Introduction
Marine phylogeographers have been attempting to reveal the formation mechanisms of genetic connectivity responsible for regional fauna (Crandall et al., Reference Crandall, Riginos, Bird, Liggins, Treml, Beger, Barber, Connolly, Cowman, DiBattista and Eble2019). Our knowledge of the genetic structures of various marine organisms has been greatly enhanced by recent advances in molecular methods. However, there is a large bias among the invertebrate taxa that have been genetically studied, and some of the neglected taxa play important roles in the marine ecosystem (Keyse et al., Reference Keyse, Crandall, Toonen, Meyer, Treml and Riginos2014). To address this research gap, which makes it difficult to compare the various marine species, it will be necessary to shed light on the level of genetic diversity and differentiation of those invertebrate taxa that remain enigmatic.
The order Polycladida (Platyhelminthes: Rhabditophora) is a group of marine flatworms living in various environments (Newman & Cannon, Reference Newman and Cannon2003; Quiroga et al., Reference Quiroga, Bolaños and Litvaitis2006; Oya & Kajihara, Reference Oya and Kajihara2019). Polycladida is thought to have about 800 species (Martín-Durán & Egger, Reference Martín-Durán and Egger2012). However, the potential species diversity of Polycladida seems to be high, since new polyclad species were described one after another by the ‘energetic polycladologists’ (a term coined by J. Bahia in Dittmann et al., Reference Dittmann, Cuadrado and Aguado2019: e.g. Bahia et al., Reference Bahia, Padula, Correia and Sovierzoski2015; Bahia & Schrödl, Reference Bahia and Schrödl2016; Tsuyuki et al., Reference Tsuyuki, Oya and Kajihara2019). Besides, molecular studies have highlighted polyclad phylogenies (e.g. Litvaitis et al., Reference Litvaitis, Bolaños and Quiroga2010; Aguado et al., Reference Aguado, Noreña, Alcaraz, Marquina, Brusa, Damborenea, Almon, Bleidorn and Grande2017; Bahia et al., Reference Bahia, Padula and Schrödl2017; Tsunashima et al., Reference Tsunashima, Hagiya, Yamada, Koito, Tsuyuki, Izawa, Kosoba, Itoi and Sugita2017). Despite such work, polyclad species are still not well studied compared with other marine animals (Keyse et al., Reference Keyse, Crandall, Toonen, Meyer, Treml and Riginos2014; Dittmann et al., Reference Dittmann, Cuadrado and Aguado2019; Litvaitis et al., Reference Litvaitis, Bolaños and Quiroga2019). Furthermore, the genetic diversity and evolutionary history of these polyclad species are relatively unknown due to a lack of population genetic studies.
While most of the polyclad species have a free-living lifestyle, some have an association with invertebrates: symbiosis in Ophiuroidea (Doignon et al., Reference Doignon, Artois and Deheyn2003), commensalism in hermit crabs (Lytwyn & McDermott, Reference Lytwyn and McDermott1976) and gastropods (Faubel et al., Reference Faubel, Sluys and Reid2007), parasitism in chitons (Kato, Reference Kato1935). Around the Japanese Pacific coastal area, the commensal relationships between a polyclad species and the intertidal gastropod have been well studied (Kato, Reference Kato1933; Fujiwara et al., Reference Fujiwara, Iwata, Urabe and Takeda2016).
