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Photo images, 3D/CT data and mtDNA of the freshwater mussels (Bivalvia: Unionidae) in the Kyushu and Ryukyu Islands, Japan, with SEM/EDS analysis of the shell

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The publisher and rights holder of this work is Biodiversity Data Journal. This work is licensed under a Creative Commons Attribution Non Commercial (CC-BY-NC) 4.0 License.

This resource has been registered with GBIF, and assigned the following GBIF UUID: 6a6ba4bc-194e-47e0-a5d0-00d83b57109d.  Biodiversity Data Journal publishes this resource, and is itself registered in GBIF as a data publisher endorsed by Participant Node Managers Committee.

3D model; Anatomy; CT scan; Digital archiving; Elemental composition; Energy dispersive X-ray spectrometry (EDS); Freshwater mussels; Morphology; Open science; Scanning electron microscope (SEM); Shell exoskeleton;; Specimen

Freshwater habitats of Kyushu and Ryukyu Islands, Japan.

All freshwater mussels (family Unionidae) distributed in the Kyushu and Ryukyu Islands were studied except Anemina arcaeformis, Hyriopsis schlegelii, and Sinanodonta sp. A shell exoskeleton that was photographically identified as A. arcaeformis was reported from Miyazaki Prefecture, Kyushu Island (Toayama and Nishi 2016), although all other details remain unclear. Hyriopsis schlegelii individuals were artificially introduced to Isahaya Bay for water purification (Ishizaki et al. 2007), even though H. schlegelii is non-native to Kyushu Island. Sinanodonta sp. (or spp.) populations were informally reported from the Ryukyu Islands, but some of the populations were likely introduced from Taiwan (Shokita 1984, Kurozumi 2003, Imai 2008).

Sampling sites of the nine freshwater mussel species in the Kyushu and Ryukyu Islands. The number attached to each circle indicates the number of individuals.

Method step description:

1. Individual photo images were taken in the field (Kano and Nakajima 2014). The specimens were fixed in 10% formalin followed by preservation in 70% ethanol. A small segment of the soft body was cut off and separately preserved in 99% ethanol for mtDNA analysis. All specimens were CT scanned (Aloka Latheta LCT-200, Hitachi Ltd., Japan), and 3D surface models (CT value: −450 to 600) were extracted from the CT data. mtDNA analysis of 16S-rRNA was conducted for 31 individuals. For PCR amplification, we used the primer pair Unio16SFwd (forward: 5′-TGCCTGTTTACCAAAAACATCG-3′) and Unio16SRev (reverse: 5′-CTTGGGGTCCTTTCGTACA-3′). PCR amplification was performed in 10-µL reaction mixtures that contained 5 µL KAPA 2G™ Robust HotStart ReadyMix (Kapa Biosystems, USA), 1 µM of each primer, 1 µL DNA template, and 2 µL sterile deionised water. The reaction mixtures were preheated at 95°C for 3 min, followed by 30 amplification cycles (95°C for 15 s, 50°C for 15 s, and 72°C for 40 s) with a final 5-min extension at 72°C. Direct sequencing of the PCR products was conducted externally (FASMAC, Japan). The nucleotide sequences were deposited in DDBJ/EMBL/GenBank (accession numbers: LC431810–LC431840). The SEM/EDS analysis was conducted for 29 individuals. A shell fragment was cut off from each specimen (from the posterior part of the shell), and the inner side of the shell was analysed by SEM (JCM-6000, JEOL Ltd., Japan) to observe the microscopic images of the pearled surface. Furthermore, EDS analysis (JED-2300, JEOL Ltd.) was conducted to determine the elemental composition of the shell fragment by targeting B, C, N, O, F, Na, Mg, Al, Si, P, S, Cl, K and Ca.