INTRODUCTION
Snails of the genus Batillaria are among the dominant groups of invertebrates inhabiting tidelands around Japan. Based on nucleotide sequences of mitochondrial DNA, a direct developing species, Batillaria attramentaria (= Batillaria cumingi; Adachi and Wada, 1997), was indicated to have a more complex geographical genetic structure than species such as Batillaria multiformis and Batillaria zonalis characterized by a planktonic larval period (Kojima et al., 2003, 2004, 2005). Among the Batillaria snails, only B. attramentaria shows a large intraspecific genetic deviation between areas along the Kuroshio and Tsushima Currents (Kojima et al., 2004). It is accordingly anticipated that these snails would provide an ideal model system to study the effects of reproductive and dispersal ecology, topography, and historical climate changes on the evolution of coastal organisms.
In Japan, many species distributed in tidelands are decreasing or are in danger of extinction owing to the reclamation of tidelands and the deterioration of coastal environments (Japanese Association of Benthology, 2012). In Tokyo Bay, approximately 90% of tidelands were reclaimed during the 1960–70s. As a result, B. multiformis and B. zonalis, which were common in the 1950s, rapidly declined and disappeared after 1995, while B. attramentaria was stably maintained (Furota, 2000). Furota et al. (2002) explained such interspecific differences as the connectivity between the local populations of species with planktonic development collapsing owing to coastal reclamation, while juveniles of the directly developing species could colonize their parents' habitat. Conserving Batillaria snails is important for maintaining the health of tidal flat ecosystems, as their feeding plays a significant role in the material cycles in tidal flats (Kamimura and Tsuchiya, 2006). Information on larval ecology is also important for the conservation of tideland species.
As the fourth Japanese species, Batillaria flectosiphonata was described based on morphological characters that differed from those of B. zonalis, B. multiformis, and B. attramentaria, including a distorted, deeply concave columella, and a siphonal canal that is deflected to the left (Ozawa, 1996). Ozawa (1996) reported that this species was distributed on the Nansei (Ryukyu) Islands and also on the western coast of Kyusyu Island, while our molecular analyses revealed the distribution of this species from the Yaeyama insular group, the southernmost area of the Nansei Islands, to the Osumi insular group, the northernmost area of the Nansei Islands (Kojima et al., 2003; Hirose, unpublished data). However, B. flectosiphonata has not been discovered in Taiwan (Kojima et al., unpublished data). Batillaria flectosiphonata is designated as a near-threatened species by Kagoshima Prefecture, which includes the Amami insular group (Kagoshima Prefecture, 2003).
Molecular phylogenetic analyses based on nucleotide sequences of 16S and 28S ribosomal RNA genes (Ozawa et al., 2009) and the mitochondrial cytochrome c oxidase subunit I (COI) gene (Kojima et al., 2001, 2003) revealed that B. flectosiphonata forms a monophyletic group with B. multiformis, which is distributed north of Okinawajima Island (Fig. 1; Kojima et al., 2003, Hirose, unpublished data). These two species can be distinguished based on nucleotide sequences of the COI gene, although the monophyly of B. flectosiphonata was supported only by a low bootstrap probability in the previous molecular phylogenetic analyses (Kojima et al., 2003). Masuda and Hayase (2000) questioned the validity of the diagnostic characteristics of B. flectosiphonata, and Kojima et al. (2003) noted that the diagnostic characteristics in B. flectosiphonata were rather gradual, with some specimens that could not be identified based on these morphological characteristics. Batillaria flectosiphonata and B. multiformis coexist in some tidelands on Amami-Oshima Island (Kojima et al., 2003), and the shell morphologies of B. flectosiphonata and B. multiformis were more similar to each other in these areas (Hirose, unpublished data). Although this phenomenon may be attributed to adaptation to the environments in habitats where they coexist, as shown for the shell color polymorphism of Batillaria snails (Miura et al., 2007), the possibility of hybridization between them cannot be denied. To confirm the validity of B. flectosiphonata, it is necessary to demonstrate the reproductive isolation between the two species within the sympatric habitat via the combined use of mitochondrial DNA and nuclear DNA markers such as microsatellites.
