Corresponding author: Benoît Dayrat (
Academic editor: Nathalie Yonow
As part of an ongoing effort to revise the taxonomy of air-breathing, marine, onchidiid slugs, a new genus,
Dayrat B, Goulding TC, Khalil M, Comendador J, Xuân QN, Tan SK, Tan SH (2019) A new genus of air-breathing marine slugs from South-East Asia (Gastropoda, Pulmonata, Onchidiidae). ZooKeys 877: 31–80.
The diversity of invertebrate species in mangrove forests of South-East Asia is still largely unknown, mainly because mangroves have not been explored well enough, which likely has to do with the fact that mangroves are not the most inviting habitats, even for savvy field naturalists: mangroves are extremely muddy, infested with malaria-carrying mosquitoes and pit vipers, and often located in remote areas. Our lack of biodiversity knowledge is a major issue not only because nobody knows exactly how many species live in the mangroves of South-East Asia, but also because mangroves are still being eradicated at a large scale across the entire region. Onchidiid slugs illustrate well this general situation: until recently, nobody knew exactly how many species of onchidiids lived in the mangroves of South-East Asia, even though they are some of the most common and diverse animals in mangroves (
Onchidiids are marine, air-breathing, true slugs. Adult onchidiids live in the intertidal zone and their larvae develop in sea water, although a few species are adapted to high elevation tropical rainforest (
In the past ten years, our laboratory has worked on a global taxonomic revision of the
In the present contribution, we describe a new genus,
The present study follows an integrative approach to taxonomy (
All specimens were collected by the authors in the last few years. Collecting parties were led by Benoît Dayrat in Brunei Darussalam, Malaysia, Northern Territory (Australia), Philippines, and Singapore, by Tricia Goulding in Queensland (Australia) and Vietnam, and by Munawar Khalil in Indonesia. We often were accompanied by local villagers or fishermen. Sites were accessed by car or by boat. 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, photographs were taken to document the kind of mangrove being visited as well as the diverse microhabitats where specimens were collected.
In the field, specimens were individually numbered and photographed in their habitat. At each site, we tried our best to sample as much diversity as possible. In addition to numbering individually the specimens that looked different, we also numbered individually many 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.
All available types of
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 anatomy of
Now that the types of intestinal loops have been reported for every species in many genera of onchidiids (
Here we provide a new approach to help reliably determine intestinal types. Because the intestinal loops found in
Intestinal loops, dorsal view
The onchidiid types of intestinal loops are defined based on the dorsal pattern of the intestine. The intestine always first appears dorsally on the right side. In a type I (Fig.
In
DNA was extracted using a phenol-chloroform extraction protocol with cetyltrimethyl-ammonium bromide (
Chromatograms were consulted to resolve rare ambiguous base calls. DNA sequences were aligned using Clustal W in MEGA 6 (
GenBank accession numbers for
Species | Individual (DNA #) | Locality | GenBank |
GenBank 16S | GenBank ITS2 | GenBank 28S |
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5137 | Halmahera, Indonesia |
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5409 | Madang, Papua New Guinea |
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1183 | Singapore |
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970 | Peninsular Malaysia |
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1011 | Singapore |
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5013 | France |
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1524 | New South Wales, Australia |
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1523 | New South Wales, Australia |
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5605 | Vietnam |
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965 | Peninsular Malaysia |
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1511 | New South Wales, Australia |
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1732 | Sumatra, Indonesia |
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696 | Okinawa, Japan |
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706 | Hawaii, USA |
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960 | Peninsular Malaysia |
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6005 | Peninsular Malaysia |
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1001 | Singapore |
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5121 | Halmahera, Indonesia |
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2740 | Ambon, Indonesia |
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1004 | Singapore |
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1005 | Singapore |
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1037 | Brunei Darussalam |
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1038 | Brunei Darussalam |
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1679 | Northern Territory, Australia |
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1681 | Northern Territory, Australia |
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1685 | Northern Territory, Australia |
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1688 H | Northern Territory, Australia |
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1729 | Sumatra, Indonesia |
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2559 | Queensland, Australia |
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2578 | Queensland, Australia |
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2593 | Queensland, Australia |
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2604 | Queensland, Australia |
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2609 | Queensland, Australia |
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2612 | Queensland, Australia |
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2692 | Queensland, Australia |
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2693 | Queensland, Australia |
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3615 | Bohol, Philippines |
