Research Article |
Corresponding author: Benoît Dayrat ( bdayrat@gmail.com ) Academic editor: Nathalie Yonow
© 2016 Benoît Dayrat, Tricia C. Goulding, Deepak Apte, Vishal Bhave, Joseph Comendador, Ngô Xuân Quang, Siong Kiat Tan, 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, Apte D, Bhave V, Comendador J, Ngô XQ, Tan SK, Tan SH (2016) Integrative taxonomy of the genus Onchidium Buchannan, 1800 (Mollusca, Gastropoda, Pulmonata, Onchidiidae). ZooKeys 636: 1-40. https://doi.org/10.3897/zookeys.636.8879
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In an effort to clarify the species diversity of onchidiid slugs, the taxonomy of the genus Onchidium Buchannan, 1800 is revised using an integrative approach. New, fresh specimens were collected in a large number of places, including type localities. The genus Onchidium is redefined here as a clade including only three species which are strongly supported by both morphological and molecular data. All three species were already named: the type species O. typhae Buchannan, 1800, O. stuxbergi (Westerlund, 1883), and O. reevesii (J.E. Gray, 1850). With the exception of a re-description of O. typhae published in 1869, all three species are re-described here for the first time. First-hand observations on the color variation of live animals in their natural habitat are provided. The anatomy of each species is described. Important nomenclatural issues are addressed. In particular, Labella Starobogatov, 1976 is regarded as a junior synonym of Onchidium and Labella ajuthiae (Labbé, 1935) and O. nigrum (Plate, 1893) are regarded as junior synonyms of O. stuxbergi. The nomenclatural status of several other species names is discussed as well. Many new records are provided across South-East Asia and precise ranges of geographic distributions are provided for the genus Onchidium and its three species. Distinctive features that help distinguish the genus Onchidium from other onchidiids are provided, as well as an identification key for the three species.
Biodiversity, Euthyneura , Indo-West Pacific, mangroves, marine slugs, South-East Asia
The systematics of the Onchidiidae, one of the higher clades of pulmonate gastropods, has been problematic for decades. Many species names were created up to the 1930s, and then the study of their diversity has been more or less abandoned. Identifications have remained nearly impossible at both generic and specific levels. As a result, there are 143 species names and 19 genus names in the literature but the actual species diversity is largely unknown (
The taxonomy of onchidiid slugs has remained problematic primarily because no malacologist has dared to study it in the past 80 years, which in turn is explained by the fact that many serious issues have made onchidiids a nightmare for taxonomists (
A few years ago, the Dayrat lab embarked on a worldwide systematic revision of the Onchidiidae. Our goal has been to integrate both traditional taxonomy and modern molecular tools (
Traditionally, especially in museum collections, any onchidiid slug from the tropical Indo-West Pacific was by default referred to as “Onchidium,” and any onchidiid slug from outside the tropical Indo-West Pacific was referred to as Onchidella. Still to this day, Onchidella has not been recorded in the tropical Indo-West Pacific but is found nearly everywhere else except for polar waters (
Our understanding of the onchidiid diversity in the tropical Indo-West Pacific has grown as new data were being gathered. Species diversity, distributions, and higher relationships have become clearer as more and more DNA sequences were obtained and more and more specimens were dissected from new places. As of November 2016, our data set for the onchidiid slugs in the tropical Indo-West Pacific includes approximately 70 species and ten clades of generic level. All species and genera are strongly supported by both DNA sequences and morphology.
After careful examination of all type materials, detailed comparisons between the original descriptions and our own observations, the nomenclatural status of nearly all existing species-group and generic-group names of the Onchidiidae is now known. In other words, it is clear which names are valid names, which names are junior synonyms, which names are nomina dubia, and which taxa require new names. So, results on the systematic revision of the Onchidiidae can now be shared.
In a first step, the nomenclature and the alpha-taxonomy of each of the clades of generic level in our data set must be clarified. The present contribution, which focuses on the genus Onchidium, marks the beginning of a series of taxonomic papers dedicated to each clade in our data set. Then, in a second step, a phylogenetic tree of the entire family will be provided (still an ongoing endeavor) and that tree used to address broader questions on onchidiid diversification, evolution, and biogeography.
