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Research Article
A new species and new records of Onchidium slugs (Gastropoda, Euthyneura, Pulmonata, Onchidiidae) in South-East Asia
expand article infoBenoît Dayrat‡, Tricia C. Goulding‡, Munawar Khalil§, Deepak Apte|, Shau Hwai Tan
‡ Pennsylvania State University, University Park, United States of America
§ Universitas Malikussaleh, North Aceh, Indonesia
| Bombay Natural History Society, Mumbai, India
¶ Universiti Sains Malaysia, Penang, Malaysia

Corresponding author: Benoît Dayrat ( bdayrat@gmail.com )

Academic editor: Nathalie Yonow
© 2019 Benoît Dayrat, Tricia C. Goulding, Munawar Khalil, Deepak Apte, Shau Hwai Tan.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Dayrat B, Goulding TC, Khalil M, Apte D, Tan SH (2019) A new species and new records of Onchidium slugs (Gastropoda, Euthyneura, Pulmonata, Onchidiidae) in South-East Asia. ZooKeys 892: 27-57. https://doi.org/10.3897/zookeys.892.39524
ZooBank: urn:lsid:zoobank.org:pub:5F543780-6E6C-4353-BF6E-FD0E252D63AE
Open Access

Abstract

A new species, Onchidium melakense Dayrat & Goulding, sp. nov., is described, bringing the total to four known species in the genus Onchidium Buchannan, 1800. Onchidium melakense is a rare species with only nine individuals found at three mangrove sites in the Andaman Islands and the Strait of Malacca (western Peninsular Malaysia and eastern Sumatra). The new species is delineated based on mitochondrial (COI and 16S) and nuclear (ITS2 and 28S) DNA sequences as well as comparative anatomy. Each Onchidium species is characterized by a distinct color and can easily be identified in the field, even in the Strait of Malacca where there are three sympatric Onchidium species. An identification key is provided. In addition, Onchidium stuxbergi (Westerlund, 1883) is recorded for the first time from eastern Sumatra, and Onchidium pallidipes Tapparone-Canefri, 1889, of which the type material is described and illustrated here, is regarded as a new junior synonym of O. stuxbergi.

Keywords

Biodiversity, integrative taxonomy, Malacca Strait, mangrove, systematic revisions

Introduction

Onchidiids are true slugs (lacking an internal shell) which breathe air with a lung and die if they are immersed in water for a few hours. Most species are found in the intertidal zone, but a few species are adapted to high-elevation rainforest up to 1,850 meters (Dayrat 2010). Onchidiids are found worldwide, but the highest species diversity is in South-East Asia, especially in mangroves where onchidiids are among the most abundant animals. Onchidiid slugs are most closely related to veronicellids, which are also true slugs but which, unlike onchidiids, are fully terrestrial, and to Stylommatophora, the land snails and slugs (Dayrat et al. 2011).

The taxonomy of the Onchidiidae has been in a state of chaos for many years (Dayrat 2009). In the past few years, the Dayrat laboratory has been revising the taxonomy of the entire family one clade at a time (Dayrat et al. 2016, 2017, 2018, 2019a, b; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c). We follow an integrative approach to taxonomy based on: 1) a re-examination of all types available and a comprehensive review of the nomenclature to address the application of all existing species- and genus-group names; 2) extensive field work to observe species in their habitat and to collect fresh material (we visited more than 300 sites worldwide); and, 3) species delineation using DNA sequences to complement comparative anatomy.

As the type genus of the family, Onchidium Buchannan, 1800 was the focus of our first revision (Dayrat et al. 2016). In the past, the generic name Onchidium was traditionally used by default for many onchidiid species, because relationships among onchidiid species were very confusing. In total, 80 species were described using Onchidium in the original binomial (Dayrat 2009). However, the revision of the genus Onchidium showed that it included only three species (Dayrat et al. 2016): the type species O. typhae Buchannan, 1800, O. reevesii (J. E. Gray, 1850), and O. stuxbergi (Westerlund, 1883). Except for Onchidium nigrum Plate, 1893 and Onchidium pallidipes Tapparone-Canefri, 1889, both junior synonyms of O. stuxbergi, all other species binomials with Onchidium as a generic name refer to species that actually belong to other onchidiid genera or to nomina dubia which cannot be placed in any onchidiid genus (Dayrat et al. 2016, 2017, 2018, 2019a, b; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c).

Onchidium slugs can be identified in the field thanks to two external features: large, conical, pointed papillae on the dorsal notum and very long and thin ocular tentacles. These two traits are synapomorphies of Onchidium which are not found in any other onchidiids. In the present contribution, we describe the new species Onchidium melakense Dayrat & Goulding. It is a rare species for which we found only nine individuals at three mangrove sites (out of the dozens of sites that we explored in the region). One individual was collected in the Andaman Islands. Eight individuals were collected in the Strait of Malacca: four individuals in the Matang mangrove near Kuala Sepatang in western Peninsular Malaysia, and four individuals in Sinaboi Island, a small uninhabited island in eastern Sumatra. Onchidium melakense is supported by mitochondrial (COI and 16S) and nuclear (ITS2 and 28S) DNA sequences and comparative anatomy. Each Onchidium species is characterized by a distinct color so the three Onchidium species that are sympatric in the Strait of Malacca can easily be identified: Onchidium melakense is characterized by a light brown dorsal notum and a perfectly white hyponotum. An identification key to Onchidium species is provided.

In addition, Onchidium stuxbergi is recorded for the first time from eastern Sumatra. Also, Onchidium pallidipes Tapparone-Canefri, 1889, of which the type material is described and illustrated here, is regarded as a new junior synonym of O. stuxbergi. Finally, for the first time, a plate illustrates precisely the range of individual variation for the intestinal loops of each Onchidium species: intestinal loops are of type II in O. typhae and of type III in the three other species.

Materials and methods

Collecting

All specimens were collected by the authors in the past few years, except six specimens from China for which sequences were obtained from GenBank (Table 1). Collecting field parties were led by Benoît Dayrat in the Andaman Islands (India) and Peninsular Malaysia and by Munawar Khalil in Sumatra (Indonesia). Sites were accessed by car or by boat. Although each site was explored for an average of two hours, the exact time spent at each site also depended on the time of the low tide, the weather conditions, etc. Photographs were taken to document the kind of mangrove being visited as well as the diverse microhabitats where specimens were collected.

Table 1.
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DNA extraction numbers and GenBank accession numbers for all the specimens included in the present study. The letter H next to an extraction number indicates the holotype.

