Species diversity of genera Peronia, Wallaconchis and Onchidella (Gastropoda: Onchidiidae) in intertidal coral reef environments of the Ryukyu Islands

doi:10.21203/rs.3.rs-5000235/v1

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Species diversity of genera Peronia, Wallaconchis and Onchidella (Gastropoda: Onchidiidae) in intertidal coral reef environments of the Ryukyu Islands

https://doi.org/10.21203/rs.3.rs-5000235/v1

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Three genera of the family Onchidiidae, Peronia, Wallaconchis and Onchidella, are known to inhabit intertidal coral reef environments in the Ryukyu Islands, however, there have been only limited studies on their diversity. This study conducted surveys around 11 islands and 40 intertidal areas of the Ryukyus, elucidating species diversity utilizing DNA barcoding of specimens. Molecular data indicated that intertidal coral reef areas of the Ryukyu Islands harbor at least eight Onchidiidae species from three genera. The highest species cohabitation in the same intertidal areas was observed around Okinawa-jima Island; five species from three genera. Peronia spp. were found from all sites investigated in this study and the distribution ranges of four species of Peronia in the Ryukyu Islands were updated from past research. Three species of Wallaconchis; W. ater, W. nangkauriensis, and Wallaconchis sp., were found in intertidal coral reef environments, mostly in flat areas with small rubble and rocks. Onchidella sp. inhabited intertidal areas and was found on many more islands of the Ryukyus than previously had been documented. All Onchidella sp. specimens collected from the Ryukyus were genetically distinct from described species. This study enhances the understanding of Onchidiidae diversity in intertidal coral reef environments of the Ryukyu Islands, providing a baseline dataset for better conservation of this understudied group.

Nansei Islands

Japan

DNA barcoding

phylogeny

Mollusca

The subtropical Ryukyu Islands are the southernmost region of Japan, and are characterized by elevated coral reefs, with fringing reefs around most of the islands that create coral reefs and hard substrate intertidal habitats (Nakazato 2013). As well, inner reefs here are generally flat, and can be up to several hundred meters wide and 0.5 to 1 m deep (Iryu et al. 1995). Particularly, intertidal areas often exhibit a combination of various environmental characteristics, including rocks, coral rubble, and receive influence from freshwater input, and marine tidal fluctuations, creating unique ecological niches (Van der Meiji et al. 2017; Koreeda and Motomura 2023), and those in the Ryukyus are no exception. However, reefs of the Ryukyus face severe threats from extensive coastal development, ongoing land reclamation, and overexploitation (Roberts et al. 2002; Masucci and Reimer 2019), driven by demands including tourism, industrial, and US military operations (Iryu et al. 1995; Okuzono et al. 1997; Maruta et al. 2011; Chapman 2013; McCormack 2020; Nakajima et al. in press). At the same time, the Ryukyu Islands hold numerous endemic species, both in freshwater and marine environments, as well as on the land (e.g. Miyagi 1992; Roberts et al. 2002; Miyagi 2016; Shinyashiki et al. 2019), and the region has been deemed a top conservation priority (Roberts et al. 2002).

Family Onchidiidae Rafinesque, 1815 is comprised of intertidal air-breathing sea slugs, inhabiting tropical to temperate regions worldwide (Britton 1984). They mostly inhabit intertidal habitats, such as mangrove forests to littoral areas, and are especially common in the Indo-Pacific (Dayrat et al. 2011a). Different genera are often found in different parts of intertidal zones, such as Peronia and Onchidella in upper intertidal zones with hard substrate shores, Wallaconchis on sandy tidal flats, Paromoinchis and Onchidium in muddy mangrove areas, and Platevindex on mangrove tree logs (Hyman 1999; Dayrat 2009; Fukuda 2014; Goulding et al. 2022). Because Onchidiidae species release their larvae into the sea, they are considered to be marine species (Britton 1984; Deshpande et al. 1979).

In the intertidal coral reef environments of the Ryukyus, species of Peronia, Fleming 1822, Onchidella J. E. Gray, 1850 and Wallaconchis Goulding & Dayrat, 2018 have been previously reported (Baba 1958; Taki 1959; Okutani 2000, 2017; Ueshima 2014; Takagi et al. 2019). Despite in situ images of living specimens and basic anatomical information in guidebooks and literature, frequent misidentifications of these groups have occurred due to taxonomic confusion (Hyman 1999; Dayrat 2009; Dayrat et al. 2011a), likely rooted in high similarities in gross morphology, as well as limited information on colour variation and papillae patterns (Fukuda 2003). Furthermore, few comprehensive studies have been conducted focusing on the diversity and distribution of intertidal onchidiids in the Ryukyu Islands, particularly using DNA barcoding techniques. As many intertidal flats have been altered by coastal development, some Onchidiidae species listed in the Red Data Book of Okinawa as near-threatened vulnerable species (Ueshima 2014). Thus, there is a pressing need to conduct assessments of the species diversity of intertidal onchidiids and to apply measures for evaluating and, if needed, mitigating extinction risks.

