An updated inventory of marine opisthobranch (Mollusca,Gastropoda) from the territorial waters of the Republic of Mauritius

Lisa AH SHEE TEE, Chandani APPADOO, Daneshwar PUCHOOA,Vishwakalyan BHOYROO

1 Department of Agricultural & Food Science, Faculty of Agriculture, University of Mauritius, Réduit 80837, Mauritius

2 Department of Biosciences & Ocean Studies, Pole of Research Excellence Sustainable Marine Biodiversity, Faculty of Science, University of Mauritius, Réduit 80837, Mauritius

Abstract Island ecosystems support diverse aquatic invertebrate communities comprising endemic taxa.Documentation of existing species is important for conservation.In this study, a checklist of marine opisthobranch from the Republic of Mauritius is presented.A combination of benthic surveys (50 m×5 m in triplicates), rover diving techniques and photo documentation were used over two years (2018-2020)within 35 sheltered and unsheltered lagoons.Morphological and molecular analysis were used for identification.Species composition within sheltered and unsheltered areas in Mauritius was estimated using the Bray-Curtis similarity.The checklist featured 117 species belonging to 61 genera and 28 families, of which 13 are new records.The findings increased the knowledge of opisthobranch diversity from the Mauritius by 15.4%.Among the listed species, the distribution range of Cyerce nigra,Actinocyclus papillatus, and Phyllidia picta extended from the Western Pacific to the South Western Indian Ocean.Molecular analysis of the undescribed Gymnodoris sp.showed it resembled Gymnodoris sp.from Hawaii and were different by a genetic distance value of 10.6%.The species richness and evenness were higher within the sheltered regions of Mauritius which harboured the food resource of opisthobranch.These areas as compared to unsheltered regions were heavily populated, suggesting the probable influence of wave actions on opisthobranch diversity and abundance.The order Nudibranchia was reported as most speciose, with 86 species.The Sacoglossa and Nudibranchia were observed only on macroalgae and sponges respectively.High abundance was also recorded on shipwrecks which are the most common form of artificial reefs.With the inclusion of observations from previous studies, 201 species belonging to 94 genera and 36 families are now known from the Mauritius.

Keyword: marine opisthobranch; inventory; new record; occurrence; Republic of Mauritius; Indian Ocean

1 INTRODUCTION

The Republic of Mauritius is a set of islands situated in the South-Western Indian Ocean, consisting of the main islands; Mauritius and Rodrigues and the outer islands; Agalega and St.Brandon.The main islands form part of the Mascarenes which is listed among the world’s top biodiversity hotspots (Thébaud et al., 2009).Studies of the malacofauna of the islands begun in 1774 comprising 125 terrestrial molluscs species of which 81 are endemic to Mauritius and 32 are unique to the Mascarenes (Griffiths and Florens,2006; Florens and Baider, 2007).Investigations of the marine malacofauna started through expeditions and systematic works in 1832 in Mauritius (Tee et al., 2019).The latest inventory within the Western Indian Ocean dated a decade ago and denoted Mauritius and Reunion islands as the most diverse with 48 opisthobranch species (Yonow, 2012).

The Republic of Mauritius has unique marine ecosystems, offering different types of reefs, fringing,atolls, reef flats and barrier reefs, seaweeds and seagrass.Mauritius is bounded mostly by discontinuous reefs, providing a lagoonal area about 1-6 m deep for about 243 km2(Bhagooli and Kaullysing, 2019).The best developed reefs are found in Rodrigues and the most pristine in St.Brandon (Turner and Klaus, 2005).Tee et al.(2019)reported a total of 105 nudibranch species belonging to 20 families in Mauritius.The sea slugs were also observed from the marine reserves of Rodrigues but no details on the species were disclosed (Desiré et al., 2011).Opisthobranch are a diverse group of beautifully coloured sea slugs comprising of the nudibranch (Nudibranchia), headshield slugs(Cephalaspidea), sea hares (Aplysiida), side-gilled(Pleurobranchida and Umbraculida) and sap-sucking slugs (Sacoglossa) (Jensen, 2013).Their ornateness has attracted the attention of many underwater photographers and SCUBA divers such that they form an economically important species for coral-reef based SCUBA diving tourism (Mehrotra and Scott, 2016).

