Spatial Dietary Shift of the Intertidal Snail, Batillaria Multiformis: Stable Isotope and Gut Content Analyses

Plankton Benthos Res 14(2): 86–96, 2019 Plankton & Benthos Research © The Japanese Association of Benthology 86

Fig. 7のdoiに関係するテキストボックスです．ノンブルを振る為に必要なので消さないこと！ Spatial dietary shift of the intertidal snail, Batillaria multiformis: stable isotope and gut content analyses

1, 2,† 3,‡ 1 Hisashi Yokoyama *, Jing Fu , Yuji Tamura & Yoh Yamashita

1 Field Science Education and Research Center, Kyoto University, Oiwake, Sakyo, Kyoto 606–8502, Japan 2 Graduate School of Global Environmental Studies, Kyoto University, Yoshida-Honmachi, Sakyo, Kyoto 606–8501, Japan 3 Shallow/Fresh Water Group, Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Kuresaki, Bungo-Takada, Oita 879–0608, Japan † Present address: East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, 300 Jungong Road, Shanghai 200090, China ‡ Present address: Research and Guidance Coordinator, Fisheries Research Division, Oita Prefectural Agriculture, Forestry and Fisheries Research Center, Tsuiura, Kamiura, Saiki, Oita 879–2602, Japan Received 4 September 2018; Accepted 6 February 2019 Responsible Editor: Gen Kanaya doi: 10.3800/pbr.14.86

Abstract: In order to identify the food sources of the intertidal snail, Batillaria multiformis, stable isotope (δ13C, δ15N) and gut content analyses were conducted. Snails and their possible food sources were collected from the river- sea transects at the mouths of the Iroha and Katsura rivers in northeastern Kyushu, Japan, which differ in land use parameters (e.g., the ratio of agricultural area in the catchment area). There was a shift in the δ13C of the snail along the transects, showing higher values at the upstream stations (−15.8 and −14.8‰) and lower values (−17.5 and −17.1‰) at the seaward stations. The δ15N showed no significant spatial gradient along the transects, but a significant difference was observed (t-test, p<0.001) between the Iroha (mean, 10.0‰) and Katsura (11.1‰) rivers. A Bayesian mixing model and the biomass of possible food sources on the tidal flats showed that (1) the major food sources were marine phytoplankton, seaweeds, and benthic microalgae, and that (2) the dependency on marine phytoplankton increased in the seaward stations. However, gut content analysis revealed that most of the identifiable dietary items across all stations were benthic diatoms, which is considered to be due to the short-term result of ingestion on the sediment surface at low tide. Increasing dependency on phytoplankton at seaward stations was considered to be due to consumption of suspended particles in the water column at high tide by filter feeding. Differences in the δ15N between the two rivers suggest the possibility of using the δ15N of snails as an indicator of watershed characteristics.

Key words: Bayesian mixing model, benthic diatom, gastropod, intertidal ﬂat

tributed to elucidate the sources and trophic transfer of Introduction organic matter in estuaries. The stable carbon isotope ratio Estuaries have been studied intensively to clarify a (δ13C) has been widely used as a natural tracer to follow mechanism of how to maintain high biodiversities and pro- the ﬂow of food sources (e.g., Fry & Sherr 1984), whereas ductivities (e.g. Daborn & Redden 2016). Such ecosystems the stable nitrogen isotope ratio (δ15N) has been used to are considered to be supported by materials including nu- show the trophic levels of consumers in food webs (e.g., trients and organic and inorganic particles that are dis- Owens 1987). Carbon isotope signatures of organic matter charged from terrestrial areas and are carried by rivers sources are determined by isotope fractionation of primary (Yamashita 2014). producers according to their photosynthetic pathways. The Carbon and nitrogen stable isotope analyses have con- 13C fractionation of aquatic plants and algae varies depending on their internal and external environments, resulting 13 * Corresponding author: Hisashi Yokoyama; E-mail, patiens07@gmail. in taking a wide range of δ C values even within each com producer group (Fogel et al. 1992, Simenstad et al. 1993, Spatial dietary shift of Batillaria 87

Fry 1996, Gervais & Riebesell 2001, Cloern et al. 2002, Usa region, northeastern Kyushu, the amount of precipita- Trudeau & Rasmussen 2003). Biotic and abiotic nitrogen tion is relatively small compared with that in other parts of are subject to various kinds of trophic transfer and/or bio- Japan (<1,600 mm yr−1, Oita Meteorological Office, http:// geochemical processes, resulting in a large spatiotemporal www.jma-net.go.jp/oita/oita-kikou.htm: June 19, 2018). In variability in their δ15N signatures in aquatic ecosystems order to develop agriculture in this region, which has low (Cifuentes et al. 1988, McCutchan et al. 2003, Middelburg rainfall and steep slopes, residents have constructed reser- & Herman 2007). The large variability in the isotopic com- voirs called ‘Tameike’ and terraced paddy fields in a stair- position of possible food sources may involve drawbacks; case pattern on very steep slopes called ‘Tanada,’ resulting