Species DNA # Voucher Locality COI 16S ITS2 28S
Onchidium melakense 1105 BNHS 94 Andaman, India – MN528066 – –
1720 UMIZ 00001 Sumatra, Indonesia MN528057 MN528067 MN527565 MN527530
1723 UMIZ 00001 Sumatra, Indonesia MN528058 MN528068 – –
1769 UMIZ 00001 Sumatra, Indonesia MN528059 MN528069 MN527566 MN527531
1771 UMIZ 00001 Sumatra, Indonesia MN528060 MN528070 MN527567 MN527532
5978 USMMC 00076 Peninsular Malaysia MN528061 MN528071 – –
5979 H USMMC 00075 Peninsular Malaysia MN528062 MN528072 MN527568 MN527533
5981 USMMC 00076 Peninsular Malaysia MN528063 MN528073 MN527569 MN527534
5982 USMMC 00076 Peninsular Malaysia MN528064 MN528074 – –
O. reevesii S871 ASTM-Mo-S871 China (22°30’N) JN543161* JN543097* – –
S831 ASTM-Mo-S831 China (24°24’N) JN543160* JN543096* – –
S853 ASTM-Mo-S853 China (27°29’N) JN543164* JN543100* – –
S821 ASTM-Mo-S821 China (33°20’N) JN543162* JN543098* – –
S802 ASTM-Mo-S802 China (34°46’N) JN543157* JN543093* – –
O. stuxbergi 971 USMMC 00006 Peninsular Malaysia KX179514* KX179531* MN527562 MN527527
1770 UMIZ 00002 Sumatra, Indonesia MN528056 MN528065 – –
1048 BDMNH Brunei KX179515* KX179532* MN527563 MN527528
3251 PNM 041199 Bohol, Philippines KX179517* KX179534* – –
3363 PNM 041202 Bohol, Philippines KX179518* KX179535* MN527564 MN527529
5602 ITBZC IM 00001 Vietnam KX179519* KX179536* – –
5605 ITBZC IM 00002 Vietnam KX179520* KX179537* MG958721* MG971211*
S891 ASTM-Mo-S891 China (19°56’N) JN543155* JN543091* – –
O. typhae 1064 BNHS 82 West Bengal, India – KX179528* – –
1089 BNHS 82-1089 Andaman, India KX179512* KX179529* – –
1109 BNHS 21-1109 Andaman, India KX179513* KX179530* – –
967 USMMC 00003 Peninsular Malaysia KX179510* KX179526* MN527560 MN527525
965 USMMC 00005 Peninsular Malaysia KX179509* KX179525* MG958720* MG971210*
1007 ZRC.MOL.6396 Singapore KX179511* KX179527* MN527561 MN527526
Alionchis jailoloensis 5137 UMIZ 00117 Indonesia, Halmahera MG953528* MG953538* MG953548* MK122918*
Marmaronchis vaigiensis 1183 ZRC.MOL.3007 Singapore MK122812* MK122854* MK122877* MK122910*
M. marmoratus 5409 MNHN-IM-2013-15764 PNG, Madang MK122838* MK122859* MK122893* MK122915*
Melayonchis aileenae 970 USMMC 00018 Peninsular Malaysia KX240033* KX240057* MK122902* MK125514*
M. annae 1010 ZRC.MOL.6502 Singapore KX240015* KX240039* MK122903* MK122919*
M. eloisae 1011 ZRC.MOL.6499 Singapore KX240026* KX240050* MK122904* MK125515*
M. siongkiati 1002 ZRC.MOL.6501 Singapore KX240020* KX240044* MK122905* MK122920*
Paromoionchis penangensis 957 USMMC 00061 Peninsular Malaysia MH055078* MH055137* MH055255* MH055293*
P. tumidus 963 USMMC 00057 Peninsular Malaysia MH054946* MH055101* MH055194* MH055266*
Onchidella celtica 5013 MNHN-IM-2014-6891 France MG958715* MG958717* MK122906* MK122921*
O. nigricans 1524 AM C468921.002 Australia, NSW MG970878* MG970944* MK122908* MK122923*
Onchidina australis 1523 AM C468918.002 Australia, NSW KX179548* KX179561* MG958719* MG971209*
Peronia sp. 706 UF 303653 USA, Hawaii HQ660038* HQ659906* MG958722* MG971212*
696 UF 352288 Japan, Okinawa HQ660043* HQ659911* MG958871* MG958883*
Peronina tenera 960 USMMC 00039 Peninsular Malaysia MG958740* MG958796* MG958840* MG958874*
P. zulfigari 924 USMMC 00048 Peninsular Malaysia MG958760* MG958816* MG958853* MG958876*
Platevindex luteus 1001 ZRC.MOL.10179 Singapore MG958714* MG958716* MG958718* MG958888*
Wallaconchis ater 3272 PNM 041222 Philippines, Bohol MG970809* MG970910* MG971132* MG971185*
W. sinanui 2740 UMIZ 00059 Indonesia, Ambon MG970713* MG970881* MG971093* MG971161*

* Sequences (including all sequences of the outgroups) from our former publications (Dayrat et al. 2011, 2016, 2017, 2018, 2019a, b; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c). Sequences from China were obtained from GenBank (Sun et al. 2014) where they are misidentified as Onchidium struma, a nomen nudum. Abbreviations: Australian Museum, Sydney (AM); Aquatic Science and Technology Museum of Shanghai Ocean University (ASTM); Brunei Darussalam Museum of Natural History (BDMNH); Bombay Natural History Society, India (BNHS); Institute of Tropical Biology, Zoology Collection, Vietnam Academy of Science and Technology (ITBZC); Muséum national d’Histoire naturelle, Paris, France (MNHN); National Museum of the Philippines, Manila (PNM); University of Florida, Gainesville (UF); Universiti Sains Malaysia Mollusc Collection, Penang, Malaysia (UMIZ); Universiti Sains Malaysia Mollusc Collection, Penang, Malaysia (USMMC); Zoological Reference Collection, Lee Kong Chian Natural History Museum, National University of Singapore (ZRC).

Specimens were individually numbered and photographed in their respective habitat. At each site, we endeavored to sample as much diversity as possible. In addition to numbering individually the specimens that looked different, we also numbered individually specimens that looked similar so that we could test for the presence of cryptic diversity. Importantly, a piece of tissue was cut for all specimens individually numbered (for DNA extraction) and the rest of each specimen was relaxed (using magnesium chloride) and fixed (using 10% formalin or 70% ethanol) for comparative anatomy.

Specimens

Eighteen specimens were already included in our revision of the genus Onchidium and are included in the molecular analyses here to demonstrate the existence of a new species and of a new record for O. stuxbergi (Table 1). Their mitochondrial COI and 16S sequences are from our revision of Onchidium, but their nuclear ITS2 and 28S sequences are new. All mitochondrial and nuclear sequences for the 10 specimens representing a new species or a new record are new. Overall, mitochondrial COI and 16S sequences are provided for 28 individuals and nuclear 28S and ITS2 sequences are provided for 12 of those 28 individuals (excluding outgroups). All DNA sequences were generated by us except for the mitochondrial sequences of six individuals from China obtained from GenBank (Table 1).

DNA extraction numbers unique to each individual are indicated in phylogenetic analyses as well as lists of material examined and figure captions (numbers are between brackets). Size (length/width) is indicated in millimeters (mm) for each specimen. Many additional specimens were examined in the context of our revision of the family, including all available types (the types of Onchidium pallidipes Tapparone-Canefri, 1889 and Onchidium multinotatum Plate, 1893, are addressed in detail in the discussion) and hundreds of onchidiids representing all the known genera and nearly all known species. The ten specimens representing a new species and a new record were deposited as vouchers in institutions in the countries of origin: Bombay Natural History Society, Mumbai, (India); Universitas Malikussaleh, North Aceh, Sumatra (Indonesia); Universiti Sains Malaysia, Penang (Malaysia).

Museum collection abbreviations

MNHN Muséum national d’Histoire naturelle, Paris, France;

NMNH National Museum of Natural History, Smithsonian Institution, Washington, DC, USA;

SMNH Swedish Museum of Natural History, Stockholm, Sweden;

UMIZ Universitas Malikussaleh, North Aceh, Sumatra, Indonesia;

USMMC Universiti Sains Malaysia, Mollusk Collection, Penang, Malaysia;

ZMB Museum für Naturkunde, Berlin, Germany;

ZMH Zoologisches Museum, Hamburg, Germany.

Anatomical preparations and descriptions

Both the external morphology and the internal anatomy were studied. All anatomical observations were made under a dissecting microscope and drawn with a camera lucida. Radulae and male reproductive organs were prepared for scanning electron microscopy (Zeiss SIGMA Field Emission Scanning Electron Microscopy). Radulae were cleaned in 10% NaOH for a week, rinsed in distilled water, briefly cleaned in an ultrasonic water bath (less than a minute), sputter-coated with gold-palladium and examined by SEM. Soft parts (penis, accessory penial gland, etc.) were dehydrated in ethanol and critical point dried before coating.