With changing oceanographic conditions and increase risk of extinction for endemic taxa especially in many isolated island ecosystems, it is important to document the existing species.Species richness among molluscs has been linked to benthic habitat diversity(Bouchet et al., 2002; Davis et al., 2017).The objective of this study was to provide an updated inventory and increase the knowledge of opisthobranch fauna distribution within the Republic of Mauritius.

2 MATERIAL AND METHOD

The rover diving technique and benthic belt transects (50 m×5 m in triplicates) were used because opisthobranch are highly diverse and least abundant (Munro, 2005; Mehrotra and Scott, 2016).The surveys were performed in reefs and non-reefs ecosystems (sites dominated by algae) for a period of two years (2018-2020).A total of 35 lagoons and 10 diving spots were surveyed in the Republic of Mauritius (25 lagoons and ten diving sites in Mauritius and ten lagoons in Rodrigues; Fig.1) using snorkeling and SCUBA diving at a maximum depth of 30 m.Both sheltered and non-sheltered lagoons were surveyed in Mauritius and photo documentation for three islets of St.Brandon were made available by Mr Eric Le Court de Billot.Coordinates of sampled areas (Fig.1) are found in Supplementary Table S1.

The rock turning method and indirect sampling(collection of dead corals and algae) were used because of the nocturnal, hiding nature and small size of opisthobranch (Brodie and Brodie, 1995).Observations of opisthobranch were recorded on a dive plate and photos taken in situ using a Canon Powershot G7 X Mark II digital camera, equipped with a WP-DC54 waterproof case.Unidentifiable sea slugs were placed in an aquarium and finer details captured using a USB microscope camera.The specimens were preserved in 96% ice-cold ethanol for DNA barcoding.Identification followed taxonomic works, field guide (Gosliner et al., 2018),online resources (South-West Indian Ocean Seaslug website and the Sea Slug Forum) and assistance of divers on social media.Total genomic DNA was extracted using the Sokolov (2000) protocol and amplified using two primer sets; the cytochrome c oxidase 1 (COI) mitochondrial gene (Folmer et al.,1994) and 16S ribosomal RNA (16S) (Palumbi,1996).PCR conditions applied include Turner and Wilson (2008), Palomar et al.(2014), Tibiri?á et al.(2020), and Bonomo and Gosliner (2020).The sequences of successfully amplified DNA were deposited in GenBank and the accession number can be found in Table 1.Alignment was performed forGymnodorissp.only (Supplementary Table S2)because it was abundant and reported as undescribed.MAFFT v.7 was used with the alignment algorithm set to auto (Katoh et al., 2019).The model of nucleotide substitution was chosen using the Jukes Cantor method (Jukes and Cantor, 1969) and genetic distances calculated using the uncorrectedpdistance and 1 000 bootstrap replicates (Felsenstein,1985) in MEGA X (Kumar et al., 2018).

Fig.1 The surveyed areas in Rodrigues (a) and Mauritius islands (b)

A data matrix was created in the Excel spreadsheet whereby the total species per regions were arranged in rows and columns.Mauritius was divided into four regions; north, south, east and west based on the influence of the South East Trade Winds.The north and west part of Mauritius were considered as the sheltered sides.All statistical analyses were performed in PRIMER 7 (Clarke and Gorley, 2015) but prior, the data was log(x+1)transformed because rare and abundant taxa will potentially skew the abundance distribution of species (McMurdie and Holmes, 2014).Similarity was estimated using the Bray-Curtis coefficient and species richness using the Shannon-WienerH′Diversity Index (Pielou, 1975) given by the formula:

wherePistands for the proportion of individuals of each species to the total number of individuals(Chang et al., 2013).Pielou’s Evenness Index (Pielou,1966) was used for species evenness and was computed using the equation below, wherebyH′ is for Shannon-WienerH′ diversity index whileH′maxrepresents the maximum of Shannon-WienerH′diversity indexes.