however, simultaneous use of dual isotopes and frequent in a unique landscape. Residents have also been utilizing sampling of food sources may compensate for such draw- deciduous fagaceous vegetation dominated by sawtooth backs and provide signatures that are integrated over a oak (Quercus acutissima) as materials for culturing Shiita- period corresponding to the turnover time of the analyzed ke mushrooms (Lentinus edodes) and producing charcoal. tissues. On the other hand, traditional gut content analy- Such a traditional farming system has been recently certi- sis provides evidence of the dietary items that individuals fied as a site for Globally Important Agricultural Heritage actually consumed, though this analysis is biased towards Systems (GIAHS) by the Food and Agriculture Organiza- recent dietary items that do not digest readily (e.g., Polito tion of the United Nations under the name of ‘Kunisaki et al. 2011). Complementary stable isotope and gut content Peninsula Usa integrated forestry, agriculture and fisheries analyses thus provide valuable ecological insights. system’ (http://www.kunisaki-usa-giahs.com/en/: June 19, Extensive data on the diets for estuarine animals are 2018). available. Of a number of these case studies, most have During field surveys around the Kunisaki Peninsula that noted the importance of benthic or pelagic microalgae aimed to verify the effectiveness of the GIAHS against (e.g., Persic et al. 2004, Kang et al. 2015). On the other production in coastal waters, we often found dense pop- hand, several studies have indicated that riverine terres- ulations (∼2,000 individuals m−2) of B. multiformis on trial organic matter (Kasai & Nakata 2005, Hoffman et al. the tidal flats of river mouths. Comparison of the snail 2008, Cole & Solomon 2012) and anthropogenic effluents populations around the Kunisaki Peninsula with those in (e.g. Hadwen & Arthington 2007) play an important role in industrialized areas of Japan where the populations have estuarine food webs. Several studies have also suggested been disappearing encouraged us consider that the dense that significant incorporation of terrestrial organic matter populations were supported by fertile GIAHS environ- by estuarine animals occurs in the upper estuarine reaches ments which were characterized by the harmonious coex- (Kikuchi & Wada 1996, Fry 1999, Antonio et al. 2012) or istence of nature and humans. We therefore tried to clarify during freshet periods (Riera & Richard 1997, Chanton & the mechanism responsible for the establishment of these Lewis 2002). Thus, there are large variations in food utili- dense populations. In the present study, we tried to clarify zation by estuarine consumers among estuaries depending what this snail feeds on by examining the δ13C and δ15N on the differences in food supply which are usually in- and gut contents of B. multiformis collected from intertidal fluenced by the mixing ratio between seawater and fresh- stations along river-sea transects at the mouths of the two water. It is, therefore, important to understand the flow of rivers, which differ in topographic, geologic, and land use organic matter and energy in estuaries in order to clarify parameters. the spatial variability in the food sources of consumers. The batillariid snail, Batillaria multiformis (Lischke, Materials and Methods 1869) (Gastropoda, Batillariidae) is distributed in estuaries in Japan (southern Hokkaido to Kyushu) (Japanese As- Study area and sample collection sociation of Benthology 2012) and Korea (Lee et al. 2016). This snail prefers sand and mud substrata in the middle The Kunisaki Peninsula is formed from a round volcano and low intertidal zones, and feeds on benthic diatoms and (diameter, ∼30 km) with a peak (Mt. Futago, 721 m above marine phytoplankton rather than terrestrial plants (Liu et sea level) at the center (Fig. 1). Much of the Kunisaki Pen- al. 2014). It produces egg capsules that hatch into plankton- insula is covered by volcanic rock that was produced by ic larvae (Furota et al. 2002). This species has been com- the Futago Volcanic Group at a geological age of 1.19 Ma monly found in Japan; however, during the latter half of (Horikawa et al. 2016). A number of short and steep rivers the 20th century, populations in many areas of Japan such occur radially and flow into Suo-nada and Iyo-nada in Seto as at the mouth of the Nanakita River (Sendai City) and in Inland Sea. In this region, plain areas are scarce, whereas Tokyo Bay (Wada 2001, Furota et al. 2002) have decreased the Nakatsu and Usa districts located to the west of the drastically due to land reclamation and water pollution. Kunisaki Peninsula have an alluvial plain created by sev- As a result, B. multiformis has been designated as a near eral rivers such as the Yamakuni and Yakkan rivers. threatened species in 2007 (Ministry of the Environment, The present study aimed to reveal the diet of B. multifor- Japan 2014). mis inhabiting the tidal flats at the mouths of the Iroha and In the area of the Kunisaki Peninsula and in the nearby Katsura rivers [Fig. 1a, b: Stations (Stns) 1–13]. This spe- 88 H. Yokoyama et al.