A complicated geographical genetic structure was reported for direct-developing B. attramentaria (Kojima et al., 2004). Batillaria flectosiphonata was also shown to be genetically differentiated among local populations inhabiting four insular groups of the Nansei Islands, namely the Amami, Okinawa, Miyako, and Yaeyama insular groups, with the exception of a few individuals with mitochondrial DNAs of the Amami-type inhabiting a restricted area on Okinawajima Island (two of the 20 individuals from the estuary of the Oura River; Fig. 1; Kojima et al., 2003). Individuals of the Osumi insular group are closely related to those of the Amami insular group (Hirose, unpublished data). Such a population structure may be attributed to the low dispersal ability of this species. However, the mode of development of B. flectosiphonata has not been identified.
Fig. 1.
Schematic illustration of the phylogenetic relationships among Batillaria multiformis and four monophyletic local groups of B. flectosiphonata and their distribution areas. Monophyly of B. flectosiphonata was not clearly supported by the previous molecular phylogenetic analysis (Kojima et al., 2003). Closed circles denote sampling sites: A, the Sakashita River, Amami-Oshima Island; B, the Okazen Tideland, Tokunoshima Island; and C, Nagahama, Yomitan, Okinawajima Island.
In this study, we examined the reproductive isolation of B. flectosiphonata and B. multiformis inhabiting a single tideland in Amami Oshima Island, using microsatellite markers developed by Itoh et al. (2015) and observed the development of B. flectosiphonata in order to obtain relevant information, not only for evolutionary and ecological studies but also from the perspective of conserving biodiversity in tidelands around Japan.
MATERIALS AND METHODS
Microsatellite analyses
In order to examine the reproductive isolation of B. flectosiphonata and B. multiformis, a total of 34 individuals of Batillaria snails were collected for microsatellite analyses in the estuary of the Sakashita River, Amami-Oshima Island (Fig. 1) and stored at –30°C. Total DNA was extracted from the head-foot region of each individual using the short PK-GR method (Böttger-Schnack and Machida, 2011). Genotyping of mtDNA was performed using a multiplex PCR-based method (Hirose et al., 2014).
Nine of the 10 microsatellite loci developed for B. flectosiphonata (Itoh et al., 2015) were used in the microsatellite analyses. Only the remaining single locus (Bflec13) in Itoh et al. (2015) could not be amplified in most individuals of B. multiformis. Each 5 μL of the multiplex PCR mixture contained 0.1 μM of each universal probe, 0.1 μM forward primer, 0.2 μM reverse primer, 2 μL 2 ×ばつ QIAGEN Multiplex PCR Master Mix (Multiplex PCR Kit, QIAGEN), and 1 μL of DNA. PCR was performed under the following conditions: initial denaturation at 95°C for 15 min, followed by 35 cycles of 94°C for 30 s, 59°C for 30 s, and 65°C for 30 s, with a final extension at 60°C for 30 min. Fragments were analyzed by electrophoresis using an ABI3130 automated DNA sequencer with GeneScan 500 Liz (Applied Biosystems). Allele size was determined using Peak Scanner Software (version 2.0; Applied Biosystems). Microsatellite data from 27 individuals of B. flectosiphonata, which were collected in the same estuary, identified based on the mtDNA, and whose microsatellites were analyzed by Itoh et al. (2015), were used in subsequent analyses.
The Deviation from the Hardy-Weinberg equilibrium (HWE) for each locus and linkage disequilibrium was tested using Genepop version 4.7 (Raymond and Rousset, 1995; Rousset, 2008) and the exact probability tests (Markov chain parameters: 10,000 dememorizations, 100 batches, and 1000 iterations per batch), with sequential Bonferroni correction (Rice, 1989). Significance of individual microsatellite allele frequency differences was determined by exact G-tests implemented in Genepop version 4.7. The average gene diversity over nine loci and effective population size (θ) under the infinite-allele model were estimated using Arlequin version 3.5.2.2. (Excoffier and Lischer, 2010).
