Introduction

General description

Project description

Sampling methods

Description: 

Freshwater mussels were collected in the wild of Kyushu and Ryukyu Islands, Japan (Fig. 1).

Figure 1.  

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.

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.

Figure 2.  

A live Nodularia douglasiae nipponensis (QUYK-08668d)

All specimens were CT scanned (Aloka Latheta LCT-200, Hitachi Ltd., Japan) and 3D surface models (Fig. 3; CT value: −450 to 600) were extracted from the CT data (Figs 4, 5).

a

b

c

d

Figure 3.

3D surface models of freshwater mussels.

a: Inversiunio yanagawensis (QUYK-06858) with "metal artifacts" noise due to the thick/hard shell.  
b: Lanceolaria grayana (QUYK-08773d).  
c: Pronodularia japanensis (QUYK-08881).  
d: Nodularia douglasiae nipponensis (QUYK-08667d).  

a

b

Figure 4.

CT digital image of a freshwater mussel (Sinanodonta japonica; QUYK-08891).

a: External shell exoskeleton.  
b: Digitally sliced shell that shows the internal soft body.  

Figure 5.  

A CT-scanned image of Obovalis omiensis (QUYK-08887).

mtDNA analysis of 16S-rRNA was conducted for 31 individuals (Fig. 6). 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).

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.

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).

a

b

c

d

Figure 7.

SEM images of the shell (inner pearled surface).

a: Multilevel wavelike structure of Sinanodonta japonica QUYK-08891 (Scale bar 100 μm).  
b: Multilevel wavelike structure of Obovalis omiensis QUYK-08872 (Scale bar 20 μm).  
c: Hexagonal microstructure of Inversidens brandtii QUYK-08915 (Scale bar 5 μm).  
d: Columnar microstructure of Pronodularia japanensis QUYK-08880 (Scale bar 5 μm).  

a

b

Figure 8.

Elemental compositions of the shell (% number of atoms) discriminated by locality.

a: Phosphorous.  
b: Calcium.  