The detailed anatomy of the type species, Onchidium typhae, can be found in our revision of Onchidium (Dayrat et al. 2016). To avoid unnecessary repetition, the description of anatomical features that are virtually identical between Onchidium species (e.g., position of female opening, position of anus, size of hyponotum relative to total width, nervous system, heart, and stomach) is not repeated here. However, all the characters that are useful for species comparison (e.g., color of live animals, radular formulae, intestinal loops, and reproductive system) are described for the new species. Special attention has been given to illustrating the holotype of the new species and its habitat, including an image of its type locality.

DNA extraction and PCR amplification

DNA was extracted using a phenol-chloroform extraction protocol with cetyltrimethyl-ammonium bromide (CTAB). The mitochondrial cytochrome c oxidase I region (COI) and 16S region were amplified using the following universal primers: LCO1490 (5’-3’) GGT CAA CAA ATC ATA AAG ATA TTG G, and HCO2198 (5’-3’) TAA ACT TCA GGG TGA CCA AAR AAY CA (Folmer et al. 1994), 16Sar-L (5’-3’) CGC CTG TTT ATC AAA AAC AT (Palumbi 1996), and the modified Palumbi primer 16S 972R (5’-3’) CCG GTC TGA ACT CAG ATC ATG T (Dayrat et al. 2011). The nuclear ITS2 region and 28S region were amplified with the following primers: LSU-1 (5’-3’) CTA GCT GCG AGA ATT AAT GTG A, and LSU-3 (5’-3’) ACT TTC CCT CAC GGT ACT TG (Wade and Mordan 2000), 28SC1 (5’-3’) ACC CGC TGA ATT TAA GCA T (Hassouna et al. 1984), and 28SD3 (5’-3’) GAC GAT CGA TTT GCA CGT CA (Vonnemann et al. 2005). The 25 μl PCRs for COI and 16S contained 15.8 μl of water, 2.5 μl of 10X PCR Buffer, 1.5 μl of 25 mM MgCl2, 0.5 μl of each 10 μM primer, 2 μl of dNTP Mixture, 0.2 μl (1 unit) of TaKaRa Taq (Code No. R001A), 1 μl of 20 ng/μl template DNA, and 1 μl of 100X BSA (Bovine Serum Albumin). The PCRs for ITS2 used the reagents in the same amounts as COI and 16S, except that water was reduced to 14.8 μl and the amount of 100X BSA was increased to 2 μl. The PCRs for 28S included 14.8 μl of water, 2.5 μl of 10X PCR Buffer, 0.5 μl of each 10 μM primer, 1 μl of dNTP Mixture, 5 μl of Q solution (which includes MgCl2) and 0.5 μl of 20 ng/μl template DNA. The thermoprofile used for COI and 16S was: 5 minutes at 94 °C; 30 cycles of 40 seconds at 94 °C, 1 minute at 46 °C, and 1 minute at 72 °C; and a final extension of 10 minutes at 72 °C. The thermoprofile used for ITS2 was: 1 minute at 96 °C; 35 cycles of 30 seconds at 94 °C, 30 seconds at 50 °C, and 1 minute at 72 °C; and a final extension of 10 minutes at 72 °C. The thermoprofile used for 28S was: 4 minutes at 94 °C; 38 cycles of 50 seconds at 94 °C, 1 minute at 52 °C, and 2 minutes 30 seconds at 72 °C; and a final extension of 10 minutes at 72 °C. The PCR products were cleaned with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) prior to sequencing. Untrimmed sequenced fragments represented approximately 680 bp for COI, 530 bp for 16S, 740 bp for ITS2, and 1000 bp for 28S.

Phylogenetic analyses

Chromatograms were consulted to resolve rare ambiguous base calls. DNA sequences were aligned using Clustal W in MEGA 7 (Kumar et al. 2016). Nineteen onchidiid species outside Onchidium were selected as outgroups from our previous studies (Dayrat et al. 2011, 2016, 2017, 2018, 2019a, b; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c): Alionchis jailoloensis Goulding & Dayrat in Goulding et al. 2018a, Marmaronchis marmoratus (Lesson, 1831), Marmaronchis vaigiensis (Quoy & Gaimard, 1825), Melayonchis aileenae Dayrat & Goulding in Dayrat et al. 2017, Melayonchis annae Dayrat in Dayrat et al. 2017, Melayonchis eloisae Dayrat in Dayrat et al. 2017, Melayonchis siongkiati Dayrat & Goulding in Dayrat et al. 2017, Onchidella celtica (Cuvier in Audouin and Milne-Edwards 1832), Onchidella nigricans (Quoy & Gaimard, 1832), Onchidina australis (Semper, 1880), Paromoionchis daemelii (Semper, 1880), Paromoionchis tumidus (Semper, 1880), Peronia sp. (Hawaii), Peronia sp. (Okinawa), Peronina tenera (Stoliczka, 1869), Peronina zulfigari Goulding & Dayrat in Goulding et al. 2018c, Platevindex luteus (Semper, 1880), Wallaconchis ater (Lesson, 1831), and Wallaconchis sinanui Goulding & Dayrat in Goulding et al. 2018b. All new DNA sequences were deposited in GenBank and vouchers deposited in museum collections (Table 1). The ends of each alignment were trimmed. Alignments of mitochondrial (COI and 16S) sequences and nuclear (ITS2 and 28S) sequences were concatenated separately in order to test whether these two data sets support the same relationships. The concatenated mitochondrial alignment included 986 nucleotide positions: 582 (COI) and 404 (16S). The concatenated ITS2 and 28S alignment included 1467 nucleotide positions: 472 (ITS2) and 995 (28S).

Two independent sets of phylogenetic analyses were performed: 1) Maximum Likelihood and Bayesian analyses with concatenated mitochondrial COI and 16S sequences; 2) Maximum Parsimony analyses with concatenated nuclear ITS2 and 28S sequences. Maximum Parsimony analyses were conducted in PAUP v 4.0 (Swofford 2002) with gaps coded as a fifth character state, and 100 bootstrap replicates conducted using a full heuristic search. Prior to Maximum Likelihood and Bayesian phylogenetic analyses, the best-fitting evolutionary model was selected for each locus separately using the Model Selection option from Topali v2.5 (Milne et al. 2004): a GTR + G model was independently selected for COI and 16S. Maximum Likelihood analyses were performed using PhyML (Guindon and Gascuel 2003) as implemented in Topali. Node support was evaluated using bootstrapping with 100 replicates. Bayesian analyses were performed using MrBayes v3.1.2 (Ronquist and Huelsenbeck 2003) as implemented in Topali, with five simultaneous runs of 1.5×106 generations each, sample frequency of 100, and burn in of 25% (and posterior probabilities were also calculated). Topali did not detect any issue with respect to convergence. All analyses were run several times and yielded the same result.

In addition, another set of analyses was performed with only COI sequences. Genetic distances between COI sequences were calculated in MEGA 7 as uncorrected p-distances. COI sequences were also translated into amino acid sequences in MEGA using the invertebrate mitochondrial genetic code to check for the presence of stop codons (no stop codon was found).

Results

Molecular phylogenetic analyses (Figs 1, 2)

DNA sequences were used to test species limits within Onchidium. The monophyly of Onchidium is recovered in all analyses. In the analyses based on mitochondrial COI and 16S concatenated sequences, four least-inclusive units are reciprocally monophyletic: O. reevesii, O. typhae, O. stuxbergi and the new species, O. melakense. The monophyly of each species is strongly supported by a bootstrap support of 98 or higher and a posterior probability of 1. Analyses with nuclear 28S and ITS2 concatenated sequences yielded similar results: O. typhae, O. stuxbergi, and the new species O. melakense are strongly supported with bootstrap values of 100. Onchidium reevesii could not be included in the nuclear analyses because ITS2 sequences for the specimens from China are not available in GenBank (Table 1).

Figure 1. 

Phylogenetic tree showing the relationships between Onchidium individuals based on mitochondrial COI and 16S DNA sequences. Numbers by the nodes are the bootstrap values (Maximum Likelihood analysis) and the posterior probabilities (Bayesian analysis); only significant numbers (> 65% and > 0.9) are indicated. All other sequences serve as outgroups. Information on specimens can be found in the lists of material examined and Table 1. The colors used for each Onchidium species are the same as those used in Figs 2–4.

