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First Record of the Slug Species Semperula Wallacei (Issel, 1874) (Gastropoda: Eupulmonata: Veronicellidae) in Japan

First Record of the Slug Species Semperula Wallacei (Issel, 1874) (Gastropoda: Eupulmonata: Veronicellidae) in Japan

BioInvasions Records (2019) Volume 8, Issue 2: 258–265

CORRECTED PROOF

Research Article First record of the Semperula wallacei (Issel, 1874) (: : ) in

Takahiro Hirano1,*, Daishi Yamazaki2, Shota Uchida2, Takumi Saito2 and Satoshi Chiba2,3 1Department of Biological Sciences, University of Idaho, Moscow, Idaho, USA 2Graduate school of Life Sciences, Tohoku University, Miyagi, Japan 3Center for Northeast Asian Studies, Tohoku University, Miyagi, Japan Author e-mails: [email protected] (TH), [email protected] (DY), [email protected] (SU), [email protected] (TS), [email protected] (SC) *Corresponding author

Citation: Hirano T, Yamazaki D, Uchida S, Saito T, Chiba S (2019) First record of Abstract the slug species Semperula wallacei (Issel, 1874) (Gastropoda: Eupulmonata: In this study, we focus on veronicellid in the Ryukyu and Ogasawara Islands Veronicellidae) in Japan. BioInvasions of Japan. We conducted phylogenetic analyses of these slugs, incorporating GenBank Records 8(2): 258–265, https://doi.org/10. data from several veronicellid species. A molecular phylogeny based on the 3391/bir.2019.8.2.07 mitochondrial COI gene revealed that three clades inhabit these islands. We report Received: 18 June 2018 here the first record of Semperula wallacei in Japan, which represents an introduced Accepted: 18 September 2018 species in this region. Published: 16 October 2018 Key words: introduced species, , , Ogasawara Handling editor: April Blakeslee Islands, phylogeny Copyright: © Hirano et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Introduction OPEN ACCESS. The Veronicellidae is a family of terrestrial slugs (Barker 2001). There are currently 23 recognized genera in this family, and they are globally distributed throughout the tropics and subtropics (Gomes et al. 2010). Veronicellid slugs have also been introduced outside of their native range for example oceanic islands (Brodie and Barker 2011; Kim et al. 2016). Some veronicellid slugs are known to be a medium to high risk to humans and feed on the leaves and stems of young dry-bean , defoliating and often killing crops (Rueda et al. 2001; Brodie and Barker 2011). In addition, veronicellid slugs are considered to be major intermediate hosts of Angiostrongylus (Brodie and Barker 2011; Kim et al. 2014), which causes the disease angiostrongyliasis (Kim et al. 2014) or rat worm. In the worst cases, angiostrongyliasis can result in death in humans. The Ryukyu Islands and Ogasawara Islands of Japan are hotspots of land species diversity (Kameda et al. 2007; Chiba et al. 2009; Hoso et al. 2010; Hirano et al. 2014; Ministry of Environment 2014; Chiba and Cowie 2016). However, many introduced species of have been recorded on

Hirano et al. (2019), BioInvasions Records 8(2): 258–265, https://doi.org/10.3391/bir.2019.8.2.07 258 First record of Semperula wallacei in Japan

Figure 1. Images of representative individuals of lineages. A. alte (Okinawa Island; photograph by H. Fukumori). B. Semperula wallacei (Chichijima Island, photo by T. Hirano). C. Veronicellidae sp. (Ishigaki Island, photo by T. Hirano). Scale bar indicates 10 mm.

both island groups (Chiba et al. 2009; Chiba and Cowie 2016; Nature Conservation Division, Department of Environmental Affairs, Okinawa Prefectural Government 2017). For example, (Férussac, 1822) was introduced on the Ryukyu Islands (Azuma 1982) and more recently in mainland Japan (Nishi and Matsuoka 2007). Here, we focus on veronicellid slugs on the Ryukyu Islands and Ogasawara Islands that differ from L. alte based on their external morphology (Figure 1). Compared with L. alte, these individuals differ in their ochre or brown body color and small body size. According to Schilthuizen and Liew (2008), L. alte is generally recognized by its dark gray or almost black body with a thin pale median dorsal line in contrast to the other slugs, which do not have this dorsal line. But, identifications of this family by external morphology are difficult (Cowie 1997; Kim et al. 2016). In particular, several veronicellid worms are similar to one another in external morphology, including plebeia (Fischer, 1868), Semperula wallacei (Issel, 1874), and cubensis (Pfeiffer, 1840). Previous molecular studies have been useful for identifying and clarifying the phylogenetic relationships within the family (Gomes et al. 2010; Kim et al. 2016). However, the phylogenetic position

Hirano et al. (2019), BioInvasions Records 8(2): 258–265, https://doi.org/10.3391/bir.2019.8.2.07 259 First record of Semperula wallacei in Japan

among veronicellid species of the Ryukyu Islands and Ogasawara Islands is unclear. Therefore, we reconstructructed the molecular phylogeny of these slug species and report, for the first time a species of Veronicellidae other than L. alte in Japan.

