Two new species of the genus Aldisa Bergh, 1878 (Gastropoda, Heterobranchia, Nudibranchia) from southern Mozambique Yara Tibiriçá, Marta Pola & Juan Lucas Cervera Marine Biodiversity ISSN 1867-1616 Mar Biodiv DOI 10.1007/s12526-017-0752-x 1 23 Your article is protected by copyright and all rights are held exclusively by Senckenberg Gesellschaft für Naturforschung and SpringerVerlag GmbH Germany. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy Mar Biodiv DOI 10.1007/s12526-017-0752-x ORIGINAL PAPER Two new species of the genus Aldisa Bergh, 1878 (Gastropoda, Heterobranchia, Nudibranchia) from southern Mozambique Yara Tibiriçá 1 & Marta Pola 2 & Juan Lucas Cervera 1,3 Received: 10 August 2016 / Revised: 7 June 2017 / Accepted: 7 June 2017 # Senckenberg Gesellschaft für Naturforschung and Springer-Verlag GmbH Germany 2017 Abstract The genus Aldisa Bergh, 1878 is relatively poorly studied. Up to now, no species have been described from the Western Indian Ocean. Two new species of the genus Aldisa are described from Zavora (Mozambique), Aldisa fragaria sp. nov. and Aldisa zavorensis sp. nov. Both species are characterized by having two oval depressions on the dorsum, a red mantle with yellowish-white patches and red rhinophores. Moreover, Aldisa fragaria sp. nov. has large round tubercles on the dorsum tipped in black and a large flattened oral glandular mass, while A. zavorensis sp. nov. has tan gills, rounded red tubercles, branchial and rhinophores sheaths distinctively serrated, and a large oral gland mass with a semi-spherical shape. Partial sequences of mitochondrial (COI and 16S) and nuclear (H3) markers of both species are provided. Both anatomical and molecular data confirm that these species are different from other known species of the genus. Communicated by V. Urgorri This article is registered in ZooBank under urn:lsid:zoobank.org:pub: 40C02E80-3198-41C4-9B08-91D43A59D9D4 * Yara Tibiriçá yaratibirica@gmail.com 1 Departamento de Biología, Facultad de Ciencias del Mar y Ambientales; Campus de Excelencia Global del Mar (CEI·MAR), Universidad de Cádiz, Av. República Saharaui s/n, 11510 Puerto Real, Cádiz, Spain 2 Departamento de Biología, Edificio de Biología, Campus de Excelencia Internacional UAM+CSIC, Universidad Autónoma de Madrid, C/ Darwin, 2, 28049 Madrid, Spain 3 Instituto Universitario de Investigación Marina (INMAR), Campus de Excelencia Internacional del Mar (CEI·MAR), Universidad de Cádiz, Avenida República Saharaui, s/n, Ap. 40, 11510 Puerto Real, Cádiz, Spain Keywords Aldisa . Cadlinidae . Nudibranchia . Heterobranchia . New species . Mozambique Introduction The taxonomic placement of the genus Aldisa Bergh, 1878 has a long and complex history. Bergh (1878) described Aldisa as a genus of the family Dorididae Rafinesque, 1815 characterized by a typical radula composed by extremely elongate teeth with a serrated margin and spatulate apex. Later, because of its aberrant radula teeth, the genus Aldisa was placed in its own family, Aldisidae (Odhner 1939). Since then, authors have included and excluded other genera in this family (Marcus and Marcus 1967; Marcus 1976; Franc 1968), but finally decided that the family Aldisidae was monotypic (see Millen and Gosliner 1985 for details). In 2002, Valdés provided a phylogenetic analysis of the cryptobranch dorids based on anatomical characters (Valdés 2002). According to his analysis, the genus Cadlina Bergh, 1878 was a sister clade of the genus Chromodoris Alder and Hancock, 1855, while the genus Aldisa was more closely related to other dorids such as the genera Doris Linnaeus, 1758, Pharodoris Valdés, 2001 and Aphelodoris Bergh, 1879. Contradictorily, recent phylogenetic analyses based on molecular data have supported the hypothesis of a sister relationship of Aldisa spp. and Cadlina spp. (Johnson 2011; Johnson and Gosliner 2012). As a result, the genus Cadlina was removed from the family Chromodorididae Bergh, 1891 and placed together with the genus Aldisa in the resurrected family Cadlinidae Bergh, 1891 (Johnson 2011). The genus Cadlina is characterized by having spicules and small tubercles on the mantle, large glands forming a submarginal row or multiple rows, simple pinnate gills trending to be secondary bi- or tripinnate, buccal armature often with bifid rodlets, oral tube and oral bulb of similar size, radular shape similar to the Author's personal copy Mar Biodiv genus Chromodoris but with rachidean teeth, most species having an armed penis and the seminal receptacle opening off the exogenous sperm duct instead of the vagina (Rudman 1984). The genus Aldisa typically has tubercles on the dorsum, often presents dorsal oval pits, mantle with spicules, multipinnate gills, smooth labial armature, atypically long and thin teeth with denticulation on the top and a lateral and armed penis (Valdés 2002). While the genera Cadlina and Aldisa share some similarities, such as the mantle covered with spicules and tubercles as well as armed penis in most of the species, the reproductive system and the radula are strikingly different. The anatomical details that make these two genera closely related are not yet understood. The new placement of the genus Aldisa within the family Cadlinidae was based on molecular analysis focused on the family Chromodorididae (Johnson 2011). Thus, that study had a clear bias in the number of Aldisa spp. versus Cadlina spp. The analysis included 11 species of the genus Cadlina (22 specimens), and only 2 species