The genus Onchidium, the type genus of the family, has been traditionally used by default for many onchidiid species from the tropical Indo-West Pacific. Therefore, it is important to give it a proper definition. The genus Onchidium is a clade including only three species, which were already named: the type species, O. typhae Buchannan, 1800, with a type locality in Bengal (Ganges delta); O. stuxbergi (Westerlund, 1883), with a type locality from Brunei Bay, north-western Borneo; and O. reevesii (J.E. Gray, 1850), with a type locality from China (exact locality unknown). With the exception of a re-description of O. typhae by
Special attention has been given to type localities. Indeed, without going back to type localities to collect fresh specimens, it can be extremely challenging, and often impossible, to address the nomenclatural status of taxon names. For that reason, new specimens have been collected from type localities as far as possible. For the present study, for instance, new specimens of O. typhae were collected from the Sundarbans, in West Bengal, India, which corresponds to the type locality, and new specimens of O. stuxbergi were collected from Brunei Darussalam, north-western Borneo, extremely close to the type locality.
Collecting. All specimens examined here were collected by our team, except for the types of existing species and a few specimens found in museum general collections. Local guides (local villagers or fishermen) also often accompanied us. Sites were accessed by car (if next to a road) or by boat (by hiring local fishermen). Local fishermen and villagers are a great source of information to find good collecting sites. They know where to find well-preserved mangroves with old trees and they also know about potential dangers (snakes, crocodiles, wasps, and even tigers in the case of the Sundarbans, West Bengal). Each site was explored for an average of two hours but the exact time spent at each site also depended on the time of the low tide, the weather, etc. At each site, many photographs were taken to keep track of the kind of mangrove being visited (e.g., thick forest of young Rhizophora trees, open forest of large Avicennia trees) as well as the diverse microhabitats where specimens were collected (e.g., surface of the mud, old and muddy log).
Specimens. All available types were examined. Some additional non-type material was collected by others and borrowed from museum collections. However, most specimens were collected by us and our new collections provided fresh material for DNA sequencing and invaluable natural history observations. All our new specimens were deposited in local institutions as vouchers. Acronyms of collections are:
BDMNH
ITBZC
USMMC
Animal preparation and anatomical description. All anatomical observations were made under a dissecting microscope and drawn with a camera lucida. In addition, organs were prepared for scanning electron microscopy (SEM). Radulae were cleaned in 10% NaOH for a week, rinsed in distilled water for at least a week, briefly cleaned in an ultrasonic water bath (less than a minute), sputter-coated with gold-palladium, and examined by SEM. Soft parts (penis and penial hooks) were dehydrated in ethanol and critical point dried before coating. When a lot included several specimens, all pieces of the dissected specimens were carefully numbered, both inside the jar and on the SEM stubs. A range of minimum to maximum animal size is provided for each lot of specimens. In addition, individualized numbers and measurements are provided for the specimens being illustrated here as well as for those comprising our molecular data set. The anatomical description of O. typhae, the type species, is fully detailed. The written description of the many anatomical features that are virtually the same between species (nervous system, heart, etc.) is not uselessly repeated three times.
DNA extraction and PCR amplification. DNA was extracted using the phenol-chloroform extraction protocol with cetyltrimethyl-ammonium bromide (CTAB). Portions of two mitochondrial genes (COI, 16S) were amplified using the following universal primers: COIF (5’-3’) GGT CAA CAA ATC ATA AAG ATA TTG G, and COIR (5’-3’) TAA ACT TCA GGG TGA CCA AAR AAY CA (Folmer et al. 1994); 16Sar (5’-3’) CGC CTG TTT ATC AAA AAC AT, and 16S 972R (5’-3’) CCG GTC TGA ACT CAG ATC ATG T (
Phylogenetic analyses. Alignments were obtained using Clustal W in MEGA 6 (
GenBank accession numbers for COI and 16S DNA sequences. All sequences are new, except for the specimens from China (
Species | Individual (DNA) | Locality | GenBank COI | GenBank 16S |
---|---|---|---|---|
Onchidella celtica | Ceuta, Northern Africa | AY345048 | AY345048 | |
Onchidella floridana | Tobago | HQ660035 | HQ659903 | |
Peroniasp. | Okinawa, Japan | HQ660043 | HQ659911 | |
Peroniasp. | Hawaii, USA | HQ660038 | HQ659906 | |
Onchidium typhae | 1064 | West Bengal, India | --- | KX179528 |
1089 | Andaman, India | KX179512 | KX179529 | |
1109 | Andaman, India | KX179513 | KX179530 | |
967 | Peninsular Malaysia | KX179510 | KX179526 | |
965 | Peninsular Malaysia | KX179509 | KX179525 | |
1007 | Singapore | KX179511 | KX179527 | |
Onchidium stuxbergi | 971 | Peninsular Malaysia | KX179514 | KX179531 |
1048 | Brunei | KX179515 | KX179532 | |
3251 | Bohol, Philippines | KX179517 | KX179534 | |
3363 | Bohol, Philippines | KX179518 | KX179535 | |
5602 | Vietnam | KX179519 | KX179536 | |
5605 | Vietnam | KX179520 | KX179537 | |
S891 | China (19°56'N) | JN543155 | JN543091 | |
Onchidium reevesii | S871 | China (22°30'N) | JN543161 | JN543097 |
S831 | China (24°24'N) | JN543160 | JN543096 | |
S853 | China (27°29'N) | JN543164 | JN543100 | |
S821 | China (33°20'N) | JN543162 | JN543098 | |
S802 | China (34°46'N) | JN543157 | JN543093 |
Molecular phylogenetic analyses. Here, the primary purpose of using DNA sequences is to test the species limits within Onchidium. The phylogenetic analyses yielded three species units that are all reciprocally monophyletic and strongly supported (Fig.