Figure 2. 

Consensus tree showing relationships between Onchidium individuals based on concatenated nuclear ITS2 and 28S sequences. Numbers by the nodes are the bootstrap values (Maximum Parsimony analysis); only significant numbers (> 50%) are indicated. All other sequences serve as outgroups. Information on specimens can be found in the lists of material examined and Table 1. The colors used for each Onchidium species are the same as those used in Figs 1, 3, and 4.

Pairwise genetic divergences (Fig. 3)

Pairwise genetic distances (between COI sequences) support the existence of four species of Onchidium as least-inclusive molecular units (Table 2). The intra-specific genetic distances are all below 3.2% (within O. stuxbergi). The inter-specific distances vary from 8.6% (between O. reevesii and O. stuxbergi) to 14.3% (between O. reevesii and O. typhae). So, overall, the distance gap between the four Onchidium species is between 3.2% and 8.6%.

Figure 3. 

Diagram to help visualize pairwise genetic distances between COI sequences within and between Onchidium species (Table 2). Ranges of minimum to maximum distances are indicated (in percentages). For instance, the intra-specific divergences within O. typhae are between 0.1 and 0.4%, while the inter-specific divergences between O. typhae and the three other species are between 10.9 and 14.3%. Overall, the distance gap between all four Onchidium species is between 3.2 and 8.6%. The colors used for each Onchidium species are the same as those used in Figs 1, 2, and 4.

Table 2.
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Pairwise genetic distances between mitochondrial COI sequences in Onchidium. Ranges of minimum to maximum distances are indicated (in percentage). For instance, the intra-specific divergences within O. typhae are between 0.1 and 0.4%, while the inter-specific divergences between O. typhae and O. stuxbergi are between 11.4 and 12.9%.

Species O. typhae O. stuxbergi O. melakense O. reevesii
O. typhae 0.1–0.4 – – –
O. stuxbergi 11.4–12.9 0.0–3.2 – –
O. melakense 10.9–11.9 10.7–13.0 0.0–0.7 –
O. reevesii 13.4–14.3 8.6–10.1 12.6–13.5 0.1–0.5

Comparative anatomy

Due to its distinctive external color, the new species was immediately recognized in the field as new to science. It also differs in internal anatomy from the three other known species. In particular, the penial sheath in the male copulatory apparatus is short and straight while coiled in the three other species (Table 3).

Table 3.
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Morphological differences among Onchidium species. All traits are subject to individual variation. Information regarding O. stuxbergi and O. typhae is from Dayrat et al. (2016). Information regarding O. reevesii is from Dayrat et al. (2016) for the holotype, and from Wang et al. (2018) for non-type material. For the type of intestinal loops, the orientation of the transitional loop (TL) is provided. For the radular formulae, the range of number of rows (e.g., 60 to 80 rows in O. melakense) and the range of number of lateral teeth per half row (e.g., 70 to 110 in O. melakense) are provided. The number of radular rows was not described in O. reevesii by Wang et al. (2018).

Species O. melakense O. reevesii O. stuxbergi O. typhae
Size Up to 45 mm Up to 67 mm Up to 55 mm Up to 65 mm
Dorsal color Light brown Brown Brown, occasionally black Brownish
Foot color Pale yellow-beige Whitish or light yellow Bright orange Grey to yellow, sometimes greenish
Hyponotum color White Light grey or beige-white Greyish to yellowish, sometimes greenish Grey to yellow, sometimes greenish
Black dots on hyponotum Absent Present Present Absent
Type of intestinal loops III, TL from 1 to 5 o’clock III, TL at 2 o’clock III, TL from 1 to 8 o’clock II, TL from 8 to 9 o’clock
Radular formulae 60/80, 70/110 62/110 (lateral teeth only) 50/70, 68/80 53/65, 65/80
Penial gland spine length Up to 1.1 mm No data available Up to 2 mm Up to 1.2 mm
Penial sheath Short and straight Long and heavily coiled in spirals Long and heavily coiled in spirals Long and heavily coiled in spirals
Insertion of retractor muscle in visceral cavity Middle Posterior third Posterior third Near the heart (India) & posterior half (everywhere else)
Anterior retractor muscle Present (occasionally absent) Absent Present (possibly occasionally absent) Absent

Systematics and anatomical descriptions

Family Onchidiidae Rafinesque, 1815

Onchidium Buchannan, 1800

Onchidium Buchannan, 1800: 132.

Labbella Starobogatov, 1970: 45; Starobogatov 1976: 211. Replacement name for Elophilus Labbé, 1935, preoccupied by Elophilus Meigen, 1803 [Diptera].

Type species

Onchidium typhae Buchannan, 1800, by monotypy.

Gender

Neuter, gender of the final component of Onchidium, a name formed from the masculine Greek word ὁ ὂγκος (mass, tumor) and the neuter Latin suffix -ium (ICZN 1999: Article 30.1.1).

Diagnosis

Body not flattened. No dorsal gills. Dorsal eyes present on notum. Large, conical, pointed papillae present on notum. Retractable, central papilla (with three or four dorsal eyes) present but not significantly larger than surrounding papillae. Eyes at tip of extremely long ocular tentacles. Male opening below right ocular tentacle and slightly to its left. Transversal protuberance on oral lobes present. Foot wide. Pneumostome medial, on average in middle between foot margin and notum margin. Intestinal loops of types II and III. Rectal gland present. Accessory penial gland present with a hollow spine but no muscular sac. Penis with hooks.

Distinctive features

In the field, Onchidium slugs differ from all other onchidiids by the presence of large, conical, pointed papillae on the dorsal notum. However, papillae can only be observed when animals remain undisturbed. In disturbed (and preserved) animals, papillae remain pointed but become minute. However, the best feature to identify Onchidium slugs in the field is the presence of very long and thin ocular tentacles (up to 20 mm). Papillae can definitely be confused between genera but Onchidium slugs are (almost) the only ones with such long eye tentacles. Very long ocular tentacles are also present in Alionchis jailoloensis but they are much thicker (in diameter) than those of Onchidium. Also, Alionchis jailoloensis is so far only known from Halmahera (where Onchidium is not found) and lacks the large, conical, pointed papillae that are typical of Onchidium. Finally, in Alionchis, the pneumostome is always located exactly at the margin of the notum. Therefore, Onchidium slugs cannot be confused with Alionchis slugs.

Distribution

From northeastern India (West Bengal) to the Philippines, including the Strait of Malacca, Singapore, Thailand, Vietnam, eastern Borneo, and China (Fig. 4).

Figure 4. 

Geographic distribution of the four known Onchidium species. Dots correspond to known records. The colors used for each Onchidium species are the same as those used in Figs 1–3.

Remarks

The diagnosis and the distinctive features provided above are slightly updated from Dayrat et al. (2016). The synonymy of Labbella (replacement name for Elophilus) with Onchidium was already discussed by Dayrat et al. (2016). In brief, Labbella ajuthiae (Labbé, 1935), the type species of Labbella, is a junior synonym of Onchidium stuxbergi (Westerlund, 1883). Therefore, both Labbella and Onchidium apply to the same clade. We remark a detail concerning the nomenclatural status of Elophilus Meigen, 1803. Under plenary powers of the Commission (ICZN 1993: 256), the generic name Elophilus Meigen, 1803 was “suppressed for the purposes of the Principle of Priority but not for those of the Principle of Homonymy.” So, even though Elophilus Meigen, 1803, was placed on the Official Index of Rejected and Invalid Generic Names in Zoology, Elophilus Labbé, 1935 remains a junior homonym of Elophilus Meigen, 1803, hence the necessity of the replacement name Labbella. Also, note that the publication date for Labbella by Starobogatov is 1970 instead of 1976 (Dayrat 2009; Dayrat et al. 2016).

