DNA Barcoding of the Freshwater Mussel, Genus Inversiunio

Biogeography 20. 55–61. Sep. 20, 2018 DNA barcoding of the freshwater mussel, genus Inversiunio and Pronodularia japanensis (Bivalvia, Unionidae) and exploration for new diagnostic characters based on soft body morphology

Tomoki Seo1*, Yuki Yashima2*, Jean Tanangonan1*

1 Program in Environmental Management, Graduate School of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan 2 Department of Environmental Management, Faculty of Agriculture, Kindai University, Nakamachi, Nara 631-8505, Japan

Abstract: Inaccurate target species designation due to taxonomic misidentification may adversely affect species conservation plans. Japanese freshwater mussels are highly susceptible to misidentification due to variations in shell form, so accurate identification with new diagnostic characters is required. In this study, we investigated the soft body morphologies and the DNA barcoding that had not been studied in the past, especially for three species of genus Inversiunio and Pronodularia japanensis which have similar shell forms, and searched for new diagnostic characters. As a result of this study, misidentification among the two strains occurred based on the shell form, but accurate identification was made using DNA barcoding and observation of the excurrent aperture form. We conclude that the form of the excurrent aperture is a valid new diagnostic character. Species identification in live specimen by the form of the excurrent aperture will enable non- invasive and accurate identification.

Key words: freshwater mussel, conservation, diagnostic character, excurrent aperture

Introduction et al., 2007). The population of the freshwater bitterling fish, which parasitically lay eggs inside the shell Freshwater mussels (Bivalvia; Unionidae) are rel- of the freshwater mussel, has also decreased because atively large-sized filter-feeding benthic organisms of loss of available host freshwater mussel (Kitamu- serving an important ecological function in freshwa- ra, 2008; Kitamura et al., 2012). Consequently, the ter ecosystems (Haag, 2012). Freshwater mussels are conservation of freshwater mussel is an urgent issue. also recognized as particularly likely to become ex- The first thing we need to do to conserve the spe- tinct among the living organisms in freshwater eco- cies is to correctly identify and recognize the species systems (IUCN, 2017; Lydeard et al., 2004). In Ja- for conservation. Identification of mollusks is mainly pan, its population has drastically decreased mainly done by shell form, but it is difficult in many cases due to water pollution and collection by enthusiasts. for freshwater mussels. It is often difficult because Loss of habitat due to concrete constructions on river the freshwater mussel easily changes its shell form banks has greatly influenced the shells from inhabit- according to environmental factors such as water ing the flowing water ecosystem causing many lotic flow velocity and sediment type (Watters, 1994; freshwater mussels in danger of extinction (Negishi Haag, 2007). Indeed, because of variability in shell ——————————————————————— form, considerable errors in species identification *Corresponding author: [email protected] occur and efforts to eliminate erroneous identifica-

