Species identification of introduced veronicellid slugs in Japan

Abstract

Reliable identification of species is important for protecting native ecosystems against the invasion of non-native species. DNA barcoding using molecular markers, such as the mitochondrial cytochrome oxidase subunit 1 (COI) gene, helps researchers distinguish species. In this study, we focused on introduced veronicellid slugs in the Ryukyu Islands and some greenhouses on mainland Japan. Some veronicellids are medium-to-high risk pest species for humans. Identifying veronicellid species by their external morphology is difficult and unreliable because there is substantial overlap between intraspecific variation and interspecific differentiation. Therefore, internal morphologies such as male genitalia have been the primary traits used to distinguish veronicellids. To identify introduced veronicellid slugs in Japan to the species level, we used morphological assessment of male genitalia and DNA barcoding of the standard COI gene fragment. We also conducted species-delimitation analyses based on the genetic data. The results showed that five evolutionarily significant units, corresponding to four nominal species inhabit the Ryukyu Islands, of which two species were also found in the greenhouses of mainland Japan, including the first record of Sarasinula plebeia in Japan. The presence of non-native slug species could increase the transmission of parasites in Japan.

Non-native terrestrial slugs from Sinaloa, Mexico: Deroceras laeve (O. F. Müller, 1774) and Sarasinula plebeia (P. Fischer, 1868) (Mollusca, Gastropoda)

“…Although it appears that their effect as agricultural pests has been reduced by improving management by farmers, it remains as a vector for parasites for rodents and other mammals and humans ( Nurinsiyah and Hausdorf 2018 ). Spatio-temporal occurrence of S. plebeia indicates an actual expansion process and their genetic convergence with Japanese and South American individuals demonstrate their invasive potential ( Hirano et al 2022 ).…”

“…In this regard, the high number of synonyms is related to the use of few morphological characters for species delimitation or superficial revision, based on external features and pigmentation patterns ( Maceira F. 2003 ). The taxonomy of the slugs requires analysing their internal anatomy of male genitalia and the use of molecular markers ( Hirano et al 2022 ), a crucial requirement to understand the dispersion of invasive species. For instance, considering that S. plebeia is one of the two species with the widest distribution ( Gomes and Thomé 2004 ), further studies on this species could use molecular data to identify clades associated with introduction routes and spreading.…”

Morphology, morpho-taxometric and molecular characterization of the invasive alien species Caribbean leatherleaf slug Sarasinula plebeia (Gastropoda: Veronicellidae): a first record in southern Philippines

“…In contrast, a considerable amount of base substitution (0.76) between S. plebeia isolate LDZS (MZ598573) and closely related sequences to the sequences from Batan island, Batanes (JQ582277, JQ582278, JQ582279). Furthermore, the Batan island slug samples (JQ582277, JQ582278, JQ582279) showed no difference (0.00) on the number of base substitutions with L. alte (Hirano et al, 2022;LC636101, LC636102, LC636103, and LC636104) from Japan. This supported that S. plebeia isolate LDZS and those from Batan island are not of the same species with the latter being closely related to L. alte having 0.00 number of base substitutions (Table 2).…”

“…Likewise, all the samples from Japan formed a sister clade with the other S. plebeia. Interestingly, S. plebeia samples from Batan island, Batanes in northern Philippines (JQ582277, JQ582278, JQ582279) previously identified using DNA barcoding showed no relationship with other S. plebeia, instead they were closely related to another veronicellid slug Laevicaulis alte (Férussac, 1822) (Hirano et al, 2022;LC636101, LC636102, LC636103, and LC636104), forming a separate clade with posterior probability value of 1 (Fig. 3).…”

“…The COI gene sequence generated from S. plebeia isolate LDZS (MZ598573, 629 bp) shared 100% similarities with S. plebeia from Brazil (Gomes et al, 2013;JX532107 and Gomes et al, unpublished;KM489378), Vietnam (KM489367) which was intercepted by USDA, and Dominica (KM489500). Furthermore, S. plebeia isolate LDZS (MZ598573) shared 99% similarity with samples from India (MH819385 and MH819384), Venezuela (KM489393), Tahiti (KM489369) intercepted by USDA, USA (KM489374), and Japan (Hirano et al, 2022; LC636113, LC636114, LC636115, LC636116, LC636117, LC636118, and LC636119). Phylogenetic relationship of S. plebeia isolate LDZS (MZ598573) with other closely related species as inferred by the COI gene sequences using Bayesian analysis showed a formation of a distinct clade with S. plebeia from Brazil, Vietnam, Dominica, India, Venezuela, Tahiti, and USA supported with a strong posterior probability of 1.…”