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

Abstract Background Freshwater mussels (Bivalvia: Unionidae), which are keystone species of freshwater ecosystems, are in global decline. In addition to ecological/genetic studies, morphological examinations are needed to help provide information for the development of additional freshwater mussel studies and eventually conservation efforts for freshwater ecosystems. The microscopic structure, which can be obtained by scanning electron microscopy (SEM) and elemental composition, which can be obtained with energy dispersive X-ray spectrometry (EDS), of mollusc shells are of interest to malacologists. However, information about freshwater mussels is still limited. Kyushu Island is the southernmost island of the four major islands of Japan. Kyushu Island is a hotspot of bitterling fishes in Japan, which simultaneously means that the island is a hotspot of freshwater mussels. The Ryukyu Islands stretch southwest from Kyushu Island to Taiwan; a freshwater mussel of unknown origin was reported from the Ryukyu Islands. Digital archiving for biology and ecology is a continuing challenge for open science. This data paper describes online published photo images, 3D/CT and mtDNA data and SEM/EDS analyses of the shell of freshwater mussels that inhabit the Kyushu and Ryukyu Islands, Japan. Our data will provide basic information regarding freshwater biology and be of public interest as open science. New information Photo images, 3D/CT data, mtDNA data, SEM images and EDS elemental analysis of freshwater mussels that inhabit the Kyushu and Ryukyu Islands (61 individuals, nine species/subspecies) were published online in a local database (http://ffish.asia/Unionidae3D), GBIF (http://ipt.pensoft.net/resource?r=unionidae3d) and DDBJ/EMBL/Genbank (LC431810–LC431840).


Introduction
Freshwater mussels (Bivalvia: Unionidae) are one of the most endangered freshwater animals in Japan and around the world. In total, 21 unionid species/subspecies have been reported from Japan, including introduced Hyriopsis cumingii from China (Masuda and Uchiyama 2004, Imai 2008, Kondo 2008, Kitano and Imai 2012, of which 13 species/ subspecies are listed as endangered (NT, VU or CR+EN) by the Red Data Book of the Ministry of the Environment, Japan (2018). Freshwater mussels have an ecological relationship with bitterling fishes, which are also generally threatened. Bitterling fishes lay their eggs in the gills of mussels; the life history/cycle of bitterling fishes is not completed without mussels (e.g. Kitamura et al. 2012). On the other hand, mussel larvae (glochidia) parasitise sympatric fishes, including bitterling fishes (Kondo 2008). Freshwater mussels also provide crucial ecosystem services by contributing to water clarity (e.g. Chowdhury et al. 2016). Thus, freshwater mussels are considered keystone species and more studies are needed to help provide information for conservation plans for freshwater ecosystems.
Kyushu Island is a hotspot for bitterling fishes in Japan (Onikura et al. 2011), which also means that the island is home to populations of freshwater mussels. The Ryukyu Islands stretch southwest from Kyushu Island to Taiwan; a freshwater mussel, Cristaria tenuis, was once reported from Ishigaki Island, one of the major Ryukyu Islands (Imai 2008). Cristaria tenuis was also found in Fukuoka Prefecture, which is in the northern part of Kyushu Island (Kitano and Imai 2012). Further studies are needed to determine whether C. tenuis is native to Ishigaki Island or Fukuoka Prefecture (Imai 2008).
Shell exoskeleton morphology is a crucial characteristic in mollusc studies related to hydrodynamics (e.g. Eagar 1978, Aldridge 1999, Denny and Blanchette 2000, Kondo 2008, whereas evaluation of the soft body has usually been overlooked. In addition to shell morphology, microscopic structure and chemical/elemental composition of the shell, which can be obtained by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS), have been of interest to malacologists (Matsuda and Hirano 1980, Carter 1990, Ubukata 1994, Fuchigami and Sasaki 2005, de Paula and Silveira 2006, Checa et al. 2016. Digital archiving is one of the progressive challenges for the open science of biology and biodiversity studies (e.g. Berquist et al. 2012, Miyazaki et al. 2014, Kano et al. 2016, Kano et al. 2018). This paper describes photo images published online, 3D/CT data (to evaluate both the shell exoskeleton and soft body) and mtDNA data of freshwater mussels that inhabit the Kyushu and Ryukyu Islands of Japan (61 individuals of nine species/ subspecies) with SEM images and EDS analysis of the shell. We expect our study will contribute valuable information to general malacology, freshwater ecosystem conservation and public interest in biodiversity conservation.

Sampling methods
Study extent: Freshwater mussels were collected in the wild of Kyushu and Ryukyu Islands, Japan ( Fig. 1).
Step description: Individual photo images were taken in the field (Fig. 2) (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.
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 (Fig. 7). 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 (Fig. 8a), S, Cl, K and Ca (Fig. 8b) (Suppl. material 1).

Figure 6.
A phylogenetic tree of nine Unionidae species/subspecies found in the Kyushu and Ryukyu Islands (mtDNA 16S-rRNA). The tree was reconstructed using the maximum likelihood method (model: GTR+G) in MEGA 7 (Kumar et al. 2016). Bootstrap values were obtained with 500 pseudoreplicates. Margaritifera falcata was used as an outgroup. The DDBJ accession numbers are shown in brackets.
Photo images, 3D/CT data and mtDNA of the freshwater mussels (Bivalvia: ...    The taxonomic rank of the most specific name in the scientificName. Recommended best practice is to use a controlled vocabulary The format the image is exposed in description (multimedia) A textual description of the content of the media item created (multimedia) The date and time this media item was taken creator (multimedia) The person who took the image, recorded the video or sound audience (multimedia) A class or description for whom the image is intended or useful rightsHolder (multimedia) A person or organisation owning or managing rights over the media item

Additional information
Note on the origin of Cristaria tenuis We cannot conclude whether Cristaria tenuis is native or introduced based on our results (Fig. 6). However, it is notable that the sequences were all the same amongst the six samples from the two distant locales; C. tenuis might have been introduced in either Ishigaki Island or Fukuoka Prefecture.

"Metal artefacts" in CT data of thick-shell individuals
Several individuals, especially Inversiunio yanagawensis individuals, had significantly thicker shells; streak-like artefacts were generated at the surface of the shell as noise and were considered "metal artefacts" (e.g. Fig. 3a).