A species list was compiled, including present observations and reports from previous studies(Table 1).The list was compared with existing literatures to determine new record for the island.Scientific names and updated nomenclature were confirmed and acquired using Bouchet et al.(2017)and the World Register of Marine Species (WoRMS,2022) online database.Doubtful identifications were marked with “cf.” and those assumed to be undescribed were marked with “sp.”

3 RESULT

3.1 Species checklist

A total of 117 opisthobranch species belonging to 61 genera and 28 families are reported.The speciose group was the Nudibranchia (86 species), followed by the Sacoglossa (12 species), Cephalaspidea (7 species),Aplysiida (5 species), Pleurobranchida (4 species),Umbraculida (1 species) and the superfamily Acteonoidea (2 species).With the addition of past observations, the Republic contained 201 species belonging to 94 genera and 36 families (Table 1).

The checklist increased the knowledge of opisthobranch diversity from Mauritius by 15.4%with 13 novel records (Fig.2), of which three were identified to the genus level (Fig.3).Also, molecular analysis ofGymnodorissp.demonstrated it resembledGymnodorissp.from Hawaii.They were different by a genetic distance value of 10.6% (Supplementary Table S3).

3.2 Species composition

Analysis of species composition in Mauritius showed a positive trend and high richness values towards the sheltered side of the island (Fig.4).A single sister clade was identified based on shared species, clustering the north and west regions together.This indicated the sister clade probably shared similarities in terms of habitat components.The north and west regions formed the sheltered sides of the island.Nudibranch are influenced by wave actions (Nybakken, 1978).The effect of the easterlies (South East Trade Winds) explained the low diversity value obtained for the east (H′=2.05)and south side (H′=3.18).High level of richness was found in the west (H′=4.34) and north (H′=3.57)regions, respectively.Among the Republic, St.Brandon had the lowest diversity value (H′=1.75).However, compared to Mauritius, the other islands were not as regularly monitored because of logistic problems.This explained the high number of species observed in Mauritius (420 nudibranch).Only five and eleven species were reported from St.Brandon and Rodrigues respectively.Hence, explaining the diversity value of 2.30 obtained for Rodrigues.The richness value is likely an underestimation of the true diversity.Pielou’s evenness index ranged from 0.96 to 0.99 for the Republic of Mauritius.

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

Table 1 Continued

3.3 Species records and substrate

The sea slugs were observed on their respective food source (Fig.5).The sacoglossan are herbivorous in nature and were found solely on the seaweedand macroalgae.TheOxynoesea slugs were observed alongside other sacoglossan such asVolvatella,PolybranchiaandElysiain shallow water, feeding on the seaweedCaulerpasp.The Aplysiida are also herbivorous and were frequently encountered in seagrass meadows.The nudibranch, mostly the phyllidiids wereobserved on sponges as they crawled during daylight.However, most of the nudibranch were found hiding under rocks (n=91) crawling in small crevices, illustrating their nocturnal nature.Within the order Nudibranchia, the sub-order Cladobranchia made up a small proportion of the total nudibranch observed.They were found mostly on hydrozoan and on shipwrecks.Figure 5 showed a total of 227 individuals were observed on food resource, 161 and 52 sea slugs were recorded under dead corals and on shipwrecks, respectively.

Within the sub-order Doridina, the Chromodorididae were highly diverse and were found either under dead corals or crawling onto it (n=126).These sea slugs were peculiarly conspicuous in appearance.The headshield sea slugs were observed in soft sediment areas such as Bain des Dames.

4 DISCUSSION

4.1 Species checklist

This study provided an updated inventory of opisthobranch species in the Republic of Mauritius.A total of 117 opisthobranch were reported and together with previous studies, the Republic contained 201 species belonging to 94 genera and 36 families(Table 1).Current findings increased the knowledge of the marine malacofauna from the Republic by 15.4% with 13 novel records (Fig.2), of which 3 were undescribed (Fig.3).

