The brachyuran crab Bythograeidae Williams, 1980 is common in hydrothermal vent fields worldwide and has recorded to sixteen species of six genera (Guinot and Segonzac, 2018). For DNA-based species identification, it is generally considered a suitable DNA barcode when sequence variation among species is 10 times greater than those within species (Hebert et al., 2003; Matzen da Silva et al., 2011). Based on these criteria, the mitochondrial cytochrome c oxidase subunit 1 (CO1) DNA barcodes for bythograeid crabs were confirmed worth supporting the traditional taxonomy and utility recognizing the existence of sibling (or cryptic) species (Mateos et al., 2012; Kim et al., 2013; Guinot and Segonzac, 2018).
Austinograea hourdezi Guinot & Segonzac, 2018 is found across wide hydrothermal vent areas in the southwestern Pacific Ocean (Guinot, 1989; Guinot and Segonzac, 2018)(Fig. 1A). It is distinguished from its relatives with spots on outer surface of chelae in males, patches of setae on inner surface of chelae, subhepatic region and meri, and ratio between the 1st and 2nd pair of gonopods (Guinot and Segonzac, 2018) (Fig. 1B-D). In this study, we determined firstly the CO1 DNA barcodes of A. hourdezi collected from the hydrothermal vents of the North Fiji Basin in southwestern Pacific Ocean. Additionally, we obtained mitochondrial 16S rDNA sequences to compare the 16S rDNA sequence of A. hourdezi retrieved from GenBank.
RESULTS AND DISCUSSION
In November 2016, six A. hourdezi individuals were collected from three hydrothermal vent regions (1 ind. from 18°49ʹS, 173°30ʹE, 2,719 m depth; 3 inds. from 18°51ʹS, 173°30ʹE, 2,721 m depth; 2 inds. from 19°03ʹS, 173°29ʹE, 2,717 m depth) (Fig. 1A) in the North Fiji Basin. Morphological identification, genomic DNA extraction, sequencing, sequence annotation, and nucleotide diversity analyses followed the methods of Guinot and Segonzac (2018) and Kim et al.(2013). All specimens and genomic DNA extracts of A. hourdezi are deposited in Korea Research Institute of Bioscience and Biotechnology.
Fig. 1. Photograph of the hydrothermal vent chimney in the North Fiji Basin of the Southwestern Pacific Ocean where Austinograea hourdezi Guinot & Segonzac, 2018 was collected. A, Overview of sampling site and the surrounding community of species including bythograeid crabs Austinograea spp., alvinocaridid shrimp species, provannid snails Alviniconcha boucheti Johnson et al., 2015 and A. kojimai Johnson et al., 2015, mytilid mussel Bathymodiolus sp., chiridotid sea cucumber Chiridota sp.; B-D, Diagnostic keys of A. hourdezi based on Guinot and Segonzac (2018). Arrowheads indicate patches of setae on inner surface of chelae (B), spots on outer surface of chelae in males (C), and patches of setae on subhepatic region and meri (D). Scale bar=20 cm.
The newly obtained sequences for A. hourdezi were registered to GenBank (accession No. MK382751-MK382753, MK359095-MK359097 for 16S rDNA; MK359098-MK 359103 for CO1) (Table 1). The lengths of alignments were 446 bp for 16S rDNA and 503 bp for CO1. All CO1 sequences were identical, while 16S rDNA sequences detected a variable nucleotide site in the single individual(MK382752). Interspecies variations of 16S rDNA and CO1 within the genus Austinograea were 4.5-7.4% and 10.3-13.3%, respectively. In the case of the genus Bythograea, interspecies variations were 0.7-9.2% for 16S rDNA and 6.6-15.7% for CO1. Two Gandalfus species showed the variations of 5.2% for 16S rDNA and 9.7% for CO1. The sequence diversity of CO1 was two times more than that of 16S rDNA. Meanwhile, the sequence variation of 16S rDNA between B. laubieri and B. vrijenhoeki was only 0.7%, which is considered to represent the most recently derived lineages or cryptic species.
Table 1. Sequence divergence among the partial sequences of mitochondrial 16S rDNA and cytochrome c oxidase subunit 1 (CO1) genes from Bythograeidae. Nucleotide sequence variations (%) were calculated using the p-distance method in Mega 10.0.5. Upper right is for 16S and lower left for CO1. The bold letters indicate interspecies variation within the genera Austinograea, Bythograea and Gandalfus
Recently, A. aff. williamsi collected in the southwestern Pacific Ocean was revised as the fifth species of Austinograea, A. hourdezi, by Guinot and Segonzac (2018). As of 8 Jan. 2019, GenBank contains the sequences of mitochondrial 16S rDNA and cytochrome b, and nuclear 28S rDNA from one single individual of A. aff. williamsi (JQ407451, JQ407421, and JQ407435), but no sequence for A. hourdezi. These results would be helpful to understand taxonomy of brachyuran crabs living in hydrothermal vent fields using CO1 DNA barcodes.
This work was a part of the R & D project  titled “Exploration for Seafloor Hydrothermal Deposits and FeMn Crusts in the Southwestern Pacific and the Indian Ocean”, funded by the Ministry of Oceans and Fisheries, Korea and the Korea Research Institute of Bioscience and Biotechnology (KRIBB) Research Initiative Program.