Psychedelics sea slugs: Observations on colour ontogeny in two nudibranch species from the genus Nembrotha (Doridina: Polyceridae)

Study location and samples information

Specimens of the genus Nembrotha were collected in Zavora Bay, Mozambique (Fig. 1A) from 2010 to 2015 by scuba diving (0-20 m deep) as part of a nudibranch diversity assessment by Tibiriçá et al. (2017)Tibiriçá Y., Pola M., Cervera J.L. 2017. Astonishing diversity revealed: an annotated and illustrated inventory of Nudipleura (Gastropoda: Heterobranchia) from Mozambique. Zootaxa 4359: 001-133. https://doi.org/10.11646/zootaxa.4359.1.1 . In 2015, we were able to set up a laboratory for aquarium observations. At that time, on two occasions, specimens of Nembrotha spp. were found on the blue lollipop ascidian Eudistoma caeruleum (Sluiter, 1909) (Fig. 1B), brought together with the ascidian to the lab and kept in captivity in a 10 L air-pumped tank for further observation (Fig. 1C). First, two adult specimens previously attributed to N. yonowae were kept in captivity for 24 days from 2 April 2015. However, after mating and laying eggs, they were euthanized due to the logistical difficulty associated with bad weather in bringing them fresh food. Second, on 26 May 2015, one specimen of N. livingstonei and two specimens of N. yonowae were found at Witch’s Hat at 17 m depth on a single individual of E. caeruleum. These specimens were collected and maintained in the tank for 100 days. To keep the specimens alive and healthy, new E. caeruleum ascidians were collected on a regular basis (every 3 to 8 days depending on the logistics and the weather) by scuba diving at Zavora Bay, and half of the tank water was changed every three days. In mid-August, the abundance of E. caeruleum on the reef was reduced, and diving conditions were unfavourable for many days, making the collection of fresh ascidians problematic. As a result, fresh food was being given more occasionally, so for logistical reasons the specimens were euthanized by freezing on 4 September 2015 for further study. Photographs were taken with Olympus PEN Lite E-PL5 with a macro lens and Olympus TG-5 cameras. Figures were created in Adobe® Photoshop® CS4. We were unable to make laboratory observations for specimens collected before 2015, and at that time, we were only able to make them on those when conditions were favourable. We tried to sequence all specimens kept in alcohol, but not all amplifications were successful (see Table 1).

Molecular studies

Tissue samples of a few specimens from Mozambique and one specimen from Bali (Table 1) were taken from the foot, and DNA was extracted using the Dneasy Blood and Tissue Kit (Qiagen, Valencia, CA). Partial sequences of the uncorrected mitochondrial cytochrome c oxidase subunit I (COI) were amplified by polymerase chain reaction (PCR) using LCO1490 and HCO2198 universal primers (Folmer et al. 1994Folmer R.O., Black M., Hoeh W., et al. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Marine Biol. Biotechnol. 3: 294-299.). Polymerase chain reactions were conducted in a 25 µL reaction volume containing 1 µL of both forward and reverse primers (10 µmol L^-1), 2.5 µL of dNTP (2 mmol L^-1), 3.5 µL of magnesium chloride (25 mmol L^-1), 0.25 µL of Qiagen DNA polymerase (5 units/µL), 5 µL of ‘Q-solution’ (5×), 2.5 µL of Qiagen buffer (10×) (Qiagen Taq PCR Core Kit) and 1-2 µL of genomic DNA. Amplification of COI was performed with an initial denaturation for 5 min at 94°C, followed by 35-36 cycles of 1 min at 94°C, 30 s at 45°C (annealing temperature) and 1 min at 72°C, with a final extension of 10 min at 72°C. Successful PCR products were sent to Macrogen, Inc. for purification and sequencing on a 3730XL DNA sequencer (Applied Biosystems). Sequences were edited in GENEIOUS v.10.2.3 (Kearse et al. 2012Kearse M., Moir R., Wilson A., et al. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28: 1647-1649. https://doi.org/10.1093/bioinformatics/bts199 ). All sequences were tested for similarity with the BLAST algorithm and aligned using MEGA7 (Kumar et al. 2016Kumar S., Stecher G., Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33: 1870-1874. https://doi.org/10.1093/molbev/msw054 ). The software jModelTest2 on XEDE, CIPRES Science Gateway (Miller et al. 2010Miller M.A., Pfeiffer W., Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), pp 1-8. https://doi.org/10.1109/GCE.2010.5676129 ), was used to select the evolutionary models for each codon position of COI under Akaike information criteria (Schwartz 1978). Evolutionary models for COI were TIM2+G, TPM1uf+I and TIM1+G for the first, second and third codon position, respectively. Bayesian inference analysis was performed using the software MrBayes on XSEDE, CIPRES Science Gateway (Miller et al. 2010Miller M.A., Pfeiffer W., Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), pp 1-8. https://doi.org/10.1109/GCE.2010.5676129 ), for ten million generations, four independent runs and a sampling frequency of 1000. New generated sequences are available in GenBank. For molecular comparison and species delimitation analysis, we used sequences downloaded from GenBank of several specimens of Nembrothinae, highlighting specimens identified as N. guttata (synonym of N. yonowae) from the Philippines (voucher code WAMS11556, accession number EF142894.1), and two specimens of N. cristata from Papua New Guinea (CASIZ191428, MF958431.1) and the Philippines (CASIZ 157483, DQ231003). Specimen details are summarized in Table 1.