A Bayesian clustering approach was adopted in Structure version 2.3.4 (Pritchard et al., 2000). The burn-in period was set to 50,000 iterations, following which a Markov chain Monte Carlo (MCMC) program was run 250,000 times. The number of clusters (K) was set to 1–7, and 10 independent runs were conducted for each K value. The K value showing the highest mean likelihood LnP(K) and ΔK values was determined to be the most suitable (Pritchard et al., 2000; Evanno et al., 2005) using the online program Structure Harvester version 0.6.94 (Earl and von Holdt, 2012). In the run with the most suitable K, the result showing the highest posterior probability (Ln P(D)) was used. The results of the independent runs for each K value were merged using a greedy algorithm in Clumpp 1.1.2 (Jakobsson and Rosenberg, 2007), and a bar plot for each K value was generated using Distruct (Rosenberg, 2004).
Observation of development
In order to reveal the development mode of B. flectosiphonata and compare it with that of B. multiformis, 48 individuals of B. flectosiphonata-like snails were collected from the Okazen Tideland, Tokunoshima Island of the Amami insular group (Fig. 1) on 11 February 2013. They were transferred to the Atmosphere and Ocean Research Institute, the University of Tokyo, in Chiba Prefecture, and housed in a tank (12 ×ばつ 22 cm w, 17 cm h) filled with seawater to 3 cm above the bottom sediment. The tank was kept at room temperature (approximately 25°C) in the laboratory. The size of the egg capsule was measured with an ocular micrometer using a stereomicroscope, and embryo development was observed. An individual juvenile was preserved in 99% ethanol.
Total DNA was extracted from the preserved juvenile using DNeasy Blood & Tissue Kits (QIAGEN, Valencia, CA, USA) and purified using Gene Releaser (BioVentures Inc., Murfreesboro, TN). A part of the mitochondrial COI gene of the juvenile was amplified by polymerase chain reaction (PCR) using the primer sets LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′; Folmer et al., 1994) and COI-6 (5′-GGRTARTCNSWRTANCGNCGNGGYAT-3′; Shimayama et al., 1990). The PCR conditions were as follows: an initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 50°C for 1 min, and extension at 72°C for 90 s, with a final extension at 72°C for 7 min. The PCR product was purified using Exo-SAP-IT (United States Biochemical, Cleveland, OH). Purified PCR products were used for the cycle sequencing reactions, carried out using a BigDye Terminator Cycle Sequencing Kit, version 3.1 (Applied Biosystems, Foster City, CA, USA) with the same PCR primers.
More than 20 individuals of B. flectosiphonata were collected from a small tideland in Nagahama, Yomitan, Okinawajima Island of the Okinawa insular group (Fig. 1) on 15 December 2016. These snails were transferred to the Atmosphere and Ocean Research Institute, the University of Tokyo, in Chiba Prefecture, and housed for 6 months under the same conditions in the laboratory, except for tank size (17 ×ばつ 22 cm w, 17 cm h).
RESULTS
Microsatellite analyses
Based on the mtDNA, 34 Batillaria snails from the estuary of the Sakashita River were identified as 32 individuals of B. multiformis and two individuals of B. flectosiphonata. For 32 individuals of B. multiformis and 29 individuals of B. flectosiphonata, the number of alleles per locus and the observed and expected heterozygosities of microsatellites are summarized in Table 1. Three loci of B. multiformis showed deviation from the Hardy-Weinberg equilibrium. No significant linkage disequilibrium was found for any pair. No polymorphisms were detected in Bflec49 in B. flectosiphonata. Fewer alleles were obtained from B. flectosiphonata (3.33 ± 2.24) than B. multiformis (5.78 ± 4.30). The average gene diversity over nine loci of B. flectosiphonata (0.307 ± 0.183) was lower than that of B. multiformis (0.425 ± 0.250), and the effective population size (θ) under the infinite allele model of the former (0.45) was lower than that of the latter (1.02).
The Bayesian clustering analysis based on nine microsatellite markers showed the highest LnP(K) and ΔK values for the model with K = 2 (see Supplementary Table S1 (zs210125_TableS1.pdf)). When K = 2, all individuals of B. multiformis were classified into a cluster that was clearly separated from another cluster, which had all B. flectosiphonata (Fig. 2, and see Supplementary Figure S1 (zs210125_FigS1.pdf)). The analysis based on only six loci without deviation from the Hardy-Weinberg equilibrium also showed the highest LnP(K) and ΔK values for the model with K = 2 and almost same clustering (see Supplementary Figure S2 (zs210125_FigS2.pdf)). In addition, exact G-test analyses revealed significant differences in the allele frequencies of the two species for all nine microsatellites scored (see Supplementary Table S2 (zs210125_TableS2.pdf)) and across all loci (P < 0.001). These results showed that strong reproductive isolation was established between the two species.
