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Phenotypic divergence in viviparid snails in a recently converted freshwater lagoon

Freshwater mollusks often show high intraspecific variability in shell morphology, which may cause incongruences between molecular phylogeny and morphology-based species taxonomy. However, it remains unclear how morphological variations in freshwater snails are associated with different habitats such as rivers, ponds, and lakes. In the present study, we investigated the morphological variation in the shells of the viviparid freshwater snail Sinotaia quadrata in the Kahoku Lagoon in Japan, which was converted from a brackish to a freshwater lagoon 60 years ago. This newly established freshwater habitat is particularly well-suited for studies on how morphology is associated with environments. We found clear differences in shell morphologies between the S. quadrata populations sampled inside and outside of the lagoon. The S. quadrata sampled in the lagoon had thicker and more elongated shells with wider apertures than those sampled outside of the lagoon. No relationship was found between shell morphology and the distance between sampling sites. This suggests that the divergence in shell morphology occurred over a fairly short time period by rapid adaptation or phenotypic plasticity. Although it is unclear whether these differences are caused by phenotypic plasticity or adaptation, the present findings suggest that the shell morphologies of freshwater snails easily change under the influence of the environment. The high flexibility in shell morphology should be carefully considered when managing or studying freshwater mollusks.


Introduction
How taxonomists identify and discriminate species affects the estimations of biodiversity and the development of ecosystem conservation programs (Mace 2004, Lenat & Resh 2006, Garnett & Christidis 2017. DNA-barcoding and environmental DNA techniques are increasingly used to determine species composition in communities; however, the taxonomy of most eukaryotes remains primarily based on morphological characteristics (Deiner et al. 2017). On the other hand, many species show incongruences between molecular phylogeny and morphology-based taxonomy (Patterson et al. 1993, Irestedt et al. 2004, Havermans et al. 2010, Covain et al. 2016, San et al. 2018. In particular, freshwater snails often show re-markable incongruences between molecular phylogeny and morphology-based taxonomy (Köhler & Glaubrecht 2006, Pfenninger et al. 2006, Lee et al. 2007, Du et al. 2013, Hirano et al. 2015, Becker et al. 2016, Köhler 2016, Saito et al. 2018a). This sometimes results in problems when estimating species diversity for conservation programs for freshwater mollusks (Saito et al. 2018b). Potential causes of these incongruences include introgressive hybridization, ancient polymorphisms, the presence of cryptic species, rapid evolution, and highly plastic morphological traits (Glaubrecht & Köhler 2004, Lee et al. 2007, Miura et al. 2013, Köhler 2016, Hirano et al. 2019). These phenomena have been suggested by phylogenetic studies; however, many of these studies have not clarified the mechanisms underlying these incongruences. In particular, environmental pressures influence the selection of genotypes that underlie the expression of suitable phenotypic traits (Preston & Roberts 2007), as well as the expression of phe-notypic plasticity, in which phenotypes change according to the environment (Hollander et al. 2006, Hoverman & Relyea 2009, Dunithan et al. 2012, Cazenave & Zanatta 2016. Nevertheless, the relationships between phenotype and environments of freshwater mollusks have not been fully considered in these phylogenetic studies. To clarify the relationship between morphology and environment, we investigated morphological variations in Sinotaia quadrata (Fig. 1). S. quadrata is broadly distributed across East Asia, and occurs in various environmental habitats (e.g., rivers, water channels, rice fields, wetlands, and lakes) (Masuda & Uchiyama 2004, Hirano et al. 2015. Based on fossil records, Japanese populations of S. quadrata were most likely introduced from the Asian continent around 4,000 years ago (Kurozumi 2013). Furthermore, a recent molecular phylogenetic study suggested that S. quadrata from the Asian continent and Japan could not be distinguished by two mitochondrial molecular markers (Hirano et al. 2015). In addition, this phylogeny also showed that S. quadrata found inside and outside of Lake Biwa in Shiga Prefecture, Japan, had quite different shell morphologies, but shared the same haplotype. However, the relationship between phenotype and environment has not been investigated quantitatively. Therefore, we focused on Kahoku Lagoon to test the morphological variation in S. quadrata between S. quadrata found inside and outside of the lagoon. Kahoku Lagoon was formed over 1,000 years ago and was originally brackish (Kahokugata Lake Institute 2013). However, this lagoon was converted to a freshwater lagoon as a result of a reclamation project that began in 1963 (Kawahara & Takahashi 2001), and subsequently, because of the desalination of the lagoon, brackish organisms disappeared from Kahoku Lagoon, and were replaced by freshwater organisms (Kahokugata Lake Institute 2013). S. quadrata currently occur inside of the current Kahoku Lagoon and in water channels connected to the lagoon (Nomura & Takahashi 2006). As such, the populations of S. quadrata within Kahoku Lagoon settled in this lagoon less than 60 years ago. This means that the desalinated Kahoku Lagoon can be considered a new habitat for S. quadrata, and therefore this lagoon provides an excellent opportunity to elucidate the relationship between phenotype and environment. Using this unique model system, we aimed to evaluate how the morphology of S. quadrata differs between S. quadrata that occur inside and outside of Kahoku Lagoon.