In order to address these gaps, this study had two main objectives via the utilization of molecular DNA barcoding analyses and basic morphological analyses:

To refine distribution ranges of Peronia spp. in the Ryukyu Islands, updating results from Mizukami et al. (2022) with data from additional islands, and

To better understand the species compositions and distribution ranges of Onchidella and Wallaconchis in the Ryukyu Islands, and obtain images of living specimens in situ along with comprehensive molecular data.

Specimen collection and morphological data analyses

Specimen collections were carried out via reef walking in intertidal zones during low tide, with one to three hours of search time per site. In this study, a total of 349 specimens of family Onchidiidae were newly collected from 11 islands of the Ryukyus; Iriomote-jima, Okinawa-jima, Ie-jima, Izena-jima, Iheya-jima, Yoron-jima, Okinoerabu-jima, Tokunoshima, Amami-Oshima, Kikai-jima and Yakushima (Fig. 1, Table 1).

Specimens were tentatively identified to genus level by gross morphology in the field. No whole specimens of Peronia or Wallaconchis were collected from Yoron-jima, Okinoerabu-jima, Tokunoshima or Kikai-jima islands, with only mantle tissues obtained and no lethal sample collections. In these cases, specimens were photographed with rulers in situ, and a 1 x 1 cm² mantle tissue portion was cut with dissection scissors and preserved in 99% ethanol for further molecular analyses. Whole specimens of Peronia and Wallaconchis from Iriomote-jima, Okinawa-jima, Ie-jima, Iheya-jima and Izena-jima, as well as all specimens of Onchidella, were photographed to examine detailed characteristics, such as colors and patterns on dorsal and ventral sides of specimens. Subsequently, mantle tissues of specimens were preserved in 99% ethanol for molecular analyses, and either 70% ethanol for future anatomical examination or frozen for future chemical analyses.

Lengths and widths from photographs of specimens that captured both scales and whole bodies were calculated to the nearest mm with scales using ImageJ (Schneider et al. 2012) (Supp. Table 1).

All specimens used in this study are deposited in Fujukan, University of the Ryukyus Museum, and the National Museum of Nature and Science, Tokyo. Collection permits were not needed for Peronia, Onchidella and Wallaconchis in Okinawa Prefecture, and collection permits for Amami Oshima National Park were covered by Kagoshima Prefecture permit Shirei Oshima Rinsui.

DNA extraction, PCR amplification and sequencing

We extracted DNA from preserved tissue specimens and/or mantle tissues from whole specimens in 99% ethanol using a Qiagen DNeasy Blood and Tissue Kit (Qiagen, Tokyo, Japan) following the manufacturer’s protocols. Polymerase chain reaction (PCR) was carried out for amplifying mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal DNA (16S rDNA), which have been previously used for species delimitation in family Onchidiidae (Chang et al. 2018; Takagi et al. 2019; Dayrat et al. 2020; Goulding et al. 2022). Primers used in this study were a universal COI primer set, LCO1490 and HCO2198 (Folmer et al. 1994), and for 16S primers, 16sar-L and 16sbr-H (Palumbi 1996).

PCR amplifications were conducted for both markers using 20 µl reaction volumes, comprising 2 µl of coral dye 10X, 0.4 µl of dNTP, 0.1 µl of Taq (Qiagen, Tokyo, Japan), 1 µl each of forward and reverse primers, 13.5 µl of H₂O, 1 µl of MgCl and 1 µl of extracted DNA template. The following thermo-cycler conditions were used to successfully amplify approximately 700 base pair (bp) regions of COI and 600 bp regions of 16S; an initial denaturation at 95°C for 5 min, further denaturation at 94°C for 30 s, annealing at 45 to 50°C for 30 s (depending on specimens), and extension at 72°C, for 1 min for 39 cycles, and final elongation at 72°C for 5 min. Amplified DNA was checked with electrophoresis and successfully amplified PCR products were cleaned and purified with Exonuclease I-Shrimp Alkaline Phosphatase (ExoSAP). Sequencing in both directions was carried out at FASMAC (Kanagawa, Japan).