Gene pairwise uncorrected p-distances for COI were obtained using MEGA7 (Kumar et al. 2016Kumar S., Stecher G., Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33: 1870-1874. https://doi.org/10.1093/molbev/msw054 ). Analyses of species delimitation were conducted on COI ingroup sequences. Bayesian Poisson Tree Process (bPTP) was carried out with the bPTP webtool (https://species.h-its.org), using the following parameters: MCMC generations = 200000, Thinning = 100 and Burn-in = 0.1. Assemble Species by Automatic Partitioning (ASAP) (Puillandre et al. 2021Puillandre N., Brouillet S., Achaz G. 2021. ASAP: assemble species by automatic partitioning. Mol. Ecol. Resour. 21: 609-620.https://doi.org/10.1111/1755-0998.13281 ) was also conducted using the webtool (https://bioinfo.mnhn.fr/abi/public/asap/asapweb.html), under the Kimura model (K80) ts/tv.

Nembrotha livingstonei Allan, 1933 (Fig. 2)

Material examined: MB28-005019, collected on 26 May 2015 at Witch’s Hat, Zavora, depth 17 m, found on E. caeruleum, length when collected 2 mm, kept in tank until 4 Sep. 2015, preserved size 18 mm. Sequenced. MB28-004737, 19 Set. 2013, Rock Pool, Zavora, Mozambique, depth 1 m, length 50 mm, on rock. Studied for external morphology (unsuccessful sequencing).

Diagnosis. As described by Pola et al. (2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 .

Colour ontogeny

Specimen MB28-005019 (Fig. 2A-G)

When collected, the specimen measured approximately 2 mm (Fig. 2A), and the background colour was blue. A light blue patch was present on the dorsal midline behind the rhinophores. Three thin light blue lines ran from this patch toward a light blue band in front of the gill. A few bright orange pustules were present on the notal border and surrounding the edge of the foot. The rhinophores and the non-retractile multipinnate gill branches were bright orange, especially bright at the tips (Fig. 2A). After one month (Fig. 2B, C), the specimen measured 6 mm, the orange pustules developed in almost complete lines (at the top of the light blue line) surrounded by a thin brown line. The light blue patches became almost bright white, except at the base of the gills, the base of the rhinophores and the end of the tail, where they remained bright light blue. The gill branches and rhinophores became less bright, becoming more reddish-brown in colour, except for the branchial rachises and rhinophoral tips. After two months, at about 10 mm (Fig. 2D), the orange lines/pustules lost their brightness, and the brown lines formed a clearer ridge texture. The bright light blue was now darker blue, clearly limited to the base of the gill branches, the tip of the rhinophoral sheaths, the oral tentacles, and the border of the entire foot. White-cream patches were limited to the circle around the gill below the blue and the area between the rhinophores, going up the rhinophore sheaths, clearly giving the typical appearance of a cross-shaped mark. In the third month the specimen grew to 12 mm (Fig. 2E, F) and the orange dorsal pustules disappeared almost completely, some becoming cream-coloured. The initial blue colour of the background had completely disappeared. Just before euthanizing, the specimen measured 12 mm long and had orangish pustules only on the anterior and posterior edge of the mantle (Fig. 2G). A much larger specimen (50 mm) was collected on the same reef in September (Fig. 2H). This specimen only had a few scattered orange pustules on its head, but it had many more creamy ones. The brown lines were darker and in greater numbers.

