Zoosyst. Evol. 99 (1) 2023, 63-75 | DOI! 10.3897/zse.99.95222 > PENSUFT. gee BERLIN Can you find me? A new sponge-like nudibranch from the genus Jorunna Bergh, 1876 (Mollusca, Gastropoda, Discodorididae) Yara Tibirica!*, Jenny Strémvoll®, Juan Lucas Cervera! 1 Departamento de Biologia, Facultad de Ciencias del Mar y Ambientales, Campus de Excelencia Internacional del Mar (CEI-MAhk), Universidad de Cadiz, Av. Republica Saharaui, s/n, 11510 Puerto Real (Cadiz), Spain 2 Instituto Universitario de Investigacién Marina (INMAR), Campus de Excelencia Internacional del Mar (CEI-MAR), Universidad de Cadiz, Ay. Republica Saharaui, s/n, 11510 Puerto Real (Cadiz), Spain 3 Back to Basics Adventures, Ponta do Ouro, Mozambique https://zoobank. org/7 DO 1LFEE-8B7D-48D5-968A-129CB317B9B2 Corresponding author: Yara Tibiriga (yara.tibirica@uca.es) Academic editor: Matthias Glaubrecht # Received 22 September 2022 # Accepted 9 December 2022 @ Published 13 January 2023 Abstract The nudibranch diversity of the western Indian Ocean is comparatively one of the least studied in the world. In this paper a sponge- like Discodoridae nudibranch Jorunna liviae sp. nov. is described. The description is based on integrative anatomy, including molecular analysis of two genes (the mitochondrial COI and the nuclear H3), dissections, electron microscopy (SEM) of buccal elements, micro tomography of the spicule’s arrangements and ecological observations. This study provides the first ever molecular data of Jorunna species from the western Indian Ocean, helping to fill the gap to further understand this apparent paraphyletic genus. Key Words biodiversity, Heterobranchia, Mozambique, new species, phylogeny, sea slugs Introduction The systematic of the genus Jorunna was revised by Camacho-Garcia and Gosliner (2008) based on morphological characters. These authors examined 246 specimens (including 30 type specimens) and described two new species. The genus Jorunna Bergh, 1876 is widely distributed with species found in the Indo- Pacific, Mediterranean, Atlantic and Eastern Pacific. Alvim and Pimenta (2013) revised the anatomy of the family Discodorididae Bergh, 1891 from Brazil and added a new species for the genus (Jorunna spongiosa Alvim & Pimenta, 2013). Recently, Neuhaus et al. (2021) provided a molecular and morphological review of the European species and described a new species (Jorunna artsdatabankia Neuhaus, Rauch, Bakken, Picton, Pola & Malaquias, 2021). Currently, 22 Jorunna species are accepted as valid (MolluscaBase eds. 2022). Nevertheless, there are still many undescribed species, particularly in the Indo-Pacific. In the field-guide of sea slugs of the Indo-Pacific, Gosliner, Valdés and Behrens (2015) illustrated 16 species of Jorunna from which only six are described: Jorunna funebris (Kelaart, 1859), Jorunna labialis (Eliot, 1908), Jorunna ramicola Miller, 1996, Jorunna rubescens (Bergh, 1876), Jorunna parva (Baba, 1938) and Jorunna alisonae Marcus, 1976. Nevertheless, the authors did not include two Australian species: Jorunna hartleyi (Burn, 1958) and Jorunna pantherina (Angas, 1864). Despite current research efforts to raise the biodiver- sity knowledge of nudibranchs from the western Indian Ocean (e.g. Manson-Parker 2015; Tibiri¢a et al. 2017a, b, 2018, 2020), this region remains comparatively far less studied than other areas of the Indo-West Pacific. The high number of undescribed species often hampers comprehensive biogeographic studies. Thus, the discov- ery of new species is of primary importance to advance our global knowledge on how biodiversity 1s formed and Copyright Tibirica, Y. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 64 how species diversity spreads across oceans. Moreover, the lack of molecular data from the western Indian Ocean (WIO) limits phylogenetic studies. Of all specimens se- quenced from the genus Jorunna so far, none are from the WIO. The present study contributes to fill this gap by providing a description of a new Jorunna species from Mozambique, including molecular, morphological and ecological data. Methods Sample collection Six specimens were collected by scuba diving in Ponta do Ouro (26°51'26"S, 32°53'4"E), Mozambique by J. Strém- voll & Y. Tibirica. All specimens were found on sponge Amphimedon brevispiculifera (Dendy, 1905), four on the reef ‘Doodles’ (26°49'50"S, 32°53'46"E) and two on the ‘Steps Reef’ (26°49'29"S, 32°53'46"E), between 15—18m depth. Sponge identification was based on a porifera as- sessment study conducted in the same area (Calcinai et al. 2020). Specimens were photographed in situ and in a tank and individually measured. The animals were then relaxed by freezing and preserved in ethanol 96%. Sam- ples were deposited in the Museu Nacional de Ciencias Naturales de Madrid (MNCN) and Museu de Historia Natural de Maputo (MHNM). Morphological study Specimens were dissected by dorsal incision under a dissecting microscope Nikon SMZ18. Their reproduc- tive system was separated, examined and drawn under a dissecting microscope Leica 80 with an attached camera lucida. Surrounding radula tissue was removed by im- mersing in 10% sodium hydroxide for about 8 hours or on a solution containing 180 mL of the tissue lysis buffer ATL with 20 mL of proteinase K-solution incubated in 56 °C for 48h (Holznagel 1998). Labial cuticle and rad- ula were then mounted for electron microscopy (SEM) examination. Imagines were obtained under a FEI Nano- SEM 450 scanning microscope at the Servicios Centrales de la Ciencia y Tecnlogia de la UCA (MEB), Universidad de Cadiz. Microcomputed tomography (uCT) was carried out to inspect the spicules arrangement by the Servicio de Técnicas No Destructivas del Museo Nacional de Cien- cias Naturales de Madrid (MNCN-CSIC). This technique uses x-ray attenuation of biological tissues in three dif- ferent planes allowing for 2D and 3D image reconstruc- tions (Ziegler et al. 2018). Images were reconstructed using VGSTUDIOMAX 2.2 and visualized in myVGL by Volume Graphics (https://www.volumegraphics.com). Spicules sizes were measured in the uCT images using the distance instrument tool. Measurements were taken from spicules that were clearly visible and from different parts of the body. zse.pensoft.net Tibirica, Y. et al.: A new sponge-like nudibranch from the genus Jorunna DNA extraction, amplification and sequence DNA extraction and amplification were conducted by the pe- ripheric services of the Instituto Universitario de Investiga- cion Marina (INMAR—UCA). Genomic DNA was extracted from a small sample of foot tissue using the Qiagen DNeasy Blood & Tissue extraction kit, following the manufacturer’s instructions. One mitochondrial gene cytochrome c oxidase subunit (COI) and one nuclear gene histone H3 (h3) were amplified by polymerase chain reaction (PCR), using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994) and H3AD-F and H3BD-R (Colgan et al. 2003), re- spectively. We tried to amplify the gene 16S using the 16S universal primers 16Sar-L and 16Sbr-H (Palumbi et al. 2002) but all attempts were unsuccessful. PCRs were performed in 25-ul reactions with 2 ul of DNA template. COI amplifica- tions 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 | min at 72 °C with a final extension of 5 min at 72 °C. H3 amplifications were performed with an initial denatur- ation 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. Once completed, successful PCR products were sent to Macrogen, Inc. (Ma- drid, ES) for purification and