Study sites and sampling
The sampling of S. quadrata was conducted around Kahoku Lagoon, Kanazawa City, Japan (36°39′05.4″N, 136°40′08.2″E). The survey sites included six locations in Kahoku Lagoon (n=54 S. quadrata) and 13 locations in the surrounding area (n=127 S. quadrata) ( Fig. 2 and Table 1). As the concentration of calcium ions in the water affects the shell morphology of freshwater mollusks (Marin et al. 1996, Rundle et al. 2004, the calcium ion concentrations were measured using an ion meter at each collection site (LAQUAtwin-Ca-11, accuracy±20%, Horiba, Japan, note that a calcium ion data of sampling site 5 was missing value). Collected S. quadrata were brought back to the laboratory within one week, boiled at 100°C for 5 minutes, and stored in 99.5% ethanol.

Quantification of shell morphology
Elliptic Fourier analysis was used to describe and quantify shell morphology (Williams et al. 2012, Doyle et al. 2018, Jackson & Claybourn 2018. Images of S. quadrata shells were acquired using a digital camera (K-S2, Ricoh, Japan), from which elliptic Fourier analysis was performed using SHAPE (Iwata & Ukai 2006). Since shell size or shell thickness are not taken into account in elliptic Fourier analysis, shell width as shell size was measured from the image using Image J (Schneider et al. 2012), and shell thickness was measured using a point micrometer (CPM15, accuracy±0.001 mm, Mitsutoyo, Japan) (Fig. 1B).

Statistical analysis
In order to investigate whether the outline of shells differ between S. quadrata sampled from inside and outside the lagoon, we constructed generalized linear mixed models (GLMMs) with Gaussian distributions. In the GLMMs, principal component scores (PC1 to PC5) obtained by elliptic Fourier analysis were included as dependent variables, and a model was constructed with each of the principal component scores. The information on whether the individual S. quadrata was collected from within the lagoon or from outside the lagoon was included as the fixed effect. The inside/outside of the lagoon status was converted to 0/1 in the analyses. To account for differences in sampled locations, the sampling site was included as a random effect. The significance of the models was examined by comparing the full model and the null model using a likelihood ratio test with chi-square distributions.
In order to determine whether shell thickness and size of S. quadrata differ inside and outside the lake, we conducted analysis using the same method (GLMMs with Gaussian distributions). The shell thickness and size (shell width) were designated as a dependent variable in each model, and the habitat (inside and outside of the lagoon) was included as the fixed effect, with sampling site included as a random effect. In addition, since shell thickness is affected by shell size, in the model that included thickness as a dependent variable, shell size was added as a fixed effect.
Different calcium ion concentrations may cause differences in shell morphology, and therefore the difference in  calcium ion concentrations between sampling sites inside and outside of the lagoon was examined using the Mann-Whitney U test.
The difference in shell morphology may be determined by the geographical distance between sampling sites, and not by differences in environmental pressures. Therefore, we conducted a Mantel test that examined the correlation between the matrices, in order to investigate whether the morphological differences between the sampling sites correlated with the geographical distances between the sampling locations. The values for morphological traits were included as principal component scores (PC1 to PC5) and morphological measurements (shell width and thickness), respectively.