Phylogenetic analyses

The quality of newly acquired sequences were checked on Geneious Prime 2024.04.01 (https://www.geneious.com), then aligned, and edited using MUSCLE 5.0 (Robert 2004). All sequences obtained were compared with previously reported GenBank sequences of Peronia, Wallaconchis and Onchidella using the BLAST function for both COI and 16S markers. Additional sequences of Veronicella cubensis were retrieved from GenBank as outgroup for phylogenetic analyses (Supp. Table 1) (Gomez et al. 2013). Moreover, previously reported GenBank sequences of 11 species of subfamily Onchidellinae and 10 species of Wallaconchis were retrieved for reconstructing the trees of each genus (Goulding et al. 2018; Goulding et al. 2022; Supp. Table 1). Consensus sequences and retrieved sequences of both markers were aligned and trimmed to the same length, and the alignments of the two markers were concatenated using Sequence Matrix version 1.8 (Vaidya et al. 2011). All newly obtained sequences were registered in GenBank under accession numbers PQ157970 to PQ158260, and PQ162847 to PQ163110 (Supp. Table 1).

Phylogenetic trees were reconstructed using alignments of COI and 16S markers, as well as the concatenated sequence dataset. The best fit model of sequence evolution was confirmed based on AIC criterion (Akaike 1974) for both COI and 16S in MEGA X (Kumar et al. 2018) in default settings. Models for both COI and 16S were HKY G + I. Bayesian inference (BI) analysis was conducted using MrBayes version 3.2.7 (Ronquist and Huelsenbeck 2003). Bayesian Markov chains were executed with two sets of three chains, each running for 2 x 10⁶ generations and continued as needed until split frequencies went between 0.01 to 0.05 convergence. Sampling every 100 generations and a burn-in of 25% was applied. Maximum likelihood (ML) analyses were constructed employing the same concatenated dataset using RaxML-NG version 1.0.2 (Kozlov et al. 2019). The “all” command was utilized to carry out tree search along with 1,000 bootstrap repetitions. For BI analyses, significance was defined as Posterior probabilities (PP) ≥ 0.9 and in ML analyses, significance was determined by bootstrap values ≥ 75%. FigTree v1.4.3 (Rambaut 2016) was employed for final visualization and coloring of trees.

A total of 11 islands and 40 intertidal sites were investigated (Fig. 1, Table 1), consisting of four sites around Iriomote-jima, 15 sites around Okinawa-jima, three sites around Ie-jima, three sites around Izena-jima, two sites around Iheya-jima, three sites around Yoron-jima, two sites around Okinoerabu-jima, two sites around Tokunoshima, two sites around Amami-Oshima, three sites around Kikai-jima, and one site around Yakushima (Supp. Table 1).

From a total of 349 specimens newly collected in this study, 229 specimens of Peronia, 19 of Wallaconchis, and 63 of Onchidella were identified to genus level. A total of 210 sequences for Peronia, 15 for Wallaconchis, and 39 for Onchidella were successfully obtained for COI, along with 225 sequences for Peronia, 17 for Wallaconchis, and 49 for Onchidella for 16S sequences. Combined with sequences generated in Mizukami et al. (2022), the final concatenated alignment was trimmed to encompass a total of 387 taxa and 782 bp: including 342 taxa with a length of 435 bp for COI and 365 taxa with a length of 347 bp for 16S rDNA.

Phylogenetic analyses

The phylogenetic trees of the three genera of Onchidiidae using the concatenated sequence dataset of COI and 16SrDNA mitochondrial marker sequences resulted in conflicting topologies between the Bayesian Inference (BI) and Maximum Likelihood (ML) methods (Supp Figs. 1 & 2). Based on these results and BI analyses using the COI dataset, we determined that the intertidal coral reef environments of the Ryukyu Islands are likely to harbor eight species from three genera of Onchidiidae (Fig. 2).

We found four species from Peronia: P. peronii (Cuvier, 1804), P. verruculata (Cuvier, 1830), P. setoensis Dayrat & Goulding, 2020 and P. okinawensis Dayrat & Goulding, 2020, among our specimens. All islands of the Ryukyus were known to harbor at least one Peronia species previously, but here we confirmed that four species of Peronia inhabit intertidal areas of Ie-jima and Iheya-jima islands. We also confirmed the presence of three species of Peronia: P. peronii, P. okinawensis and P. setoensis from Izena-jima and Okinoerabu-jima islands. Similarly, P. verruculata, P. peronii and P. setoensis were found around Tokunoshima, and P. verruculata, P. peronii, and P. okinawensis were found around Yoron-jima. As well, the presence of P. setoensis from Kikai-jima was confirmed for the first time. Moreover, P. peronii was observed in Yakushima, extending the northernmost distribution range of this species. Similarly, P. okinawensis was observed in Iriomote-jima for the first time, representing the southernmost distribution range of this species.