Onchidium melakense Dayrat & Goulding, sp. nov.

http://zoobank.org/D7C0CD7A-C888-407B-9E36-C1544835B355
Figs 5, 6, 7, 8, 9, 10, 11, 13G, H

Type material

Holotype. Malaysia • holotype, designated here, 45/25 mm [5979 H]; Peninsular Malaysia, Kuala Sepatang; 04°50.605'N, 100°38.133'E; 28 Jul 2016; B Dayrat and field party leg.; st 258, old forest with tall Rhizophora trees, high in the tidal zone (ferns), in educational mangrove preserve; USMMC 00075.

Additional material examined

India – Andaman Islands • 1 specimen 25/15 mm [1105]; Middle Andaman, Rangat, Shyamkund; 12°28.953'N, 92°50.638'E; 11 Jan 2011; B Dayrat and field party leg.; st 57, by a large river, deep mangrove with tall trees, small creeks, and many muddy logs; BNHS 94. Malaysia – Peninsular Malaysia • 3 specimens 30/20 mm [5978], 35/18 mm [5981], and 35/30 mm [5982]; same collection data as for the holotype; USMMC 00076. Indonesia – Sumatra • 4 specimens 27/20 mm [1723], 25/22 mm [1720], 35/20 mm [1769], and 40/20 mm [1771]; Pulau Sinaboi; 02°18.145'N, 100°59.309'E; 8 Oct 2012; M Khalil and field party leg.; st 73, mangrove forest with medium Rhizophora and Avicennia trees, logs, hard mud; UMIZ 00001.

Distribution

(Fig. 4). Western Peninsular Malaysia (type locality), eastern Sumatra (Indonesia), and Andaman Islands (India).

Etymology

Onchidium melakense is named after the Strait of Malacca or ‘Selat Melaka’ in Malay: melakense is a Latinized adjective that agrees in gender (neuter) with the generic name (ICZN 1999: Art. 31.2). The mangrove gastropod diversity of the Strait of Malacca is extraordinarily rich. For instance, three of the four known Onchidium species are sympatric there: O. typhae, O. stuxbergi, and the new species O. melakense. The fourth species, O. reevesii, is restricted to the Chinese coast (Fig. 4).

Habitat

(Fig. 5). Onchidium melakense was found under a log (type locality, Peninsular Malaysia), inside crevices of a muddy log (Sumatra) and on the cemented wall of a bridge over a mangrove creek (Andaman Islands). Most individuals were hidden and could not have been found if logs had not been turned over and thoroughly searched inside. This search, however, should be done with caution because pit vipers often like to rest near logs in mangroves (Fig. 5D). Onchidium melakense does not seem to particularly favor the habitat where O. typhae and O. stuxbergi are most commonly found, i.e., the surface of muddy trunks, logs, and Thalassina lobster mounds. Even though O. typhae and O. stuxbergi can be found at the same sites as O. melakense (they are found in the Matang mangrove, where the type locality of O. melakense is located), they do not share exactly the same micro-habitats. Clearly, all these species hide in crevices at high tide but, unlike O. typhae and O. stuxbergi, O. melakense appears to remain hidden at low tide as well.

Figure 5. 

Habitats, Onchidium melakense A type locality, Peninsular Malaysia, Kuala Sepatang, old forest with tall Rhizophora trees, high in the tidal zone (ferns), in educational mangrove preserve (st 258) B Sumatra, Pulau Sinaboi, mangrove forest with medium Rhizophora and Avicennia trees, dead logs, hard mud (st 73) C old log with crevices which O. melakense typically favors (st 73) D mangrove pit viper (arrow) resting by a log (st 73).

Abundance

Onchidium melakense is a rare species. In total, we found only nine individuals: four individuals at the type locality in Peninsular Malaysia, four individuals at one site in eastern Sumatra, and a single individual in the Andaman Islands.

Color and external morphology of live animals

(Figs 6, 7). Live animals are not covered with mud and the color of their dorsum can normally be seen. The dorsum is homogenously light brown. The hyponotum is distinctly white. The foot is pale yellow-beige. The ocular tentacles are dark grey and are extremely long (up to 2 cm) when animals are undisturbed. The head is grey. Large, conical, pointed papillae (which are typical of Onchidium species) are present but can only be seen when the animal remains undisturbed for a long time. Some of these papillae bear dorsal eyes. When animals are disturbed, papillae immediately retract and become minute (although they remain pointed). A central papilla (with three or four dorsal eyes), fully retractable within the dorsal notum, is also present but is not particularly more prominent than surrounding papillae. Crawling individuals are up to 45 mm long. Preserved specimens no longer display the distinct color seen in live animals: the dorsal notum remains light brown and the hyponotum remains white, but the foot of preserved animals is whitish.

Figure 6. 

Live animals, Onchidium melakense A dorsal view, holotype, 45 mm long [5979], Peninsular Malaysia (USMMC 00075) B dorsal view, 30 mm long [5978], Peninsular Malaysia (USMMC 00076) C dorsal view, 35 mm long [5981], Peninsular Malaysia (USMMC 00076) D dorsal view, 35 mm long [1769], Sumatra (UMIZ 00001) E ventral view, same as A F ventral view, same as C G dorsal view, 40 mm long [1771], Sumatra (UMIZ 00001).

Figure 7. 

External morphology and digestive system, Onchidium melakense, Peninsular Malaysia A–C holotype [5979] (USMMC 00075) D [5978] (USMMC 00076) A dorsal, anterior view B posterior, ventral view (dotted lines indicate where the foot was cut to show the anus) C digestive system, dorsal view D digestive system, dorsal view. Abbreviations: a anus, ddg dorsal lobe of digestive gland, f foot (pedal sole), fo female opening, h hyponotum, i intestine, mo male opening, ol oral lobe, ot ocular tentacle, p pneumostome, pdg posterior lobe of the digestive gland, ppg peripodial groove, rg rectal gland, st stomach, tp transversal protuberance (on oral lobe). Scale bars: 5 mm (A–C), 3 mm (D).

Digestive system

(Figs 7, 9). Examples of radular formulae are presented in Table 4. The median cusp of the rachidian tooth is always present; its lateral cusps (on its lateral sides) can be conspicuous. The intestine is of type III, with a transitional loop oriented to the right, approximately from 1 to 5 o’clock (for a comparison of intestinal types between Onchidium species, see the Discussion).

Figure 8. 

Radula, Onchidium melakense, Peninsular Malaysia, holotype [5979] (USMMC 00075) A rachidian and innermost lateral teeth B right lateral teeth C right lateral teeth. Abbreviations: 1llt first left lateral tooth, 1rlt first right lateral tooth, bls basal lateral spine, hlt hook of lateral tooth, lc lateral cusp of rachidian tooth, mc median cusp of rachidian tooth, rt rachidian tooth. Scale bars: 10 μm (A,) 20 μm (B, C).

Figure 9. 

Radula, Onchidium melakense, Sumatra (UMIZ 00001) A left, half rows, [1720] B rachidian and innermost lateral teeth, [1720] C lateral teeth, [1723] D outermost, lateral teeth, [1723]. Abbreviations: 1llt first left lateral tooth, 1rlt first right lateral tooth, 2llt second left lateral tooth, 2rlt second right lateral tooth, hlt hook of lateral tooth, lc lateral cusp of rachidian tooth, mc median cusp of rachidian tooth, rt rachidian tooth. Scale bars: 200 μm (A), 10 μm (B), 60 μm (C), 20 μm (D).

Table 4.
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Radular formulae for Onchidium melakense. Each formula follows the same format: number of rows × number of lateral teeth per left half row – 1 (rachidian tooth) – number of lateral teeth per right half row. Each DNA extraction number corresponds to one individual. The letter H next to an extraction number indicates the holotype.