Fig. 1. Study area. (a) Mouth of the Iroha River showing sampling stations (Stns 1–6). (b) Mouth of the Katsura River showing sampling stations (Stns 7–13). (c) The Nakatsu and Usa districts and the Kunisaki Peninsula showing a sampling station for marine particulate organic matter in Suo-nada (Stn 14). cies occurs as an exclusive dominant species on the tidal In order to collect a sufficient amount of benthic mi- flats in these two rivers as well as in other rivers in the croalgae, we looked for surface sediments that had turned Nakatsu and Usa districts and the Kunisaki Peninsula area. yellowish brown as a sign of high growth of benthic mi- We considered that this snail is a model species that might croalgae on the tidal flat in IR and KR on four occasions: demonstrate the watershed and estuarine linkage. February 19, March 20, April 19, and April 21 in 2015. We The Iroha River (hereafter, IR) flows in the Nakatsu collected approximately 500 mL of surface sediments from and Usa districts where the ratio of agricultural area in the whole area of the tidal flats, and brought the sediments the catchment area is relatively high (25.8% vs. 17.0% in to the laboratory and extracted the microalgae following the Katsura River area), whereas the water catchment area the procedure described by Couch (1989) as modified by of the Katsura River (hereafter, KR) is located mainly in Yokoyama & Ishihi (2003) (total number of samples=8). Kunisaki Peninsula where forested land covers a large pro- Living seaweeds growing on the pebbles and rocks on portion of the area (79.1% in the catchment area vs. 67.1% the tidal flats or on seawalls mainly around Stns 1–4 (IR) in IR). Batillaria multiformis were collected from six sta- and Stns 7–10 (KR) and the debris of terrestrial plant ma- tions in IR (Fig. 1a, Stns 1–6) and seven stations in KR terial composed mostly of broad-leaved trees and dead (Fig. 1b, Stns 7–13) on April 20 and 21, 2015. These sta- leaves and stems of the common reed, Phragmites austral- tions were arranged along the river-sea transects in the is (Cav.) Trin. ex Steud. were collected from the two tidal mediolittoral zone to evaluate the spatial dietary shift in flats on three occasions, i.e., February 19, March 20, and B. multiformis. The surface sediments at the stations were April 21 in 2015. A living salt marsh plant, Zoysia sinica composed of sand or muddy sand. Hance, 1869 was collected on the same three occasions, The tidal flats in the study area are covered by a variety whereas such vegetation was not found in IR. This plant of potential food sources including autochthonous (ben- covered a small part (∼580 m2) of the high intertidal zone thic microalgae, various seaweed species, and salt marsh at the KR mouth (Fig. 1b). On the tidal flats, we could not plants) and allochthonous (terrestrial plants, reeds, marine find any debris that originated from this plant during the phytoplankton, and seagrasses) primary producers. In or- survey. A small amount of drifted seagrass fragments was der to determine the diet of B. multiformis, we collected occasionally found on the tidal flats. The seagrass Zostera seven primary producers from the tidal flats monthly, over marina Linnaeus, 1753 was collected from the IR tidal flat a 2-month period from February to April in 2015, preced- in February and from the KR tidal flat in April, whereas ing the animal collection, assuming that the majority of the a fragment of Z. japonica Ascherson & Graebner, 1907 snail’s tissues are formed during this period. was collected from the KR tidal flat in April alone (a total Spatial dietary shift of Batillaria 89 of four fragments of Zostera species collected from two Adoption of a mixing model tidal flats during a total of 6 hours in the sampling period). These materials were stored in a freezer, defrosted, rinsed The Bayesian stable isotope mixing model, SIAR (Sta- with distilled water, dried at 60°C, and ground to a fine ble Isotope Analysis in R: Parnell et al. 2010) was used powder. to estimate the contributions of seven different primary We collected seawater including particulate organic producers to the B. multiformis diet. This model enables matter (POM) in Suo-nada from a depth of 5 m at Stn 14 calculating the contribution ratios of food sources to the (approximately 11 m deep, Fig. 1c) that is located 3 km consumer’s diet as credible intervals, even if the number from the mouth of KR on three occasions: February 4, of sources is more than (n+1) relative to the number of March 2, and April 13 in 2015. This sampling location and isotopes used (n). We used the means and standard