Ecological notes. This species is relatively rare in Zavora. It has only been seen on two occasions over seven years of intensive field research in shallow reefs (up to 17 m).

Remarks and discussion. Pola et al. (2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 reports different morphotypes for this species, including a dark specimen with green spots. However, the early juvenile is described here for the first time. We confirmed that the pale, cross-shaped mark is present among the rhinophores from juvenile to adult and, at present, it can be considered a diagnosis character regardless of life stage. Similarly, the oral tentacles and foot were lined with blue and did not lose colour with growth. All adults specimens reported for Mozambique show the same blue colouration, but the Philippines specimens observed by Pola et al. (2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 had bright orange oral tentacles and foot edge. Notably, in the specimen observed in the tank, the gill branches and rhinophores changed from bright orange to red and brownish. The edge of the rhinophore sheath was orange-pink pigmented in the large specimens and in the photographs provided by Pola et al. (2008, Fig. 14)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 , but not in the juvenile kept in captivity. Additionally, the background changed from blue to creamy. The species delimitation analysis (Fig. 3) and uncorrected COI p-distances clearly indicate that this species is different from N. yonowae, with a minimum distance of 9.5% (Table 2).

Nembrotha yonowae Goether and Debelius, 1992Goethel H., Debelius H. 1992. Nacktschnecken der Maldiven, mit der Beschreibung einer neuen Art. DATZ (Die Aquarien- und Terrarienzeitschrift), 8: 512-519.

Material examined: Thirteen specimens in total. MB28-005020 and MB28-005021, 26 May 2015, Witch’s Hat, Zavora, Mozambique, depth 17 m, found on Eudistoma caeruleum, length 2 and 30 mm when collected, kept in tank until 4 Sep. 2015, length 28 and 42 mm when preserved, respectively. Both sequenced and dissected. MB28-005003 and MB28-005004, 27 May 2015, Vascos, Zavora, Mozambique, depth 15 m, found on E. caeruleum, length 22 mm and 28 mm, respectively, collected by Mona Andskog, kept in tank for 24 days. MB28-004421, 31 May 2010, Witch’s Hat, Zavora, Mozambique, depth 16 m, length 42 mm, on E. caeruleum. Dissected. MB28-004422, 07 Jun. 2010, Witch’s Hat, Zavora, Mozambique, depth 18 m, length 25 mm, substrate not recorded. MB28-004424, 27 May 2010, Witch’s Hat, Zavora, Mozambique, depth 18 m, length 18 mm, crawling on the reef. MB28-004547, 20 May 2012, Launch area reef, Zavora, Mozambique, depth 2 m, length 38 mm, substrate not recorded. Dissected. MNCN15.05/91069, 9 Dec. 2012, Area 51, Zavora, Mozambique, depth 12 m, length 32 mm, on E. caeruleum. Sequenced and dissected. MNCN15.05/91070, 12 Dec. 2012, Area 51, Zavora, Mozambique, depth 12 m, length 6 mm, on E. caeruleum. Sequenced and dissected. MB28-004707, 06 Aug. 2013, Launch area reef, Zavora, Mozambique, depth 0.5 m, length 16 mm, between blue tunicate (Clavelina sp.). MHN-YT 1506, 02 Apr. 2015, Vascos, Zavora, Mozambique, depth 15 m, length 21 mm, on E. caeruleum. MB28-004980, 3 Apr. 2015, Vascos, Zavora, Mozambique, depth 17 m, length 16 mm, on E. caeruleum.

Colour ontogeny

Specimen MB28-005020 (Fig. 4A-D)

When collected, the specimen measured 2 mm, the background colour was dark blue with bright scattered orange pustules (Fig. 4A). The base of the rhinophores and the four non-retractile multipinnate gill branches were lighter blue. The sheaths, the rhinophore clubs and the oral tentacles were same blue as the body. The foot sole was dark blue with a dark line on the “tail” and light blue edge. At one month, the specimen reached 5 mm (Fig. 4B). The background colour was still blue, but not so dark, and the overall number of spots increased, these being more and more intensely orange in colour; a line of spots was well formed around the edge of the mantle and on the upper part of the foot. Traces of orange pustules appeared on the rhinophores’ sheaths. The outer rachises of the gill branches maintained a light blue tonality, but the pinnae turned dark blue. The rhinophores maintained their blue but a light blue tip appeared. The foot sole was translucent with blue edge and darker tip (Fig. 4B). In the second month the specimen grew to 10 mm (Fig. 4C). The background colour became lighter, with a light purple colouration, and the orange pustules became very bright. The rhinophores’ lamellae and the gill pinnae changed to the same colour as the body ground. The rhinophores’ tip and stalk, as well as the outer gill rachises turned a lighter blue, almost white. The edge of the elevated anus turned bright orange, as did most of the edge of the rhinophore sheath (Fig. 4C). In the third month, at about 25 mm size, the background colour was light purple, almost brownish, and the pustules stayed orange but less vivid. The pustules were bordered by a darker purple/brownish line. The rhinophores’ lamellae and the gill plumes turned the same shade of purple/brownish. The rhinophores were tipped in white and the rhinophores’ sheaths lost their pale colour, turning brown but bordered in orange. The tail, oral veil and foot line lost the vivid blue turning into a grey-light blue (Fig. 4D).