We also confirmed the presence of Wallaconchis ater (Lesson, 1831) units #1 and #2 (Goulding et al. 2018), W. nangkauriensis (Plate, 1893), and another, genetically distinct species, here designated as Wallaconchis sp., based on phylogenetic analyses (Figs. 2 & 3 & 4). The literature for Wallaconchis was scarce for the Ryukyus, but it is likely these represent the first records of W. ater and W. nangkauriensis from Izena-jima. Wallaconchis sp. was located among W. melanesiensis unit #1 and unit #2, and formed a unique clades with 13–16% and 20–22% genetic distances from its nearest relatives for each marker, respectively (Supp. Figure 3).

We also found one species from Onchidella in our specimens from intertidal coral reef environments of the Ryukyus (Figs. 2 & 4, Supp. Figures 1 & 2). All Onchidella specimens collected in this study fell into one monophyletic clade, different from any other sequenced Onchidella species (Supp. Figure 4). 19–20% genetic differences were observed in COI and 16S markers between Onchidella sp. and previously reported GenBank sequences of the Japanese species Onchidella kurodai (Iw. Taki 1935). We refer to this species as Onchidella sp. in this study. Our specimens represent new records for Onchidella around Okinoerabu-jima, Tokunoshima and Kikai-jima islands.

Species distributions

Species distributions across the Ryukyus

Peronia was observed in all sites of the Ryukyus investigated in this study (Table 2). P. verruculata was the most numerous among our specimens from Okinawa-jima and Iriomote-jima islands, was found from all sites investigated at both islands, except for one site around Iriomote-jima. However, P. verruculata was significantly lower in numbers or absent around the remaining nine Ryukyu Islands, and only a total of seven specimens were collected from these nine islands. On the other hand, total numbers of P. peronii, P. okinawensis and P. setoensis collected from these nine islands were similar; 46 specimens of P. okinawensis and P. setoensis, and 54 specimens of P. peronii (Table 2). P. okinawensis was most numerous around Ie-jima, Iheya-jima, and Yoron-jima islands, and P. peronii was most abundant around Okinoerabu-jima and Yakushima islands. P. setoensis had its highest abundance around Kikai-jima in this study.

For genus Wallaconchis, W. ater had the highest abundance, with eight specimens collected from two sites around Okinawa-jima and one site around Izena-jima. Both intertidal areas had numerous small coral rubble and rocks (Table 2). Five specimens of W. nangkauriensis were exclusively found at the same intertidal area of Izena-jima, and six specimens of Wallaconchis sp. were collected from Okinawa-jima, Tokunoshima and Kikai-jima. Wallaconchis sp. had slightly a wider distribution range than the other Wallaconchis species, but they were not collected from Izena-jima, where W. ater and W. nangkauriensis sympatrically coinhabited in large numbers. Wallaconchis sp. coinhabited with W. ater around Okinawa-jima.

Specimens of Onchidella sp. were found at five sites around Okinawa-jima, one site around Okinoerabu-jima, one site around Tokunoshima, one site around Amami-Oshima, and two sites around the Kikai-jima islands (Table 2). At these islands, they appeared in large numbers when found in the intertidal area.

Overlapping distribution ranges

Two or more genera coexisted in many intertidal areas we investigated. Onchidiidae species displayed patchy distributions within intertidal areas, and multiple species often were present on exactly the same rock, whether small or big, covered by Monostroma sp. macroalgae (Fig. 5, a,b).

Peronia spp. exhibited the widest distribution ranges among the three genera, inhabiting the southern (Iriomote-jima) to northern Ryukyu Islands (Yakushima). Onchidella spp. exhibited slightly smaller distribution ranges, from Okinawa-jima to Amami-Oshima and Kikai-jima islands. Also, Onchidella specimens were always found in large numbers in intertidal areas co-existing with Peronia spp. (Fig. 5, a). Wallaconchis had the lowest number of observations, with the exception of at Izana Beach, Izena-jima, which harbored significantly high abundances (Fig. 5, c,d). Wallaconchis spp. also often coinhabited with Peronia spp. (Fig. 5, b).

There were some sites (28/40 sites) where only Peronia was observed, but there were no sites where only Wallaconchis or Onchidella were observed. The most commonly observed cooccurrences were Peronia + Onchidella in the same area (7/40 sites), then Peronia + Wallaconchis (3/40 sites). There were two sites where three genera coinhabited sympatrically in the same intertidal area; Nakagusuku Mall Beach in Okinawa-jima, and Shitooke Beach in Kikai-jima (Table 2). However, the abundance of Wallaconchis was significantly lower than Peronia and Onchidella spp. at both sites.