− 55 − New diagnostic characters of Japanese freshwater mussel Tomoki Seo, Yuki Yashima, Jean Tanangonan tion are needed for conservation and management inhabits the lotic environment have higher conser- planning (Shea et al., 2011). Many misidentifications vation priority (Ministry of the Environment, 2014). have occurred in citizen surveys (Shea et al., 2011), Aside from being often sympatric, genus Inversiunio so a simple, easy and accurate species identification and P. japanensis are difficult to identify because is needed for participatory conservation activities. their shell forms are similar. Despite the possible uncertainty of identification in In this study, genus Inversiunio and P. japanensis freshwater mussels, shell form tends to be empha- are identified by DNA barcoding methods, and their sized and the soft body morphologies has not been soft body morphologies examined to clarify diag- utilized as a taxonomic character. Recently, research nostic characters for the purpose of contributing to have been carried out in which detailed analysis is conservation by enabling easy and accurate species performed on differences in aperture form as an ef- identification. fective taxonomic character (Klishko et al., 2017). Harada & Nishino (1995) reported that it is possible Materials and Methods to identify Japanese species of Corbicula by inhalant siphonal papillae, but the possibility of similarly We collected freshwater mussel samples from identifying Japanese freshwater mussel species has 33 sites extending from southern Hokkaido south not been studied. The morphological features of the throughout Japan in 2016 to 2017. Collected sam- soft body as a taxonomic character of the freshwater ples are simply identified as genus Inversiunio or mussel in Japan has not been examined. In addi- P. japanensis from each survey site. A total of 47 tion, if it is a gravid female, it is possible to classify individuals were used for analysis. Sampling points between subfamilies depending on the form of the are not specified in this paper as a precaution against glochidia (Kondo, 1982). On the other hand, molec- over-collection. The collected individuals were ular identification using molecular markers has been lightly boiled in water, and a portion of the adductor performed in recent years for species that are dif- muscle or foot was cut and placed in 99% ethanol. ficult to identify by shell form, suggesting that this The remaining soft body parts were fixed in 8% for- technique could be effective for freshwater mussel malin. identification (Zieritzet al., 2016). Total genomic DNA was extracted by general Genus Inversiunio belonging to Unioninae con- phenol chloroform method from a part of the foot sists of three species I. reinianus, I. jokohamensis, or the adductor muscle stored in ethanol. According and I. yanagawensis. One species - I. reinianus lives to the method of Froufe et al. (2016), primers of only in Lake Biwa, while I. jokohamensis ranges LCO22me2 + HCO700dy2 (Walker et al., 2006) from southern part of Hokkaido to the Tokai region. were used for PCR in the cytochrome c oxidase I. yanagawensis is ectopically distributed westward subunit I (COI) region of F type mitochondrial DNA from the Kinki District. Pronodularia japanensis and amplification made at annealing temperature of belongs to Gonideinae and is widely distributed 50°C. The PCR product was purified and the nucle- throughout Japan south of Aomori (Kondo, 2008). I. otide sequence was determined by delegating it to jokohamensis, I. yanagawensis and P. japanensis in- Macrogen using the same primer. The newly deter- habit mainly the lotic environment and are sympatric mined sequence was compared from GenBank using depending on the habitat (Negishi et al., 2007). All the Basic Local Alignment Search Tool (BLASTN; species of genus Inversiunio and P. japanensis are http://blast.ncbi.nlm.nih.gov). threatened with extinction, but I. reinianus which is As a result, some sequences showed 96-99% sim- endemic to Lake Biwa, and I. yanagawensis which ilarity with P. japanensis F type mtDNA sequence

− 56 − New diagnostic characters of Japanese freshwater mussel Tomoki Seo, Yuki Yashima, Jean Tanangonan

(KX 822659). For that reason, the F type mtDNA parallel to the excurrent aperture. More than a few sequence (KX822659) of P. japanensis and the se- nodular protrusions were seen as knobs or bumps quence of Margaritifera laevis (KU763221) as an on the inner mantle surface in the excurrent aperture out group were used for comparative analysis from (Fig. 1A). In the live specimen, the papillae in the the DNA database, GenBank. excurrent aperture was white and very short and The sequence data was aligned using Clustal W had a bump shape (Fig. 1B). In Type II, pigment (Thompson et al., 2002) in MEGA 7.0 (Kumar et al., coating without papillae in the excurrent aperture 2016). The aligned data set was trimmed and finally was yellow and black striped color perpendicular to 614 bp was used for analysis. A Neighbor-joining the excurrent aperture. The inner mantle surface of (NJ) tree was constructed by NJ method based on the excurrent aperture was smooth (Fig. 1C). In the K2P distance (Kimura, 1980) by MEGA 7.0. Boot- live specimen, the papillae in the excurrent aperture strap probability was obtained by resampling and analyzing 1000 replicates. Sequence divergences A B (uncorrected p-distance) were assessed using MEGA 7.0.

