Elysia aowthai, Mehrotra & Gutiérrez & Scott & Arnold & Monchanin & Chavanich, 2020

Mehrotra, Rahul, Gutierrez, Manuel Caballer, Scott, Chad M., Arnold, Spencer, Monchanin, Coline & Chavanich, Suchana, 2020, On the Plakobranchidae (Gastropoda, Sacoglossa) from soft sediment habitats of Koh Tao, Gulf of Thailand, with descriptions of two new species, ZooKeys 969, pp. 85-121 : 85

Elysia aowthai sp. nov. Figures 8F-J , 9 , 10A-C , 11

Elysia cf. japonica : Bass 2006 (Guam)

Elysia amakusana : Wägele et al. 2010 (Lizard Island, Australia)

Elysia sp. 1: Mehrotra and Scott 2016: fig. 2B, C (Koh Tao, Thailand)

Elysia cf. japonica : Mehrotra et al. 2019: fig. 1C (Koh Tao, Thailand)

Type material.

Holotype: adult, 14 mm long (alive), collected from soft sediment habitats at 12 m depth, Leuk Bay (type locality) 10°4'15.23"N, 99°50'32.86"E, Koh Tao, Thailand, 20 December 2015, deposited in MNHN (IM-2000- 35326). Paratype: adult, 16 mm long (alive), collected from soft sediment habitats at 16 m depth, Tao Tong 10°3'58.13"N, 99°49'4.76"E, Koh Tao, Thailand, 19 June 2017, deposited in MNHN (IM-2000- 35327). Paratype dissected: reproductive system studied, and jaw, radula, and penis mounted for optical microscopy.

Paratype: adult, 14 mm long (alive), collected in silty sand at 25 m depth, Tao Tong, 10°3'58.13"N, 99°49'4.76"E, Koh Tao, Thailand, 17 February 2020, deposited in RBRG (EcjIV-NR012).

Supplementary observations.

More than 100 individuals, ranging in size 5 mm-16 mm, observed in regular diving surveys between 2012 and 2019, Leuk Bay 10°4'15.23"N, 99°50'32.86"E; Suan Olan Artificial Reef 10°4'6.70"N, 99°50'26.29"E; Shark Bay 10°3'39.75"N, 99°50'4.43"E; Tao Tong 10°3'58.13"N, 99°49'4.76"E; Sai Nuan 10°4'43.24"N, 99°48'48.51"E; Twins 10°7'1.93"N, 99°48'44.26"E; Hin Wong Bay 10°6'12.30"N, 99°50'58.63"E; Laem Tien 10°5'19.13"N, 99°51'17.64"E Koh Tao, Thailand; not collected.

Description.

Length alive up to 16 mm. Body translucent white, with tips of rhinophores deep blue to purple, fading to the base and lacking tubules of digestive gland. Opaque white specks all over the body, concentrated on the edge of parapodia, renopericardial prominence, and dorsal surface of head and rhinophores. Digestive gland variable from reddish brown to light green, forming a characteristic reticulated pattern of thin tributaries. Eyes black, conspicuous, comma-shaped, behind rhinophores, bearing 75 µm diameter lentil-shaped crystalline lenses. Rhinophores long, pointed at the tip, with groove along entire length. Lateral groove on right side from anterior border of right parapodium to foot. Foot’s transversal groove at same height as end of lateral groove, very subtle, almost invisible, dividing foot in two. Anterior foot corners slightly extended (in preservation), bluntly pointed. Body and parapodia in some specimens with sparse small papillae which disappear upon preservation. Tail pointed, extending beyond parapodia (Fig. 8 ).

Renopericardial prominence long and narrow, oval anteriorly, slightly constricted in the middle, and straight posteriorly. Posterior end with two pairs of major dorsal sinuses: first short, perpendicular, fading at the middle of the parapodium; second long, oblique, curved, orientated towards but not reaching edge of parapodium, ending before the tail. Area between second pair of dorsal sinuses appearing translucent in preserved specimens (Fig. 9B ) and probably corresponding to the 'smooth trench’ mentioned by Eliot (1913: 47) describing E. japonica (KR Jensen, pers. comm.). Renal pore at anterior right side of pericardium. Anus in groove separating the right parapodium from the neck (Fig. 9 ).