Consequently, the highest species numbers were recorded from Nakagusuku Mall Beach, Okinawa-jima, where five species from three genera sympatrically coinhabited; two Peronia (P. verruculata, P. peronii), two Wallaconchis (W. ater, Wallaconchis sp.), and one species of Onchidella (Onchidella sp.) (Table 2). The intertidal area was dominated by Peronia and Onchidella with Wallaconchis spp. lesser in numbers.

Morphological analyses

The largest and the smallest specimens of each species collected in this study were; P. verruculata (largest: length 70 mm/ width 37 mm, smallest: 1.5 mm/ 1 mm, length average: 35.65 ± 12.58, n = 101), P. peronii (137/82 mm, 14/8 mm, 85.68 ± 32.33 mm, n = 53), P. okinawensis (96/57 mm, 7/6 mm, 37.46 ± 16.45 mm, n = 69), P. setoensis (36/25 mm, 4/3 mm, 18.86 ± 7.56 mm, n = 76), W. ater (39/18 mm, 18/8 mm, 26.14 ± 6.87 mm, n = 7), W. nangkauriensis (30/20 mm, 27/18 mm, 28.5 ± 2.12 mm, n = 2), Wallaconchis sp. (55/42 mm, 13/5 mm, 26.33 ± 15.1 mm, n = 6), and Onchidella sp. (12.8/7.5 mm, 2.3/1.5 mm, 6.9 ± 2.3 mm, n = 57) (Supp. Table 1).

From this study, the intertidal coral reef environments of Ryukyu Islands harbored a higher diversity of onchidiid species than previously thought (Takagi et al. 2018; Goulding et al. 2022). The high species diversity of Onchidiidae in this region may be due to the influence of Kuroshio Current in their larval dispersal, as well as unique characteristics of the Ryukyu Islands’ humid subtropical climate, different from other subtropical regions (Miyagi 1992). These conditions are likely suitable for Onchidiidae to inhabit intertidal coral reef environments.

Species diversity of Onchidiidae in intertidal coral reef environments of the Ryukyus

Genus Peronia

Peronia spp. are air-breathing sea slugs inhabiting intertidal reef flats, especially on hard substrates (Hamaguchi and Yoshioka 2002; Dayrat et al. 2020; Goulding et al. 2022). Peronia spp. can be found across tropical and subtropical the Indo-Pacific, from South Africa to Hawaii (Britton 1984), and some species have been frequently observed in temperate to subtropical regions of Japan (Katagiri & Katagiri 2007; Okutani 2000, Wardiatno et al. 2015). Among family Onchidiidae only genus Peronia has branchial gills on the back of the notum, which allows easy distinction from other genera (Dayrat et al. 2009). Four species of genus Peronia have been previously recorded from around the Ryukyu Islands; P. peronii, P. verruculata, P. setoensis and P. okinawensis (Mizukami et al. 2022).

Our previous study showed that the most numerous species around Okinawa-jima was P. verruculata, with P. okinawensis the most abundant around Amami-Oshima (Mizukami et al. 2022). These past observations led to the hypothesis that P. verruculata had a comparatively higher tolerance to disturbed intertidal areas, while P. okinawensis showed a preference for pristine sites. However, this idea was based on the fact that Okinawa-jima is the most densely populated island among the Ryukyus, with an area of 1208 km² and a population of 1.3 million people (= 1,076 people/km², Okinawa Prefectural Office, 2024; Statistics Bureau, Japan 2023). In contrast, Amami-Oshima is listed as a UNESCO Natural World Heritage site and is much more protected as a national park (Amami-Oshima 2021). Moreover, Mizukami et al. (2022) did not observe any species other than P. verruculata in Iriomote-jima, even though the island is also registered as a UNESCO Natural World Heritage Site, with pristine coastlines (Ministry of Environment 2021). Thus, this hypothesis needed to be re-evaluated with specimens from additional islands of the Ryukyus, as well as more sampling effort across Iriomote-jima, Okinawa-jima, and Amami-Oshima islands.