Geographic coverage

Taxonomic coverage

Traits coverage

Temporal coverage

Collection data

Usage rights

Data resources

Additional information

Acknowledgements

References

Aldridge D (1999)
The morphology, growth and reproduction of Unionidae (Bivalvia) in a fenland aterway
.
Journal of Molluscan Studies
65
(
1
):
47
‑
60
. https://doi.org/10.1093/mollus/65.1.47
Berquist R, Gledhill K, Peterson M, Doan A, Baxter G, Yopak K, Kang N, Walker HJ, Hastings P, Frank L (2012)
The digital fish library: Using MRI to digitize, database, and document the morphological diversity of fish
.
PLOS One
7
(
4
):
e34499
. https://doi.org/10.1371/journal.pone.0034499
Carter JG (Ed.) (1990)
Skeletal biomineralization: patterns, processes and evolutionary trends. Vol. 1.
Van Nostrand Reinhold
,
New York
,
832
pp.
Checa A, Macías-Sánchez E, Harper E, Cartwright JE (2016)
Organic membranes determine the pattern of the columnar prismatic layer of mollusc shells
.
Proceedings of the Royal Society B: Biological Sciences
283
(
1830
):
20160032
. https://doi.org/10.1098/rspb.2016.0032
Chowdhury G, Zieritz A, Aldridge D (2016)
Ecosystem engineering by mussels supports biodiversity and water clarity in a heavily polluted lake in Dhaka, Bangladesh
.
Freshwater Science
35
(
1
):
188
‑
199
. https://doi.org/10.1086/684169
Denny MW, Blanchette CA (2000)
Hydrodynamics, shell shape, behavior and survivorship in the owl limpet Lottia gigantea.
Journal of Experimental Biology
203
:
2623
‑
2639
.
de Paula S, Silveira M (2006)
Microstructural characterization of shell components in the mollusc Physa sp.
Scanning
27
(
3
):
120
‑
125
. https://doi.org/10.1002/sca.4950270303
Eagar RMC (1978)
Shape and function of the shell: a comparison of some living and fossil bivalve molluscs
.
Biological Reviews
53
(
2
):
169
‑
210
. https://doi.org/10.1111/j.1469-185x.1978.tb01436.x
Fuchigami T, Sasaki T (2005)
The shell structure of the recent Patellogastropoda (Mollusca: Gastropoda)
.
Paleontological Research
9
(
2
):
143
‑
168
. https://doi.org/10.2517/prpsj.9.143
Imai H (2008)
The first record of Cristaria discoidea (Lea, 1834) (Bivalvia: Unionidae) from Ishigakijima Island, the Ryukyu Archipelago, Japan
.
The Biological Magazine Okinawa
46
:
65
‑
70
. [In
Japanese with English abstract
].
Ishizaki S, Ura N, Migata U (2007)
Water purification of detention pond in Isahaya-Bay land reclamation using Biwa pealy mussel (H. schlegeli)
.
Annual report of Nagasaki Prefectural Institute for Environmental Research and Public Health
53
:
47
‑
52
. [In
Japanese with English abstract
].
Kano Y, Nakajima J (2014)
Non-killing simple photography techniques of small–middle freshwater fishes in the field
.
Japanese Journal of Ichthyology
61
:
123
‑
125
. [In
Japanese
].
Kano Y, Musikasinthorn P, Iwata A, Tun S, Yun L, Win SS, Matsui S, Tabata R, Yamasaki T, Watanabe K (2016)
A dataset of fishes in and around Inle Lake, an ancient lake of Myanmar, with DNA barcoding, photo images and CT/3D models.
Biodiversity Data Journal
4
:
e10539
. https://doi.org/10.3897/BDJ.4.e10539
Kano Y, Nakajima J, Yamasaki T, Kitamura J, Tabata R (2018)
Photo images, 3D models and CT scanned data of loaches (Botiidae, Cobitidae and Nemacheilidae) of Japan
.
Biodiversity Data Journal
6
:
e26265
. https://doi.org/10.3897/bdj.6.e26265
Kitamura J, Nagata N, Nakajima J, Sota T (2012)
Divergence of ovipositor length and egg shape in a brood parasitic bitterling fish through the use of different mussel hosts
.
Journal of Evolutionary Biology
25
(
3
):
566
‑
573
. https://doi.org/10.1111/j.1420-9101.2011.02453.x
Kitano T, Imai H (2012)
The first record of Cristaria tenuis (Gray in Griffith & Pidgeon, 1833) (Bivalvia: Unionidae) from Fukuoka Prefecture, Kyushu, Japan
.
Bulletin of the Biogeographical Society of Japan
67
:
193
‑
196
. [In
Japanese
].
Kondo T (2008)
Monograph of Unionoida in Japan (Mollusca: Bivalvia). Special Publication of the Malacological Society of Japan No. 3
. [
日本産イシガイ目貝類図譜
].
The Malacological Society of Japan
,
Tokyo
.
Kumar S, Stecher G, Tamura K (2016)
MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets
.
Molecular Biology and Evolution
33
(
7
):
1870
‑
1874
. https://doi.org/10.1093/molbev/msw054
Kurozumi T (2003) Mollusks. In: Nishida M, Shikatani N, Shokita S (Eds)
The Flora and Fauna of Inland Waters in the Ryukyu Islands
.
Tokai University Press
,
Tokyo
,
167-180
pp. [In
Japanese
].
Masuda O, Uchiyama R (2004)
PISCES Ecological Field Guide Series 2: Freshwater Mollusks of Japan
. [
日本産淡水貝類図鑑 ２汽水域を含む全国の淡水貝類
].
PISCES
,
Tokyo
. [In
Japanese
].
Matsuda F, Hirano M (1980)
Chemical composition of some modern marine pelecypod shells
.
Science Reports of the Institute of Geoscience University of Tsukuba Section B Geological Sciences
1
:
163
‑
177
.
Ministry of the Environment, Japan (2018)
Redlist 2018
. https://www.env.go.jp/nature/kisho/hozen/redlist/RL2018_5_180604.pdf. Accessed on: 2018-9-05.
Miyazaki Y, Murase A, Shiina M, Naoe K, Nakashiro R, Honda J, Yamaide J, Senou H (2014)
Biological monitoring by citizens using Web-based photographic databases of fishes
.
Biodiversity and Conservation
23
(
9
):
2383
‑
2391
. https://doi.org/10.1007/s10531-014-0724-4
Onikura N, Nakajima J, Miyake T, Kawamura K, Fukuda S (2011)
Predicting distributions of seven bitterling fishes in northern Kyushu, Japan
.
Ichthyological Research
59
(
2
):
124
‑
133
. https://doi.org/10.1007/s10228-011-0260-0
Sano I, Shirai A, Kondo T, Miyazaki J (2017)
Phylogenetic relationships of Japanese Unionoida (Mollusca: Bivalvia) based on mitochondrial 16S rDNA sequences
.
Journal of Water Resource and Protection
9
:
493
‑
509
. https://doi.org/10.4236/jwarp.2017.95032
Shokita S (1984) Naturalized animals.
Organisms in Okinawa
.
Japan Association of Biology Education Okinawa
,
Naha
,
377-384
pp. [In
Japanese
].
Toyama M, Nishi K (2016)
Records of Anemina arcaeformis from Miyazaki Prefecture, sourthern Kyushu, Japan
.
Bulletin of the Miyazaki Prefectural Museum of Nature and History
36
:
1
‑
5
. [In
Japanese with English abstract
].
Ubukata T (1994)
Architectural constraints on the morphogenesis of prismatic structure in Bivalvia
.
Palaeontology
37
(
2
):
241
‑
261
.

Supplementary material