DNA extraction number Voucher Radular formula Specimen length (mm)
5979 H USMMC 00075 80 × 110-1-110 45
5982 USMMC 00076 80 × 90-1-90 35
1723 UMIZ 00001 75 × 90-1-90 27
5981 USMMC 00076 65 × 70-1-70 35
1720 UMIZ 00001 60 × 85-1-85 25

Reproductive system

(Fig. 10A). The receptaculum seminis (caecum) is bent, ovate and elongated. The spermatheca is spherical-ovate and connects to the oviduct through a short duct with one loop. The oviduct and the deferent duct are narrow and straight. A vaginal gland is absent.

Figure 10. 

Reproductive system, Onchidium melakense A, B Peninsular Malaysia, holotype [5979] (USMMC 00075) C Sumatra, [1723] (UMIZ 00001) A posterior, hermaphroditic, reproductive parts B anterior, male, copulatory parts C anterior, male, copulatory parts. Abbreviations: ag accessory penial gland, arm anterior penial retractor muscle, dd deferent duct, fgm female gland mass, hg hermaphroditic gland, ov oviduct, ps penial sheath, rm penial retractor muscle, rs receptaculum seminis, sp spermatheca, v vestibule. Scale bars: 4 mm (A), 5 mm (B), 2 mm (C).

Copulatory apparatus

(Figs 10, 11). The male anterior organs consist of the penial complex (penial papilla, penial sheath, deferent duct, and retractor muscle) and the accessory penial gland (flagellum and hollow spine). The penial complex and the accessory penial gland share the same vestibule and male opening. The flagellum of the penial gland is coiled. Distally, it ends in a hard, hollow spine. The hollow spine is narrow, elongated, and slightly curved. Its length varies from 0.8 to 1.1 mm. Its diameter is approximately 50 μm for most of its length (but approximately 140 μm at its conical base). The hollow spine does not open directly into the proximal region of the vestibule. There is a transversal, flat disc at the distal end of the flagellum (approximately 0.4 mm in diameter) through which the hollow spine must protrude in order to be outside and shared with the partner (Fig. 11D).

Figure 11. 

Male, anterior, copulatory parts, Onchidium melakense A, C Sumatra, [1723] (UMIZ 00001) B, E, F Peninsular Malaysia, holotype [5979] (USMMC 00075) D, G, H Peninsular Malaysia, holotype [5982] (USMMC 00076) A stalk and penial hooks B penial hook C penial hooks D flat disc at distal end of flagellum of penial accessory gland (the arrow indicates the hole through which the hollow spine protrudes) E hollow spine F hollow spine tip G hollow spine H hollow spine tip. Scale bars: 300 μm (A), 10 μm (B, C, F), 100 μm (D, G, H), 200 μm (E).

The penial sheath is short (less than 5 mm) and straight, not coiled in spirals. The (posterior) retractor muscle is longer than the penial sheath and inserts at about the middle of the visceral cavity floor. An additional, anterior retractor muscle is present (and occasionally absent) in the distal part of the penial sheath. The deferent duct is highly convoluted with many loops. The penis is made of two distinct parts. The proximal part is a hollow, solid, flexible stalk with no hooks; its length varies from 1.2 to 1.8 mm and its diameter from 100 μm to 200 μm. The distal part is short (up to approximately 0.8 mm long), soft, and covered with penial hooks internally. Penial hooks are inside the tube-like penis when the penis is retracted inside the penial sheath. During copulation, the penis is everted like a glove and the hooks are then on the outside. Penial hooks are conical, curved, pointed, and up to 60 μm long.

Diagnostic features

Externally, Onchidium melakense differs from all other Onchidium species by its color. Onchidium melakense is the only known species with a light brown dorsal notum, a pale yellow-beige foot, and a white hyponotum (see Table 3 and the Identification key). Internally, O. melakense is the only known species with a short and straight penial sheath, while in other species the penial sheath is long and coiled in spirals (Table 3). Other traits are helpful as well but may not be as diagnostic as the penial sheath. For instance, intestinal loops help distinguish O. melakense from O. typhae but not from O. stuxbergi (Table 3).

Remarks

A new species name is needed because no existing name applies to the species described here, based on the examination of all the type specimens available in the Onchidiidae, a careful study of all the original descriptions, and our ongoing taxonomic revision of every genus of the family (Dayrat et al. 2016, 2017, 2018, 2019a, b; Dayrat and Goulding 2017; Goulding et al. 2018a, b, c). Moreover, based on its known distribution (Andaman Islands, eastern Sumatra, western Peninsular Malaysia), O. melakense is expected to be found in other places, such as the Nicobar Islands. However, O. melakense is rare, at least in comparison to its two sympatric species, O. typhae and O. stuxbergi. Large populations (with dozens of individuals) of O. typhae were encountered (Dayrat et al. 2016). In the field, O. typhae and O. stuxbergi can be found by looking at the muddy surface of trunks, logs, and lobster mounds, while O. melakense can be found only if one actively searches for it under and inside logs.

Onchidium stuxbergi (Westerlund, 1883)

Figs 12B, C, 13C–E

Vaginulus stuxbergi Westerlund, 1883: 165; Westerlund 1885, 191–192, pl. 2, fig. 2a–c.

Onchidium stuxbergi (Westerlund, 1883): Dayrat et al. 2016: 21–32, figs 9–16.

Onchidium pallidipes Tapparone-Canefri, 1889: 329–331. Syn. nov.

Onchidium nigrum Plate, 1893: 188–190, pl. 8, fig. 31a, pl. 10, fig. 53, pl. 11, fig. 75; Hoffmann 1928: 78; Labbé 1934: 223–224, figs 58–61.

Elophilus ajuthiae Labbé, 1935: 312–317, figs 1–3. Elophilus Labbé, 1935, preoccupied by Elophilus Meigen, 1803 [Diptera], was replaced by Labbella Starobogatov, 1970.

Type material

Lectotype and paralectotypes (Vaginulus stuxbergi). Brunei DARUSSALAM • lectotype, 43/25 mm; Brunei Bay, northwestern Borneo; SMNH 1334. • 11 paralectotypes, 35/30 to 15/12 mm; SMNH 1334, SMNH 7523. For detailed information, see Dayrat et al. (2016: 22).

Lectotype and paralectotypes (Onchidium pallidipes). Myanmar • lectotype, 15/12 mm, designated here; Moulmein, Tenasserim [now Mawlamyine, Tanintharyi]; USNM 127328. • 1 paralectotype, 12/9 mm; same collection data as for the lectotype; ZMH 27467/1. • 1 paralectotype, 10/5 mm; same collection data as for the lectotype; ZMB/Moll 47190. The lectotype is poorly-preserved but its dorsal notum bears some faint traces of what could have been dorsal papillae similar to those found in Onchidium; its copulatory apparatus and its digestive system are drawn for the present study (Fig. 12B, C). One paralectotype is completely destroyed (ZMB/Moll 47190): it likely dried and it cannot be identified. The other paralectotype is an immature specimen with no male or female reproductive system (ZMH 27467/1), but its intestinal loops are exactly identical to those of the lectotype. Labels of the three type specimens indicate Moulmein as locality. All three type specimens seem to be from the same locality according to the original description (Tapparone-Canefri 1889: 330), and are preserved in three different museum collections.

Figure 12. 

Name-bearing types of Onchidium multinotatum and Onchidium pallidipes A digestive system, type III with a transitional loop at 2 o’clock (based on marks left by the intestine in the digestive gland), dorsal view, holotype, O. multinotatum (ZMB/Moll 240117) B digestive system, type III with a transitional loop at 2 o’clock, dorsal view, lectotype, O. pallidipes (NMNH 127328) C anterior, male, copulatory parts, lectotype, O. pallidipes (NMNH 127328). Abbreviations: ag accessory penial gland, dd deferent duct, ddg dorsal lobe of digestive gland, i intestine, pdg posterior lobe of the digestive gland, ps penial sheath, rg rectal gland, rm penial retractor muscle, st stomach, v vestibule. Scale bars: 3 mm (A, B), 2 mm (C).