Consistent with Mizukami et al. (2022), P. okinawensis was the most abundant species around many islands of the Ryukyus, namely Ie-jima, Iheya-jima, Yoron-jima islands, all of which have well-preserved intertidal areas. As well, Okinawa-jima had a large number of P. verruculata, where it was the most abundant species. Moreover, P. verruculata was the only species that inhabited all intertidal coral reef environments investigated in this study. For example, P. verruculata was found at the Awase tidal flat, a site with an ongoing (since 2002) reclamation project creating an artificial island (Okinawa Prefecture 2014). Also, unlike other Peronia species, P. verruculata was found directly on artificial structures at Gushikawa Beach, Okinawa-jima. These results further suggest that P. verruculata has a higher tolerance for degraded environments, while P. okinawensis prefers more pristine intertidal areas. However, P. okinawensis was also found at Sunabe and Baba Park, Okinawa-jima, both intertidal areas that are heavily armored and considered as medium to high impacted sites (Masucci and Reimer 2019; DiBattista et al. 2020). Additionally, although P. okinawensis was reported for the first time from Iriomote-jima in this study, the most abundant species in Iriomote-jima was still P. verruculata. Since our study indicates possible relationships between species diversity and artificial coastlines, future studies should investigate coastal development and species compositions of Peronia in the Ryukyu Islands.

There were clear differences in species compositions between sampling months, particularly for P. okinawensis and P. setoensis. In this study, P. okinawensis appeared in samples from May to December, and numbers were highest during June to September and lowest in May and December. These differences in species compositions may be considered as geographical differences and investigated sites, however, sampling in Yoan, Amami-Oshima was conducted in February 2024 and the only Peronia species collected was P. setoensis. Meanwhile, during surveys in Tomori, Amami-Oshima, in November 2021, four Peronia species were collected (Mizukami et al. 2022). The surface water temperature in Amami-Oshima Island during February was approximately 20℃, and 24℃ degrees in November. Similar findings were observed in Baba Park, Okinawa-jima, where only P. setoensis was collected in December 2021, but three species (P. verruculata, P. okinawensis, P. setoensis) were found in September 2023. The average differences between mean maximum and minimum surface water temperatures in the Ryukyu Islands are approximately 7℃ to 8℃ degrees (Iryu et al. 1995). P. setoensis was described from Wakayama, located in the temperate region of mainland Japan, and previously considered as endemic to the southern Honshu area, from Boso Peninsula to Nagasaki (Katagiri and Katagiri 2007; Ueshima 2007; Dayrat et al. 2018), supporting the idea that P. setoensis tolerates colder water than the other species (Dayrat et al. 2018). The distribution of P. setoensis ranges from Okinawa-jima to Boso Peninsula, Chiba, and P. okinawensis from Iriomote to Amami-Oshima. Our results suggest that potential seasonal influences in the behavior or active time could also play a role in the habitat suitability of genus Peronia in the Ryukyu Islands, especially for P. setoensis and P. okinawensis.

Furthermore, Dayrat et al. (2020) reported that P. peronii is a nocturnal species as fewer individuals appeared during the daytime. The highest number of P. peronii specimens collected in this study was in Kurio, Yakushima (eight specimens), followed by Kasaishi Beach, Okinoerabu-jima (seven specimens). Both collections were carried out during the lowest night tides in March and December, respectively. However, all sampling in this study was conducted based on low tides, which occur during the daytime in the summer and at night in the winter in the Ryukyu Islands, and were not conducted based on circadian rhythms. Species occurrences in the intertidal areas of the Ryukyus might also be correlated with lowest tides, as well as corresponding seasonal tide differences, and thus the circadian and tidal rhythms of each species require further study.

Genus Wallaconchis

Wallaconchis is a recently described genus within Onchidiidae, and all species in this genus were originally placed in Onchidium Buchannan, 1800 (Goulding et al. 2018). Japanese literature often misidentifies this genus as genus Paraoncidium Starobogatov, 1976 with the misspelling “Paraonchidium”. According to guidebooks and literature, the Ryukyu Islands harbor three intertidal Paraoncidium species; P. fungiforme (Stantschuinsky, 1907) (Kiboshi Awamochi), P. keinense (Hoffmann, 1926) (Hime-kiboshi Awamochi), and Paraonchidium sp. (Goshiki Awamochi). The specimen pictures and descriptive anatomical information in these reports, particularly oviduct morphology, match well with the description of Wallaconchis provided by Goulding et al. (2018). Based on this information, we hereby identify Paraonchidium sp. (Goshiki awamochi) as Wallaconchis nangkauriensis and Paraonchidium keinense (Hime-kiboshi awamochi) as Wallaconchis ater (Ueshima 2014; Okutani 2017). In the description of a third species, P. fungiforme (Kiboshi Awamochi), it is mentioned that: “they inhabited at intertidal flat, the dorsal color ranges from yellow-brown to cream, with irregular black or reddish-brown spots often forming indistinct longitudinal stripes in pairs, and oviduct is narrow. Distributed in Japan to Australia” (Japanese translation of Ueshima 2014; Okutani 2017). This description does not match with any Wallaconchis (Goulding et al. 2018), and instead likely refers to Onchidina (Dayrat and Goulding 2017). However, P. fungiforme likely belongs to Wallaconchis as it co-occurred with P. keinense (= W. ater), and was mentioned as “very similar to P. keinense and the only difference was the lengths of oviduct” (Ueshima 2014).