Fig.2 Among the 13 novel records, 9 are presented Berthellina delicata (a) (size: 40 mm); Cyerce nigra (b) (size: 18 mm);Elysia tomentosa (c) (size: 35 mm); Elysia sp.(d) (size: 8 mm); Facelina sp.(e) (size: 15 mm); Actinocyclus papillatus(f) (size: 45 mm); Atagema spongiosa (g) (size: 80 mm); Tayuva lilacina (h) (size: 50 mm); Dendrodoris sp.(i) (size:140 mm)

Within the checklist, 18 were undescribed and 13 were marked as “cf.” as likely to require further investigation.Among the undescribed species,Elysiasp.(Fig.3) matched the description ofElysiasp.36 from Gosliner et al.(2018) except for the rhinophores which were not acutely pointed.It was observed on a shipwreck in the south-west of Mauritius similar toTenelliasp.which resembledTenelliasp.6 from Gosliner et al.(2018).Tenelliasp.6 is distributed in the Indian and Western Pacific Oceans but never reported from Mauritius.Genetic analysis was performed only forGymnodorissp.because it was observed in large populations in the south-east region of the island nearby a fish farm.It resembledGymnodorissp.45 from Gosliner et al.(2018) which was reported from Philippines and Indonesia only.Nevertheless, genetic distance analysis showed it was more closely related toGymnodorissp.from Hawaii (10.6%) (Supplementary Table S3).Further anatomical and molecular analysis are needed to confirm the identities of undescribed species.

This study extended the distribution ofCyerce nigra,Actinocyclus papillatus,andPhyllidia pictafrom the Western Pacific Ocean (Gosliner et al.,2018) to the South Western Indian Ocean.Among the records, the Nudibranchia was the speciose order(86 species), followed by the Sacoglossa (12 species),Cephalaspidea (7 species), Aplysiida (5 species),Pleurobranchida (4 species), Umbraculida (1 species)and the superfamily Acteonoidea (2 species).Within this diverse order, the Chromodorididae, Phyllidiidae,and Polyceridae were the dominant groups,belonging to the suborder Doridina.This suborder is generally considered as the most abundant in many regions (Chavanich et al., 2013).

Fig.3 Species identified to the genus level Elysia sp.(a) (size: 8 mm); Facelina sp.(b) (size: 15 mm); Janolus sp.(c) (size:35 mm); Tenellia sp.(d) (size: 8 mm); Goniobranchus sp.(e) (size: 45 mm); Doridoidea sp.(f) (size: 220 mm);Halgerda sp.(g) (size: 43 mm); Doris sp.(h) (size: 40 mm); Dendrodoris sp.(i) (size: 140 mm); Gymnodoris sp.(j) (size: 30 mm)

Fig.4 Bray-Curtis similarity between species composition of four regions in Mauritius

Fig.5 Record of sea slugs on their respective substrate by order

The frequently encountered family was the Phyllidiidae.They were observed both during daylight and night surveys, possibly because of their tendency of exposing themselves and their high activity (Brunckhorst, 1991; Su et al., 2009).Similar observations were made by Papu et al.(2020) and Su et al.(2009) wherebyPhyllidiella pustulosawas reported as the dominating species, with a total of 39 individual records and was sighted in 18 out of 20 surveys respectively.In this study, the most common species werePhyllidiella meandrina,Phyllidia marindica, andPhyllidia alytawhich are distributed only in the Indian Ocean (Gosliner et al.,2018).Phyllidiella meandrinawas first collected in Mauritius and was known to be a frequent species in the Mascarenes (Rudman, 2002).Compared to this family, the suborder Cladobranchia accounted only a quarter to one third of the total records and were least abundant.This proportion of Doridina to Cladobranchia was reported both in Indonesia and Mauritius (Yonow and Hayward, 1991; Undap et al.,2019).

4.2 Species composition

The analysis of species composition in Mauritius showed a positive trend and high richness value towards the sheltered side of the island.The highest similarity was found between the north and west part of the island which form the sheltered side (H′=4.34 for the West andH′=3.57 for the North of Mauritius).The number of nudibranch are influenced by wave actions (Nybakken, 1978), currents and tides (Costello, 1938; Willows, 1999).They are generally heavily populated in regions receiving strong tidal wash and no heavy silt deposit (Miller,1961).This explained the low richness value for the south and east side of the island which are impacted by the South East Trade Wind.Nevertheless,different factors can explain the species composition of opisthobranch.Their sudden disappearance and appearance were linked to a diversity of theories such as migration from subtidal to intertidal areas for mating and spawning.Nevertheless, the diversity of opisthobranch can also be explained by the environmental needs of food sources (Papu et al.,2020).