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914 | Peninsular Malaysia |
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915 | Peninsular Malaysia |
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5520 | Peninsular Malaysia |
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5609 | Vietnam |
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5610 | Vietnam |
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1656 | Northern Territory, Australia |
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1616 | Northern Territory, Australia |
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1617 | Northern Territory, Australia |
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1618 | Northern Territory, Australia |
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1621 | Northern Territory, Australia |
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1652 | Northern Territory, Australia |
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1657 H | Northern Territory, Australia |
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1659 | Northern Territory, Australia |
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1666 | Northern Territory, Australia |
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1673 | Northern Territory, Australia |
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1692 | Northern Territory, Australia |
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1693 | Northern Territory, Australia |
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1694 | Northern Territory, Australia |
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2743 | Ambon, Indonesia |
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2753 | Ambon, Indonesia |
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6061 | Ambon, Indonesia |
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6063 | Ambon, Indonesia |
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6064 H | Ambon, Indonesia |
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6065 | Ambon, Indonesia |
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978 | Singapore |
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979 | Singapore |
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980 | Singapore |
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983 | Singapore |
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985 | Singapore |
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1783 | Sumatra, Indonesia |
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1784 | Sumatra, Indonesia |
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1785 | Sumatra, Indonesia |
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2230 | Sulawesi, Indonesia |
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3343 | Bohol, Philippines |
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3616 | Bohol, Philippines |
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5627 | Vietnam |
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5646 | Vietnam |
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5958 H | Peninsular Malaysia |
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5959 | Peninsular Malaysia |
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5960 | Peninsular Malaysia |
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5961 | Peninsular Malaysia |
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5963 | Peninsular Malaysia |
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5965 | Peninsular Malaysia |
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Four independent sets of phylogenetic analyses were performed: 1) Maximum Likelihood and Bayesian analyses with concatenated mitochondrial
In addition, another set of analyses was performed with only
DNA sequences were used to test species limits within
Phylogenetic relationships within
Maximum parsimony consensus tree within
Phylogenetic relationships within
Maximum parsimony consensus tree within
Pairwise genetic distances (between
Intra- and inter-unit pairwise genetic distances between the four mitochondrial units of
Species |
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0.0–2.5 | |||
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8.6–10.4 | 0.0–1.2 | ||
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7.5–8.5 | 6.1–7.8 | 0.0–0.0 | |
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7.5–9.5 | 5.3–6.1 | 3.9–5.5 | 0.0–2.5 |
Diagram to help visualize the data on pairwise genetic distances between
In the field, slugs were numbered individually without being assigned to any particular species because onchidiid species are commonly cryptic externally. As anticipated,
Summary of traits that can help distinguish the two species of
Species | Retractor muscle (penis) | Retractor muscle (penis) attachment site | Distal muscle fibers | Accessory penial gland spine size (mm) | Penial hooks (μm) | Distribution |
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Strong and long | Posterior end of visceral cavity (by the rectum) | yes | 0.7 to 1 | 60 to 160 | Peninsular Malaysia, Indonesia (Sumatra), Singapore, Brunei, Vietnam, Philippines (Bohol), Australia (Northern Territory, Queensland) |
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Very short | Anterior third of the visceral cavity | no | 0.75 to 1 | 20 to 35 | Australia (Northern Territory) |
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Absent or vestigial | – | no | 0.35 to 0.75 | 20 to 45 | Indonesia (Ambon) |
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Absent or vestigial | – | no | 0.43 to 0.57 | 15 to 40 | Peninsular Malaysia, Indonesia (Sulawesi, Sumatra), Singapore, Philippines (Bohol), Vietnam |
The new genus described here,
Combination of
Gender masculine of
Body not flattened. No dorsal gills. Dorsal eyes present on notum. Retractable, central papilla (usually with four dorsal eyes) present, often raised above dorsal surface. Eyes at tip of short ocular tentacles. Male opening below right ocular tentacle (or below it and very slightly to its left). No transversal protuberance on oral lobes. Foot wide. Pneumostome median, on ventral hyponotum. Intestinal loops exactly between types I and II (with a transitional loop on average descending at 6 o’clock). Rectal gland absent. Accessory penial gland present with a hollow spine and a muscular sac. Penis with hooks: numerous, densely arranged next to each other, and pointed.