sequencing. All sequences were revised and examined in Gene- ious v.10.2.4 (Kearse et al. 2012). Possible contamina- tion was verified using the Basic Local Alignment Search tool (BLAST) web server (https://blast.ncbi.nlm.nih.gov/ Blast.cgi, Altschul et al. 1990). New sequences were up- loaded in Genbank, NCBI and ascension numbers are provided in Appendix 1. Outgroup sequences and other Jorunna spp. sequences were obtained from GenBank. The outgroup selection followed Neuhaus et al. (2021). Additionally, one species of each available genus of Dis- codorididae Bergh, 1891 from GenBank was included in the analysis with preference given for type species. When available, up to three sequences of each morpho-species of Jorunna from GenBank, NCBI were included in the phylogeny. Preference was given to specimens with COI and H3. Sequences were aligned in Geneious (https:// www.geneious.com) using Muscle and default settings. Phylogenetic analysis Maximum likelihood (ML) and Bayesian inference (BI) were used to infer evolutionary relationships. Analyses were conducted for individual genes as well as for the concate- nated COI+16S. JModeltest was used to estimate the best fit-evolutionary model by applying the Akaike information criterion (AIC) for each gene. The model chosen was the GTR+I+G for COI and H3. Bayesian inference was per- formed via MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2003) and run for 5,000,000 generations and four chains, with unlinked parameters, partitioned by genes and a burn- in of 25%. Node support was assessed based on the posterior probability (PP) and considered strongly supported when PP Zoosyst. Evol. 99 (1) 2023, 63-75 > 0.95 (Alfaro et al. 2003). Maximum likelihood analyses were performed in RAXML v8.2.4 implemented in the Cy- pres portal, applying 5,000 bootstrap (https://www.phylo. org, Miller et al. 2010). Support for nodes in the ML analysis was assessed with non-parametric bootstrapping (BP) using RAXML v.7.06 (Zhang et al. 2013). Maximum likelihood values of 70 or higher were considered statistically signif- icant (Huelsenbeck and Rannala 2004). The trees obtained were visualized and collapsed (PP = 0.5) in TreeGraph2 (http://treegraph. bioinfweb.info, Stover and Muller 2010) and edited in Adobe Illustration 2021 v.25.2 (https:/Awww. adobe.com/products/illustrator.html). Species delimitation Three molecular species delimitation analyses were conduct- ed to aid the species hypothesis. Firstly, Species by Automat- ic Partitioning (ASAP) was performed on the in-group COI dataset applying the Kimura two Parameter (K2P) and the default setting parameters (Puillandre et al. 2021). Secondly, the Poisson Tree Processes model (bPTP) was implemented in the bPTP web server (https://species.h-its.org) applying default settings in the COI and concatenated tree resulted from the BI phylogeny (Zhang et al. 2013); and, third, the minimum COI p-distance was calculated applying default settings on Mega X version 10.2.4 (Kumar et al. 2018). Results Systematics Order Nudibranchia Cuvier, 1817 Superfamily Doridoidea Rafinesque, 1815 Family Discodorididae Bergh, 1891 Genus Jorunna Bergh, 1876 Jorunna liviae Tibirica, Strémvoll & Cervera, sp. nov. https://zoobank.org/5B6809CC-32EE-456F-8302-4B8A3D4E05 13 Jorunna sp.: Stromvoll, J. & Jones, G. (2019): pg.49. Material examined. Holotype: MNCN15.05/200187 (dissected and sequenced), 12.04.2022, Doodles, Ponta do Ouro, Mozambique, depth 15 m, length 20 mm. Paratypes: MNCN15.05/200188 (dissected and se- quenced), 12.04.2022, Doodles, Ponta do Ouro, Mozam- bique, depth 17 m, length 11 mm. MNCN15.05/94693 (sequenced and tomography), 12.04.2022, Doodles, Pon- ta do Ouro, Mozambique, depth 15 m, length 20 mm., size 5 mm. MNCN15.05/200189 (dissected and sequenced), 14.04.2022, Doodles, Ponta do Ouro, Mozambique, depth 18 m, length 13 mm. MHNM.MOL.2022.0002, (2 specs.), 23.06.2022, Steps Reef, Ponta do Ouro, Mozam- bique, depth 16 m, length 30 mm (both). Type locality. Ponta do Ouro, (26°51'26"S, 32°53'4"E). Mozambique 65 Habitat. Specimens were collected on submerged subtropical compressed sandstone reefs in Ponta do Ouro, Mozambique. Diagnosis. Body elongate-ovulated. Dorsum pale gray to pink, covered on highly dense caryphyllidia; rhino- phores short, with up to nine lamellae, ending in a knob apex; six to nine bipinnate branchial leaves encircling the anal pore. Radula with five to seven very thin pectinated outermost teeth bearing long bundled fibrous denticles. Labial cuticle smooth. Copulatory spine with bifid apex. Etymology. This species is dedicated to Livia Renée Cornelius, daughter of the second author of this paper. Description. External morphology (Figs 1, 2). Length varied from 11 to 30mm. Body elongate-ovulated, with gritty texture (Fig. 1A). Mantle covered on highly dense caryophyllid, evenly distributed on the dorsum (Fig. 2A). Caryophyllidia elongated, formed by five to eight spic- ules, projecting over tip, forming a crown of approximately 140 um on the dorsum, taller on the margin of gill sheath (= 280 um). Rhinophoral and branchial sheaths low, mar- gin covered by caryophyllidia (Fig. 1D). Rhinophores short, retractable, with six to eight diagonal lamellae with a knob protruding apex (Fig. 1E). Gill with six to nine retractile, bipinnate branchial leaves, held vertically and forming a closed circle around the anal pore (Fig. 1F). Foot narrower than mantle, bilabiate anteriorly, upper lip bifurcate at center (Fig. 1B). Side of the foot covered by spicules (~ 60 um), spicules absent on foot sole (Figs 1B, C, 2B, C). Feet do not project beyond mantle in natural crawling position. Oral tentacles small and conical. Dorsum color pale pink to gray. Some specimens covered by pinkish-brown minute dots forming spots distributed on the notum. Gill and rhinophores translucent pinkish-white. Oral tentacle white. Upper lip translucent white with brownish dots. Foot pinkish-white. Internal morphology. (Figs 3, 4) The visceral mass is enveloped by a translucent-white tissue covered by brownish dots. Eye spots are visible by transparency. Digestive system. Smooth labial cuticle (Fig. 3A). Oral tube long, about twice the size of oral bulb, with a pair of retractor muscles (Fig. 4A). Buccal bulb ovate, short, radular sac small and ovate, protruding ventrally, with a pair of strong retractor muscles (Fig. 4A). Radular formula difficult to determinate as outermost teeth are very thin and overlapping each other (Fig. 3B). Approxi- mate radular formula 1s: 24 x 5—7,22.0.22.5—7 for the 13 mm specimen MNCN15.05/200189 and 38 x 6—7.26.0.26.6—-7 for the 20 mm specimen MNCN15.05/200187. Rachidian tooth absent. Innermost and lateral teeth are single cusped, hamate, lacking denticles (Fig. 3C). Lateral teeth gradually increase in size from the inner teeth (~ 25 um) toward the external margin (outermost teeth ~ 100 um). Five to seven outermost teeth highly differentiated, very thin, pectinate, bearing 5 to 9 long bundled fibrous denticles (Fig. 3D, E). Oesophagus passing through nerve ring, where it folds. Pair of salivary glands, relatively short, uniform, near the base of oesophagus (4A). Oesophagus con- nects to oval stomach. Intestine about half of oesoph- agus diameter. Caecum locate ventrally to stomach. zse.pensoft.net 66 Tibirica, Y. et al.: A new sponge-like nudibranch from the genus Jorunna Figure 1. Jorunna liviae sp. nov. (MNCN15.05/200187) external morphology. A. Dorsal view; B. Ventral view; C. SEM photogra- phy