of the genus Aldisa (1 specimen of Aldisa banyulensis Pruvot-Fol, 1951 and 1 specimen of Aldisa sp.). Johnson (2011) mentioned that the genera Aldisa and Cadlina share some common characteristics such as tubercles on the mantle and a differentiated stomach; however, details of the anatomical features that supported this clade were not cited. Moreover, as shown by Johnson (2011), taxon-sampling might have a great influence in the phylogenetic results; therefore, adding more specimens of the genus Aldisa is important to confirm the taxonomical status of the family. One of the main difficulties with studying Aldisa spp. is that most of them are relatively rare or nocturnal (Millen and Gosliner 1985). Hence, several species remain undescribed (Debelius and Kuiter 2007; Coleman 2008; Gosliner et al. 2008, 2015). Gosliner et al. (2015) suggested that there are at least nine undescribed species in the Indo-Pacific. Moreover, the number of specimens of the genus Aldisa sequenced to date is very low (only four species have been included in GenBank). In this paper, we describe two new species of the genus Aldisa from southern Mozambique and provide partial sequences of mitochondrial (COI and 16S) and nuclear (H3) markers for both species, contributing to the increase in baseline knowledge necessary to understand the phylogenetic relationships and characteristics of the family. Material and methods Several specimens of two putative undescribed species of the genus Aldisa were examined. The first author collected all of them by snorkelling or SCUBA diving in the same area, a rock pool located in Zavora Bay, Inhambane Province, Mozambique (24°31’09″S, 35°12’27″E). Individual information on date of collection and size are given under the descriptions. After collection, individuals were photographed and relaxed in a solution of MgCl2 7% and preserved in 96% ethanol or 4% formalin. Anatomy Specimens were dissected under a stereomicroscope by dorsal incision. Special attention was giving to the buccal bulb and reproductive system. Drawings of the dissected specimens were made with the assistance of a camera lucida and improved in Photoshop CS5. Scanning electronic microscope (SEM) photographs were taken of radulae and penis. For the radulae, the buccal mass was immersed in a solution of 10% sodium hydroxide to dissolve soft tissues, washed in water and mounted for SEM. For the penis, the penial bulb was separated and the penis was critical point-dried before being mounted for SEM. Type specimens were deposited at the Museu Nacional de História e da Ciência de Lisboa (MB). Duplicates, when available, were deposited at the Museu de História Natural de Maputo (MHN, catalogue number not available), Mozambique. Molecular markers DNA extraction, amplification and sequencing Two specimens of Aldisa sp. (1) and one specimen of Aldisa sp. (2) were sequenced (Table 1). Partial sequences of three molecular markers were obtained: the mitochondrial cytochrome c oxidase subnit I (COI) and 16S rRNA and the nuclear Histone 3 (H3). All three markers were successfully amplified for all specimens except the 16S for one specimen (MB28–004392). DNA samples were extracted from a small piece of the foot with DNeasy Blood and Tissue Kit (Qiagen) using universal primers from Folmer et al. (1994) for COI, Palumbi (1991) for 16S and Colgan et al. (2000) for H3. PCRs were performed in 25-μl reactions with 2 μl of DNA template. COI amplifications were performed with an initial denaturation for 3 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 30 s at 46 °C and 1 min at 72 °C with a final extension of 5 min at 72C. 16S amplifications were performed with an initial denaturation for 3 min at 94–95 °C, followed by 40 cycles of 30 s at 94 °C, 30– 45 s at 48–51 °C (annealing temperature), 1–2 min at 72 °C, with a final extension of 5–7 min at 72 °C. H3 amplifications were performed with an initial denaturation for 3 min at 95 °C, followed by 25 cycles of 45 s at 94 °C, 45 s at 50 °C (annealing temperature) and 2 min at 72 °C, with a final extension of 10 min at 72 °C. Successful PCR products were purified and sequenced by Macrogen. Sequences were edited, aligned and concatenated using Geneious R6 (v.6.1.8) (http://www.geneious.com, Kearse et al. 2012). All sequences were checked for contamination using Blast in GenBank (Altschul et al. 1990). Alignments were generated by MUSCLE using default sets (Edgar 2004). For protein-coding, sequences alignment translations Author's personal copy Mar Biodiv Table 1 Specimens used in this study with respective voucher number and accession number Specimen Voucher no. 16S COI H3 Aldisa fragaria sp. nov.* Aldisa fragaria sp. nov.* MB28–004392 MB28–004393 – MF288006 MF288004 MF288003 MF327390 MF327389 Aldisa zavorensis sp. nov.