Results
Results of the principal component analysis of the elliptic Fourier analysis showed that at least 83% of the variation in S. quadrata shell morphology was explained by the first five principal components (PC1: 54.9%; PC2: 10.5%; PC3: 9.26%; PC4: 4.83%; PC5: 4.03%). The outline from each principal component was reconstructed using the results of the elliptic Fourier analysis (Fig. 3). GLMMs, which tested whether PC scores were different inside and outside the lagoon, showed that the values for PC2 and PC3 differed significantly between sampling sites located inside and outside of the lagoon, respectively (PC2: P=0.002; PC3: P=0.047, Table 2, Fig. 4). These principal component scores were lower for the shells of S. quadrata sampled inside than those sampled outside of the lagoon ( Table 2, Fig. 4). However, the other principal components were not significantly different between sampling sites located inside and outside of the lagoon (PC1: P=0.857; PC4: P=0.423; PC5: P=0.616) ( Table 2).
After measuring the shells, we found that the average shell size of S. quadrata sampled from sites inside the lagoon was 18.54±0.22 mm (mean±s.e.) and the average shell size of S. quadrata sampled outside the lake was 18.97±0.21 mm (mean±s.e.) mm. The shell size was not significantly different between S. quadrata sampled from sites located inside and outside the lagoon (P=0.979, Table  2). Shell thickness showed a significant positive correlation with shell size (P=0.001, Fig. 5, Table 2). Additionally, the shells tended to be thicker in S. quadrata sampled within the lagoon than S. quadrata sampled outside the lagoon, although the difference was not statistically significant (P=0.052, Fig. 5, Table 2). Calcium ion concentrations did not differ between sites located inside and outside of the lagoon (P=0.320) (Table 1), and, therefore, calcium ion concentration was not related to the morphological variations in S. quadrata shells.
There was no correlation between S. quadrata shell morphology and geographical distance between sampling sites (Table 3).