4.3 Species record and substrate

As reported by Miller (1961), nudibranch is abundant in regions of abundant food organisms.Nevertheless, the nudibranch-food organism relationship is very specialized such that this kind of preference is likely to influence the distribution of species (Miller, 1961).In this study, the cladobranch were found typically in areas dominated by hydrozoan preys and most dorids were observed under coral rubbles (Fig.5;n=139) which form an important habitat and supplies a wide array of food items (Papu et al., 2020).The sacoglossan and sea hares were found in close association to their food resources; seagrass and seaweed.Some were recovered under rocks, indicating their hiding and nocturnal nature (Fig.5).An assemblage of species consisting of the genusVolvatella,Polybranchia, andElysiawere observed only onCaulerpasp.patches at Le Morne, Mauritius.The type and amount of substrate ultimately affected the density of sacoglossan sea slugs (Trowbridge et al., 2008; Mielke, 2015).

On contrary, the Aplysiida were abundant in shallow water and the distribution of their population was clumped as reported by Singh and Vuki (2015).The cephalaspidean were dominant in areas rich in fine-sediment such as Bain des Dames.Their abundance can be related to feeding purposes.They are physically adapted to inhabit such areas,equipped with a broad head, also referred to as headshield to help in pushing through the soft sediment.Additionally, many species were observed on structures such as shipwrecks (n=52) which are considered as biodiversity hotspots (Lengkeek et al.,2013).Some have been sunk intentionally and eventually became artificial reefs (Hamdan et al.,2021).They provide an entirely new habitat and increase the structural complexity of seabed(Badalamenti et al., 2002; Knott et al., 2004).Meyer et al.(2017) stated they also shaped the biogeographic distribution of macro-organisms.While many sea slugs hid themselves in crevices, theUmbraculum umbraculumwas spotted both on seagrass bed and on sand.The meadow could be serving both as shelter and a food source to this species (Garcia-Bonilla et al., 2020).

Comparison of food sources, the sponges were frequently observed in both shallow and deep waters than the hydrozoan.This could explain the low sights of cladobranch.The latter were particularly abundant on shipwrecks.Further, the abundant seagrass in the west of Mauritius explained the large number of sacoglossan observed.Aside from Mauritius, the remaining islands (St.Brandon and Rodrigues) were not as regularly monitored, explaining their low richness value.The record from the Republic is thus, an underestimation of the true number of opisthobranch.The remaining islands require further sampling efforts.

5 CONCLUSION

This study increased the knowledge of opisthobranch diversity from the Republic of Mauritius by 15.4% and formed a baseline for future biodiversity studies and monitoring projects.Current findings reported 117 species with a total of 13 novel records.The distributional ranges of three species(Cyerce nigra,Actinocyclus papillatus,andPhyllidia picta) now extend from the Western Pacific to the South Western Indian Ocean.The distribution of opisthobranch particularly sacoglossan and aplysiid were influenced by food sources.Comparison of sequences forGymnodorissp.from Mauritius showed it is more closely related toGymnodorissp.from Hawaii with a genetic distance of 10.6%.The current findings are however, an underestimate of the true records of opisthobranch from the Republic.This is because islands such as St.Brandon and Rodrigues were not as regularly sampled as Mauritius and require further investigations.Additionally, the sea slugs are small and demonstrate remarkable mimics of their prey such that with increase sampling effort,the record of opisthobranch is expected to increase.

6 DATA AVAILABILITY STATEMENT

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

7 ACKNOWLEDGMENT

The first author would like to thank the University of Mauritius for a postgraduate scholarship and logistics and Mr.Le Court de Billot for his support.The authors also thank the Ministry of Defence and Rodrigues for sampling permits within the Republic of Mauritius and the staff of the South East Marine Protected Areas (SEMPA) for their help in sample collection.