Holotype (Onchidium nigrum). Borneo • holotype, 40/30 mm, by monotypy; unidentified area on the island of Borneo; ZMB/Moll 22749. For detailed information, see Dayrat et al. (2016: 23).

Syntypes (Labbella ajuthiae). Thailand • 3 syntypes 20/17 mm, 20/15 mm, and 20/14 mm; Chao Phraya River, Ayutthaya Province; brackish waters; MNHN-IM-2000-22965. For detailed information, see Dayrat et al. (2016: 23).

Additional material examined

Indonesia – Sumatra • 1 specimen 23/14 mm [1775]; Dumai; 01°42.838'N, 101°23.286'E; 9 Oct 2012; M Khalil and field party leg.; st 74, mangrove forest just behind abandoned buildings, high intertidal, with many Thalassina mounds and small creeks; UMIZ 00003.

Distribution

(Fig. 4). Myanmar (type locality of O. pallidipes, new record), and eastern Sumatra (new record). Other known records are in Singapore, Sabah and western Peninsular Malaysia (Malaysia), Brunei Darussalam, Bohol (Philippines), Vietnam, Thailand (Gulf of Thailand), and southern China up to 22°10'N (Dayrat et al. 2016: 24).

Habitat

In eastern Sumatra, O. stuxbergi was found on a muddy log, one of the habitats in which it is known to live (Dayrat et al. 2016: 24). In the original description of O. pallidipes, it is indicated that the slugs were found under the plant debris of sugar cane (Tapparone-Canefri 1889: 330), which is an unusual but possible habitat.

Abundance

The present record from eastern Sumatra confirms that O. stuxbergi is not found in high densities (a few individuals at most) even though it is found at many sites across its distribution range.

Remarks

Given its known records on the other side of the Strait of Malacca (western Peninsular Malaysia) and Singapore, Onchidium stuxbergi was expected to be present in eastern Sumatra. Anatomically, Onchidium stuxbergi in Sumatra is indistinguishable from the individuals found elsewhere. Also, the DNA sequences of the individual from eastern Sumatra are nested within the rest of the species (Fig. 1).

A detailed discussion on the synonymy of Labbella ajuthiae and Onchidium nigrum with O. stuxbergi can be found in Dayrat et al. (2016). The type material of Onchidium pallidipes was briefly addressed in a study on the genus Melayonchis Dayrat & Goulding in Dayrat et al. 2017. At the time, it was thought that O. pallidipes was a nomen dubium. However, the dissection of far more onchidiid species in the past few years has revealed that the coiled penial sheath of the lectotype of O. pallidipes (Fig. 12C) is typical of what is observed only in Onchidium (except for the new Onchidium species described here, in which the penial sheath is short and straight). Also, the poorly-preserved dorsal notum of the lectotype of O. pallidipes bears some faint traces of what could have been papillae similar to those found in Onchidium. So, now, it is considered that the name Onchidium pallidipes applies to an Onchidium species. Note that this application is exclusively based on the lectotype (designated here) because a paralectotype is destroyed and the other paralectotype is an immature specimen.

Onchidium typhae is supposedly present in Myanmar because it is known from West Bengal eastward all the way to Singapore (Fig. 4). However, O. pallidipes cannot apply to O. typhae because the intestinal loops of O. typhae are always of type II (see below, Fig. 13A, B). Given the intestinal loops of type III of its lectotype (Fig. 12B), O. pallidipes applies to O. stuxbergi, also characterized by intestinal loops of type III (Fig. 13C–E). The hollow spine of the accessory penial gland of the lectotype of O. pallidipes is 2.7 mm long, which is slightly outside the range known so far in O. stuxbergi (0.5 to 2 mm), but that character is expected to vary. No additional retractor muscle fibers were found in the distal part of the male apparatus of the lectotype of O. pallidipes (Fig. 12C), even though they are known to be present in O. stuxbergi (Dayrat et al. 2016: fig. 11C). However, the lack of an anterior, retractor muscle in the lectotype of O. pallidipes can be explained by the fact that it is relatively small (15 mm long) and poorly-preserved. Also, this trait was found to vary in O. melakense and it is possible that it also varies in O. stuxbergi, especially among small individuals. Finally, it is worth pointing out that Tapparone-Canefri (1889: 330) selected the specific name pallidipes to refer to the “pale foot” of the preserved specimens he examined for the original description. Tapparone-Canefri (1889: 330) did not have access to information on live animals but he suggested that the foot was “probably ocher in living specimens,” which fits well with O. stuxbergi (of which the foot is bright orange).

Figure 13. 

Types of intestinal loops in the genus Onchidium. Small black arrows indicate the direction of the intestinal transport, which starts in the blue loop. A blue loop turns clockwise. A yellow loop turns counterclockwise. A green loop is transitional in between a blue loop and a yellow loop. The orientation of the transitional (green) loop is indicated with a red arrow. Details on individuals of O. reevesii, O. typhae, O. stuxbergi can be found in Dayrat et al. (2016) A O. typhae, type II with a transitional loop at 9 o’clock, [1007] B O. typhae, type II with a transitional loop at 8 o’clock (from Dayrat et al. 2016: fig. 5E) C O. stuxbergi, type III with a transitional loop at 3 o’clock (from Dayrat et al. 2016: fig. 11B) D O. stuxbergi, type III with a transitional loop at 1 o’clock, (PNM 041200) E O. stuxbergi, type III with a transitional loop at 8 o’clock, [5605] F O. reevesii, holotype, type III with a transitional loop at 2 o’clock (from Dayrat et al. 2016: fig. 14A) G O. melakense, type III with a transitional loop at 1 o’clock, holotype [5979] (USMMC 00075) H O. melakense, type III with a transitional loop at 5 o’clock, [5978] (USMMC 00076). Scale bars: 3 mm (A, H), 5 mm (B, C, E–G), 4 mm (D).

In the future, if fresh material collected from the type locality of O. pallidipes is shown to form its own reciprocally-monophyletic unit using both mitochondrial and nuclear DNA sequences, and if it is shown to be anatomically fully compatible with the lectotype of O. pallidipes (especially regarding the length of the spine of the accessory penial gland), then O. pallidipes could become a valid name for a distinct Onchidium species endemic to the eastern Andaman Sea. This hypothesis cannot be completely ruled out at this stage. However, given the data currently available and all the reasons given above, we regard O. pallidipes as a junior synonym of O. stuxbergi.

The name Onchidium multinotatum Plate, 1893 needs to be briefly discussed. Its type locality is Cavite, Manila, in Luzon, Philippines. The original description is quite detailed, as often with Plate, but the holotype (30/15 mm), by monotypy (ZMB/Moll 240117), is very poorly preserved because it likely dried for a while. Only a few destroyed pieces of the digestive system remain in the empty body wall. Some of the features that Plate described could unfortunately not be checked (rectal gland present, accessory penial gland present, penial gland spine 4 mm long, penis 30 mm long). Plate separated the intestine from the digestive gland but described intestinal loops of type II. However, marks left by the intestine on the dorsal aspect of the digestive glands suggest that the intestinal loops of O. multinotatum were of type III (Fig. 12A). Given the critical uncertainty regarding the type of intestinal loops and that traits described by Plate cannot be checked, Onchidium multinotatum is regarded as a nomen dubium. Onchidium multinotatum could apply to O. stuxbergi, but that is not certain. We collected many onchidiids from the Philippines, including in Batangas, just south of Manila in Luzon, and the only species that could match the anatomy of O. multinotatum (acknowledging some uncertainty) is O. stuxbergi. Unfortunately, the type locality of O. multinotatum is in a part of Manila which is now completely developed and we could not collect onchidiids there.

Identification key

A key based on external characters is provided to help identify Onchidium slugs in the field. Information on the color of live individuals of O. typhae and O. stuxbergi is from Dayrat et al. (2016). Information on the color of live individuals of O. reevesii is from Wang et al. (2018).