Phylogenetic tree. Relationships within the genus Onchidium based on COI and 16S sequences. Numbers above branches are the bootstrap values (Maximum Likelihood analysis) and below are the posterior probabilities (Bayesian analysis); only significant numbers (> 80% and > 0.9) are indicated. Onchidella and Peronia sequences serve as outgroups. Numbers for each individual correspond to unique identifiers in our DNA collection. All sequences of Onchidium specimens are new, with the exception of the specimens from China which were all misidentified as Onchidium “struma” by
Pairwise genetic divergences. The pairwise genetic distances unambiguously support the existence of three species of Onchidium (Table
Intra- and inter-species pairwise genetic distances. Ranges of minimum to maximum distances are indicated (in percentage). For instance, within O. typhae, individual sequences are between 0% to 0.5% divergent, and individual sequences between O. stuxbergi and O. typhae are minimally 21.7% and maximally 26.2% divergent.
Species | O. typhae | O. stuxbergi | O. reevesii |
---|---|---|---|
O. typhae | 0.0–0.5 | ||
O. stuxbergi | 21.7–26.2 | 0.0–5.1 | |
O. reevesii | 26.9–28.3 | 15.0–18.1 | 0.0–0.7 |
Onchidium typhae Buchannan, 1800, by monotypy.
Labella Starobogatov, 1976: 211. New synonym. Type species, by monotypy, Labella ajuthiae (Labbé, 1935); replacement name of Elophilus Labbé, 1935, preoccupied by Elophilus Meigen, 1803 [Diptera].
The synonymy of Labella ajuthiae (Labbé, 1935) with Onchidium stuxbergi (Westerlund, 1883) is discussed in the remarks on O. stuxbergi. Labella is a junior synonym of Onchidium because the two generic names Labella and Onchidium apply to the same clade.
Body not flattened. No marginal glands in the notum. No dorsal gills. Dorsal eyes present on notum. Fully retractable, central papilla (with three dorsal eyes) present. Long eye tentacles. Male opening inferior to the right ocular tentacle, slightly to its left. Pneumostome medial. Intestine of types II and III. Rectal gland present. Accessory penial gland and hollow spine present. Penis with hooks.
Onchidium differs from all other onchidiids by the presence of unmistakably, large, conical, pointed papillae on the dorsum of live animals. Disturbed live animals and preserved animals are retracted and their dorsal papillae are significantly smaller. The identification to the genus level can then be more challenging.
From north-eastern India (West Bengal) to the Philippines, including the Strait of Malacca, Singapore, Thailand, Vietnam, eastern Borneo, and China (Fig.
Geographic distribution of the genus Onchidium and its three valid species. The colored dots correspond to the known records for each species. Colored areas correspond to hypothetical ranges proposed based on those known records. Naturally, details about the distribution of each species remain uncertain. For instance, O. typhae may or may not be found on the western coasts of Thailand by the Andaman Sea.
Onchidium
typhae
Buchannan, 1800: 132–134, plate V, figs 1–3;
“Bengal.” Bengal is a vast region of eastern India (and Bangladesh) around the delta of the Ganges. Collecting specimens in West Bengal was the best that could be done to try to go back to the type locality.
The original type material could not be located and is likely lost. Given that the identity of O. typhae is no longer problematic, there is no need to designate a neotype.
Bangladesh, Sundarbans, delta, October 1927, 1 specimen [25/15 mm], leg. Konietzko, det. as Onchidium (
(Fig.
(Fig.