Discussion
The results of the elliptic Fourier analysis showed that PC2 and PC3 of S. quadrata were significantly different between sites located inside and outside of the lagoon (Table 2). Lower values for PC2 indicate a more elongated, cylindrical shape, while higher values for PC2 reflect a more conical shape (Figs. 3, 4). PC3 represents differences in the bulge of the outer edge of the shell s aperture; higher values indicate wider apertures (Figs. 3, 4). Therefore, because these PC scores reflect the expansion rate of shell aperture size with growth, the S. quadrata inside the lagoon have elongated cylindrical shells, while the S. quadrata outside the lagoon have conical shells. Shell size did not differ between the sites located inside and outside of the lagoon ( Table 2), suggesting that the lagoon environment has no effect on S. quadrata shell size. On the other hand, regarding shell thickness, the S. quadrata sampled inside of the lagoon tended to have thicker shells than those sampled outside the lagoon (P=0.052) (Fig. 5) (Table 2). In addition, shell thickness correlated positively with shell size (Fig. 5, Table 2). Furthermore, our Mantel tests showed that there was no significant correlation between shell morphology and geographic distance (Table 3), and this suggests that morphological differences do not reflect spatial autocorrelation. In summary, our results suggest that   the S. quadrata shells are the same size both inside and outside of the lagoon, but the shells of S. quadrata sampled inside the lagoon are thicker than those of S. quadrata sampled outside the lagoon. The different environmental pressures inside and outside of the lagoon potentially led to the observed morphological differences in S. quadrata shells. Predators represent one such environmental pressure. Different shell morphologies result in several defense mechanisms against predators. For example, in freshwater snails in the genus Physa, globular shells interfere with predation from fish and elongated shells from crayfish (Dewitt et al. 2000). Furthermore, the narrowness of the shell s aperture and its thickness affect shell-breaking predators (Appleton & Palmer 1988, Trussell 2006, Konuma & Chiba 2007. In recent years, there was a huge outbreak of crayfish in Kahoku Lagoon (Kahokugata Lake Institute 2013). Furthermore, cyprinid fishes also occur in Kahoku Lagoon (Kahokugata Lake Institute 2013), and these fish feed on freshwater mollusks (Imamura & Hashitani 1957, Correia et al. 2005, Moriyama 2007). Thus, crayfish and cyprinid fishes, which can be predators of freshwater mollusks, may have caused differences in shell morphology between the sampling sites located inside and outside of Kahoku Lagoon.
Furthermore, water physical environments are another factor that may underlie the morphological differences in S. quadrata shells. Some morphological features of the shell function may reduce the stress caused by wave and/ or water current effects (Way et al. 1993, Kihira 1996, Urabe 1998, Le Pennec et al. 2017). There are waves inside the lagoon (Shirai & Kawahara 2005), and there are many environments in which water flows in one direction outside the lagoon. Due to the large differences in these physical properties, morphological differences in S. quadrata shells may occur.
Additionally, as freshwater mollusks produce shells using calcium ions in the water, the concentration of these ions in the environment affects shell thickness (Marin et al. 1996, Rundle et al. 2004). However, this effect can be ignored in the present study because there were no differences in the calcium ion concentrations inside and outside of the lagoon. Alternatively, fast-growing snails develop thin, globular shells that accommodate more body mass than their thicker, more elongated counterparts (Kemp & Bertness 1984). If the S. quadrata in the lagoon grow slowly, they are likely to have thick, elongated shells. This mechanism may be able to explain the morphological differences observed in this study.
Our results show that S. quadrata sampled within the lagoon had elongated cylindrical shells. The S. quadrata population within the lagoon has been established in the last 60 years because of the history of the lagoon. The origin of the lagoon population is not clear; however, it is highly possible that the lagoon population was introduced from the surrounding area, because freshwater habi-tats within and outside the lagoon are connected by water channels (Fig. 2). In addition, even if the S. quadrata population within the lagoon and the S. quadrata population outside the lagoon have different origins, it is likely that the two populations have a certain gene flow due to these water channels. The dispersal ability of freshwater mollusks including Viviparidae in small and connected habitats (Ribi 1986, Ribi & Arter 1986, Statzner et al. 2008, Kappes & Haase 2012) may also facilitate this. Furthermore, our Mantel tests showed that there was no significant correlation between shell morphology and geographic distance (Table 3), and this suggests that morphological differences do not reflect the origin and/or primitive history of immigration of each population. Considering these factors, the morphological differences between S. quadrata sampled within the lagoon and outside the lagoon are likely to have developed after the desalination of the lagoon. That is to say, the observed changes may be a consequence of either phenotypic plasticity or rapid morphological evolution. To distinguish between these two mechanisms underlying morphological change, further studies using highresolution genetic markers are required. Nevertheless, the present findings suggest that morphological variation occurred in S. quadrata as a result of their habitat differences between flowing water and the lagoon. A similar qualitative relationship between shell morphology and habitat has also been observed in S. quadrata sampled inside and outside of Lake Biwa (Hirano et al. 2015). This relationship may be general in S. quadrata, although further indepth studies are needed. Thus, the consistent relationship between morphological variation and habitat suggests that the environment of the lake affects the morphology of viviparid mollusks. Furthermore, the presumed flexibility in shell morphology of freshwater mollusks may emphasize the importance of considering habitat environment in species assessment, identification, and studies that examine shell morphology.