No external diagnostic feature unambiguously distinguishes
Intestinal loops between types I and II, with a transitional loop on average oriented at 6 o’clock, could almost be regarded as diagnostic of
A new generic name is needed because no existing name applies to the clade described here. Based on the examination of all the type specimens available in
AUSTRALIA • holotype, designated here, 30/20 mm [1688 H]; Northern Territory, Darwin, end of the Channel Island Road;
AUSTRALIA –
(Fig.
Geographical distribution of the two species of
(Fig.
Habitats,
(Table
(Figs
Live animals,
Live animals,
Generally, the dorsal notum of any given slug can rapidly change from almost perfectly smooth to densely covered by many papillae. However, when slugs are not disturbed, the dorsum is usually covered by papillae of various sizes. In some slugs, larger papillae may be arranged in two longitudinal ridges on either side of the median line, but those ridges can appear and disappear rapidly. Some papillae bear dorsal eyes at their tip (most papillae bear three eyes). The number of papillae with dorsal eyes is variable (between 8 and 12, on average) and they mostly are on the central part of the notum. Their tip can be pale yellow, but not always. A central, much larger papilla, which also bears three dorsal eyes, is entirely retractable within the notum. In addition to the large papillae, the notum is covered by smaller, rounded papillae, which can make it look very granular.
(Fig.
External morphology and nervous system,
The heart, enclosed in the pericardium, is on the right side of the visceral cavity, slightly posterior to the middle. From the anterior ventricle is an anterior vessel supporting several anterior organs such as the buccal mass, the nervous system, and the copulatory complex. The auricle is posterior. The kidney is more or less symmetrical, the right and left parts being equally developed. The kidney is intricately attached to the respiratory complex. The lung is in two left and right, more or less symmetrical, parts.
(Figs
Digestive system,
Radula,
Radular formulae for the two species of
Species | Radular formula | Specimen length (mm) | Voucher | DNA extraction number |
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43 × 50-1-50 | 30 | 1688 H | |
57 × 90-1-90 | 31 |
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1037 | |
55 × 70-1-70 | 31 | 5609 | ||
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55 × 75-1-75 | 23 | 1657 H | |
50 × 75-1-75 | 32 | 1666 | ||
45 × 65-1-65 | 25 | 1692 | ||
43 × 60-1-60 | 19 | 1673 | ||
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45 × 65-1-65 | 17 | 6064 H | |
50 × 50-1-50 | 18 | 6063 | ||
45 × 60-1-60 | 15 | 6065 | ||
40 × 55-1-55 | 15 | 6061 | ||
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42 × 60-1-60 | 15 | 5958 H | |
55 × 70-1-70 | 25 | 3343 | ||
45 × 60-1-60 | 12 | 5960 | ||
41 × 55-1-55 | 15 | 5959 | ||
37 × 45-1-45 | 8 | 5965 |
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 into four chambers. The first chamber, which follows the esophagus, receives the ducts of the dorsal and lateral lobes of the digestive gland. The second chamber, posterior, receives the duct of the posterior lobe of the digestive gland. The third chamber is funnel-shaped and lined by ridges internally. The fourth chamber is continuous and externally similar to the third. The intestine is long, narrow, and the intestinal loops are exactly between types I and II, i.e., with a transitional loop on average oriented at 6 o’clock, though the orientation of the transitional loop ranges between 5 and 7 o’clock (Figs
(Fig.
(Fig.
Reproductive system,
(Figs
Penial hooks,
Spine of the accessory penial gland,
The penial sheath is narrow and elongated. The penial sheath protects the penis for its entire length. The beginning of the retractor muscle marks the separation between the penial sheath (and the penis inside) and the deferent duct. The retractor muscle is strong, shorter than the penial sheath, and inserts at the posterior end of the visceral cavity. In addition, there is a cluster of retractor muscle fibers on the distal part of the penial sheath, near the vestibule. The deferent duct is highly convoluted with many loops. Inside the penial sheath, the penis is a narrow, thin, elongated, hollow tube, with numerous and densely-arranged (next to each other) hooks in its distal part. Penial hooks are pointed and measure from 50 to 100 μm. When the penis is retracted inside the penial sheath, the hooks are inside the tube-like penis; during copulation, the penis is everted like a glove and the hooks are then on the outside.
The accessory 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. Near its distal part, the flagellum is enlarged into a muscular sac. Distally, the flagellum ends in a hard, hollow spine protected by a sheath which opens into the vestibule. The hollow spine is narrow, straight, elongated. Its base is conical. Its diameter is ca. 50 μm except at the base where it is larger (ca. 100 μm). The diameter of the opening at the tip measures ca. 30 μm. Its length ranges from 0.7 mm [1037] (
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
AUSTRALIA • holotype, designated here, 23/18 mm [1657 H]; Northern Territory, Darwin;
See below for each subspecies.