Results

Based on the shell form category of Kondo (2008) and Masuda & Uchiyama (2004), the collected indi- C viduals were separated into genus Inversiunio or P. japanensis, with 6 individuals identified as I. reinianus, 11 as I. jokohamensis, 15 as I. yanagawensis, and 15 individuals as P. japanensis (Table 1). In the rear part of the soft bodies of freshwater mussel there is an incurrent aperture, an excurrent aperture and an anal aperture. After examining the Fig. 1. Detail of the excurrent aperture. A, Preserved specimen of form of the excurrent aperture, two morphological Type I. B, Live specimen of Type I. C, Preserved specimen of Type II. D, Live specimen of Type II. features were observed in genus Inversiunio and Abbreviations: exa, excurrent aperture; pg, pigment P. japanensis. Pigment coating without papillae in coating without papillae in the excurrent aperture; pexa, pigmentation of excurrent aperture; k, knobs or bumps on the excurrent aperture of Type I was roughly black the inner mantle surface in excurrent aperture. Scale bar: and changed to yellowish brown as it goes inside in A-C, 1 mm; B-D, 2 mm.

Table 1. Result of Identification by shell morphology, Anatomy and Genetic analysis in genus Inversiunio and P. japanensis. Numerals represent the number of individuals.

enetic nalsis orm of ecurrent aperture dentified shell morpholog lade Tpe Tpe r a

Total Identified by shell morphology: Pj, Pronodularia japanensis; Ij, Inversiunio jokohamensis; Ir, Inversiunio reinianus; Iy, Inversiunio yanagawensis.

− 57 − New diagnostic characters of Japanese freshwater mussel Tomoki Seo, Yuki Yashima, Jean Tanangonan was yellow, elongated and spindle shaped (Fig. 1D). to KX 822659. The maximum p-distance between In addition to the 15 individuals identified by shell Clade A and Clade B was 18.6%. Since this is clear- form as P. japanensis, a total of 24 individuals – ly lower than the value of p-distance between F type including 2 individuals identified as I. jokohamensis mtDNA and M type mtDNA of P. japanensis (Dou- and 7 individuals identified as I. yanagawensis had cet-Beaupré et al., 2010), Clade B is not formed by P. the morphological features of Type I (Table 1). Out japanensis M type mtDNA. Based on morphological of the 32 individuals identified by shell form as feature, Clade A was formed by 24 individuals in- genus Inversiunio, 23 had Type II morphological cluding 2 individuals identified as I. jokohamensis, features and the remaining 9 individuals had charac- 7 individuals identified as I. yanagawensis, and all teristics of Type I (Table 1). The characters of Type 15 individuals identified as P. japanensis (Table 1). I and Type II were stable; there were no individuals Clade B was formed by 23 individuals identified with an intermediate form, no difference according to genus Inversiunio by shell form but excluding 9 to geographical variation was observed. On the other individuals contained in Clade A (Table 1). Based on hand, no clear interspecific difference was observed shell form, Clade Ba consists of 9 individuals identi- within genus Inversiunio. fied as I. jokohamensis, and Clade Bb consists of 14 The clade was mainly divided into two, Clade individuals from I. reinianus and I yanagawensis. A and Clade B. Although each was monophyletic, Clade B further comprised two subclades of Clade Discussion Ba and Bb (Fig. 2). Bootstrap values between each clade were all supported with high reliability. Twen- Type I was confirmed in 24 individuals consisting ty-four individuals formed Clade A, and 23 formed of 15 individuals identified as P. japanensis and 9 Clade B. Clade A included the sequence data of F individuals identified as genus Inversiunio. Clade A type mtDNA of P. japanensis. All of the sequenc- is assumed to be a clade corresponding to P. japan- es that formed Clade A showed 96-99% similarity ensis, because this clade was formed by individuals

100 A Pronodularia japanensis

Inversiunio reinianus 99 Ba Inversiunio yanagawensis 100

100 Bb Inversiunio jokohamensis

Margaritifera laevis KU763221

0.02 Fig. 2. Neighbor joining tree of genus Inversiunio and Pronodularia japanensis and outgroups based on Kimura 2-parameter distances. Numbers next to major nodes represent bootstrap support (1,000 replicates).