Specimen MB28-005021 (Fig. 4E-H)

When collected, the specimen measured about 30 mm (Fig. 4E) and was like the one described above after two months (Fig. 4C). Over the days, the specimen gradually lost brightness (Fig 4F). By the third month (approx. 50 mm). most pustules were pale and the oral tentacles and foot line pale bluish grey. The white tip of the rhinophores and the orange edge of the rhinophore sheath disappeared, and the gills and rhinophores turned dark brown (Fig. 4G-H). The specimen lost their vivid colour when the food supply was reduced, suggesting that colour is probably related to diet.

Specimens MB28-005003 and MB28-005004 (Fig. 5A-C)

These specimens, 22 mm and 28 mm in length respectively, were kept in the tank for 24 days. They were very similar to the ones described above with roughly the same size. However, both bore a few creamy marks under the orangish pustules, particularly on the side of the mantle. In one specimen the creamy pigmentation was also present between the rhinophores (Fig. 5A). Moreover, in these specimens, the colouration of the rhinophores and gill was much lighter. They mated in the tank (Fig. 5A) and laid eggs two days later (Fig. 5B). The eggs were photographed under an optical microscope 15 days later (Fig. 5C).

Several other specimens were collected and preserved for further studies (Table 1). These specimens were very similar to those observed in captivity (Fig. 5D-H). We noted that the orange edge on the rhinophore sheath, anus and kidney pore were variable, as was the presence and abundance of creamy spots (Fig. 5E-G). Some of these specimens were sequenced and dissected (Table 1) and, as predicted, they all belonged to the same species, with maximum uncorrected COI p-distance of 1% (Table 2). This maximum genetic distance is between the two specimens kept together in the aquarium (MB28-005003 and MB28-005004), which had mated and laid eggs (Fig. 5A-C), as well as between one of the previous specimens (MB28-005004) and the one from Bali (MNCN15.05/91071). The anatomy of some of them (see Table 1) was also studied, and no internal differences were found. Delimitation analysis of both ASAP and bPTP species also confirmed that these specimens belonged to the same species (Fig. 3).

Ecological notes. This species was relatively abundant in the reef in Zavora and most often associated with Eudistoma caeruleum. Interestingly, at 15 m (without flash) this species appeared dark brown with glowing red/orange pustules and a glowing rhinophore sheath (Fig. 5E). On the surface or under flash/strobe, the glowing parts were not as vivid. Red was the first depth-filtered colour, suggesting that this specimen fluoresced. On another occasion, a specimen of N. kubaryana was collected, observed and filmed in the laboratory in the dark with blue light, revealing red fluorescence on the rhinophores, gill sheath, oral tentacles and end of the gill branches and around the edge of the foot (Supplementary material). The specimen released a purple ink when it was disturbed.