Takagi et al. (2018) indicated that there are three species of Paraoncidium around the northern part of Okinawa-jima based on mitochondrial COI haplotype groups. Based on their registered COI sequence data in the DNA Data Bank in Japan and compared with other available GenBank sequences, these three species are likely W. ater unit #1, unit #2 and Wallaconchis sp., but no anatomical data were provided in these previous reports. Takagi et al. (2018) did not identify the different haplotype groups to species level, but found that W. ater unit #1 and #2 sympatrically existed in several intertidal areas, while Wallaconchis sp. was solely observed from only one site.

Furthermore, accurate identification of Wallaconchis species requires not only mitochondrial DNA, but also nuclear loci and anatomical information as incongruences have been observed in their mitochondrial sequences (Goulding et al. 2018). According to mitochondrial COI and 16S sequences, two specimens in our study matched with W. ater unit #1 while five specimens matched with W. ater unit #2 (Goulding et al. 2018), with 18–19% genetic distances between these two units for both markers. Goulding et al. (2018) also observed 25.7–28.9% genetic distances between W. ater units #1 and #2 based on mitochondrial markers. However, the phylogenetic tree from nuclear ITS (ITS1 and part of 5.8S and ITS2) sequences revealed that W. ater should not be separated into two units, and instead is a single species (Goulding et al. 2018). These results were also congruent with their morphological analyses (Goulding et al. 2018). This mitochondrial sequencing incongruency also occurs in other species including W. melanesiensis and W. graniferum, and it has been hypothesized to be due to maternal inheritance of ancestral sequences (Goulding et al. 2018).

Wallaconchis sp. collected in this study from Okinawa-jima, Tokunoshima, and Kikai-jima formed a distinct clade along with sequences extracted from Takagi et al. (2018), that was separated from both W. ater and W. nangkauriensis (Fig. 3). Wallaconchis sp. is located among W. melanesiensis unit #1 and unit #2 in the phylogenetic tree based on mitochondrial markers, with 13–16% and 20–22% genetic distances between species, respectively (Supp. Figure 3). However, these results are also not reliable since Goulding et al. (2018) found that W. ater units #1 and #2 did not form a sister clade in mitochondrial markers. To definitively determine if Wallaconchis sp. is a cryptic species or an already described species, incorporating anatomical information and additional nuclear marker data are needed.

Wallaconchis spp. are often observed in inner bay areas and near river mouths on smaller rocks and rubble to muddy substrates in the Ryukyus (Ueshima 2014; Takagi et al. 2021). During this study, high numbers of W. ater and W. nangkauriensis were also collected from flat intertidal areas, particularly under rubble and small rocks, rather than coral reef intertidal areas. In the Red Data book for Okinawa (2014), P. fungiforme and P. keinense were listed as threatened vulnerable (VU) species in Okinawa-jima, especially in the Awase tidal flats (Ueshima 2014). We conducted surveys during daytime low tide in August 2020 and August 2023, and at night in December 2021 in Awase, but we did not observe any Wallaconchis species in this area. Despite the claimed conservation efforts alongside the artificial island project in this area (Okinawa Prefecture 2014), there are reports across several taxa that the biodiversity of the area has already been negatively affected (Sakai et al. 2006; Kubo 2017). Thus, it is possible that Wallaconchis spp. have been extirpated from the Awase tidal flats, or that abundances have been significantly reduced in this area since 2014. Furthermore, it is likely that higher numbers of species or higher abundances of Wallaconchis inhabit mangrove forest to tidal flats in the Ryukyus. We strongly recommend that future studies examining diversity of Wallaconchis should explore not only coral reef areas but also other types of intertidal areas such as mangrove ecosystems.

Genus Onchidella

Onchidella belongs to subfamily Onchidellinae Labbe, 1934 within family Onchidiidae (Goulding et al. 2022). The distinct characteristics of Onchidella are evenly distributed marginal yellow-ish patterns and lack of dorsal eyes (Dayrat et al. 2011b). Onchidella are often found in crevices of intertidal coral reef areas (Baba 1958; Cumming 2014), but are also found on the sponge Halichondria okadai in mainland Japan (Ohgaki et al. 2011), and on bull-kelp Durvillaea spp. in the southern hemisphere (Cumming 2014).