devia- layer was determined from the high salinity (>32) and tions (SD) of the seven primary producers and the widely at an intermediate layer of 6 m above the seabed, which applied fractionation values, 1.0‰ for 13C (DeNiro & Ep- suggest minimal influence of freshwater, high growth stein 1978, Fry & Sherr 1984) and 3.4‰ for 15N (DeNiro & rate of phytoplankton (mean±SD of chlorophyll a in col- Epstein 1981, Minagawa & Wada 1984), which were deter- lected seawater=2.7±0.6 µg L−1, http://www.pref.oita.jp/ mined by analyzing the non-defatted whole-body samples soshiki/15090/jigyouhoukoku.html: November 9, 2018), a of consumers. Contributions of dietary sources (propor- low level of resuspension of sediments, and lack of any tion, %) were represented as 95%, 75%, and 25% credible estuarine turbidity maximum (ETM). Therefore, we re- intervals (CI) and as the mean and mode values for propor- garded the collected POM as a representative of marine tional source contributions. end-members (marine phytoplankton). The collected sea- Gut content analysis water was passed through a 0.125 mm mesh net to remove large particles and was filtered on a pre-combusted (450°C, The stomach and intestine of B. multiformis were ex- 2 hours) Whatman GF/F glass fiber filter, which was then tracted from 1–5 individuals collected from each of the washed with 1.2 N HCl, rinsed with distilled water, and Stns 1–13 (excluding Stn 3) and mixed with distilled wa- dried at 60°C. ter. The gut contents were observed, diatom cells were identified to the genus level, and their number of cells was Sample processing and analysis counted under an optical microscope. We distinguished The collected snails were kept frozen at −20°C. Before benthic and pelagic diatoms as described by Round et al. analysis, three individuals from each station were defrost- (1990). We did not include formless detrital particles as an ed, the shell was physically removed using diagonal cut- analysis target. ting pliers, the removal of shells and inorganic carbon con- tained in the snail tissues was then confirmed by soaking Results in 1.2 N HCl for a few minutes, the gut contents were removed, whole soft tissues were rinsed with distilled water, Gut content of B. multiformis dried at 60°C, and ground to a fine powder. Examination of acid treatment on the δ15N of snails showed a significant Gut content analysis of B. multiformis collected from increase in value (two-sample t-test, p=0.025, n=5), how- the two river mouths showed no traces of terrestrial plants, ever, the difference (mean±SD=0.24±0.15‰) was small. reeds, seaweeds, seagrasses, salt marsh plants, or animal Therefore, we used the δ13C and δ15N values obtained from tissues and indicated that the only identifiable primary pro- acidified samples. ducer was diatoms. We confirmed the occurrence of 32 The δ13C and δ15N of B. multiformis (18 individuals from species/species groups, which were classified as 27 spe- IR, shell height=25.2–33.3 mm; 21 individuals from KR, cies/species groups of benthic diatoms, four species/spe- shell height=20.8–28.9 mm) and its possible food sources cies groups of pelagic diatoms, and species belonging to were determined using a Thermo Fisher delta V plus mass the genus Odontella, for which we could not determine the spectrometer connected with an Elemental Analyzer (EA) benthic or pelagic type (Table 1). The species compositions equipped at the Center for Ecological Research, Kyoto in IR and KR were similar; in both river mouths, (1) ben- University. Analytical errors (±SD of the working stan- thic diatoms accounted for the majority of cells, i.e., 95.6% dard) were 0.06‰ (δ13C) and 0.04‰ (δ15N). in IR and 98.0% in KR, whereas the composition of pe- In order to test the statistical differences in the δ13C and lagic diatoms was quite small, 4.2% (range=1.0% to 7.0%) δ15N of B. multiformis among the station groups in the two in IR and 1.2% (0% to 4.4%) in KR, and there was no tidal flats, we used one-way ANOVA, and then applied the gradient in the composition of benthic and pelagic diatoms multiple comparison test (Tukey–Kramer test) to verify the along the river-sea transects, (2) Navicula spp., unidenti- difference between stations in each river. We also exam- fied Diatomaceae, and Nitzschia spp., all benthic diatoms, ined the differences in the δ15N between IR and KR using were the dominant species groups, and (3) there was no the two-sample t-test. P values less than 0.05 were consid- gradient in the species composition except for the compo- ered statistically significant. sition of Diatomaceae in IR, which showed lower values at 90 H. Yokoyama et al.