* MB28–004397 MF288007 MF288005 MF327391 Aldisa banyulensis Aldisa albotrossae – – – KP871679.1 AY345039.1 KP871632.1 – KP871655.1 Aldisa smaragdina – KJ022806.1 KF992175.1 KJ022914.1 Aldisa sp. Aldisa zetlandica CASIZ175733 – EU982818.1 – – KU695603.1 – – Ardeadoris egretta CASIZ157481 EU982762.1 EU982713.1 – Cadlina sparsa Cadlina rumina CASIZ182932 CASIZ175456 EU982776.1 EU982775.1 EU982726.1 EU982725.1 – – Chromodoris magnifica Discodoris cebuensis CASIZ144119 CASIZ185141 JQ727731.1 KP871687.1 JQ727852.1 KP871639.1 – KP871663.1 Hypselodoris zephyra CASIZ175555 JQ727797.1 JQ727905.1 – Berthella sideralis – AJ225181.1 AJ223257.1 – *Specimens sequenced for this study, all others sequences were obtained from GenBank into amido acids were carried out in Genious using the translation tool (Genetic Code: Invertebrate Mitochondrial) to confirm accuracy. We tested the 16S alignment with all gap regions and excluded most of them. In the final analyses, we used the one excluding the gaps because, despite both alignments showing similar results for the BL analyses, the ML tree was poorly resolved when the gaps were included. Saturation testing was carried out by plotting the absolute number of transitions and transversions at each condon position against p distance in an Excel plot graph. In order to compare the genetic distances between species, the pairwise uncorrected p distance for COI was calculated using MEGA (v.6.06-mac). Finally, all sequences were deposited in Genbank. Accession codes and museum vouchers are shown in Table 1. Phylogenetic analyses A phylogenetic tree was carried out using Bayesian inference and Maximum likelihood (ML) to verify the relationship between the new species and others Aldisa spp. available in GenBank. We tested all the marks separately and combined. The best-fit evolutionary model was chosen in JModeltest (v.2.1.7) using AIC selection (Akaike 1974). The evolutionary model obtained for the concatenated analysis (COI+16S) was TVM+I+G. Bayesian Inference (BI) analysis was accessed using the software MrBayes v.3.2.6 and 6 substitutions (nst = 6) (Ronquist and Huelsenbeck 2003). The analysis ran for 5,000,000 generations MCMC; the first and last 1250 generations were discarded. The node support for the ML analysis was assessed with non-parametrica bootsrap (BS) with 5000 replicates, random starting trees and parameters estimated according to the model selected in J Modeltest. Mr. Bayes and ML tree were visualized, collapsed (PP ≥ 0.90, BS ≥ 75) and edited in TreeGraph (v.2.7.1; Müller and Müller 2004). Final editions were carried out in Photoshop CS5. Results SYSTEMATIC DESCRIPTIONS Class GASTROPODA Cuvier, 1797 Subclass HETEROBRANCHIA Burmeister, 1837 Order Nudibranchia Cuvier, 1817 Suborder Euctenidiacea Tardy, 1970 Infraorder Doridacea Thiele, 1931 Superfamily Doridoidea Rafinesque, 1815 Family Cadlinidae Bergh, 1891 Genus Aldisa Bergh, 1878 Aldisa fragaria sp. nov. (Figs. 1a, b, c,d, e, f, 2a, b, c, 3a, b, c, d) Diagnosis Mantle red with whitish-yellow patches. Rhinophores and gills red. Large round red tubercles on dorsum. Two oval depressions (pits) on dorsum ringed by tubercles. Numerous elongate teeth, which are very thin, serrated on sides and folded on tips. Armed penis. Differential diagnosis Tubercles tipped in black. Large flattened oral glandular mass. Derivatio nominis. The specific name refers to the external similarity with strawberries (Fragaria spp.). Author's personal copy Mar Biodiv Material examined Description Holotype, depth 1 m, 37 mm in length alive (MB28–004392), 22 Apr. 2015, preserved in ethanol 96%, GenBank (pending number), dissected and sequenced, penis and radula mounted for SEM; Paratype 1, depth 0.3 m, 34 mm in length alive (MB28–004393), 05 Jan. 2014, preserved in alcohol 96%, GenBank (pending number), dissected and sequenced, penis and radula mounted for SEM; Paratype 2, depth 1 m, 25 mm in length alive (MB28–004394), 05 Nov. 2010 preserved in 4% formalin, dissected; Paratype 3, depth 1 m, 24 mm in length alive (MB28–004395), 26 May 2013, preserved in alcohol 96%, dissected; Paratype 4, depth 1 m, 33 mm in length alive (MB28–004396), 26 May 2014, preserved in 96% alcohol, dissected; Paratype 5, one exemplar deposited in the Natural History Museum of Maputo (no voucher number available); depth 2 m, 10 Dec. 2011, 14 mm in length preserved (immature), preserved in 4% formalin, not dissected.. Type locality: Zavora Bay, Inhambane Province, Mozambique (24°31’09″S, 35°12’27″E). This species has been registered in ZooBank under the name Aldisa fragaria sp. nov. urn:lsid:zoobank.org:act: B9D75718-1DF0-4903-A849-D9795C0C4B4F External anatomy (Fig. 1a–c). The body shape is oval, rigid, with two depressions on the dorsum. Each depression is ringed by tubercles. The tubercles are rounded, larger on the dorsum but smaller closer to the margin of the individuals (Fig. 1a, b). Under a dissecting microscope (×250), minute spicules can be seen covering the mantle. The oral tentacles are short and oval, near the mouth (Fig. 1c). The perfoliate rhinophores bear from 13 to 19 lamellae with slightly elevated sheaths. There are five bipinnate and retractile branchial leaves located dorsally in the posterior region of the dorsum. The anus is mid-dorsal, located in the centre of the branchial tuft. The foot is narrower than the mantle. Coloration. The mantle is red with creamy-yellow patches on the sides. The tubercles are red with the top dark brown to black. A thin creamy-yellow ring often surrounds the tubercles. The rhinophores sheaths are red with creamy edges. The rhinophores are completely red. The branchial leaves are red. The underside of the mantle and the foot are also red (Fig. 1a– c). Internal organs coloration. One specimen (MB28– 004392) was dissected on the same day of collection Fig. 1 Aldisa fragraria sp. nov.: a living animal in the field (MB28–004396, Paratype 4); b dorsal view (MB28–004393, Paratype 1); c ventral view (MB28–004393, Paratype 1); d dorsal view showing internal coloration (MB28–004392, Holotype); d buccal mass showing internal coloration (MB28–004392, Holotype); e egg mass (MB28–004394, Paratype 2). bg blood