Remarks and discussion. The naming history of N. yonowae is complicated and is clearly summarized by Pola et al. (2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 , who considered N. guttata Yonow, 1994Yonow N. 1994. Opisthobranchs from the Maldive Islands, including descriptions of seven new species (Mollusca: Gastropoda). Revue Française DÁquariologie Herpetologie, 20: 97-130. as a junior synonym of N. yonowae. This species, with type locality in the Maldives Islands, was described by Goether and Debelius (1992)Goethel H., Debelius H. 1992. Nacktschnecken der Maldiven, mit der Beschreibung einer neuen Art. DATZ (Die Aquarien- und Terrarienzeitschrift), 8: 512-519., Yonow (1994Yonow N. 1994. Opisthobranchs from the Maldive Islands, including descriptions of seven new species (Mollusca: Gastropoda). Revue Française DÁquariologie Herpetologie, 20: 97-130., 2012)Yonow N. 2012. Opisthobranchs from the western Indian Ocean, with descriptions of two new species and ten new records (Mollusca, Gastropoda). Zookeys, 197: 1-129. https://doi.org/10.3897/zookeys.197.1728 and Pola et al. (2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 for specimens of 40, 31, 35 and almost 60 mm long, respectively. In this size range, N. yonowae were described with black ground colour covered with strong orange-red pustules all over, 30% of them with a green border, black rhinophores with orange-red lamellae and rhinophoral sheaths bordered in orange. Gills black with green rachises. This exact pattern was always seen in adult specimens reaching at least 30 mm (Fig. 6A). However, other specimens resembling that colouration but not exactly, much more similar to some of the specimens described in this study, have been identified as Roboastra rubropapillosa in Ono (1999)Ono A. 1999. Opisthobranchs of Kerama Islands TBS-Britannica Co., Ltd. Tokyo, Japan, 184 pp., Nembrotha nigerrima Bergh, 1877 in Debelius and Kuiter (2007)Debelius H., Kuiter R. 2007. Nudibranchs of the world. IKAN-Unterwasserarchiv. Frankfurt, Germany., N. mullineri Gosliner and Behrens, 1997 in Gosliner et al. (2018)Gosliner T.M., Valdés Á., Behrens D.W. 2018. Nudibranchs and Sea Slug Identification Indo-Pacific. New World Publications, Inc., Jacksonville, Florida, 451 pp., or most commonly as undescribed species (Nembrotha sp. 1, Nembrotha sp. 2, Nembrotha sp. 3 in Gosliner et al. (2008)Gosliner T.M., Valdés Á., Behrens D.W. 2018. Nudibranchs and Sea Slug Identification Indo-Pacific. New World Publications, Inc., Jacksonville, Florida, 451 pp.; Nembrotha sp. 2, Nembrotha sp. 3, Nembrotha sp. 4 in Human and DeLoach (2010)Human P., Deloach N. 2010. Reef Creature Identification Tropical Pacific. New World Publication, inc. ISBN, 978-1878348.; Nembrotha sp. 2, Nembrotha sp. 4 in Nakano (2018)Nakano R. 2018. Field Guide to Sea Slugs and Nudibranchs of Japan. Bun-ichi Co. Ltd. Tokyo, Japan, 544 pp.; Nembrotha sp. 1, Nembrotha sp. 3, Nembrotha sp. 4, Nembrotha sp. 5, Nembrotha sp. 6 in Debelius and Kuiter (2007)Debelius H., Kuiter R. 2007. Nudibranchs of the world. IKAN-Unterwasserarchiv. Frankfurt, Germany.; Nembrotha sp. A in Tonozuka (2003)Tonozuka T. 2003. Opisthobranchs of Bali and Indonesia, Hankyu Communications Co., Ltd., 165 pp. from Bali; Nembrotha sp. cf. guttata in Nakano (2004)Nakano R. 2004. Opisthobranchs of Japan Islands. Rutles, Inc. 304 pp. from Japan or even Gymnodoris sp. 4 in Wells and Clayton (1993)Wells F., Clayton W.B. 1993. Sea slugs and their relatives of Western Australia. Western Australia Museum: 1-184 pp. from western Australia. Coleman (2008, p. 345)Coleman N. 2008. Nudibranchs enciclopedia, catalogue of Asia/Indo-Pacific sea slugs. Neville Coleman’s Underwater Geographic Pty Ltd. 416 pp., identified specimens similar to ours as Nembrotha guttata (synonymy of N. yonowae).

In this paper we confirm that Nembrotha yonowae changes colouration as it grows, and that colouration depends on the diet. As the individuals grow, the number of pustules and their brightness increase, until the food disappears and then their colouration fades away. The studied specimens were found and fed in Mozambique with E. caeruleum, a composed blue ascidian with a recorded distribution on the KwaZulu-Natal and Mozambique coasts, the west coast of Madagascar, and Mayotte in the Comoros islands (http://www.biodiversityexplorer.info/mm/tunicates/eudistoma_caeruleum.htm). Most likely, due to its proximity (the two farthest regions are between 25 and 38 degrees apart in latitude), it is also present in the Maldives, although no records or images are available to date.