Habitats for O. typhae. A India, West Bengal, very soft mud on the shore with recently-planted Avicennia trees (station 49) B India, Andaman Islands, by a large river, deep mangrove with tall trees, small creeks, and plenty of old muddy logs (station 57) C Malaysia, Matang, old and open Rhizophora forest with tall trees, hard mud, creeks, and many old logs (station 28) D Malaysia, Langkawi Island, open forest with high mud lobster (Thalassina) mounds (station 26).
In the Sundarbans, O. typhae was found in two of the four mangrove sites that were visited, but for a total of only six specimens. In the Andaman Islands, it was found in four of the five mangrove sites visited. The mangrove site where O. typhae was not found was not at all muddy but rather, comprised a sandy patch of Avicennia by a coral rubble sandy beach. It was especially abundant at two sites (many specimens were observed but not collected), mostly on or near old logs. In Malaysia, O. typhae was found in only five of the 18 mangroves that were visited and it was abundant (17 specimens collected) only at one site, which happens to be one of the best, oldest, most pristine, and most diverse mangrove forests we have ever seen anywhere. In Singapore, O. typhae was found in three of the five mangroves that were visited (the two sites mentioned above, and one site by the Mandai River where one specimen was collected but ultimately escaped). However, it is rare there (only five specimens collected in total).
(Fig.
Live specimens, O. typhae. A Dorsal view, 45 mm long, India, West Bengal, station 48 (
When animals are not disturbed, the dorsum is typically covered by large pointed papillae that rest on larger, hemispherical bases. Those pointed papillae are unique to Onchidium and extremely useful for identification in the field. For a long time (before we realized those slugs were Onchidium in the strict sense), we called them the “spiky” slugs to refer to the unique pointed papillae. Those papillae may seem to be arranged in two to four longitudinal ridges (each with five to ten papillae), but this is not the rule. They bear from one to four black “dorsal eyes” at their tip but some papillae do not bear dorsal eyes (especially on the dorsum margin). As in many other onchidiids, there is a central peduncle entirely retractable within the notum. The central peduncle bears three or four “dorsal eyes” but its size is similar to the large pointed papillae (i.e., it is not significantly larger than the other large papillae). The large pointed papillae are surrounded by small papillae as well, which may be rounded or pointed. As soon as the animal is disturbed (by walking on the mud on which it crawls or by touching its dorsum), all dorsal papillae rapidly retract and the animal looks completely different. It then is evenly covered with minute pointed papillae. The body of disturbed animals also is more humped and their ocular tentacles are entirely retracted. One has to patiently wait for a long time for a disturbed slug to relax again (easily 10 or 15 minutes) so the rule is “take a picture first and then touch it!” Interestingly, the appearance of the dorsum of live, disturbed animals is very close to the appearance of preserved animals, which was quite useful when examining type materials of existing species and recognizing them as Onchidium species. Crawling individuals can easily measure 30 to 40 mm in length (largest individuals measured 65 mm).
(Figs
External morphology and digestive system, O. typhae. A Anterior region, ventral view, Singapore, scale bar 8 mm (
(Fig.
(Figs
The esophagus is narrow and straight, with thin internal folds. The esophagus enters the stomach anteriorly. Only a portion of the posterior aspect of the stomach can be seen in dorsal view because it is partly covered by the lobes of the digestive gland. The dorsal lobe is mainly on the right. The left, lateral lobe is mainly ventral. The posterior lobe covers the posterior aspect of the stomach. The stomach is a U-shaped sac divided in four chambers. The first chamber, which receives the esophagus, is delimited by a very thin layer of tissue, and receives the ducts of the dorsal and lateral lobes of the digestive gland. The second chamber, posterior, is delimited by a thick muscular tissue and receives the duct of the posterior lobe of the digestive gland. It appears divided externally but consists of only one internal chamber. The third, funnel-shaped chamber is delimited by a thin layer of tissue with high ridges internally. The fourth chamber is continuous and externally similar to the third, but it bears only low, thin ridges internally. The intestine is long, narrow, and of type II. A rectal gland is present. It is a long, narrow, and coiled tube that opens in the left portion of the pulmonary complex. Its function is unknown.
(Fig.
Nervous and reproductive systems, O. typhae. A Nervous system, Malaysia, Langkawi Island, scale bar 3.8 mm (USMMC 00002, #1) B Hermaphroditic (female), posterior parts, Singapore, scale bar 3.8 mm (
(Fig.