gland; dg + hg digestive gland complex + hermaphrodite gland; es oesophagus; in intestine; ob oral bulb; og oral gland mass; ot oral tube; r radula; rep sys reproductive system; sg salivary gland; ve ventricle Author's personal copy Mar Biodiv allowing us to observe the internal coloration (Fig. 1d, e). The blood gland is salmon in colour (Fig. 1d). The oral tube is salmon. The oral bulb, oral glandular mass and radular sac have a creamy coloration. The salivary glands are light cream (Fig. 1e). The female gland is dark salmon, the prostate yellowish, the deferent duct and penis are white (not shown). The vagina and receptacle seminal are creamy and the bursa copulatrix salmon with grey (not shown). The digestive gland, the ventricle and intestine are yellowish. General internal anatomy (Figs. 1d, e, 2). A large blood gland is located in front of the central nervous ring (Fig. 2a). The eyespots are connected to the cerebral ring by a short optic nerve, visible when the blood gland is removed. There is a distinctive large flattened oral glandular mass connected to the distal part of the oral tube. Such glandular mass covers most of the ventral surface of the oral bulb. The radular sac is round. There are two salivary glands connected at each side of the oesophagus. They are triangular at the base and thin at the end (Figs. 1e, 2b). The stomach is slightly dilated, but not clearly differentiated (Fig. 2a). The muscular oral tube is larger and wider than the oral bulb (Figs 1e, 2b). Radula (Fig. 3a–d). The radula is typical of Aldisa spp. with extremely narrow teeth. As in other species of the genus (e.g. A. binotata Pruvot-Fol, 1953; A. smaragdina Ortea, Perez and Llera, 1982; A. pikokai Bertsch and S. Johnson, 1982; Aldisa andersoni Gosliner and Behrens, Fig. 2 Aldisa fragraria sp. nov.: general internal anatomy of the Holotype (MB28–004392). a, dorsal view; b, buccal mass; c, reproductive system. am ampulla; at aortic trunk; bc bursa copulatrix; bg blood gland; dd deferent duct; dg + hg digestive gland complex + hermaphrodite gland; es oesophagus; fg female gland; in intestine; mo mouth; ob oral bulb; og oral gland mass; ot oral tube; p penis; pr prostate; r radular sac; rep sys reproductive system; rv renal vesicle; sg salivary gland; st stomach; rs receptaculum seminis; ud uterine duct; v vagina; ve ventricle 2004), the radular formula was impossible to determine, as the teeth are too numerous, thin and overlapping (Fig. 3a–c). In the 24-mm specimen (MB28–004395), the teeth measure around 420 μm in length. Each tooth bears from 10 to 15 long sharp lateral serrations (Fig. 3c). The teeth are triangular in the base and the apex is curved and has from 4 to 10 folded long denticles (Fig. 3d). Reproductive system (Fig. 2c). The reproductive system is triaulic. The hermaphrodite duct leads to a bent ampulla. The ampulla is located ventrally, close to the prostate. The ampulla narrows in a short postampullatory duct, which forks into the prostate and oviduct. The oviduct enters into the female gland. The prostate has a U-shape with two different parts; the portion of the prostate connected to the postampullatory duct is narrower and darker than the second part of the prostate. The prostate narrows into the muscular part of the deferent duct. The distal part of the deferent duct leads to a small penial bulb, which opens in a common atrium with the vagina. The vagina is thin and long, forking into three: a very short uterine duct, a short duct that leads to the bursa copulatrix and a duct that ends in the receptaculum seminis. The bursa copulatrix is oval and bigger than the bean-shaped receptaculum seminis. The penis is armed with 11 rows of at least 18–24 spines each (Fig. 4). Each spine has a triangular shape; the base is wide, narrowing off to a thin tip (Fig. 4a, b). Some of the basal spines are fused on the base and bifurcate in two hooks (Fig. 4d). Author's personal copy Mar Biodiv Fig. 3 Aldisa fragaria sp. nov., Zavora, Mozambique (MB28– 004395, Paratype 3) SEMs of radula: a outer side showing overlapping of the teeth; b radular teeth; c teeth showing lateral serrations; d detail of the apex of the teeth Natural history Differential diagnosis This uncommon species was found in a large rock pool from 0.3 to 2 m deep. The egg mass colour is red (Fig. 1f). Uniformly tan branchial leaves. Large oral gland mass with semi-spherical shape. Derivatio nominis. The specific name Bzavorensis^ refers to the tidal rock pool of Zavora, the location where the holotype was found. The rock pool is a natural tidal pool approximately 110 m in diameter where more than 80 species of nudibranchs have been found (personal observation). Unfortunately, the high biodiversity of this area is currently under threat due to the illegal use of gill nets inside the pool. We named this species after the rock pool with the hope that it will bring more attention to this area and to the importance of conserving this fragile, easily accessible and rich environment. This species has been registered in ZooBank under the name Aldisa zavorensis sp. nov. urn:lsid:zoobank.org:act: 2B442691-7796-4879-8E65-FEC3F70C5403 Distribution Mozambique (Gosliner et al. 2015, as Aldisa sp. 3; present study). Aldisa zavorensis sp. nov. Material examined Holotype, depth 0.5 m, 16 Apr. 2014, 19 mm in length alive (MB28–004397), preserved in ethanol 96%, GenBank (pending number), dissected and sequenced, penis mounted on slide and radula mounted for