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

Study Extent

Freshwater mussels were collected in the wild of the Kyushu and Ryukyu Islands, Japan.

Sampling

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.

Quality Control

Identification followed Masuda and Uchiyama (2004), Kondo (2008) and Sano et al. (2017).

Method steps

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.

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).

1. Animalia
   common name: Animals rank: kingdom
2. Mollusca
   common name: Molluscs rank: phylum
3. Bivalvia
   common name: Bivalves rank: class
4. Unionoida
   common name: Freshwater mussels and pearl mussels rank: order
5. Unionidae
   common name: Freshwater mussels rank: family
6. Cristaria tenuis
   common name: "Dobu-gai-modoki" rank: species
7. Inversidens brandti
   common name: "Obaeboshi-gai" rank: species
8. Inversiunio yanagawensis
   common name: "Nise-matsukasa-gai" rank: species
9. Lanceolaria grayana
   common name: "Tongari-sasanoha-gai" rank: species
10. Nodularia douglasiae nipponensis
    common name: "Ishi-gai" rank: subspecies
11. Obovalis omiensis
    common name: "Kataha-gai" rank: species
12. Pronodularia japanensis
    common name: "Matsukasa-gai" rank: species
13. Sinanodonta lauta
    common name: "Numa-gai" rank: species
14. Sinanodonta japonica
    common name: "Ta-gai" rank: species

Freshwater habitats of Kyushu and Ryukyu Islands, Japan.