Buccal bulb 500 μm long (in three 10, 15, and 16 mm long specimens). Uniserial radula with seven teeth including ghost teeth in ascending row, 8-11 in descending row. Discarded teeth driven out of buccal bulb through a short tube; 12-30 in the ascus, varied in size, some broken, stacked in several groups, some loose. Teeth narrow, blade-shaped. Cutting edges smooth or finely denticulated. Denticulation difficult to observe with the light microscope and random (Fig. 10 ). First teeth in ascending series 72-76 μm long, 22-25 μm high. Active teeth 71-80 μm long, 27-30 μm high. Last teeth in descending series 50-76 μm long, 20-29 μm high.

Penis unarmed, pseudo-conical with a narrow and curved end (chilli-shaped), 500 μm long. Vas deferens long and convoluted, passing through the mucus gland. Male genital opening below right rhinophore, next to right eye. Vagina fusiform, narrow at the extremes, passing through the mucus gland. Bursa copulatrix connected to the vagina. Mucus gland quasi-oval, translucent, 900-1000 μm long, located under the cardiac area. Ducts from the follicles, prostate, and albumen gland coming from the parapodia meet in the mucus gland, but the connections between them are unclear (Fig. 11 ).

Biology. All animals were observed year-round at different locations around the island, exclusively in deeper soft sediment habitats at Koh Tao. Animals were always found moving along the surface of the sediments, rarely stationary and with no specific ecological relationship with any other species. Not observed to be in association with any particular prey algae, but often found in the vicinity of a currently unknown filamentous red algae, which may contribute to the reddish colouration seen in most specimens. Specimens with green, brown, purple, and unpigmented digestive glands are likely due to some degree of variability in prey; however, further investigations are required. No observations were made shallower than 10 m depth or in the vicinity of coral reef or reef edge habitats, deeper observations up to 24 m.

Derivatio nominis.

Elysia aowthai in reference to the type locality of the species in the Gulf of Thailand, 'Aow Thai’ in the local vernacular language, and to honour the local populations that speak it.

Distribution.

Gulf of Thailand (present study), Guam (as E. cf. japonica , Bass 2006), and Australia (as E. amakusana , Wägele et al. 2010) based on our molecular analyses.

Remarks.

The general appearance of Elysia aowthai sp. nov. places it within the E. japonica species complex. The problem with this complex has its origins in the incomplete description of E. japonica itself, but also in the provenance of the type material and the subsequent specimens captured, sometimes associated with descriptions and illustrations and sometimes not. Elysia japonica was described based on preserved specimens, which makes the external anatomy and living colouration very difficult to untangle, collected from an unknown location in Japan. The complete description by Eliot (1913) is as follows:

" 18 specimens. Locality - unknown. The largest is about 20 mm long and the wings are moderately ample. In two specimens which were dissected, the radula was found to contain 5 teeth in the ascending row, 15 in the descending and about 20 more various sizes lying in a heap. The structure and the shape of the teeth is as usual in the genus. No denticles are to be seen. I think that this form is probably a new species distinguished by the following characters:

1) Colour: In all specimens, the rhinophores and the tip of the tail are conspicuously black or dark brown. Otherwise the colour is uniform and in the best preserved specimens is yellowish brown. The wings have no coloured borders and the head and the pericardium are of the same colour as the dorsal surface.

2) The arrangement of the dorsal surface. This is similar in all specimens and I have not seen it in any other species. The pericardium is not ovate but is constricted in the middle. Its length is greater than its breadth but it is short in comparison with the length of the whole animal. The dorsal ridges which run into it are very distinct and the two hindmost, which run backwards towards the tail, are parallel to one another and enclose an area which is differentiated from the back and forms a smooth trench. "

