to avoid possible PCR dropouts from eDNA extracts. Moreover, a detailed elucidation of the brittle star fauna in the examined area, and the accurate identification of brittle star species in the current DNA databank is required

A new species of Xenoturbella from the western Pacific Ocean and the evolution of Xenoturbella

BackgroundXenoturbella is a group of marine benthic animals lacking an anus and a centralized nervous system. Molecular phylogenetic analyses group the animal together with the Acoelomorpha, forming the Xenacoelomorpha. This group has been suggested to be either a sister group to the Nephrozoa or a deuterostome, and therefore it may provide important insights into origins of bilaterian traits such as an anus, the nephron, feeding larvae and centralized nervous systems. However, only five Xenoturbella species have been reported and the evolutionary history of xenoturbellids and Xenacoelomorpha remains obscure.ResultsHere we describe a new Xenoturbella species from the western Pacific Ocean, and report a new xenoturbellid structure - the frontal pore. Non-destructive microCT was used to investigate the internal morphology of this soft-bodied animal. This revealed the presence of a frontal pore that is continuous with the ventral glandular network and which exhibits similarities with the frontal organ in acoelomorphs.ConclusionsOur results suggest that large size, oval mouth, frontal pore and ventral glandular network may be ancestral features for Xenoturbella. Further studies will clarify the evolutionary relationship of the frontal pore and ventral glandular network of xenoturbellids and the acoelomorph frontal organ. One of the habitats of the newly identified species is easily accessible from a marine station and so this species promises to be valuable for research on bilaterian and deuterostome evolution

Molecular Phylogeny of the Genus Nipponnemertes (Nemertea: Monostilifera: Cratenemertidae) and Descriptions of 10 New Species, With Notes on Small Body Size in a Newly Discovered Clade

Nemerteans, or ribbon worms, have been reported from intertidal to hadal depths, often showing bathymetrically wide distribution in genus levels. Although current nemertean systematics practices require to provide DNA sequences and infer phylogenetic relationships with suitable molecular markers, previous molecular systematics on nemerteans are mostly biased toward shallow-water species. Members in the genus Nipponnemertes occur worldwide, from tropical to polar waters and intertidal to bathyal waters. Molecular phylogenetic studies are scarce for the genus; only six shallow-water species of 18 species in the genus were subject to molecular phylogeny. Thus, Nipponnemertes is one candidate that needs to be assessed by genetic approaches. In this study, we performed molecular phylogenetic analyses using 59 specimens in 23 species based on partial sequences of two mitochondrial (16S rRNA and cytochrome c oxidase subunit I) and three nuclear gene markers (18S rRNA, 28S rRNA, and histone H3). Our extensive sampling from intertidal to bathyal waters in the Northwest Pacific significantly updated the fauna of Nipponnemertes in this region from four to 17 species. We herein establish 10 new species and provide an updated species list concisely summarizing all the congeners known from the world. Our phylogenetic tree indicated three major lineages within the genus (herein referred to as “Clade A, B, and C”), each presumably characterized by the combination of morphological characters in the head region. Members in Clade A are: Nipponnemertes pulchra (Johnston, 1837), Nipponnemertes ogumai (Yamaoka, 1947), and several unidentified congeners, characterized by having demarcated head without cephalic patches; members in Clade B are: Nipponnemertes crypta sp. nov., Nipponnemertes jambio sp. nov., Nipponnemertes neonilae sp. nov., and Nipponnemertes ojimaorum sp. nov., species having demarcated head with cephalic patches; members in Clade C are: Nipponnemertes ganahai sp. nov., Nipponnemertes kozaensis sp. nov., Nipponnemertes lactea sp. nov., Nipponnemertes notoensis sp. nov., Nipponnemertes ornata sp. nov., Nipponnemertes sugashimaensis sp. nov., and two unidentified forms collected off Jogashima (Japan) and Guam (USA), species with non-demarcated head lacking cephalic patches. Furthermore, we discuss the evolution of remarkably small body size retained among Clade C

Dendrochirotid holothurians (Echinodermata: Holothuroidea: Dendrochirotida) including four new species, from off Misaki, Japan

Yamana, Yusuke, Kohtsuka, Hisanori (2018): Dendrochirotid holothurians (Echinodermata: Holothuroidea: Dendrochirotida) including four new species, from off Misaki, Japan. Zootaxa 4455 (3): 429-453, DOI: https://doi.org/10.11646/zootaxa.4455.3.

FIGURE 4 in Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda)

FIGURE 4. Aora biarticulata sp. nov. All except for F, SM and JM: holotype, male, 6.3 mm (OMNH-Ar-12429); F: paratype, female, 6.2 mm (OMNH-Ar-12433); SM: paratype, small male, 5.1 mm (OMNH-Ar-12430), slender setae omitted; JM: paratype, juvenile male, 3.6 mm (OMNH-Ar-12432), slender setae omitted. Scales: 0.1 mm.Published as part of Ariyama, Hiroyuki & Kohtsuka, Hisanori, 2022, Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda), pp. 393-413 in Zootaxa 5159 (3) on page 399, DOI: 10.11646/zootaxa.5159.3.5, http://zenodo.org/record/678179

Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda)

Ariyama, Hiroyuki, Kohtsuka, Hisanori (2022): Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda). Zootaxa 5159 (3): 393-413, DOI: https://doi.org/10.11646/zootaxa.5159.3.

FIGURE 4 in Metarhachotropis parva, a new genus and species of Eusiridae (Crustacea: Amphipoda) from Sagami Bay, central Japan

FIGURE 4. Metarhachotropis parva sp. nov. Holotype, male, 2.1 mm (OMNH-Ar-12515). Inner ramus of pleopod 2 lost. Scales: 0.1 mm.Published as part of Ariyama, Hiroyuki & Kohtsuka, Hisanori, 2022, Metarhachotropis parva, a new genus and species of Eusiridae (Crustacea: Amphipoda) from Sagami Bay, central Japan, pp. 95-100 in Zootaxa 5188 (1) on page 99, DOI: 10.11646/zootaxa.5188.1.6, http://zenodo.org/record/708744

Metarhachotropis parva, a new genus and species of Eusiridae (Crustacea: Amphipoda) from Sagami Bay, central Japan

Ariyama, Hiroyuki, Kohtsuka, Hisanori (2022): Metarhachotropis parva, a new genus and species of Eusiridae (Crustacea: Amphipoda) from Sagami Bay, central Japan. Zootaxa 5188 (1): 95-100, DOI: https://doi.org/10.11646/zootaxa.5188.1.

FIGURE 2 in Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda)

FIGURE 2. Aora biarticulata sp. nov. Holotype, male, 6.3 mm (OMNH-Ar-12429). Scales: 0.05 mm.Published as part of Ariyama, Hiroyuki & Kohtsuka, Hisanori, 2022, Three new species of the family Aoridae collected from Sagami Bay, central Japan (Crustacea: Amphipoda), pp. 393-413 in Zootaxa 5159 (3) on page 397, DOI: 10.11646/zootaxa.5159.3.5, http://zenodo.org/record/678179