Table 1.
Number of alleles per locus (R) and the expected (He) and observed (Ho) heterozygosities for individuals of Batillaria flectosiphonata and B. multiformis. Significant deviations from the Hardy-Weinberg equilibrium (HWE) after sequential Bonferroni correction (P < 0.05) are highlighted in bold.
ta_zs210125_001.gif
Development mode of B. flectosiphonata
Reared B. flectosiphonata-like snails from Tokunoshima Island deposited egg capsules around 8 June 2013. The capsules were not sessile but cohesive. Each capsule contained only a single egg, and the veliger larval stage was completed within the egg capsules. The maximum diameter of the egg capsules was 480–710 μm (N = 30). Their larval features were lost before they hatched as crawling juveniles with coiled shells (Fig. 3A). The crawling and floating behaviors of juveniles after hatching was often observed (Fig. 3B). The nucleotide sequence of the COI gene (1020 bp) of the juvenile was determined and deposited in the DNA Data Bank of Japan (DDBJ), European Molecular Biology Laboratory (EMBL), and National Center for Biotechnology Information (NCBI) GenBank databases under the accession number LC652444. This sequence was almost identical (923/924 bp) to the reported sequence of B. flectosiphonata from Amami-Oshima Island (DDBJ accession number, AB845832).
Fig. 2.
Bayesian clustering of 29 individuals of Batillaria flectosiphonata and 32 individuals of B. multiformis from the estuary of Sakashita River, Amami-Oshima Island into two genetically distinguishable clusters. Each individual is indicated by a vertical bar colored according to the frequency of the genetic characters of the clusters.
Fig. 3.
A juvenile of Batillaria flectosiphonata from Tokunoshima Island within an egg capsule (A) and crawling after hatching (B); and a deposited egg capsule (C) and a veliger larva within an egg capsule (D) obtained from individuals collected in Okinawajima Island. Scale bars: 500 μm. Abbreviations: c, egg capsule; e, egg; es, eye spot; f, foot; j, juvenile; sh, shell; sn, snout; v, veliger larva; vl, velar lobe.
Spawning of the individuals from Okinawajima Island was confirmed on 25 March 2017 (Fig. 3C). Egg capsules were similar in shape and size (540–630 μm in maximum diameter, N = 4) to those of snails from Tokunoshima Island. Each capsule contained a single egg, which developed into a veliger larva of up to 190 μm in shell diameter without hatching (Fig. 3D). Unfortunately, these adult snails and larvae accidentally died. As a result, subsequent developmental processes could not be observed.
DISCUSSION
The present microsatellite analyses of sympatric populations of B. flectosiphonata and its sister species B. multiformis in a tideland on Amami-Oshima Island revealed that these species are reproductively isolated from each other, confirming the validity of B. flectosiphonata, whose monophyly was not clearly supported by the previous molecular phylogenetic analysis (Kojima et al., 2003). Kojima et al. (2003) suggested allopatric speciation between the two species based on molecular phylogenetic analysis. They hypothesized that the formation of the Tokara Gap between the Osumi and Amami insular groups promoted divergence of B. multiformis and B. flectosiphonata in the Pliocene or Pleistocene. After isolation from the Nansei Islands, the ancestor of B. multiformis genetically diverged on the Japanese Islands and might have secondarily colonized the Amami and Okinawa insular groups during the glacial period, coinciding with a period in which the Kuroshio Current was suggested to have weakened and/or have not passed through the Tokara Gap (Gallagher et al., 2015).