Table 1. Species composition of diatoms in the gut contents of Batillaria multiformis collected from tidal flats at the mouths of the Iroha and Katsura rivers (the number of cells per total number of diatom cells, %). Species Iroha Katsura Benthic diatoms Melosira sp. 0 0.6 Hydrosera sp. 0 0.1 Eunotogramma sp. 2.0 0.4 Fragilaria sp. 1.2 0 Grammatophora spp. 0.4 0 Opephora sp. 0.6 0.2 Rhaphoneis sp. 0 0.4 Ulnaria sp. 0 0.3 Diatomaceae 26.8 18.7 Achnanthes sp. 0 1.1 Achnanthidium spp. 0.8 5.2 Cocconeis spp. 8.8 3.0 Planothidium spp. 3.0 1.7 Amphora spp. 2.8 4.1 Catenula sp. 1.4 0.6 Cymbella sp. 0 0.7 Diploneis sp. 0.4 0 Encyonema sp. 0 0.2 Gomphonema spp. 0 1.6 Gomphonemopsis sp. 1.4 1.2 Navicula spp. 26.2 42.6 Reimeria sp. 0 0.6 Fig. 2. Composition of diatoms in the gut contents of Batillaria Rhoicosphenia sp. 0.2 0.1 multiformis collected from the Iroha (a) and Katsura (b) rivers Trachyneis sp. 0 0.1 during the ebbing tide. Composition ratios of benthic and pelagic Hantzschia sp. 0 0.4 diatoms determined from the identification of genera are shown Nitzschia spp. 19.4 13.9 above each bar in italics. Surirella sp. 0.2 0 Total of benthic diatoms 95.6 98.0 δ15N were found in seagrasses (−14.4‰ and 6.4‰ in IR, Pelagic diatoms −11.0±0.6‰ and 5.6±1.3‰ in KR, respectively) and salt Aulacoseira spp. 0 0.2 marsh plants (−14.5±0.5‰ and 6.6±3.0‰ in KR alone). Cyclotella spp. 1.2 0.1 The δ15N of marine phytoplankton (6.9±0.7‰), seaweeds Thalassiosira spp. 2.8 0.9 (5.7±1.4‰ in IR and 6.8±1.4‰ in KR) and benthic micro- Thalassionema sp. 0.2 0 algae (7.1±2.3‰ in IR and 4.5±4.6‰ in KR) showed simi- Total of pelagic diatoms 4.2 1.2 lar values to the seagrasses and salt marsh plants. Among Benthic or pelagic diatoms these three kinds of algae, marine phytoplankton showed Odontella spp. 0.2 0.8 the lowest δ13C (−22.7±0.7‰), followed by seaweeds (−19.5±5.2‰ in IR and −19.3±5.4‰ in KR), whereas benthic microalgae showed the highest value (−14.6±2.3‰ in more seaward stations along the river-sea transects (Fig. 2). IR and −17.0±0.2‰ in KR). The mean δ13C values of seaweeds were determined Isotopic compositions of potential food sources based on the 26 species collected from IR and 12 species Among the possible food sources, terrestrial plants collected from KR, which showed wide ranges of the mean showed the lowest δ13C and δ15N values (Fig. 3a, b: δ13C of each species i.e., from −35.1‰ of Plocamium lep- mean±SD=−30.6±1.1‰ and 2.8±1.0‰ in IR, −29.8±0.7‰ tophyllum Kützing, 1849 (No. 21, an open circle in Fig. 3c) and 0.6±0.6‰ in KR, respectively). The reeds had high- to −13.4‰ of Ulva sp. (No. 3, an open circle) in IR and er δ13C and δ15N values (−25.8±2.4‰ and 4.1±1.3‰ in from −36.3‰ of P. leptophyllum (No. 21, a filled circle) to IR, −25.7±1.6‰ and 5.5±0.7‰ in KR, respectively) than −13.9‰ of Gracilaria vermiculophylla (Ohmi) Papenfuss, those of terrestrial plants. The highest levels of δ13C and 1967 (No. 23, a filled circle) in KR, resulting in large SD Spatial dietary shift of Batillaria 91

Fig. 3. Dual isotope plots of Batillaria multiformis and possible food sources. Batillaria multiformis, the diet of this species (1–13, sampling stations), and possible food sources on the tidal flats at the mouth of the Iroha (a) and Katsura (b) rivers. Seaweed species and benthic microalgae collected from the two river mouths (c). Error bars (possible food sources only) are SD of the δ13C and δ15N values. Numerals in italics in Fig. 3a, b indicate the sample size. Numerical codes in Fig. 3c indicate seaweed species: 1 Monostroma nitidum, 2 Ulva pertusa, 3 Ulva sp., 4 Ulva intestinalis, 5 Ulva compressa, 6 Blidingia minima, 7 Codium fragile, 8 Cutleria multifida, 9 Colpomenia sinuosa, 10 Undaria pinnatifida, 11 Hizikia fusiformis, 12 Sargassum horneri, 13 Sargassum thunbergii, 14 Sargassum muticum, 15 Pyropia sp., 16 Gelidium divaricatum, 17 Gelidium elegans, 18 Hyalosiphonia caespitosa, 19 Grateloupia asiatica, 20 Gloiopeltis furcata, 21 Plocamium leptophyllum, 22 Gracilaria textorii, 23 Gracilaria vermiculophylla, 24 Champia parvula, 25 Acrosorium sp., 26 Heterosiphonia japonica, 27 Polysiphonia senticulosa, and 28 Neorhodomela