SEM; paratype: depth 1 m, 08 Feb 2012, 22 mm in length alive (MB28–004398), preserved in 4% formalin, dissected and radula mounted for SEM. Type locality: Zavora Bay, Inhambane Province, Mozambique (24°31’09″S, 35°12’27″E). Diagnosis Red mantle and rhinophores. Yellowish specks. Tan gills. Rounded tubercles. Smaller tubercles close to the margin of the specimens. Two pits on dorsum. Branchial and rhinophore sheaths with deeply serrated borders. Elongate teeth. Description External anatomy (Fig. 5a–e). The body shape is elongated oval, the profile is low and it is rigid to the ouch, with two depressions on the dorsum ringed by small tubercles. The dorsum is covered by round tubercles. The tubercles are small closer to the edge of the mantle, larger on the sides and slightly smaller on the top of the dorsum (Fig. 5a). The oral tentacles are very short and rounded. The perfoliate rhinophores bear from 15 to 18 lamellae with slightly elevated sheaths. The Author's personal copy Mar Biodiv Fig. 4 Aldisa fragaria sp. nov. SEM pictures of the penial spines: a general view of A. fragaria sp. nov. (MB28–004393, Paratype 1) penial spines; b general view of A. fragaria sp. nov. (MB28–004392, Holotype) penial spines; c top view of the penial spines; d detail of a basal bifurcated spine (MB28–004392, Holotype) rhinophores and branchial sheaths have a deeply scalloped edge, forming small triangles (Fig. 5c–e). There are five bipinnate and retractile branchial leaves located in the posterior portion of the dorsum, posterior to the second pit. The anus is mid-dorsal located in the centre of the branchial circle in the mantle. The foot is narrower than the mantle (Fig. 5b). Coloration. The mantle is red with yellowish speckles on the dorsum, which concentrate in a few areas forming creamy- Fig. 5 Aldisa zavorensis sp. nov. (MB28–004398): dorsal (a) and ventral (b) view of the Paratype; c dorsal view showing details of the rhinophores and gill sheaths; d gill leaves and gill sheath details; e rhinophore and rhinophore sheath details Author's personal copy Mar Biodiv Fig. 6 Aldisa zavorensis sp. nov. internal anatomy of the Holotype (MB28–004397): a buccal mass; b reproductive system; c photography of the penial bulb; d detail of the armed penis. am ampulla; bc bursa copulatrix; dd deferent duct; es oesophagus; fg female gland; hd hemaphrodite duct; mo mouth; ob oral bulb; og oral gland mass; ot oral tube; p penis; pr prostate; r radular sac; sg salivary gland; st stomach; rs receptaculum seminis; ud uterine duct; v vagina yellow patches. The edge of the mantle is orange-red. The tubercles are red. The rhinophores sheaths are red with creamy patches. The rhinophores are red. The red pits on the dorsum have orange edges (Fig. 5a). The branchial leaves are uniformly tan. The underside of the mantle is red with orange-red edges. The foot is orange-red (Fig. 5b). General internal anatomy (Fig. 6a–d). The blood gland covers the nerve ring but not the eyes, which are connected by optical nerves. The oral tube is muscular and half-globular, with a large oral gland mass connected directly at its posterior side. The oral gland mass is quite thick and lies alongside the flat side of the oral bulb. The oral bulb has a semi-hemispheric shape, flat on the dorsum side and oval on the ventral part with a small round radular sac. There are two flat salivary glands connected at each side of the oesophagus (Fig. 6a). The stomach forms a small caecum clearly visible on the surface of the digestive gland. Radula (Fig. 7a–d). The radular formula was impossible to determine. Around 62 rows could be seen through the dorsal view of the radula during dissection. The teeth are extremely long and overlapping (Fig. 7a, b). In the 19-mm individual, the teeth measured approximately 1.2 mm in length (Fig. 7b). The teeth are triangular at the base and very thin, tending to fold throughout their length (Fig. 7b, c). Therefore, it was impossible to observe lateral serrations in the two radulae examined. The distal end is wider and folds at the tip. This end is curved and has from 4 to 6 folded long denticles (Fig. 7d). Reproductive system (Fig. 6b). The reproductive system is triaulic. The hermaphrodite duct leads to a thick, bent ampulla. The ampulla narrows into a very short postampullatory duct, which forks into the prostate and the oviduct. The oviduct enters into the female gland. The curved thick prostate narrows abruptly into the muscular part of the deferent duct. The distal part of the deferent duct leads to the penial bulb that opens in a common atrium with the vagina. The short vagina leads to the bursa copulatrix. The receptaculum seminis is connected to the bursa copulatrix by a very short duct. The uterine duct, very short, leads the bursa copulatrix between the receptaculum seminis and the vagina to the female gland. The bursa copulatrix and the receptaculum seminis are both oval and of similar size, the receptaculum seminis is slightly more curved than the bursa copulatrix (Fig. 6b). The penis is armed with diminutive hooks. Unfortunately, the penis was inverted and because of the fragility of the diminutive hooks, we decided to not risk losing the material by trying to carry out the technique of the critical point for the SEM. Instead, we took photographs under an optical microscope (×1000). The penis contains at least six rows of spines with 15–17 spines each (Fig. 6c, d). Natural history This species is very rare and has only been seen