Materials and methods We collected five individuals of unidentified Veronicellidae species (Table 1) from the Ryukyu Islands and Ogasawara Islands (Figure 2) and a single individual of L. alte from Amami Island in the Ryukyu Islands. We also obtained sequence data of 25 individuals of 14 veronicellid species from GenBank (Table 1). We used two individuals from Onchidiid genera as outgroups (Dayrat et al. 2011). A fragment of the foot muscle of each living individual was stored in 99.5% ethanol for DNA extraction. Total DNA from the six individuals was extracted using a NucleoSpin Tissue Kit (Macherey-Nagel), following the manufacturer’s standard protocol. To estimate the phylogenetic relationships among the collected slugs, we sequenced fragments of the Cytochrome oxidase I (COI) mitochondrial gene. Polymerase chain reaction (PCR) conditions and the primers used are listed in Table 2. The PCR products were purified using Exo-SAP-IT (Amersham Biosciences, UK). The sequencing was performed using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and electrophoresed using an ABI 3130xl sequencer (Applied Biosystems). The newly generated sequences have been deposited in the DDBJ/EMBL/GenBank databases (Table 1). The COI sequences were aligned using MUSCLE v3.8 (Edgar 2004), and summarized to limit node density artifacts (Fitch and Bruschi 1987; Fitch and Beintema 1990). Three individuals from the Ogasawara and Miyako Islands, and Semperula wallacei from GenBank (DQ897673) shared the same haplotype. Based on 470 bp of the sequence, we generated a maximum likelihood (ML) phylogenetic tree using MEGA6 (Tamura et al. 2013). Prior to the ML analysis, we selected the appropriate model (HKY+G+I) for sequence evolution also using MEGA6. Nodal support for the ML analysis was assessed using bootstrap analysis with 1000 replications. Only bootstrap values higher than 75% were considered to be strongly supported.

Results The phylogenetic analysis recovered seven major clades (Figure 3). Clades A, B, and D only included one each (, Veronicella, and Sarasinula, respectively). Clade C was composed of the genera and . Semperula wallacei from the GenBank data (2 individuals), and four individuals from Okinawa Island (1), the Ogasawara (1) and Miyako Islands (2) constituted a well-supported clade F. However, the lone

Hirano et al. (2019), BioInvasions Records 8(2): 258–265, https://doi.org/10.3391/bir.2019.8.2.07 260 First record of Semperula wallacei in Japan

Table 1. Slug sampling sites and GenBank accession number of DNA sequences.

Sampling site Accession Specimen ID No. Name number Samples collected by authors Laevicaulis alte 1 Amami, Kagoshima, Japan HC2445 LC415570 Semperula wallacei 2 (Miyako Islands) Taira, Miyakojima, Okinawa, Japan HC6220 LC415572 3 (Miyako Islands) Irabuikemasoe, Miyakojima, Okinawa, Japan HC6221 LC415573 6 (Chichijima Island) Chichijima, Ogasawara, Tokyo, Japan HC7454 LC415571 5 (Okinawa Island), Naha, Okinawa, Japan HC7455 LC415574 Veronicellidae sp. 4 (Ishigaki Island) Nagura, Ishigaki, Okinawa, Japan HC7456 LC415569 GenBank Colosius propinquus Imbabura, Ecuador JX532115 C. pulcher Pichincha, Ecuador JX532116 Napo, Ecuador JX532117 Colosius sp.1 Colombia JX532113 Napo, Ecuador JX532114 L. alte India KY774830 L. natalensis Natal, South Africa JX532110 L. sp. South Africa HQ660052 Phyllocaulis boraceiensis Sao Paulo, Brazil JX532111 Sarasinula linguaeformis Minas Gerais, Brazil JX532108 S. plebeia Iquitos, Loreto, Peru KM489401 , USA KM489499 El Hatillo, Venezuela KM489393 S. sp. 1 Chapeco, Santa Catarina, Brazil KM489501 Sao Paulo, Sao Paulo, Brazil KM489446 Pinhalzinho, Sao Paulo, Brazil KM489448 S. sp. 2 Jatai, Goias, Brazil KM489452 Jatai, Goias, Brazil KM489454 Jatai, Goias, Brazil KM489456 Semperula wallacei Sabah, Malaysia DQ897673 Tutuila, American JX532109 Vaginulus taunaisii Brazil HQ660056 Hawaii, USA HQ660057 St. Paul Parish, Antigua and Barbuda JX532112 Winfried Gibbons Nature Reserve, Devonshire Parish, South Road Bermuda KC206184 Outgroups floridana HQ660035 Onchidium vaigiense HQ660040

Hirano et al. (2019), BioInvasions Records 8(2): 258–265, https://doi.org/10.3391/bir.2019.8.2.07 261 First record of Semperula wallacei in Japan

Figure 2. Map of the veronicellid sampling sites. Site 1–5 are in the Ryukyu Islands, and 6 is in the Ogasawara Islands. The numbers correspond to the site number in Table 1.