Stylochoplana pusilla, distributed in the Japanese Pacific coastal area from southern Hokkaido to Kyushu, is found associated with certain intertidal snails (Kato, Reference Kato1933, Reference Kato and Okada1965). After hatching, S. pusilla passes through the planktonic larval phase (7 days) and settles (Deguchi et al., Reference Deguchi, Sasaki, Iwata and Echizen2009). Next, they begin to use intertidal snails as refuge sites in the intertidal zone. At low tide, they hide in the mantle cavity of the intertidal snails and protect themselves from desiccation and predation (Fujiwara et al., Reference Fujiwara, Iwata, Urabe and Takeda2016). In fact, once S. pusilla leaves the snail in the sublittoral zone, S. pusilla is soon preyed upon by predators such as a large flatworm species and gobies (Fujiwara et al., Reference Fujiwara, Urabe and Takeda2014). However, S. pusilla does not use all intertidal snails equally. Fujiwara et al. (Reference Fujiwara, Urabe and Takeda2014) demonstrated that S. pusilla showed a specific preference for Monodonta confusa, although S. pusilla was found in several intertidal snails in Mutsu Bay, the northern part of Honshu, Japan. The range of available species is known to influence the genetic population structure of the user (Li et al., Reference Li, Jovelin, Yoshiga, Tanaka and Cutter2014). However, to our knowledge, no population genetic studies have yet been conducted in polyclad species, including S. pusilla, which have a unique association with certain snails.
Here, we aimed to show the genetic population structure of a Polycladida species. Our model species is S. pusilla. Since data about its dispersal ability is needed to discuss the formation process of the population genetic structure, S. pusilla is a suitable study model. The genetic diversity and level of genetic differentiation among populations were estimated using the mitochondrial Cytochrome c Oxidase subunit I (COI) marker developed by Oya & Kajihara (Reference Oya and Kajihara2017), because the resolution of it was also suitable for the evaluation of the intraspecific variation. Also, we surveyed the intertidal snail species used by S. pusilla. The reason is that although Fujiwara et al. (Reference Fujiwara, Urabe and Takeda2014) studied the preference of S. pusilla in Mutsu Bay, in the whole distribution area of S. pusilla it is unknown if and how S. pusilla makes use of snail species other than M. confusa; despite the distribution of snails potentially available for S. pusilla around the Japanese coastal area (Genus Monodonta and Genus Tegula: Sasaki, Reference Sasaki and Okutani2017; Yamazaki et al., Reference Yamazaki, Hirano, Uchida, Miura and Chiba2019). Our data will help to bridge the research gap of enigmatic invertebrate taxa that are insufficiently genetically studied.
Materials and methods
We collected S. pusilla samples from 17 locations in the Japanese mainland (Table 1, Figure 1). Our study area extends from the north of Honshu to Kyushu within about 2000 kilometres. We recorded what kinds of intertidal snails were used by S. pusilla in all locations. When sampling, the collected snails were placed into species-specific plastic bags containing seawater, and then checked for whether they were used by S. pusilla, which is known to go out into the seawater when snails are submerged (Fujiwara et al., Reference Fujiwara, Iwata, Urabe and Takeda2016). The collected S. pusilla were stored in 99.5% ethanol for subsequent molecular analyses.
Fig. 1. Map of the survey and sampled locations. Numbers in black circles indicate the locations used for genetic population structure analyses (calculation of the genetic diversity indices, AMOVA and pairwise F ST values).
Table 1. Genetic information of each population of Stylochoplana pusilla
N, number of individuals; nH, number of haplotypes; HD, Haplotype diversity; ND, Nucleotide diversity.
Six populations (locality numbers: 1, 5, 9, 10, 12 and 14) were used for population genetic analyses.
The NucleoSpin® Tissue kit (TaKaRa, Shiga Pref., Japan) was used to extract DNA from the tissue, according to the manufacturer's instructions. Fragments of the COI gene were amplified using the primers Acotylea_COI_F (5′-ACTTTATTCTACTAATCATAAGGATATAGG-3′) and Acotylea_COI_R (5′-CTTTCCTCTATAAAATGTTACT ATTTGAGA-3′) (Oya & Kajihara, Reference Oya and Kajihara2017). Polymerase chain reaction (PCR) was performed for the COI gene following the protocol described in Oya & Kajihara (Reference Oya and Kajihara2017): 94°C for 5 min; 35 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 1.5 min; 72°C for 7 min. The PCR products were subsequently purified using Exo-SAP-IT (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK). Cycle sequencing was performed using the PCR primers with the BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA), and the products were directly sequenced from both directions using an ABI 3130xl automated sequencer (Applied Biosystems).