twice during 5 years of sampling in Mozambique, both times in a larger rock pool around 0.5 m depth. Distribution Mozambique (present study). Author's personal copy Mar Biodiv Fig. 7 Aldisa zavorensis sp. nov., Zavora, Mozambique (MB28– 004397, Holotype), SEMs of radula: a general view of the radula; b one isolated tooth; c triangular base of the teeth; d detail of the apex of the teeth Molecular results All codons were considered but no signal of saturation was observed for any of the marks (not shown). The tree topology with best resolution was obtained with the combined 16S and COI alignment (1055 pb) (Fig. 8). In general, the BI topology was similar to the ML topology; however, ML showed lower resolution. The monophyly of the genus Aldisa was not retrieved using only COI (not shown), but it was obtained using 16S (not shown), as well as the combined 16S and COI dataset (Fig. 8). The concatenate tree using the three marks did not show good resolution probably due the low number of H3 sequences (not shown). The phylogenetic analyses confirms that the clade comprised by Cadlina spp. is sister to the clade constituted by Aldisa spp. Figure 8 shows that A. zavorensis sp. nov. is Fig. 8 Molecular phylogeny of available sequenced Aldisa spp. based on the combined dataset (COI + 16S) inferred by Bayesian analysis. Support values shown represent posterior probabilities from Bayesian interference and maximum likelihood (PP/ML). GB GenBank closely related to an undescribed species of the genus Aldisa from GenBank (PP = 1, BL = 100) and these two sp ecie s are sister to A. fr aga ria sp . no v. fr om Mozambique ( PP = 1, BL = 96). M oreover, A. zetlandica (Alder and Hancock, 1854) and A. albatrossae Elwood, Valdés and Gosliner, 2000 from GenBank are closely related (PP = 0.96; BL = 79), and A. smaragdina Ortea, Pérez and Llera, 1982 and A. banyulensis from GenBank (PP = 1; BL = 100) seem to be the same species. The p-uncorrected distance for COI between species of the genus Aldisa reached values from 10.05% (A. smaragdina/ A. banyulensis vs. A. albatrossae) up to 16.58% (A. fragaria sp. nov. vs. A. zetlandica), except in the case of A. smaragdina vs. A. banyulensis (1.01%). The COI genetic divergence between A. fragaria sp. nov. and A. zavorensis sp. nov. is 15.08% (Table 2). Author's personal copy Mar Biodiv Table 2 Minimum pairwise uncorrected p-distances for COI between Aldisa spp. 1 1. A. smaragdina Ortea, Perez and Llera, 1982 2. A. banyulensis Pruvot-Fol, 1951 1.01% 2 3 - 4 - 5 - - 3. A. albatrossae Elwood, Valdés and Gosliner, 2000 10.05% 10.05% - - - 4. A. zetlandica (Alder and Hancock, 1854) 5. A. fragaria sp. nov. 12.06% 12.31% 12.06% 11.81% 11.06% 14.32% 16.58% - 6. A. zavorensis sp. nov. 13.57% 14.07% 14.57% 16.08% 15.08% Discussion So far, 18 species of the genus Aldisa have been described, but many are still waiting to be described and discovered (Debelius and Kuiter 2007; Coleman 2008; Yonow 2008; Gosliner et al. 2008, 2015). The Western Indian Ocean (WIO) is recognized as a hotspot for marine biodiversity with the second highest diversity of corals in the world. However, the diversity of marine invertebrates of the WIO is far less studied than in the Pacific Ocean (Obura 2012; Wilson and Kirkendale 2016). The two nudibranch species regarded here are the two first species of the genus Aldisa to be described with material collected from the Western Indian Ocean. Anatomy Several colours are found in the genus Aldisa, but two are dominant: whitish-blue and orange-red. Over eight species of red Aldisa spp. have been recorded for the Indo-Pacific (e.g. Gosliner et al. 2015), but only Aldisa pikokai has been described. Table 3 compares the different species of red Aldisa with those described here. A. pikokai was described from specimens from Hawaii Islands and clearly differs from all others described Aldisa spp. by having elevated reticulate ridges instead of the typical tubercles or papillae (Bertsch and Johnson 1982). Aldisa fragaria sp. nov. and A. zavorensis sp. nov. have rounded tubercles and two depressions on the dorsum, while A. pikokai has three craters and ridges, which are slightly elevated on the junctions (Bertsch and Johnson 1982). Another species that resembles the species studied here is Aldisa sanguinea (Cooper 1873) described from specimens from San Diego Bay (eastern Pacific). This species has oval dark spots, which are similar to a depression (MacFarland 1905; Millen and Gosliner 1985). Millen and Gosliner (1985) reviewed A. sanguinea and state that the preserved animals sometimes had flattened tubercles. Both species described here are geographically far apart and have their tubercles prominent alive or preserved, and therefore could not be A. sanguinea. The remaining red species with oval depressions on the dorsum are from the Eastern Atlantic and Mediterranean: Aldisa binotata, Aldisa smaragdina and Aldisa banyulensis. A. banyulensis was described based on external anatomy of a single specimen from Banyuls-sur-Mer (France, Mediterranean) (Pruvot-Fol 1951). Millen and Gosliner (1985) refer to this species as having uniform notum without depression; however, both the illustration from Pruvot-Fol (1951; pl. 2, fig. 20) and the review by García et al. (1986) show a species with two depressions on the dorsum. This species differ externally from the one described here by having unipinnate gills and smaller tubercles. A. banyulensis and A. binotata are very similar species. They