(Figs
Male, anterior, copulatory parts, O. typhae. A Penial hooks, Malaysia, Matang, scale bar 30 µm (USMMC 00005, #1) B Flat disc at distal end of flagellum of penial accessory gland, Malaysia, Langkawi Island, scale bar 100 µm (USMMC 00002, #1) C Tip of hollow spine, penial accessory gland, Singapore, scale bar 4 µm (
The penial gland is a long, tube-like flagellum with a proximal dead end. The length of the flagellum of the penial gland varies among individuals but it is always heavily coiled. Distally, the flagellum ends in a hard, hollow spine protected by a sheath which is fused distally with the vestibule. The hollow spine is narrow and elongated, slightly curved. Its diameter is between 20 and 30 µm but it narrows down distally. The diameter of the opening at its tip is between 4 and 6 µm. Its length ranges from 0.7 mm (
The penial sheath is long and very strongly coiled in spirals. In less mature individuals, the coils may not be as strong and numerous but they are present. The penial sheath protects the penis for its entire length. The insertion of the retractor muscle marks the separation between the penial sheath (and the penis inside) and the deferent duct. The retractor muscle is shorter than the penial sheath and runs straight to the posterior half of the visceral cavity. The insertion of the retractor muscle varies among individuals: in the posterior half of the visceral cavity in all specimens but those from India; in the anterior half of the visceral cavity (just anterior to the heart) in the specimens from India. The deferent duct also is highly convoluted with many loops. In immature specimens, the deferent duct is significantly less coiled. The penis is elongated, round, narrow, and hollow; its diameter is less than 200 µm, and its distal part covered with hooks. When the penis is retracted inside the penial sheath, the hooks are inside the tube-like penis. During copulation, the penis is exerted like a glove and the hooks are then on the outside. Hooks are very densely packed inside the penis, with multiple, irregular rows of an average of 15 hooks around the circumference of the penis. Hooks are conical, slender, sharply pointed, and measure up to 300 µm in length.
Externally, O. typhae differs from other Onchidium species by the color of the hyponotum, which is not white but instead varies between grayish, yellowish, and even greenish (see below the dichotomous identification key, before the final conclusion). The color of the foot (yellowish, not bright orange) is not diagnostic. Internally, the spirally coiled penial sheath is not diagnostic. However, O. typhae is the only Onchidium species known so far with an intestine of type II.
The original description of O. typhae by Buchannan was brief but it was based on first-hand observation of live animals, which is quite unusual for onchidiids since most onchidiid species were described based on preserved material with no information on shape and color of live animals. Even though the type material is likely lost, two features described and illustrated by Buchannan support the identification of the material described here as O. typhae. First, and most importantly, the long eye tentacles (only Onchidium species have such long eye tentacles) and the dorsal papillae of various sizes (Buchannan’s “glandular tubercles”).
Semper accepted Stoliczka’s anatomical re-description of O. typhae and added some detail on the anterior male parts. In particular, Semper illustrated some penial hooks and the spine of the penial accessory gland. However, the sizes described by Semper (a maximum size of 170 µm for the penial hooks and a length of 4.5 mm for the spine of the penial accessory gland) are not really compatible with the sizes observed for the present study (penial hooks up to 300 µm and a spine less than 1.2 mm long). Therefore, Semper likely examined individuals of a different species, which cannot be identified at this stage.
Finally, a search for potential synonyms of O. typhae revealed no synonyms (the available type materials of all onchidiid species were personally examined) and it does seem that it was named only once.
Vaginulus
stuxbergi
Westerlund, 1883: 165;
Oncidium
nigrum
Plate, 1893: 188–190, pl. 8, fig. 31a, pl. 10, fig. 53, pl. 11, fig. 75;
Elophilus ajuthiae Labbé, 1935: 312–317, figs 1–3. New synonym.
(V. stuxbergi). “Borneo in silva, ad flum Kalias” means that the slugs were found in forests by a river now called the Klias River. The latter runs into the Brunei Bay, which is a small bay bordered by Brunei Darussalam in the South, by Sabah (Malaysia) in the north, and by the small island of the Labuan Territory (Malaysia) in the west. Several of the labels of the type material indicate Labuan as the locality. So, it is possible that the type material is a mix of specimens collected at Labuan Island itself and on the shore of Borneo, facing Labuan. Here is what the different labels read for the first jar: “Vaginulus stuxbergi Westerlund, 1885. Borneo, Labuan. On the beach, mangroves. Leg. Vega Exp 1878-1880, sta. 633.