The forward and reverse sequences were assembled using CLUSTALW (Thompson et al., Reference Thompson, Higgins and Gibson1994). We checked the validity of the sequences using a chromatogram viewer and the quality scores of each base using the software package 4Peaks (Griekspoor & Groothuis, Reference Griekspoor and Groothuis2005). The obtained sequences were aligned using MUSCLE v3.8 (Edgar, Reference Edgar2004).
To reconstruct the population genetic structure of S. pusilla using six locations where more than 10 individuals of S. pusilla were collected (locality numbers: 1, 5, 9, 10, 12 and 14), we calculated two genetic indices (haplotype and nucleotide diversity) and estimated the hierarchical analysis of molecular variance (AMOVA; Excoffier et al., Reference Excoffier, Smouse and Quattro1992) and pairwise F ST among populations with 1000 permutations, using Arlequin v. 3.5 for statistical analyses (Excoffier & Lischer, Reference Excoffier and Lischer2010). To visualize the geographic distribution pattern of haplotypes obtained from all S. pusilla individuals, haplotype networks were reconstructed using a TCS network implemented in PopART (Leigh & Bryant, Reference Leigh and Bryant2015).
Results
The snails used by S. pusilla in the 17 locations we surveyed are shown in Table 2. Although S. pusilla mainly used M. confusa at all 17 sampling locations, it also used eight other snail species. Stylochoplana pusilla used three families (Trochidae, Tegulidae and Muricidae) and mainly used two genera, Monodonta and Tegula.
Table 2. The host snails used by Stylochoplana pusilla in the 17 locations surveyed.
In total, 116 S. pusilla COI sequences were obtained, and the alignment length was 615 base pairs. We identified 72 haplotypes from the 116 sequences, and the data are available from GenBank (Supplemental Table S1; accession numbers: LC515251–LC515366). The haplotype diversity and nucleotide diversity of S. pusilla are shown in Table 1 (locality numbers: 1, 5, 9, 10, 12 and 14). The haplotype diversity of six populations was higher than 0.90 (0.921 (locality number 1: Aomori, Aomori) – 0.989 (locality number 5: Miura, Kanagawa)). Nucleotide diversity was 0.00454 (locality number 1: Aomori, Aomori) – 0.01152 (locality number 12: Hiji, Oita). The hierarchical AMOVA showed the existence of a genetic structure in six S. pusilla populations (ΦST = 0.056, P < 0.0001; Table 3). Table 4 shows pairwise F ST among the six populations of S. pusilla. While the population of Hiji, Oita (locality number 12) was genetically differentiated from other populations, significant genetic differentiation among the population was not detected after Bonferroni correction. The haplotype network exhibited no clear geographic structure and showed a complex network, including some dominant haplotypes (Figure 2). However, locality number 12 (Hiji, Oita) had two genetically distantly related haplotypes (12 nucleotide substitution).
Fig. 2. Haplotype network inferred from 116 individuals and 72 haplotypes of Stylochoplana pusilla.
Table 3. Result of hierarchical analysis of molecular variance (AMOVA) among the six populations (locality number: 1, 5, 9, 10, 12 and 14).
Table 4. Pairwise F ST among the six populations of Stylochoplana pusilla
*P < 0.05.