The first illustration showing the external anatomy of a specimen assumed to be Elysia japonica was provided by Baba (1949), with a description of the species. However, this was later changed in a supplement to the previous paper wherein the same specimen ( Baba 1949: plate IX, fig. 27) was reidentified and claimed as a new species ( Baba 1955), Elysia amakusana Baba, 1955, based exclusively on the presence of finely denticulated teeth, which are (theoretically) not present in E. japonica . Also, in the same supplement, Baba described Elysia abei Baba, 1955, which also has finely denticulated teeth but is differentiated by being green in colour with fine orange-red spots ( Baba 1949: plate VIII, fig. 26; Baba 1955). Afterwards, however, Baba determined all three species as valid in a summarised inventory of Elysia species from Japan ( Baba 1957). A year later, Elysia furvacauda Burn, 1958 was described from Australia ( Burn 1958), based entirely on the external anatomy (therefore lacking any mention of radular detail), as a red-brown animal with small blue spots and rhinophores, and tail tipped in black. The type material for this species was reported as lost by Jensen (1985), but it is actually deposited in Museums Victoria Collections (MVC 2020, registration number F19467) (KR Jensen, pers. comm.).

Subsequently, Marcus (1980) and later Jensen (1985) both pointed out the similarity in radular teeth between E. amakusana and E. abei , with Jensen providing a description of three different colour morphs of E. japonica from Hong Kong, a comprehensive analysis of the taxonomic confusion to date, and concluding that E. amakusana and E. abei are junior synonyms of E. japonica . Importantly, Jensen showed that the morphology of specimens of three different external colourations was remarkably similar and that radular teeth have 'blunt tips and are finely serrulate’. Between 1999 and 2002, discussions on the Sea Slug Forum (hosted by the Australian Museum) between Rudman, Jensen, and others ( Rudman 2001) generally supported the view that some or all of E. abei , E. amakusana , and E. japonica were likely synonyms, although Rudman suggested that the name Elysia japonica may need to be abandoned due to the lack of type material or information on the type locality. Burn (2006) restated that E. furvacauda was distinct from E. japonica which was supported by an external illustration of the former (Fig. 8 ), although no internal anatomy was discussed. Trowbridge et al. (2010, 2011) also discussed in detail the complexity and need for clarification between these species.

The first molecular evidence on any species in the complex was provided by Händeler and Wägele (2007), who sequenced a portion of the mitochondrial 16s rRNA for a species they called Elysia amakusana (from Australia) as part of a larger analysis. While this analysis provided no illustration, description, or discussion of the species in particular, an illustration of a specimen identified as E. amakusana from Lizard island (Australia) was published a year before by the second author ( Wägele et al. 2006: 41, fig. 8H), with this specimen looking similar to Elysia aowthai sp. nov. A more thorough molecular assessment regarding the presently discussed species was later provided by Takano et al. (2013), where a comparison between specimens they considered representative of E. abei , E. amakusana , and E. cf. japonica was carried out. These authors provided colour photographs of E. abei and E. amakusana from Japan ( Takano et al. 2013: fig. 3A, B) matching Baba´s (1949: plate VIII, fig. 26; plate IX, fig. 27) illustrations. Takano et al. (2013: fig. 3C) also provided an illustration of a specimen determined as E. cf. japonica from Japan that matches the general appearance of a species in the complex of E. japonica , and shows a character that could have gone unnoticed in the original description of the species due to the state of preservation of the specimens: big rounded blotches of orange pigment (different from the orange dots of E. abei ). In their analysis, these authors concluded that E. abei and E. amakusana were likely synonyms despite their morphological variability, but E. cf. japonica appeared distinct. Takano´s et al. (2013) species identifications were based on external colouration and morphology alone. However, if the internal anatomy of their E. cf. japonica would match that of the original description, E. japonica could be re-described, which would help to clarify the taxonomic/systematic problems around the species complex in the area. Nonetheless, the fact is that there are at least two lineages in Japan corresponding to the external morphology of E. abei / E. amakusana and (potentially) E. japonica . An extensive phylogeny by Krug et al. (2015) showed specimens determined as E. abei (Japan), E. amakusana (Australia), and E. furvacauda (Australia) as distinct species, clustered with species they call Elysia sp. 5 (Hawaii) and Elysia sp. 30 (Japan), but again the external anatomy of the species is not shown nor discussed, which makes it impossible to draw conclusions on the validity of the different taxa.

After this review it seems clear that, within the complex, several groups may be present based on the external anatomy: one composed of specimens similar to E. abei / E. amakusana with a wide range of morphological variability, another with unclear characteristics belonging to E. japonica sensu stricto, and a third including E. furvacauda . However, the reliability of species delimitation using external colouration in this group is questionable when used alone.