Two species of Onchidella from Japan were described by Taki (1935); O. kurodai Iw. Taki, 1935 (Hime Awamochi) from Gogoshima and Minatoyama, Ehime, and O. orientalis Iw. Taki, 1935 (Yamato Hime Awamochi), from Tonda-mura, Wakayama. The gross morphological differences between these two species are that O. orientalis is more elongated than O. kurodai but slightly smaller, the minute papillae on O. orientalis are much numerous than O. kurodai, and that there are grey round patches on the dorsal side of O. orientalis that vary in size, which are not observed in O. kurodai.

In the Ryukyu Islands, the first observations of O. orientalis were reported from intertidal coral reef areas of Yoan, Amami-Oshima and Nakanoshima Island, Tokara (Baba 1958; Taki 1959). However, no photographic records were given, and identification criteria were not specifically mentioned for specimens. Additional reports were made for both O. kurodai and O. orientalis from mainland Japan, south of the Boso Peninsula to Ariake Bay (e.g. Nishimura 1992; Okutani and Soyama 1994; Okutani 2000, 2017; Takagi et al. 2019), O. orientalis in northern Taiwan (Cheng et al. 2005)d kurodai in Jindo Island, South Korea (Kil et al. 2005).

In Okutani and Soyama (1994) and Okutani (2000, plate 404, O. orientalis), an image of O. orientalis from Kushimoto, Wakayama includes both O. kurodai and O. orientalis, based on dorsal patterns described by Taki (1935). Later, Okutani (2017) mentioned that these two species are likely to be the same species. Recently, O. orientalis was synonymized with O. kurodai due to no anatomical differences and little genetic variation in mitochondrial COI, 16S, and 12S, and nuclear ITS2, 28S and H3 DNA sequences observed in specimens collected from both type localities (Goulding et al. 2022). However, Goulding et al. (2022) examined only three specimens of O. kurodai and O. orientalis from mainland Japan. Moreover, specimens from the Ryukyu Islands were missing from Goulding et al. (2022), and it was mentioned that Onchidella spp. are largely absent from subtropical and tropical Indo-West Pacific regions (Goulding et al. 2022). Thus, the newly collected specimens from the Ryukyus are particularly important to understand Onchidella species diversity in Japan.

Genetic distances between Onchidella specimens collected in this study from the Ryukyus and O. kurodai (Takagi et al. 2018; Goulding et al. 2022) were 19–20%, and Onchidella sp. formed a separate clade distinct from the other previously described species of Onchidella (SFig. 4). The average size of Onchidella sp. was smaller than as described for O. kurodai and is more similar to the size of O. orientalis in Taki (1935). Dorsal patterns with grey round patches were often present as in O. orientalis, but varied among individuals and some specimens did not have any patterns. Further studies with anatomical information and more genetic markers are needed to determine whether O. orientalis needs to be resurrected, or our Onchidella sp. instead represents a cryptic, undescribed species.

Onchidella spp. smaller sizes often make them challenging to notice in the field. Thus, despite our focus on this group, we feel it likely that this study overlooked Onchidella spp. at many intertidal areas, and we expect them to have much wider distribution ranges within the Ryukyu Islands than has been noted here and in previous research.

This study focused on species diversity of Onchidiidae in intertidal coral reef environments of the Ryukyu Islands. We detected at least eight species from three genera of onchidiid species present across the islands, with a maximum of five species coinhabiting the same intertidal area. Our observations, utilizing DNA barcoding techniques with mitochondrial sequences, included many first records of species for many islands. In the future, the calculation of more accurate phylogenetic positions for Wallaconchis sp. and Onchidella sp. utilizing nuclear markers, as well as gathering additional anatomical information, are needed. It is hoped that the information provided in the current study will enhance the understanding of the species diversity of family Onchidiidae across the Ryukyu Islands by providing a baseline dataset for future reference.

Acknowledgement

We thank all Molecular Invertebrate Systematics and Ecology Laboratory (MISE) members at the University of the Ryukyus, particularly Geminne Manzano for help in sample collection. We also thank Dr. Yuka Kushida (Rissho Uni) and Megumi Nakano (NACS-J) for their assistance in obtaining sampling permissions in Amami Oshima. CJLF thanks the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) for a scholarship.

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Species diversity of genera Peronia, Wallaconchis and Onchidella (Gastropoda: Onchidiidae) in intertidal coral reef environments of the Ryukyu Islands

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Abstract

Figures

Introduction

Materials And Methods

Specimen collection and morphological data analyses

DNA extraction, PCR amplification and sequencing

Phylogenetic analyses

Results

Phylogenetic analyses

Species distributions

Species distributions across the Ryukyus

Overlapping distribution ranges

Morphological analyses

Discussion

Species diversity of Onchidiidae in intertidal coral reef environments of the Ryukyus

Conclusion

Declarations

Acknowledgement

References

Tables

Supplementary Files

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