Discussion
Our present study is the first aimed at clarifying the mitochondrial genetic structure of the polyclad species S. pusilla, which has a commensal association with the intertidal snail M. confusa. Although genetic differentiation among local populations of S. pusilla is significant (AMOVA), the level of differentiation is relatively low as no genetically differentiated population pairs were detected after Bonferroni correction and no geographic structure is observed. Also, high genetic diversity within populations is found in S. pusilla. However, some marine species such as fish and intertidal snails along the Japanese coast are genetically differentiated in accordance with geographic structure, such as splitting oceanic currents (fish: Hirase et al., Reference Hirase, Ikeda, Kanno and Kijima2012; snails: Kojima et al., Reference Kojima, Segawa and Hayashi1997; Yamazaki et al., Reference Yamazaki, Miura, Ikeda, Kijima, Van Tu, Sasaki and Chiba2017). In the case of S. pusilla, the low level of genetic differentiation indicates that it possesses sufficient dispersal ability to maintain the opportunities of gene flow among populations (7 days of planktonic larval duration: Deguchi et al., Reference Deguchi, Sasaki, Iwata and Echizen2009).
The present study also revealed that S. pusilla used several intertidal snail species in their whole distribution area. The snails used by S. pusilla are known to exhibit a variety of habitat preferences. For instance, M. confusa, the main available snail for S. pusilla, can inhabit wider coastal environments compared with other congeneric species (Takenouchi, Reference Takenouchi1985; Yamazaki et al., Reference Yamazaki, Miura, Ikeda, Kijima, Van Tu, Sasaki and Chiba2017). Besides, S. pusilla used other intertidal snails such as Tegulidae and Muricidae. Among tegulid species, Tegula xanthostigma lives in an exposed shore while its sister species, Tegula sp., prefers sheltered habitats, like the inner bay (Yamazaki et al., Reference Yamazaki, Hirano, Uchida, Miura and Chiba2019). This suggests that S. pusilla can live in a wide range of coastal exposures due to the utilization of various snails. If populations of the available snail tend to be connected due to a wide range of available habitat, S. pusilla populations are also likely to be connected and weaken the levels of genetic differentiation among the populations. In general, species that can use various host species showed a lower level of genetic differentiation than host-specific species (Li et al., Reference Li, Jovelin, Yoshiga, Tanaka and Cutter2014). In the marine environment, various types of ecological interactions have been reported, and commensal relationships have often been observed (e.g. Williams & McDermott, Reference Williams and McDermott2004). In the case of polyclad species, many are free-living, but some have commensal relationships with other marine organisms (Kato, Reference Kato1933; Lytwyn & McDermott, Reference Lytwyn and McDermott1976; Faubel et al., Reference Faubel, Sluys and Reid2007; Fujiwara et al., Reference Fujiwara, Iwata, Urabe and Takeda2016). However, to date, knowledge on the genetic structure of organisms that have an ecological relationship with other species is missing in marine taxa compared with terrestrial species. The present finding indicates that the association with other marine species might not prevent genetic connectivity among populations of S. pusilla. The wide range of available snail species may help the genetic connectivity among populations. To better understand the relationship between these lifestyles and genetic structure, we have to perform a comparative study using a polyclad species which has an association with marine invertebrates.
We detected the two distant haplotypes of Hiji, Oita (locality number 12). Although the lack of examples of population genetic studies in polyclad species makes it difficult to discuss the cause of these distantly related haplotypes, this implies ancestral polymorphisms (Dillon & Robinson, Reference Dillon and Robinson2009). It is necessary to carry out genetic studies on other polyclad species using not only mitochondrial but also other highly variable nuclear markers, such as microsatellite DNA data and genome-wide SNPs.
In conclusion, the present study showed the low level of genetic population structure of S. pusilla due to their sufficient dispersal ability. Also, a wide range of available snail species may help the above trend. At present, there are no population genetic studies of Polycladida. This study thus could be a framework for future genetic studies. Although detailed genetic and ecological studies are needed, the present study provides important knowledge to help bridge the gap of invertebrate taxa, which have been insufficiently studied.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315420000570.
Acknowledgements
We thank T. Saito and T. Hirano for helpful advice on this study. We also thank T. Seo, S. Uchida, O. Kagawa, S. Ito and K. Endo for sampling and useful information.
Financial support
Daishi Yamazaki was supported by the Sasakawa Scientific Research Grant from The Japan Science Society (Research number: 2018-5017).