Nembrotha cristata Bergh, 1877 is very similar to N. yonowae, but all its pustules and pigmentation around rhinophores, oral tentacles, gill and foot are consistently green. This species has been reported mostly from the Western Pacific, with fewer observations in the Indian Ocean (Tanzania, Mauritius, Maldives Islands) (Yonow 1994Yonow N. 1994. Opisthobranchs from the Maldive Islands, including descriptions of seven new species (Mollusca: Gastropoda). Revue Française DÁquariologie Herpetologie, 20: 97-130., 2012Yonow N. 2012. Opisthobranchs from the western Indian Ocean, with descriptions of two new species and ten new records (Mollusca, Gastropoda). Zookeys, 197: 1-129. https://doi.org/10.3897/zookeys.197.1728 , Pola et al. 2008Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 , Anderson 2022Anderson J. 2022. Maldives nudibranchs and Others Sea Slugs. A photographic identification guide. Available at http://www.nudibranch.org/iBooks/identification%20guides.html.). In most of its recorded distribution, N. cristata has been observed feeding on Sigillina signifera (Sluiter, 1909) (http://www.seaslugforum.net/showall/nembcris), a dark green colonial tunicate (Fig. 6B). However, it has also been observed feeding on other ascidians (Rudman 2007Rudman W.B. 2007. Comment on Nembrotha cristata feeding from Raja Ampat by Mike Krampf. [Message in] Sea Slug Forum. Australian Museum, Sydney. Available from http://www.seaslugforum.net/find/19177.), suggesting that it may feed on a variety of ascidians. Yonow (2012)Yonow N. 2012. Opisthobranchs from the western Indian Ocean, with descriptions of two new species and ten new records (Mollusca, Gastropoda). Zookeys, 197: 1-129. https://doi.org/10.3897/zookeys.197.1728 includes a photograph of N. yonowae together with N. cristata in the Maldives (Fig. 6C), and Debelius and Kuiter (2007, p. 58)Debelius H., Kuiter R. 2007. Nudibranchs of the world. IKAN-Unterwasserarchiv. Frankfurt, Germany. show a picture of two specimens identified as Nembrotha sp. 3 that resemble N. yonowae (identified as Nembrotha guttata) mating with N. cristata, but the latter had a few orange pustules in addition to the green ones (Fig. 6D). Finally, we show a small individual with intermediate colour pattern (Fig. 6E), with some green and some bright yellow-orangish pustules. This specimen was studied and sequenced here. Sequences of N. cristata specimens previously studied by Pola et al. (2006Pola M., Cervera J.L., Gosliner T.M. 2006. Taxonomic revision and phylogenetic analysis of the genus Tambja Burn, 1962 (Mollusca, Nudibranchia, Polyceridae). Zool. Scr. 35: 491-530. https://doi.org/10.1111/j.1463-6409.2006.00241.x , 2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 and Hallas et al. (2017)Hallas J.M., Chichvarkhin A., Gosliner T.M. 2017. Aligning evidence: concerns regarding multiple sequence alignments in estimating the phylogeny of the Nudibranchia suborder Doridina. R. Soc. Open Sci. 4: 171095. https://doi.org/10.1098/rsos.171095 were downloaded from GenBank and compared with our specimens of N. yonowae from Mozambique and Bali, as well as one N. yonowae sequence (EF142894.1) from the Philippines. The maximum uncorrected COI p-distances between N. yonowae and N. cristata was 1.3% (Table 2), and species delimitation analysis (Fig. 2) included all N. yonowae specimens as belonging to the same species. Based on all the observations and analysis presented in this study as well as the results obtained by Pola et al (2007Pola M., Cervera J.L., Gosliner T.M. 2007. Phylogenetic relationships of Nembrothinae (Mollusca: Doridacea: Polyceridae) inferred from morphology and mitochondrial DNA. Mol. Phylogenet. Evol. 43: 726-742. https://doi.org/10.1016/j.ympev.2007.02.003 , 2008)Pola M., Cervera J.L., Gosliner T.M. 2008. Revision of the Indo-Pacific genus Nembrotha (Nudibranchia: Dorididae: Polyceridae), with a description of two new species. Sci. Mar. 72: 145-183. https://doi.org/10.3989/scimar.2008.72n1145 , we finally synonymized N. yonowae as a junior synonym of N. cristata Bergh, 1877.

It is important to comment on the effect that the fact that these specimens are in an aquarium could have. Obviously, for one thing, this is the only way we can see how these changes are taking place. It could be thought that it is an effect of their maintenance and the conditions in which the aquarium is located, but because these intermediate colorations are frequently observed in nature, we are quite sure that the aquarium effect is minimal, and these changes simply reflect actual ontogenetic changes that depend on growth and diet.