munita. values. Isotopic compositions of B. multiformis along river-sea The δ15N of benthic microalgae showed large temporal transects variations, as shown by the wide ranges from 5.2 to 10.3‰ in IR and from 0.7 to 10.9‰ in KR (Fig. 3c). In April, One-way ANOVA showed significant differences in when samples were collected on two occasions at a two- the δ13C of B. multiformis among the station groups in day interval, the difference in the δ15N between these two IR (F=10.6, p<0.001) and in KR (F=9.6, p<0.001). The occasions reached 3.3‰ in IR and 6.0‰ in KR. The δ13C Tukey–Kramer test shows that (1) the mean δ13C values at of benthic microalgae in IR also showed a wide fluctuation the upstream stations in IR (Stns 1–3) ranged from −16.1 range (4.3‰). to −15.8‰, which were significantly different from the 92 H. Yokoyama et al.

marsh plant (−14.5‰ in KR) and seagrasses (−14.4‰ in IR, −11.0‰ in KR). At more seaward stations, the dietary δ13C shifted to values nearer to marine phytoplankton in both the localities. The SIAR mixing model showed the contribution (proportion, %) of the six producers in IR and the seven producers in KR to the diet of B. multiformis (Figs. 5, 6). The contribution of terrestrial plants was very low in both IR and KR, as shown by the low mean (4–7% in IR, 5–7% in KR) and mode (1% in IR and KR) values, although the 95% credible interval (95% CI) showed a relatively large variation (0–17% in IR, 0–18% in KR) (Figs. 5a, 6a). The reed also showed a minor contribution (95% CI=0– 23% in IR, 0–25% in KR; mean=6–10% in IR, 7–12% Fig. 4. Isotopic composition of Batillaria multiformis at each station. δ13C (a, b) and δ15N (c, d) values (mean±SD, all n=3) for in KR; mode=1–2% in IR, 1–4% in KR) (Figs. 5b, 6b). B. multiformis collected from the 13 stations on the tidal flats at the The contribution of marine phytoplankton was relatively mouth of the Iroha (a, c) and Katsura (b, d) rivers. Different letters low at upstream stations in IR (Stns 1–3: 95% CI=0–34%, denoted beside bars indicate significant differences (p<0.05) cal- mean=14–16%, mode=6–14%) and in KR (Stns 7–9: 95% culated from the Tukey–Kramer test. CI=0–26%, mean=10–12%, mode=2–5%), whereas at more seaward stations, their contribution increased (Stns values obtained from the seaward stations (−17.5‰ and 5–6 in IR: 95% CI=1–42%, mean=21–23%, mode=23– −17.4‰ at Stns 5 and 6) (Figs. 3a and 4a); (2) at the more 26%. Stns 13 in KR: 95% CI=0–29%, mean=15%, seaward stations, the δ13C in KR also showed a gradual de- mode=18%) (Figs. 5c, 6c). The 95% CI, mean, and mode creasing tendency from –15.2‰ (Stn 7) or –14.8‰ (Stn 8) for the contribution of seaweeds in IR and KR were with- to –17.1‰ at the seaward station (Stn 13) (Figs. 3b and 4b). in the range of 0–33% and 0–28%, 11–15% and 10–14%, Regarding the δ15N of B. multiformis, there was a signif- and 3–8% and 2–14%, respectively (Figs. 5d, 6d). In IR, icant difference among the stations in IR (one-way ANO- there was no clear trend in the spatial pattern of seaweed VA, F=4.6, p=0.01); however, the significant difference contribution, whereas at the seaward station in KR (Stn was found only between Stn 1 and Stns 3 and 4 (Tukey– 13), the mean and mode (both 14%) showed higher values Kramer test, Fig. 4c). One-way ANOVA showed no sig- than those at other stations. The highest values of the con- nificant difference in the δ15N of B. multiformis among the tribution were found in seagrasses (IR: 95% CI=5–65%, stations in KR (F=0.21, p=0.97). Since a gradient of δ15N mean=29–35%, mode=27–32%. KR: 95% CI=2–50%, along the river-sea direction was not found for the two riv- mean=19–28%, mode=20–28%) (Figs. 5f, 6f). The second ers, values obtained from each river were compiled, and highest values were found in benthic microalgae (IR: 95% the two-sample t-test was used to evaluate the difference CI=0–46%, mean=18–26%, mode=19–26%. KR: 95% between the two rivers, resulting in a significant difference CI=0–37%, mean=15–17%, mode=8–18%) (Figs. 5e, 6e). (p<0.001) between IR (mean±SD=10.0±0.3‰) and KR In KR, the salt marsh plant showed high contributions (IR: (11.1±0.5‰). 