(Fig.
(Figs
Habitats,
(Table
(Figs
Live animals,
(Figs
Digestive system, dorsal view,
Radula,
(Fig.
Posterior, hermaphroditic (female) reproductive system,
(Figs
Anterior, male, copulatory apparatus,
Penial hooks,
Spine of the accessory penial gland,
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
(1) The three units within
(2) Ranking the three units within
(3) As of today, the three units within
The type locality and the holotype of the nominotypical subspecies
AUSTRALIA –
(Fig.
(Fig.
See above, the species
(Table
(Fig.
(Figs
(Fig.
(Figs
See above, the remarks on the species
INDONESIA • holotype, designated here, 17/16 mm [6064 H]; Ambon, Lateri;
INDONESIA –
(Fig.
(Fig.
The subspecies
(Table
(Fig.
(Figs
Radula,
(Fig.
(Figs
See above, the remarks on the species
MALAYSIA • holotype, designated here, 15/9 mm [5958 H]; Peninsular Malaysia, Matang, facing fishermen’s village on the other side of river;
INDONESIA –
(Fig.
(Fig.
Habitats,
The subspecies
(Table
(Fig.
Live specimens,
(Figs
(Fig.
(Figs
Penial hooks,
Spine of the accessory penial gland,
See above the remarks on the species
A few preliminary remarks can be made here regarding the types of intestinal loops, even though a more detailed discussion will be provided after our revisions of
(1) Nearly all onchidiid species are characterized by only one intestinal type. Some intra-specific variation exists, which can be evaluated based on the orientation of the transitional loop. However, the presence of more than one intestinal type in an onchidiid species remains exceptional, such as in
(2) Nearly all onchidiid genera are characterized by only one or two intestinal types. For instance,
(3) Intestinal loops are quite useful to identify genera. For instance, all known species of
(4) There must be some reasons explaining why intestinal types are not randomly distributed across onchidiid species and genera; however, the exact reasons are still unclear at this stage. Evolutionary history is possibly involved. For instance, the fact that all
Onchidiids are notoriously difficult to identify, both at the genus and species levels.
The two known species of
The two new species described here are widespread and can be locally common. That they are discovered only now is not so surprising.
We are grateful to associate editor Nathalie Yonow and reviewers Adrienne Jochum and Pierre Lozouet for constructive comments that helped improve the manuscript. We are grateful to all the people who helped us with field work in various ways, by hosting us at their institutions, helping with logistics, or accompanying us in the field. Our study would have been impossible without their generous help and efforts: Teddy Chua in Brunei Darussalam; Neil Bruce in Queensland; Adam Bourke and Richard Willan in the Northern Territory; Vivian Ang, Don Dumale, and Marivene Manuel in the Philippines. Accessing mangrove sites would have been impossible without help from local fishermen and villagers. We also thank the collection managers of various institutions for accepting to host our material in their collections and sending us specimens on loan: Brunei Museum, Natural History, Brunei Darussalam; Institute of Tropical Biology, Zoology Collection, Vietnam Academy of Science and Technology, Ho Chi Minh City, Vietnam; Museum of Tropical Queensland, Townsville, Queensland, Australia; Museum and Art Gallery of the Northern Territory, Australia; National Museum of the Philippines, Manila, Philippines; Universitas Malikussaleh, North Aceh, Sumatra, Indonesia; Mollusk Collection, Universiti Sains Malaysia, Penang, Malaysia; Zoological Reference Collection, Lee Kong Chian Natural History Museum, National University of Singapore. Specimens were collected following local regulations, as overseen by Shau Hwai Tan (Malaysia), Marivene Manuel (Philippines), Munawar Khalil (Indonesia), and Quảng Ngô Xuân (Vietnam). Collecting in Brunei, New South Wales, Queensland, and the Northern Territory was done with permits from local institutions. A research permit was issued to Benoît Dayrat in Singapore (#NP/RP10-020). We thank the Ministry of Research, Technology and Higher Education, Republic of Indonesia (Ristek-Dikti) that issued a research permit to Benoît Dayrat (Ristek #134/SIP/FRP/E5/Dit.KI/VI/2017). We also wish to thank the Universitas Malikussaleh for being our home base 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 from the US National Science Foundation (DEB 1419394).