of dorsal caryophyllids; D. Rhinophores sheath details; E. Rhinophore; F. Gill branches. zse.pensoft.net Zoosyst. Evol. 99 (1) 2023, 63-75 Figure 2. Microcomputed tomography (uCT) of /. /iviae sp. nov. (MNCN15.05/94693). A. Exterior view: dorsal (top) and anterior (bottom); B. Internal arrangement of the spicules: dorsal (top) and ventral (bottom); C. General internal arrangement of spicules: green = top right; blue = middle nght; red = bottom right. zse.pensoft.net Tibirica, Y. et al.: A new sponge-like nudibranch from the genus Jorunna Figure 3. SEM photographs of Jorunna livae sp. nov. A. labial cuticle (MNCN15.05/200188); B. entire view of the radula; C. Half- row of posterior part of the radula; D. Outermost teeth; E. Detail of the outermost teeth; F. Copulatory spine. zse.pensoft.net Zoosyst. Evol. 99 (1) 2023, 63-75 Digestive gland cone-shaped, occupying approximate- ly 30% of visceral mass. Anus opening at the center of gill circle. Central nervous system. Central nervous system par- tially covered by blood gland. This is divided into two parts, anterior part about half the size of posterior part. Cerebral ganglia about half the size of pleural ganglia. Cerebral ganglia and pleural ganglia fused. Pedal ganglia ventrally located connected by a simple pedal commis- sure. Buccal ganglia short, ventrally located. Rhinophoral ganglia bulb-shaped, about 30% the size of cerebral gan- glia. Eyes connected to cerebral gland by short rhinopho- ral nerve (Fig. 4B). C 1mm 69 Reproductive system. Hermaphroditic duct leading to an ampulla long and convoluted, located between female gland and accessory gland. Ampulla branching into short oviduct and prostate. Flattened and ovulated prostate nar- rowing into a thin deferent duct, expanding into ejacu- latory portion. Penis unarmed. Accessory gland size and shape varied according to the specimen, from pear-shaped and similar size to the female gland MNCN15.05/200187 to elongated and half of the size MNCN15.05/200189; in all specimens it narrows into a very thin, highly convolut- ed tube. Copulatory spine in accessory gland of approx- imately 1.25 mm (Fig. 3F). Vagina with similar length and width than deferent duct, leading to an oval bursa Ig cg 500um Figure 4. Jorunna livae sp. nov. internal anatomy. A. Oral mass; mo — mouth; rm — retractor muscles; ob — oral bulb; oe — oesopha- gus; ot — oral tube; rs — radular sac; sg — salivary gland; B. Central system (blood gland removed); eg — cerebral ganglia; cp — pedal commissure; ey — eye; gp — pedal ganglia; pl — pleural ganglia; rg — rhinophoral ganglia; C. Reproductive system; ag — accessory gland; amp — ampulla; be — bursa copulatrix; es — copulatory spine; dd — deferent duct; pr — prostate; sr — seminal receptacle; ud — uterine duct; vag — vagina. zse.pensoft.net 70 copulatrix. Thin duct near the vagina leads to oval sem- inal receptacle, about 2/3 of the size of the bursa copu- latrix, which connects to a large female gland by a short uterine duct (Fig. 4C). Natural history. This species has only been seen as- sociated with the sponge A. brevispiculifera, on which the species is very cryptic (Fig. 5A, B). They are usually found at the base of the sponge branches but they have also been seen on other parts. When removed from the host sponge, the Jorunna liviae sp. nov. stretches the body curling the mantle toward the middle of the foot, similar to what Miller (1996) observed for J. ramicola. Perhaps this behavior aims to protect the sole of the foot which lacks caryophylliid. The white egg mass 1s also found on the same sponge and forms a close spiral ribbon of ap- proximately five coils (Fig. 5F). A likely undescribed spe- cies of nudibranch egg-eater Favorinus sp. has been seen feeding on the