95% CI=0–46%, mean=18–24%, mode=20–24%) (Fig. 6g). There was no clear trend in the spatial pattern of the Contribution of primary producers to the B. multiformis contribution by these three sources (benthic microalgae, diet seagrasses, and salt marsh plant). In accordance with the presumptive 13C and 15N fractionation of consumers (1.0‰ and 3.4‰, respectively), the Discussion estimated mean δ13C and δ15N values of the diet for B. multiformis at each station should be −18.5‰ to −16.8‰ Large differences in the δ13C between B. multiformis and 6.1‰ to 6.8‰ in IR, and −18.1‰ to −15.8‰ and and terrestrial plants or reeds and the SIAR mixing mod- 7.5‰ to 8.0‰ in KR, respectively (Fig. 3a, b). There was el showed minimal contributions of terrestrial plants and a ≥7.2‰ difference in the δ13C between the estimated diet reeds to the diet of this snail. Vascular plants including of B. multiformis and the reed and terrestrial plant debris. detrital terrestrial plants and reeds have previously been On the other hand, differences in δ13C between the esti- considered to have little nutritional importance for inter- mated diet and other primary producers were generally tidal and/or estuarine animals due to the presence of a within 7.0‰ in general and the dietary δ13C values were large amount of refractory substances such as cellulose between the relatively low value of marine phytoplankton and lignin (e.g., Fontugne & Jouanneau 1987, Cividanes et (mean=−22.7‰) or seaweeds (−19.5‰ in IR, −19.3‰ in al. 2002). The model also showed that the mean and mode KR) and the high values of primary producers such as values of seagrasses and salt marsh plants were very high. benthic microalgae (−14.6‰ in IR, −17.0‰ in KR), a salt However, the credible intervals (CI) found in these produc- Spatial dietary shift of Batillaria 93

Fig. 5. Proportional contribution of six primary producers to the diet of Batillaria multiformis at six stations (1–6) in the Iroha River. The SIAR mixing model was used for the calculation. Dark Fig. 6. Proportional contribution of seven primary producers gray in the center, light gray in the middle, and white areas in the to the diet of Batillaria multiformis at the seven stations (7–13) outer area indicate 25%, 75%, and 95% credible intervals, respec- in the Katsura River. The SIAR mixing model was used for the tively. Means and modes of contributions are indicated by lines and calculation. Dark gray in the center, light gray in the middle, and filled circles in the boxes and numerals in roman (mean) and italics white areas in the outer area indicate 25%, 75%, and 95% credible (mode) beside the boxes, respectively. intervals, respectively. Means and modes of contributions are indicated by lines and filled circles in the boxes and numerals in roman (mean) and italics (mode) beside the boxes, respectively. 94 H. Yokoyama et al. ers showed large variabilities (2–65% and 0–46%, respectively), indicating the possibility of scarce contribution. In addition to this, the biomasses of these producers were small, and the biochemical composition of these plants is similar to that of terrestrial vascular plants. It is thus diffi- cult to conclude that seagrasses and salt marsh plant made a notable contribution to the diet. The SIAR mixing model (Figs. 5, 6) and observations on the biomass of living and fragmentary primary producers on the tidal flats showed that (1) the major food sources were marine benthic microalgae, phytoplankton, and seaweeds, and (2) the dependency on marine phytoplankton increased at the seaward stations. On the other hand, gut content analysis revealed that the only identifiable dietary items were diatoms and that most of them were benthic diatoms across all stations (Fig. 2). Visual observations Fig. 7. Photograph showing trails of Batillaria multiformis on the sediment surface at the mouth of the Katsura River, taken at Stn in the daytime on the KR tidal flat showed that B. multi- 11 on April 21, 2015. Note the lower left of the photograph where formis left gray trails on the sediment surface that looked the change in color from yellowish brown to gray after scraping the yellowish brown (Fig. 7). Kikuchi (1993) described that surface sediments with a