are accepted as two separate species in the World Register of Marine Species (WoRMS) and by some authors (Calado and Urgorri 1999; Ávila et al. 2000), but as synonyms by others (Millen and Gosliner 1985; Cervera et al. 2006). Further anatomical and molecular studies are needed to clarify this issue. Independently, A. fragaria sp. nov. differs from all the above Aldisa spp. by the large black-tipped tubercles and from A. zavorensis sp. nov. by the tan colour of the gills. A striking internal characteristic of A. fragaria sp. nov. and A. zavorensis sp. nov. is the unusual massive granular oral gland mass connected to the posterior end of the oral tube. This gland has not been mentioned for any of the other red Aldisa spp., except for A. pikokai. In both Mozambican species, the gland is located ventrally and extends beyond the oral bulb covering part of the radular sac. In A. fragaria sp. nov., the granular mass is ventrally flatted, while in A. zavorensis sp. nov., it is much thicker with a semi-hemispherical shape. This gland seems to be characteristic of many species of the genus (Gosliner and Behrens 2004). However, not much attention has been given to it, particularly in earlier descriptions. Millen and Gosliner (1985) briefly mention a glandular projection in the oral tube of A. albomarginata Millen in Millen and Gosliner, 1985, A. tara Millen in Millen and Gosliner, 1985 and A. pikokai, but no drawing or picture was provided. Later, Gosliner and Behrens (2004) described A. andersoni Gosliner and Behrens, 2004, highlighting and illustrating a large gland behind the oral tube. They then reviewed A. williamsi Elwood, Valdés and Gosliner, 2000 and A. albatrossae and found the same kind of gland but much smaller (Gosliner and Behrens 2004). The gland illustrated by Gosliner and Behrens (2004) for A. andersoni is considerably smaller than that observed in A. fragaria sp. nov. and A. zavorensis sp. nov. This aberrant gland is not mentioned in Species Distribution Aldisa Pacific side of sanguinea North (J. G. America Cooper, 1863) Aldisa binotata Morocco, Pruvot-Fol, North 1953 Atlantic Ocean Buccal Oral Mass Rhinophores Radula Stomach Penis hooks with caecum Gills Ecology Not mentioned Red-orange, 12–15 lamellae 70 ×100.0.70–100 Yes 5–6 regular rows with aprox. 14 spines per row 8–10 orange-red Intertidal, peach-orange spawn Scattered Not rounded; two mendarker red tioned spots in slight depression 8–12 lamellae yellow, 4–5 70 × 60.060 No deeply scalloped lobes 30–35 denticles along around the margin of the top, tip is not the rhinophores sheath folded 7–8 regular rows of recurved hooks 5 μm long No pit; Not around gills mengranulated, tioned the rest of mantle fine granulated Ridges instead Yes of tubercles; 3 depressions 8–10 lamellae 60 × 40.0.40 large radular sac 7–13 μm; 6 regular row 8–9 Intertidal. tripinnate, Red spawn red-orange (5 to 9 in Millen and Gosliner 1985) 5–8 Subtidal unipinnat9–24 m e, red-orange Red-orange Teeth from 60 to 260 μm Tubercles of Not different mensizes, 2 darker tioned round areas (pits) Conical with no Not pits mentioned Red, 12–13 lamellae Tubercles Red-orange with densely dotted From conical to black spots (original desc.) In flattened Millen & Gosliner (1985), 2 mid-dorsal black spots Red-orange with a pair of creamy diagonal stripes Eastern Red-orange with light Atlantic and brown and white dots Mediterrean Aldisa pikokai Hawaii Bertsch & S. Johnson, 1982 Red Aldisa Mediterranean smaragdina to Atlantic Ortea, Pérez Ocean & Llera, (Canary 1982 Islands) Aldisa expleta Mediterranean to Atlantic Ortea, Pérez Ocean & Llera, (Canary 1982 Islands) Aldisa Atlantic side of benguelae South Gosliner in Africa Millen & Gosliner, 1985 Aldisa fragaria So far only sp. nov. known from Red with tan dots and few white patches Red with several small white patches Red-orange Red often with whitish-creamy specks Rounded, no pit Not mentioned Yes Red, 16 lamellae Yes Present 10 μm; 6 irregular Cream-white Subtidal, inside row Bright red, the upright digescoil tive gland 50 rows impossible to Yes 12 rows; 17 5 tripinnate Red spawn, estimate; 30 lateral curved hooks orange intertidal serration and per row with 4–5 at the tip distributed white tip 350 μm irregular 60 × 80.0.80: teeth Stomach 8 rows of curved 5 tripinnate ? 190-250 μm no hooks with 15 red with dilated per row white tip 9–10 lamellae, 4–11 tubercles around the margin of the rhinophores sheath 35 × 75.0.75; teeth Yes from 80 to 750 μm; 28–40 serration on folded tip and side 12 rows of curved 6 tripinnate, spines, 12 μm red long and 8 μm wide at the base Subtidal 13–19 lamellae Indeterminate 11 rows of 18–24 hooks Intertidal 5 tripinnate, red Author's personal copy Aldisa banyulensis Pruvot-Fol, 1951 Coloration Mar Biodiv Table 3 Comparative table of red-colored Aldisa spp. Aldisa sanguinea: Cooper (1863), Millen and Gosliner (1985); Aldisa binotata: Pruvot-Fol (1953), Millen and Gosliner (1985); Aldisa banyulensis: Pruvot-Fol (1951), Garcia et al. (1986); Aldisa pikokai: Bertsrh and Johnson (1982); Aldisa smaragdina and A. expleta: Ortea, Pérez and Llera (1982); Aldisa benguelae: Millen and Gosliner (1985) Author's personal copy Indeterminate Stomach slightly dilated Yes Diminutive hooks, 5 bipinnate, tan min. 6 rows with 14–17 hooks each Round, red with black top and two pits Round, red, two Yes pits Tubercles Buccal Oral Mass Rhinophores 15–18 lamellae Radula Stomach Penis hooks with caecum Gills Ecology Intertidal Mar