DNA Barcoding of the Freshwater Mussel, Genus Inversiunio
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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 diagnos- tic 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 bitter- ling 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. rein- ianus, 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