J. /iviae sp. nov. egg mass (Fig. 5C, D). Curiously, most of the time the egg ribbons are found on the tip of the sponge. Perhaps this strategy provides some protection against encrusting organisms due the higher water flux in this part of the sponge. Mating has been ob- served through July between specimens of different sizes and tonalities (Fig. SE). Jorunna liviae sp. nov. seems to prefer sandy reefs with predominantly hydroids, soft cor- al and sponges. In Southern Mozambique, the flatter sand reefs have a higher density of sponges than the reefs with predominantly hard coral. Molecular study and phylogeny. We successfully amplified the gene COI and H3 of four Jorunna liviae sp. nov. specimens. The phylogenetic trees constructed Table 1. COI inter- and intraspecific uncorrected p-distances. Jorunna artsdatabankia Jorunna artsdatabankia Jorunna tomentosa LA 10.30 Jorunna tomentosa LB 9.08 3.65 Jorunna liviae sp. nov. 14.29 14.74 Jorunna onubensis 12.61 10.74 Jorunna funebris 16.92 17.78 Discussion The phylogenetic relationships within the family Discodoridae are poorly solved. Most of the type species of Discodoridae genera are not sequenced which hinders our capacity to further understand the family. Jorunna 1s one of the few genera of the Discodoridae family which has its type species (J. tomentosa) sequenced. However, a recent study based on three genes (COI+16S+H3) reveals that it is uncertain if J. tomentosa represents two distinct lineages (Neuhaus et al. 2021). In our phylogenetic analysis, J. tomentosa is divided into two sub-clades (lineage A and B), which form a clade which is sister of J. artsdatabankia and related to J. onubensis and J. liviae sp. nov. Additionally, the genus Jorunna appears paraphyletic as J. funebris did not nest within the large Jorunna clade. In Camacho-Garcia and Gosliner’s (2008) zse.pensoft.net Jorunna tomentosa Jorunna tomentosa Tibirica, Y. et al.: A new sponge-like nudibranch from the genus Jorunna by BI and ML analyses of single gene datasets (Sup- pl. material 1) were not conflictive but differed in the ability to resolve phylogenetic relationships. The sin- gle gene H3 analysis retrieved the lowest resolution and the concatenate dataset the highest. Nevertheless, all Jorunna species were recovered with more than 50% support in all analysis. In general, the BI analysis better solved the relationship between species, while the ML analysis appears to reflect populational struc- ture. Therefore, the results discussed below are based on the concatenated analysis (Fig. 6), except when stated otherwise. The family Discodorididae formed a large polyto- my. The genus Jorunna was divided in two paraphylet- ic clades, one containing all specimens of J. funebris (PP = 1; BS = 94) and another clade with the remaining Jorunna species (PP = 0.99; BS = 74). The COI inter-specific variation (uncorrected p-dis- tance) within the genus varied from 9.08% between J. tomentosa \ineage B (LB) and J. artsdatabankia to up 16.92% between J. funebris and J. tomentosa \in- eage A (LA) (Table 1). The COI intra-specific varia- tion of Jorunna liviae sp. nov. ranged from 0.16% to 1.08%. The closest species to Jorunna liviae sp. nov. was J. tomentosa lineage B with a minimum p-distance of 13.06%. ASAP retrieved 10 partitions, in both anal- ysis (COI and concatenate) the partitions with high- er score (asap-score 1.50-—3) Jorunna liviae sp. nov. was retrieved as a distinct taxonomic unit. Curiously, J. funebris were retrieved as a species complex in all possible partitions. Jorunna liviae Jorunna onubensis Intra-specific sp. nov. 0-0.15% 0.15-0.68% 0.15-0.92% 13.06 0.16-1.08% 10.45 12.31 N/A 17.78 16.92 16.92 0.46-14.18% morphological study, J. funebris nested on a clade together