spatula is shown. intertidal benthic diatoms move to the sediment surface or near-surface during the daytime, resulting in yellowing of the sediment surface. These observations suggest that the It has been reported that isotopic compositions of con- snails grazed on the yellowish sediments including benthic sumers shift along the estuarine gradient, accompanied by microalgae while crawling on the sediment surface. Thus, shifts in isotopic values of food sources (Chanton & Lewis it is a non-controversial fact that microalgae on tidal flats 1999, Fry 1999) or in the relative contribution of different are an important food source for B. multiformis. However, sources (Kikuchi & Wada 1996, Kasai & Nakata 2005, it should be noted that the gut content analysis is biased to- Dias et al., 2014). These studies show that the estuarine wards recent dietary items that do not digest readily (Polito gradients extend over several kilometers or several tens of et al. 2011). Accordingly, the role of phytoplankton and kilometers. In contrast, the present study showed a shift of seaweeds as a diet for this species needs further discus- the δ13C in the snail tissues over relatively short distances sion. (<400 m). These findings suggest that the relative contri- Batillaria species are reported to be deposit feeders bution of benthic microalgae and marine phytoplankton and grazers of benthic diatoms (Morton and Morton 1983, to the diet of snails shifts even at such short distances Koike et al. 1989, Ozawa 1996). However, Kamimura and and that the behavior range of snails is relatively small Tsuchiya (2004) observed ctenidial filter feeding behav- throughout their whole benthic life. ior of Batillaria zonalis occurring in subtropical tidal flat Estimated δ15N values of the snail’s diet (6.1–6.8‰) in habitats. Batillaria zonalis in seawater sucks suspended IR calculated from the currently accepted 15N fraction- particles through the siphonal canal, forms a mucus cord ation (3.4‰) are close to the mean values of marine phyto- to entangle particles, and then ingests it. Itoh et al. (2018) plankton (6.9‰) and benthic microalgae (7.1‰), suggest- found a similar feeding behavior in B. multiformis. These ing the direct consumption of these primary producers by findings indicate that B. multiformis has an ability to con- the snail. On the other hand, estimated δ15N values of the sume living phytoplankton in the eutrophic water column snail’s dietary items (7.5–8.0‰) in KR are higher than the at high tide. It is also possible that snails consume the values of the three producers (4.5–6.9‰). One possible ex- detrital phytoplankton accumulated in sediments as well planation for the difference in the estimated δ15N values as living phytoplankton by their filter-feeding behavior. may be that the snails consume these producers through The proportion of phytoplankton-derived organic matter intermediate animals such as foraminifers, copepods, and in estuarine sediments has been found to increase at more the eggs of turbellarians, which has been previously dem- seaward areas (e.g., Yokoyama et al. 2006). The gradient onstrated in the intertidal snail Cerithium atratum (Mar- in the concentration of detrital phytoplankton may also be cus and Marcus 1964). It appears likely that these animals related to the observed spatial shift in the snail’s δ13C. Re- were not recognized during the gut content analysis be- garding the utilization of seaweeds as the diet of B. multi- cause the animal tissues had been broken by the radulae formis, we did not observe any behavior indicating that the of the snails and/or the tissues had been digested. Another snails grazed on living or fragmentary seaweeds during explanation is that the δ15N values of detrital organic mat- the survey; however, the large biomasses of seaweeds in IR ter may increase in comparison with those of the original and KR and the SIAR analysis indicate the possibility of primary producers under deoxygenated and enriched envi- consumption of detritus originating from seaweeds. ronments (e.g. Koba et al. 1997). Spatial dietary shift of Batillaria 95

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