Biodiv early descriptions for many other species, such as A. sanguinea (MacFarland 1905, 1906), A. cooperi Robilliard and Baba, 1972 (Robilliard and Baba 1972), A. expleta Ortea, Pérez and Llera, 1982 (Ortea et al. 1982) and A. puntallanensis Moro and Ortea, 2011 (Moro and Ortea 2011). It is unlikely that a large gland such as the one found in A. fragaria sp. nov. and A. zavorensis sp. nov. went unnoticed by other authors, but as they can be quite small (Gosliner and Behrens 2004), it might need further review to confirm its absence in other species of the genus. Phylogenetic analyses To date, the number of specimens sequenced of the genus Aldisa is relatively low. The use of small taxon sampling can lead to a misinterpretation of the results, particularly with regards to complex questions (Tholesson, 2000; Johnson 2011). Therefore, the analyses performed here only give preliminary information about the relationship of the two new species and previously sequenced Aldisa spp., and in order to confirm the specific identity of both new species. Our study also reveals that the sequences of A. smaragdina and A. banyulensis deposited in Genbank appear to be from the same species. It is true that A. smaragdina and A. banyulensis share several common features (Ortea et al. 1982; Millen and Gosliner 1985); however, the type locality of A. banyulensis is the Banyuls sur mer (France, Mediterraneo), thus, without additional material from the Mediterranean and anatomical examination, which is beyond the scope of this study, it is not possible to synonymize them, but only to call attention to this issue. Our results agree with Johnson (2011) supporting the relationship between the genera Cadlina and Aldisa. Genetically, the closest species to A. zavorensis sp. nov. appears to be an undescribed species from Malaysia (PP = 1, BS = 100). So far only Red with whitish specks known from Mozambique Aldisa zavorensis sp. nov. Mozambique Distribution Species Table 3 (continued) Coloration Distribution Specimens from other part of the world could not be examined. Thus, based only on external appearance, it is possible that both A. fragaria sp. nov. and A. zavorensis sp. nov. have an Indo-Pacific distribution. For A. fragaria sp. nov., photograph records of similar specimens exist for Reunion Island (http://seaslugs.free.fr/nudibranche/a_aldisa_sp2.htm), Australia (Marshall 1999; Debelius and Kuiter 2007; Coleman 2008; Gosliner et al. 2008) and the Philippines (Coleman 2008; Gosliner et al. 2008, 2015). Diagnostic features of the external anatomy such as the whitish-yellow patch, two dorsal depressions ringed by tubercles and large round red tubercles tipped in black can be seen in these photographs. Controversially, Gosliner et al. (2015) considered a specimen from the Pacific (Aldisa sp. 4, pg. 169, bottom left photo) as a different species from A. fragaria sp. nov. Author's personal copy Mar Biodiv (specimen examined by us and illustrated on p. 168, bottom right photo as Aldisa sp. 3), but no clear external difference could be seen between them, except that, in the Mozambican specimen illustrated in Gosliner (2015), the lighter ring surrounding the tubercles (which is often present in A. fragaria sp. nov.) are not so visible as in the Pacific specimen. For A. zavorensis sp. nov., photograph records of specimens of similar coloration exist for Reunion Island (http:// seaslugs.free.fr/nudibranche/a_aldisa_sp4.htm), Guam (http://www.nudipixel.net/photo/00022022/), Taiwan (http:// www.nudipixel.net/photo/00007497), and Malaysia (Gosliner et al. 2008). Despite the colour similarity, key diagnostic features such as the deeply serrated borders of the branchial and rhinophores sheaths could not be seen in any of the photographs. Nevertheless, our molecular study shows that the specimen from Malaysia, illustrated in Gosliner et al. 2008 (p. 160, second bottom), appears to be the closest species to A. zavorensis sp. nov. Unfortunately, the COI sequence of this material is not available to verify the genetic divergence between these two specimens (Johnson 2011). Therefore, with such limited information, it is not possible to confirm whether or not the Malaysian specimen is A. zavorensis sp. nov. Despite the external similarities between the photograph records with the species examined here, the broader distribution of A. fragaria sp. nov. and A. zavorensis sp. nov. can only be confirmed after molecular and/or anatomical examination of additional material from other regions. Conclusion Our phylogenetic and anatomical studies both indicate that Aldisa fragaria sp. nov. and Aldisa zavorensis sp. nov. are two new different species. They differ from all other described species both externally and internally. Externally, A. fragaria sp. nov. has distinctive round tubercles tipped in black, while A. zavorensis sp. nov. has distinctive rhinophores and gill sheaths, as well as tan gill branches. Internally, the most striking feature is the massive granular oral gland mass, which no other red Aldisa spp. with tubercles has, at least of such size. Moreover, the minimum genetic distance of the species described here, compared with all other sequenced Aldisa spp., was as high as 11.81%, which clearly distinguishes them from the other sequenced species of the genus. Nevertheless, some anatomical details such as the lateral border of the teeth of A. zavorensis sp. nov. could not be analysed. 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