(O. nigrum). “Borneo” is the only geographic information provided by
(E. ajuthiae). The “Province d’Ajuthia (Siam)” is the province of Ayutthaya, in Thailand, approximately 80 kilometers north of Bangkok, which used to be the capital of the Kingdom of Siam. Because that province is inland, Labbé assumed that the slugs had been collected in fresh water. However, the Chao Phraya River of the basin than runs through the province of Ayutthaya actually is under the influence of the tide year round. The salt front (brackish water) goes up to 75 and 175 kilometers from the river mouth in the wet and dry seasons, respectively, and it was even more so the case in the past when the river side was still not developed (syntypes collected by M. Bocourt in 1862 according to the label of the type material). In the province of Ayutthaya, the river is approximately at its kilometer 140 (
(V. stuxbergi). One lectotype hereby designated (43/25 mm; entire and never dissected;
(O. nigrum). Holotype, by monotypy (
(E. ajuthiae). Three syntypes (
Malaysia, Peninsular Malaysia, Kuala Sepatang, 04°50.434N, 100°38.176E, 18 July 2011, 1 specimen (42/24 [DNA 971] mm), leg. B. Dayrat & T. Goulding, [station 27, old forest with tall, old Rhizophora trees, high in the tidal zone (ferns), following boardwalk in educational preserve, reached a creek lower in the tidal zone, with mud] (USMMC 00006); Brunei Darussalam, Mentiri, Jalan Batu Marang, 04°59.131N, 115°01.820E, 29 July 2011, 3 specimens (33/18 [#1], 20/16, and 16/13 [DNA 1048] mm), leg. T. Goulding & S. Calloway, [station 36, old mangrove with tall Rhizophora trees with high roots and Thalassina mounds] (BDMNH); Philippines, Bohol, Inabanga, 10°04.255'N, 124°04.416'E, 13 July 2014, 3 specimens (from 30/20 [DNA 3251] to 25/17 mm), leg. J. Comendador, B. Dayrat & T. Goulding, [station 187, mostly Nypa palms with Thalassina mounds] (
(Fig.
(Fig.
Habitats for O. stuxbergi. A Malaysia, Kuala Sepatang, old forest with tall, old Rhizophora trees, high in the tidal zone (station 27) B Philippines, Bohol, mostly Nypa palms with Thalassina mounds (station 187) C Vietnam, Can Gio, open mangrove with large Avicennia trees, soft mud, some old logs (station 231) D Vietnam, Can Gio, hard mud with trees spread out by a small road and then a steep bank to the soft, deep mud (station 221).
Onchidium stuxbergi is a rare species. Only one individual was found in Malaysia (where 18 mangrove sites were explored), three individuals at one site in Brunei Darussalam (7 sites), nine individuals at four sites in Bohol (17 sites), and 12 individuals at four sites in Vietnam (19 sites). Even though it will need to be confirmed in the future, it seems that O. stuxbergi tends to be slightly more common (although still rare, overall) in more northern latitudes (Vietnam and Philippines).
(Fig.
Live specimens, O. stuxbergi. A Dorsal view, 33 mm long, Brunei Darussalam (BDMNH, #1) B Dorsal view, 27 mm long, Philippines, Bohol (
(Figs
External morphology and internal anatomy, O. stuxbergi. A Anterior region, dorsal view, Malaysia, Kuala Sepatang, scale bar 4.4 mm [DNA 971] (USMMC 00006) B Digestive system, dorsal view, Philippines, Bohol, scale bar 5 mm [DNA 3363] (
Radula, O. stuxbergi. A Rachidian and innermost lateral teeth, Brunei Darussalam, scale bar 40 µm (BDMNH # . B Rachidian and innermost lateral teeth, Philippines, Bohol, scale bar 20 µm (
Male, anterior, copulatory parts, O. stuxbergi. A Penial hooks, Philippines, Bohol, scale bar 40 µm [DNA 3363] (
The oviduct is narrow, short, and straight. The hollow spine of the penial accessory gland is slender and slightly curved. It measures between 0.5 (USMMC 00006) and 1.4 mm (
The penial sheath is long (to the posterior third of the visceral cavity) and coiled in a few spirals. In less mature individuals, the coils may not be as marked. The retractor muscle is short and inserts into the posterior third of the visceral cavity. There is an additional retractor muscle attaching the anterior portion of the penial sheath to the left wall of the visceral cavity, near the buccal mass. In some individuals, that left additional retractor muscle is very thick and strong. The deferent duct is highly convoluted with many loops, but less so in immature specimens. The penis is elongated, round, narrow, and hollow. Its diameter is less than 200 µm. Its distal part is covered with hooks. When the penis is retracted inside the penial sheath, the hooks are inside the tube-like penis. During copulation, the penis is exerted like a glove and the hooks are then on the outside. Hooks are very densely packed inside the penis, with multiple, irregular rows of an average of 15 hooks around the circumference of the penis. Hooks are conical, slender, sharply pointed, and measure from 40 µm up to 300 µm in length. The longer they are the more slender they are.