The present study showed that B. flectosiphonata produces benthic juveniles through the direct development of individuals on Tokunoshima Island, the Amami insular group. The egg capsule of individuals from Okinawajima Island differed from that of the planktotrophic B. multiformis (Furota et al., 2002). The egg capsule of B. multiformis is elliptical with a maximum diameter of 620 ± 51 μm and the egg capsules connect to form a filamentous string; each capsule contains five–nine eggs (Furota et al., 2002). These findings corroborate the direct development of the individuals of Okinawajima, similar to those of Tokunoshima, although their crawling juveniles were not observed in the present study. Batillaria flectosiphonata inhabiting the Amami and Okinawa insular groups belong to different lineages that are the most phylogenetically distinct within the species (Kojima et al., 2003), strongly suggesting that this developmental mode is a characteristic of this species. A molecular phylogenetic analysis based on the mitochondrial COI gene showed that local populations of B. flectosiphonata of four insular groups consisted exclusively of unique sets of monophyletic haplotypes, with a single exception (Kojima et al., 2003). Kojima et al. (2003) highlighted that the local populations of each insular group have been isolated from each other and that they expected that the dispersal ability of this species is not very high. The present results reveal that B. flectosiphonata produces benthic juveniles without a planktonic larval period, which supports this expectation. The direct development as well as the isolation by long seaway distances between insular groups are thought to have promoted the clear geographic population structure of this species.
Molecular phylogenetic analyses by Kojima et al. (2001) and Ozawa et al. (2009) revealed that B. flectosiphonata forms a monophyletic group with not a direct developer, B. attramentaria, but rather with B. multiformis, which has a planktotrophic larval period of approximately 20 days (Furota et al., 2002; Kimura. personal communication). Another Japanese species, B. zonalis, also produces planktotrophic larvae, with a larval period of 7 days (T. Kimura, personal communication). The transitions of developmental modes of gastropods have occurred from the planktotrophic to non-feeding developmental modes containing direct development in most cases (Hart, 2000; Hookham and Page, 2016). In particular, few examples of the transition from the direct to planktotrophic developmental mode are known in gastropod lineages of species that lay a single egg in a capsule without nurse eggs (Hookham and Page, 2016), such as B. flectosiphonata (Fig. 3) and B. attramentaria (Adachi and Wada, 1997; Furota et al., 2002). Loss of the planktonic larval stage is relatively common and occurs frequently among many lineages of mollusks (Li and Foighil, 2016; Wiggering et al., 2020). Direct development is thus thought to have evolved twice within batillariids in Japanese waters, which form a monophyletic lineage (Ozawa et al., 2009). The genetic and environmental background of the evolution of direct development in Batillaria snails is a very interesting issue.
The geographical range of B. flectosiphonata is confined to the Nansei Islands and this species consists of four genetically distinct local groups (Fig. 1). On the other hand, local populations of B. multiformis are connected over its distributional range, from the northernmost part of Honshu Island (the Japanese mainland) to the Okinawa insular group, via the dispersal of planktonic larvae with a long larval period (Kojima et al., 2003). The present results show the significant effects of developmental modes on the genetic diversity of coastal species. The number of alleles and genetic diversity of B. multiformis were larger than those of B. flectosiphonata in the sympatric habitat. These results might be attributed to the repetitive colonization of planktotrophic larvae from many remote habitats via long-distance dispersal.
The taxonomic status and information on genetic diversity are very important from the viewpoints of not only biology, but also the conservation of representative benthic groups of Japanese tidelands. Batillaria flectosiphonata has low dispersal ability owing to its direct development and indeed, genetically deviates into independent lineages (Kojima et al., 2003). The geographic range of each lineage is markedly restricted relative to that of other Japanese Batillaria species (Kojima et al., 2003, 2004, 2005). Although direct developers are thought to be relatively resistant to a decrease in tidelands (Furota et al., 2002), the local lineages might be fatally affected by catastrophic natural disturbances along with the destruction of the coastal environment by human activities, such as reclamation and runoff of red soil. For conservation of biodiversity, accurate recognition of valid species is indispensable (Dubois, 2003; Ely et al., 2017). The present approach using sympatric populations is very promising for such a purpose and information about the developmental mode of coastal species is also important for conservation planning and practices. As B. flectosiphonata is one of the dominant species of tidelands in the Nansei Islands, conservation of its genetic diversity will help to sustain healthy environments in this region.