On the other hand, the integrated analysis of the COI of numerous species/specimens within the complex conducted here reveals a clear geographical structure. There are at least two separate lineages in Japan (Fig. 2 , clade D) corresponding to the true E. abei / E. amakusana group and (potentially) to the true E. japonica sensu stricto (whose type appears to be lost: Trowbridge et al. 2011). The remainder of the groups within the complex (Fig. 2 , clades A-C) include E. furvacauda from Australia and at least two undescribed species within the range of morphological variability observed for E. abei / E. amakusana scattered in the Indo-West Pacific (I-WP).

In this work, to contribute to the untangling of the E. japonica complex, we describe one of these apparently widely distributed species scattered in the I-WP, E. aowthai sp. nov., by providing detailed morphological (external and internal) and molecular evidence. Future findings including precise sampling coordinates, complete diagnoses, colour images, and molecular data will resolve the identities of the members of this species complex and its distribution.

Despite the fact that the radular teeth of E. aowthai sp. nov. are indeed finely serrated, two points must be noted: these appear to be rapidly worn away with use leaving smoother edges in older teeth, and this feature is not visible under light microscopy alone and required the use of SEM observations to confirm their presence. Hence, the use of this feature alone does not have enough diagnostic significance in distinguishing the species of the complex.

Elysia aowthai sp. nov. is not conspecific to the specimens studied by Jensen (1985) from Hong Kong and determined as E. japonica : there are some differences that should be noted such as the morphology of the pericardium which is shorter, wider, and bearing four anastomosed dorsal sinuses in Jensen´s specimens, and the shape of the teeth, which are blade-shaped in E. aowthai sp. nov. instead of having the rounded blunt tips observed in Jensen´s specimens ( Jensen 1985: fig. 2C) which appear significantly more rounded than those of E. aowthai sp. nov. The reproductive apparatus of Jensen´s (1985) specimens were described as 'very similar’ to that of Elysia verrucosa Jensen 1985, but this statement is insufficient to establish any comparison.

Figures

Figure 2. Phylogenetic hypothesis for the Elysia japonica complex based on COI sequences. Sequences obtained in this study in bold. Bootstrap values from ML shown above branches and PP values from BI below branches. Tree rooted to Costasiella coronata (not shown).

Figure 8. Illustrations and images of specimens belong to the Elysia japonica complex A-E illustrations taken from the original descriptions of species belonging to the Elysia japonica complex A, D Elysia abei Baba, 1955 B Elysia amakusana Baba, 1955 C Elysia furvacauda Burn, 1958 E illustration of radula of Elysia japonica Eliot, 1913 by Baba (1949) F-J Elysia aowthai sp. nov. showing variation in colouration, 12 mm (F), 9 mm (G), 11 mm (H, I), 8 mm (J).

Figure 9. Elysia aowthai sp. nov., Koh Tao Thailand A-F 16 mm alive A right side view, the black dot at the end of the parapodia represents the vaginal opening B dorsal view, the shaded area probably being the same as that mentioned by Eliot (1913: 47) C ventral view with a detail of the head D detail of the eyes E buccal bulb F last teeth of the ascending series and active teeth G, H 10 mm alive G ascus H last teeth of the ascending series and active teeth.

Figure 10. SEM images of radulae of Elysia aowthai sp. nov. and Plakobranchus noctisstellatus sp. nov. A-C Elysia aowthai sp. nov., Koh Tao, Thailand, 15 mm alive A general view of the radula without the ascus B active teeth from the radula with smooth cutting edge C sixth teeth of the descending series with denticulated cutting edge D-F Plakobranchus noctisstellatus sp. nov. Koh Tao, Thailand, 21 mm alive D general view of the radula without the ascus E lateral view of the descending series F detail of the cutting edge of a tooth.

Figure 11. Genitalia and reproductive system of Elysia aowthai sp. nov., Koh Tao, Thailand, 15 mm alive. Abbreviations: ag - to the albumen gland; bc - bursa copulatrix; f - to the follicles; mg - mucus gland; p - penis; pr - to the prostate; v - vagina; vd - vas deferens; vo - vaginal opening.