Externally, O. stuxbergi differs from other Onchidium species by the color of the foot, which is bright orange (see below the dichotomous identification key, before the final conclusion). Internally, O. stuxbergi is the only Onchidium species known so far (and the only onchidiid species, for that matter) with a strong, additional retractor muscle attaching the anterior penial sheath to the left, anterior wall of the visceral mass, near the buccal mass.
The status of O. stuxbergi has been problematic from the start because Westerlund unknowingly based his original description on specimens that are part of two distinct species (see Type materials, above): eleven former syntypes (now paralectotypes) are Platevindex and another former syntype (now the lectotype) is an Onchidium (in the strict sense, as defined here). Two years after the original description,
However, as a direct consequence of Westerlund’s ambiguous original description and type material, many authors have proposed synonymies between O. stuxbergi and some species names that clearly belong to Platevindex. Those cases are briefly discussed here, but they will be discussed in more detail in our revision of the genus Platevindex.
There is no doubt that O. nigrum, which is only known from the holotype, belongs to the genus Onchidium as re-defined here: the mantle of the preserved holotype bears the typical papillae of Onchidium. Also, Plate described both a rectal gland and an accessory gland, which are found in all three known Onchidium species. Plate did not mention the presence of an additional, left, retractor muscle for the penial sheath. He only mentioned that the insertion of the retractor muscle is of “type II” (i.e., near the pericardium). According to Plate, the penial hooks are from 14 to 87 µm long and the spine of the penial accessory gland is 1.2 mm long. The penial hooks observed here are from 40 to nearly 300 µm in length. It is possible that Plate, who observed only one specimen, could not fully evaluate the variation of penial hooks. Also, penial hooks are extremely challenging to extract and observe without SEM. However, Plate’s description of the penial accessory gland spine is fully compatible with our observations (from 0.5 to 1.2 mm long). Finally, Plate described the intestine loops of O. nigrum as of a unique and exceptional pattern, which he referred to as “type III.” The latter, as illustrated by
The three syntypes described as Elophilus ajuthiae by
The name Onchidium struma, introduced by
Onchidella
reevesii
J.E. Gray, 1850: 117, pl. 181, fig. 5–5a;
Onchidium
reevesii
:
China. Onchidella reevesii was not described by Gray. That name, which appears on page 117, simply referred to figures 5 and 5a of the plate 181). On page 117, there is no indication of the geographic origin, and there is no indication of geographic origin on the label of the holotype either. However, on page 36, those same figures are referred to as “Onchidium —. Mr. Reeve’s drawings. China,” clearly indicating that the animal illustrated on the fig. 5 (and 5a for the ventral side) of the plate 181 is from China.
Holotype (43/25 mm), by monotypy (
Internal anatomy, holotype, O. reevesii (
(Fig.
(Fig.
Externally, O. reevesii differs from other Onchidium species by its ventral color, i.e., a whitish hyponotum and foot (see the dichotomous key below). Internally, the spirally coiled penial sheath of O. reevesii is not diagnostic (as in O. stuxbergi and O. typhae). However, O. reevesii lacks the additional, anterior, left retractor muscle of the penial sheath that is unique to O. stuxbergi (the only species with which O. reevesii may be shown later to overlap in the southernmost part of its distribution).
Semper transferred Onchidella reevesii to Onchidium probably by default (i.e., as a non-Onchidella species) because he did not give any explanation and he did not examine any new material. At any rate, it just so happens that Onchidium reevesii is the correct combination, because the notum of the type specimen bears the long papillae that are typically found in Onchidium as defined here. The presence of a rectal gland and of an accessory penial gland also supports that Onchidella reevesii is an Onchidium, even though those traits are found in other genera and even though the accessory penial gland can be both present and absent within a genus. That being said, a rectal gland and an accessory gland are found in all other Onchidium species. There is a small disc at the distal end of the accessory gland of the holotype of O. reevesii, a structure which we found so far only in Onchidium.
There are actually only two species in China with both a rectal gland and an accessory penial gland, and both species belong to the genus Onchidium. In the study by
Onchidium reevesii and O. stuxbergi, of which the distribution ranges do not seem to overlap, differ externally and internally. A few pictures of live animals of O. “struma” from Shangai, northern China (
A key is provided here to help identify the three known species of Onchidium. The key is based on external characters because they are the most readily available. However, internal diagnostic features also distinguish the species (see species descriptions). DNA sequences provided in the present contribution can also help clear any potential confusion, because, to date, DNA sequences of Onchidium have yielded unambiguous results.