Table 2. Information on primers and PCR conditions. Primer Sequence 5'-3' References PCR conditions LCO1490 GGT CAA CAA TCA TAA AGA TAT TGG Folmer et al. 1994 94°C 3 min (94°C 30 sec, 40°C HCO2198 TAA ACT TCA GGG TGA CCA AAA AAT C Folmer et al. 1994 30 sec, 72°C 1 min) × 34, 72°C 5 min

individual from Ishigaki Island was genetically different from all of the other samples (clade E). In addition, Laevicaulis constituted clade G, which included the L. alte we had collected.

Discussion Our study represents the first record of Semperula wallacei in Japan. Molecular phylogenetic analyses revealed that S. wallacei in Japan shared the same haplotype as S. wallacei of Malaysia (DQ897673) and phylogenetic relationships did not reflect a geographical distribution pattern, indicating that S. wallacei has been introduced to both the Ryukyu and Ogasawara Islands. According to a prior study, S. wallacei originated from Southeast and was recently recorded in several Pacific (Gomes et al. 2010) and Caribbean islands (Gomes and Thomé 2004). Here we only collected five specimens, so further study using a larger sample size and multiple genes is needed to clarify the evolutionary history and of these Japanese veronicellids. In addition, we detected a sample from Ishigaki Island that was genetically distinct from S. wallacei (Figure 3). Though this individual is morphologically similar to S. wallacei, genetic information suggests that it

Hirano et al. (2019), BioInvasions Records 8(2): 258–265, https://doi.org/10.3391/bir.2019.8.2.07 262 First record of Semperula wallacei in Japan

Figure 3. Maximum likelihood tree of the veronicellid slugs based on 470 bp of the COI gene. Each OTU label represents the GenBank accession number followed by a species name. All Japanese individuals were indicated in bold. Unidentified Japanese species were indicated by only the GenBank accession number. The numbers in brackets next to the specimen ID indicate the sampling site number in Figure 2. Numbers on branches indicate maximum likelihood bootstrap values. Scale bar indicates 0.1 substitutions per site.

may represent a distinct species. In the present study, we did not perform dissections, but genital morphology may be useful to test the relationships recovered in our phylogenetic analysis (Kim et al. 2016). Single-gene phylogenetic analysis can reflect incomplete lineage sorting (Glaubrecht and Köhler 2004) and introgressive hybridization (Glaubrecht and von Rintelen 2008). Nonetheless, we designate this individual as “Veronicellidae sp.” for convenience (Table 1). Many species, including land snails, have been introduced to the Ogasawara and Ryukyu Islands (Hirano et al. 2017). In the Osagawara Islands, individuals resembling those now identified as S. wallacei have been present on Chichijima Island at least since 2009 (S. Wada, personal communication). But, this introduced species has only been found on Chichijima Island and has not yet been recorded in other islands of the

Hirano et al. (2019), BioInvasions Records 8(2): 258–265, https://doi.org/10.3391/bir.2019.8.2.07 263 First record of Semperula wallacei in Japan

archipelago. However, it is not clear how long ago S. wallacei was discovered in the Ryukyu Islands. Through the nursery trade, some species have been transferred from the Ryukyu Islands to the Ogasawara Islands (Shimozu and Tabata 1991; Kato and Sugai 2009). Moreover, endemic land snails of the Ryukyu Islands have been recorded in the Ogasawara Islands (Chiba et al. 2009). Perhaps S. wallacei invaded the Ryukyu Islands sometime before 2009. It may have then invaded the Ogasawara Islands after having established association with garden plants. It is necessary to be aware of coming from other countries and inhabited landscapes, like offshore Japanese islands and mainland Japan (Hirano et al. 2017). In Japan, land snails, including L. alte infected with Angiostrongylus have been reported in Okinawa (Asato et al. 2004). In addition, Angiostrongylus is already in the Ogasawara Islands (Tokiwa et al. 2013), and rats of the Ryukyu and Ogasawara Islands are common definitive hosts (Asato and Kishimoto 1976; Tokiwa et al. 2013). It therefore is possible that S. wallacei could be a competent intermediate host of harmful parasites in these islands, but experiments would be required to confirm this. Given this new invasion in Japan, more research is needed to understand the slug’s fundamental biology and role in of the Ryukyu and Ogasawara Islands.

Acknowledgements

We thank S. Wada for information about S. wallacei on Chichijima Island, and H. Fukumori for the photograph of L. alte. We thank for H. Takagi and R. Takagi for sample collection. We also thank A. Gonzalez and J. Phillips for English editing. Finally we are grateful to A.M.H Blakeslee, B. Rowson, J. Capinera and an anonymous referee for constructive comments on the manuscript. References

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