1 The foot is bright orange O. stuxbergi*
– The foot is not bright orange 2
2 The hyponotum is pure white and the dorsum is light brown O. melakense**
– The hyponotum is not pure white and the dorsum is brown 3
3 The hyponotum is light grey or beige-white, the foot is whitish or light yellow, and the dorsum is brown O. reevesii***
– The hyponotum and the foot vary between greyish and yellowish, and sometimes even greenish, and the dorsum is brown O. typhae****

Discussion

Onchidium slugs can easily be identified in the field at the generic and specific levels. Indeed, all live Onchidium slugs are characterized by two external features that distinguish them from other onchidiids: large, conical, pointed papillae, and very long and thin ocular tentacles (easily up to 20 mm). Also, each Onchidium species is characterized by a distinct color and, even though O. stuxbergi, O. typhae, and O. melakense are sympatric, they cannot be confused (see the Identification key above, and Table 3). The only other genus in which species can be easily distinguished in the field is Melayonchis Dayrat & Goulding in Dayrat et al. 2017, but, in most other onchidiid genera, such as Peronina, Wallaconchis, or Paromoionchis, species are cryptic externally. This could suggest that Onchidium and Melayonchis species are relatively older and that there has been enough time for external differences to accumulate. Finally, the discovery of O. melakense suggests that additional, rare, endemic Onchidium species possibly still are unknown, especially in the region of the Strait of Malacca, which seems to be its center of highest diversity.

Our molecular phylogenetic analyses (Figs 1, 2) indicate that O. stuxbergi and O. reevesii are most closely related, which is supported by the fact that their hyponotum bears black dots (absent in O. typhae and O. melakense, Table 3). As of today, O. stuxbergi and O. reevesii do not overlap geographically even though they get very close in southern China (Fig. 4). Their speciation is possibly related to adaptation to warm (O. stuxbergi) and colder (O. reevesii) waters.

Our knowledge of O. reevesii is based on the re-description of the holotype (Dayrat et al. 2016: 32–35) as well as a recent re-description of fresh material by Wang et al. (2018). The latter study begs discussion here. The foot sole of O. reevesii is said to be “whitish or light yellow” within the body of the species description (Wang et al. 2018: 2). In the discussion, some individuals from Cixi City, Zhejiang Province, are also mentioned with a yellow foot (Wang et al. 2018: 6). It is unclear whether those specimens from Cixi City belong to O. reevesii. However, it cannot be excluded that the color of the foot sole of O. reevesii might vary from white to yellow, instead of light yellow. More importantly, according to Wang et al. (2018: 6): “On the basis of COI sequences we misidentified two distinct species as Onchidium ‘struma’ (Sun et al. 2014). These were O. reevesii and O. hongkongense.” That is incorrect: Dayrat et al. (2016: 35) demonstrated that Sun et al. (2014) applied the nomen nudum Onchidium ‘struma’ to O. reevesii and O. stuxbergi. Also, Dayrat et al. (2019a: 22) showed that Onchidium hongkongense Britton, 1984 is a junior synonym of Paromoionchis tumidus (Semper, 1880) and therefore does not apply to an Onchidium species. Finally, the individuals misidentified as Paraoncidium reevesii (J.E. Gray, 1850) by Sun et al. (2014) actually belong to Paromoionchis tumidus (Dayrat et al. 2019a: 44): the combination Paraoncidium reevesii is erroneous because the species described as Onchidium reevesii by J.E. Gray (1850) belongs to the genus Onchidium, based on the re-description of its holotype (Dayrat et al. 2016: 32–35), and also because Paraoncidium Labbé, 1934 is a junior synonym of Onchidina Semper, 1882 (Dayrat and Goulding 2017: 123).

In onchidiids, types of intestinal loops are defined based on the pattern of the intestine on the dorsal aspect of the digestive gland. Plate (1893) first distinguished four types of intestinal loops (types I to IV) and Labbé (1934) later added a type V. Only the types II and III are found in Onchidium (Table 3). The type species, O. typhae, is characterized by intestinal loops of type II, and the three other species are characterized by intestinal loops of type III. The different types of intestinal loops and their individual variation are best revealed by coloring with a different color different sections of the intestine (Dayrat et al. 2019b): a clockwise intestinal loop is colored in blue, a counterclockwise intestinal loop is colored in yellow, and a transitional loop between them is colored in green (Fig. 13).

The intestine first appears dorsally on the right side and starts by forming a clockwise (blue) loop (Fig. 13). In intestinal loops of type II, the clockwise (blue) loop makes approximately a complete circle. As a result, the transitional (green) loop is oriented to the left, typically at 9 o’clock (horizontal red arrow, Fig. 13A). In Onchidium, intestinal loops of type II are found only in O. typhae, in which the orientation of the transition loop varies approximately between 8 and 9 o’clock (Fig. 13A, B). In intestinal loops of type III, the clockwise (blue) loop is longer and rotates more than in a type II. As a result, the transitional (green) loop is oriented to the right, typically at 3 o’clock (horizontal red arrow, Fig. 13C). In O. stuxbergi, the orientation of the transitional (green) loop varies from 1 to 8 o’clock (red arrow, Fig. 13C–E). In O. reevesii, the orientation of the transitional (green) loop is approximately at 2 o’clock (red arrow, Fig. 13F). In O. melakense, the orientation of the transitional (green) loop varies from 1 to 5 o’clock (red arrow, Fig. 13G, H). A few preliminary remarks on the distribution of types of intestinal loops in genera of onchidiid slugs can be found in Dayrat et al. (2019b). A more thorough discussion regarding types of intestinal loops will be provided after our revisions of Peronia and Platevindex are published (in preparation).

Acknowledgements

We are grateful to associate editor Nathalie Yonow and reviewers Adrienne Jochum and Pierre Lozouet for constructive comments that helped improve the manuscript. Accessing field sites would have been impossible without help from local fishermen and villagers. We would like to thank the collection managers and curators of various institutions for sending us specimens on loan for examination in the present study: Virginie Héros (Muséum national d’Histoire naturelle, Paris); Chad Walter (Smithsonian Institution, Washington DC); Karin Kronestedt, Elin Sigvaldadottir, Emily Dock Åkerman, Anna Persson, and Anders Warén (Swedish Museum of Natural History, Stockholm); Thomas von Rintelen, Christine Zorn, and Matthias Glaubrecht formerly (Museum für Naturkunde, Berlin); Bernhard Hausdorf (Zoologisches Museum, Hamburg). Collecting was overseen by Deepak Apte in India, by Munawar Khalil in Indonesia, and by Shaw Hwai Tan in Malaysia. We are grateful to Rahul C. Salunkhe and Yogesh Shouche (Bombay Natural History Society, Mumbai, India, and National Center for Cell Science, Pune, India) for their help with the DNA sequencing of the specimens from India, and we thank Vishal Bhave for his help collecting slugs in the Andaman Islands. We thank the Ministry of Research, Technology and Higher Education, Republic of Indonesia (Ristek-Dikti) for issuing a research permit to BD (Ristek #134/SIP/FRP/E5/Dit.KI/VI/2017). We also wish to thank the Universitas Malikussaleh, North Aceh, Sumatra, Indonesia, for being our homebase institution in Indonesia. This work was supported by the Eberly College of Science at the Pennsylvania State University and by a REVSYS (Revisionary Syntheses in Systematics) award to BD from the US National Science Foundation (DEB 1419394).

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1 Known distribution: Myanmar, western Peninsular Malaysia, eastern Sumatra, Thailand (Gulf of Thailand), Vietnam, eastern Borneo, Philippines, and southernmost tropical China (up to 22°10'N).
2 Known distribution: Andaman Islands, western Peninsular Malaysia, and eastern Sumatra.
3 Known distribution: subtropical China, from 22°30'N to 34°36'N.
4 Known distribution: West Bengal, Andaman Islands, western Peninsular Malaysia, and Singapore.
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