1 | The foot is bright orange | O. stuxbergi (known distribution: Peninsular Malaysia, Thailand (Gulf of Thailand), Vietnam, eastern Borneo, Philippines, southernmost tropical China). |
– | The foot is not bright orange | 2 |
2 | The hyponotum is white and the foot is whitish | O. reevesii (known distribution: subtropical China, from 22°30’ to 34°36’) |
– | The hyponotum (and the foot) is not white but varies between grayish and yellowish, and sometimes even greenish | O. typhae (known distribution: Bengal, Andaman Islands, Malaysia, Singapore). |
Naturally, new species of Onchidium may be discovered in the future. However, our data currently support the existence of only three species. It is worth pointing out that Onchidium is not found in eastern Indonesia and is also absent in recent collections from Madang, Papua New Guinea, by the Paris Museum (
Based on current data, the distribution ranges of O. stuxbergi and O. reevesii do not overlap. If they do overlap, it may simply be over a very small area around Hong Kong. The geographic distribution of O. reevesii is typically subtropical (from 22°30’ to 34°36'N) and tolerance for different water temperatures may have largely participated in the speciation between O. stuxbergi and O. reevesii, which are most closely related in our phylogenetic analysis (Fig.
Several authors in the past (e.g.,
Because the new limits to the genus Onchidium are much more restricted than its traditional meaning, many specific names traditionally combined with Onchidium must be combined with different generic names (
Onchidium aberrans Semper, 1885 is a nomen dubium because its type locality (Singapore) was mentioned as uncertain in the original description. Also, no type material could be located. Onchidium griseo-fuscum (Tapparone-Canefri, 1874), originally described as Onchidella griseo-fusca from Singapore and for which no type material could be located, could not be associated with any of the species we collected in Singapore. It could belong to Peronia but it is uncertain. As a result, it is here regarded as a nomen dubium. Onchidium hardwickii (J. E. Gray, 1850) was originally described as Onchidella hardwickii from an unknown locality and is thus regarded here as a nomen dubium. The holotype (by monotypy,
The present contribution illustrates well some of the complicated and relatively common issues faced in taxonomy and the possible ways to address them within an integrative approach. Integrative taxonomy is more than simply comparing morphological data and molecular data: nomenclatural issues are at the core of integrative taxonomy (
We are extremely grateful to all the people that helped us with field work in various ways, either by hosting us, assisting with some logistics, or accompanying us in the field. Our study would have been impossible without their generous help and efforts: Sudhir Sapre and CR Sreeraj in India; Peter Ng (Lee Kong Chian Natural History Museum, National University of Singapore) in Singapore; Teddy Chua in Brunei Darussalam; Marivene Manuel, Vivian Ang, and Don Dumale (all from the National Museum of the Philippines, Manila) in the Philippines. Accessing mangrove sites would have been also impossible without the help from anonymous local fishermen and villagers (who also kept us safe on more than one occasion, advising us for instance not to go in the water when they knew crocodiles had been recently spotted). We are grateful to Dr. Rahul C. Salunkhe and Dr. Yogesh Shouche (National Center for Cell Science, Pune, and Bombay Natural History Society, Mumbai) for their help with the DNA sequencing of the specimens from India. We also thank the collection managers of various institutions for sending us specimens on loan: Brunei Museum, Natural History, Brunei Darussalam; Institute of Tropical Biology, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam; Muséum national d’histoire naturelle, Paris, France; Natural History Museum, London, United Kingdom; National Museum of the Philippines, Manila, Philippines; Naturmuseum Senckenberg, Frankfurt-am-Main, Germany; Swedish Museum of Natural History, Stockholm, Sweden; Universiti Sains Malaysia, Penang, Malaysia; Zoologisches Museum, Berlin, Germany; Zoologisches Museum, Hamburg, Germany; Zoological Reference Collection, Lee Kong Chian Natural History Museum, National University of Singapore. The SEM pictures were taken at the Zeiss SIGMA FE-SEM (Field Emission Scanning Electron Microscopy) at the Huck Institutes of the Life Sciences, the Pennsylvania State University. Stefan Achleitner helped with the translation of some German articles. We thank Nathalie Yonow (subject editor), António M. de Frias Martins and Adrienne Jochum (reviewers) for providing constructive comments that greatly improved the manuscript, as well as the whole editorial team from Zookeys. Finally, this work was supported by the Eberly College of Science at the Pennsylvania State University and by a REVSYS (Revisionary Syntheses in Systematics) award from the US National Science Foundation (DEB 1419394).