− 58 − New diagnostic characters of Japanese freshwater mussel Tomoki Seo, Yuki Yashima, Jean Tanangonan

identified as P. japanensis with F type mtDNA. All the excurrent aperture cannot be a taxonomic char- 24 individuals that made up Clade A were Type I, acter that reflects the phylogenetic relationship, even and no individual with Type II form was identified. between subfamily and species. For example, in our The 9 individuals that were identified as genus In- study, P. japanensis showed knobs or bumps on the versiunio but had the Type I form were also included inner mantle surface in excurrent aperture, but Klish- in Clade A. For this reason, it is reasonable to think ko et al. (2018) reported bumps in Unioninae. In that the difference in the result of these 9 individuals genus Inversiunio, clear distinct species differences resulted from misidentifying P. japanensis as genus could not be confirmed in the form of the excurrent Inversiunio. And it is suggested that Type I is an aperture. In this way, several examples have been autopomorphic character for P. japanensis. In addi- reported in which there is no species differences in tion, 23 individuals identified as genus Inversiunio the excurrent aperture (Araujo et al., 2005; Klishko - but excluding the 9 individuals suggested to be et al., 2017). Based on the above, the form of excur- misidentified were all Type II. Clade B is assumed rent aperture is an effective character for correctly to be a clade corresponding to genus Inversiunio, identifying genus Inversiunio and P. japanensis. as it is clear that it is not a clade formed by the M Especially in identification made based on the shell type mtDNA of P. japanensis based on the value of form, the variation was large, and misidentification p-distance. For this reason, it is suggested that Type occurred, whereas the form of excurrent aperture II is an autopomorphic character for genus Inversiu- was stable and there was no misidentification. This nio. Individuals identified as I. jokohamensis formed seems to be significant in grasping species diversity Clade Ba, with not one individual from I. reinianus in the future for genus Inversiunio and P. japanensis. and I. yanagawensis. Shell form and molecular iden- It should also be noted that differences in the form of tification results were consistent to identify Clade Ba the excurrent aperture were clearly observed in the as corresponding to I. jokohamensis and Clade Bb to living specimen. This time we dissected specimens I. reinianus and I. yanagawensis. Formation of the to conduct detailed anatomical observation, but same clade by I. reinianus and I. yanagawensis is non-invasive identification methods are needed in possible because I. yanagawensis is a paraphyletic the future for endangered species of genus Inversiu- group as pointed out by Sano et al. (2017). It is rea- nio and P. japanensis. In the case of freshwater mus- sonable to assume that research conducted in the past sel, it is possible to observe the form of the excurrent may include results based on misidentification, and aperture in a relatively short time when it is placed future revalidation based on correct identification is in water in a small plastic container. For this reason, necessary. For example, although I. yanagawensis it is expected that identification by excurrent aper- has higher conservation priority than other species, ture form in living specimen can be used as a simple, some individuals have been erroneously identified as non-invasive and reliable identification method for P. japanensis with lower conservation priority. This environmental assessment and citizen surveys. On indicates the possibility that it does not accurately the other hand, since it is impossible to identify reflect the distribution of species and the state of the the form of the excurrent aperture without a live population, and accurate grasp of species diversity is specimen, search for new diagnostic characters by urgently needed. detailed analysis of shell form is needed to identify Clear difference was found in the form of the museum specimens, where only shells are preserved. excurrent aperture of genus Inversiunio and P. japa- Sayenko et al. (2017) performed both genetic and nensis, and it became clear that it is an effective di- detailed shell form analysis and found new taxonom- agnostic character. However, it should be noted, that ic characters from shell form. A detailed analysis of

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