with J. rubescens, J. parva and J. pardus, which is sister of the clade containing the remaining Jorunna species studied by the authors. Unfortunately, to date no other species from the J. funebris clade has been sequenced. Consequently, the lack of molecular data from several Jorunna species hampers any further conclusion about the phylogeny of the genus. Nevertheless, it is clear that the species here described belongs to the genus Jorunna, as it forms a clade with the type species. In addition, the new species fits all the morphological diagnosis characters of the genus (see Camacho-Garcia and Gosliner 2008). Interesting, all the species delimitation analyses suggest that J. funebris is a species complex, or alternatively, as proposed by Ip et al. (2019), there is an identification error in their sequences. Jorunna liviae sp. nov. is similar in appearance to the Atlantic species J. spongiosa and J. tomentosa. This Zoosyst. Evol. 99 (1) 2023, 63-75 71 Figure 5. Jorunna liviae sp. nov. in situ. A. Hosting sponge Amphimedon brevispiculifera (Dendy, 1905); B. Jorunna liviae sp. nov. resting on sponge; C. Jorunna liviae sp. nov. near its egg mass, and Favorinus sp. feeding on it; D. Close-up of Favorinus sp.; E. Jorunna liviae sp. nov. mating; F. Details of Jorunna liviae sp. nov. egg mass. zse.pensoft.net 72 Tibirica, Y. et al.: A new sponge-like nudibranch from the genus Jorunna Glossodoris hikuerensis Felimida binza Chromodoris celinae Halgerda nuarrensis Atagema notacristata Diaulula nayarita Taringa telopia Tayuva lilacina 1 Thordisa albomacula 68 0.98 Asteronotus cespitosus 63 Hoplodoris desmoparypha 0.99 Carminodoris flammea Geitodoris heathi 0.97 Platydoris argo 0.72 79 Sclerodoris tuberculata Paradoris liturata Peltodoris atromaculata Rostanga elandsia PTP bPTP ASAP col a 1 Jorunna funebris CPIC00633 94 1 100 'Jorunna funebris [P0302 } Jorunna sp MNCN15.05/200187 99 Jorunna sp MNCN15.05/94693 0.9/86 Jorunna sp MNCN15.05/200189 0.99 1/89' Jorunna sp MNCN15.05/200188 a Jorunna onubensis ZMBN125474 F Eo i Jorunna artsdatabankia NTINUVM58891 | | 34 1/99 0.0 0.1 Jorunna funebris [P0011 2.94 | Jorunna artsdatabankia ZMBN125946 Jorunna artsdatabankia ZMBN127749 Jorunna tomentosa LA ZMBN125512 Jorunna tomentosa LA ZMBN127710 1 | !Jorunna tomentosa LA ZMBN127711 mia Jorunna tomentosa LB ZMBN127709 73 |}Jorunna tomentosa LB NINUVM588&88& Jorunna tomentosa LB ZMBN132446 Figure 6. Bayesian inference tree based on the concatenate sequence dataset (COI+H3) collapsed (PP< 0.5). Numbers at the top of nodes indicate Bayesian Posterior probability (PP) and on the bottom bootstrap support from the maximum likelihood analysis (BS). Colored bars on the right represent the results of the species delimitation analyses on the Jorunna spp., from left to right: ASAP on COI dataset, PTP on COI dataset, bPTP on COI+H3 dataset. latter is typically found in European waters (Atlantic and Mediterranean), but few records exist from South Africa and none of them from the Indian Ocean side (Camacho- Garcia and Gosliner 2008; Neuhaus et al. 2021). Apart from the geography and genetic distance, these three species can be clearly distinguished by their radulae, in particular by the shape of the outermost teeth. These are very thin and pectinate in J. Jiviae sp. nov., hooked with small branches on J. spongiosa and slender hamate with up to 8 short denticles in J. tomentosa. In fact, the outermost pectinate teeth of Jorunna liviae sp. nov. are quite unique, and only similar to J. parva, a species also found in the WIO but easily distinguishable by the yellow background and dark caryophyllidia. Camacho- Garcia and Gosliner (2008) provided detailed anatomical descriptions and comparative tables of Jorunna species by region. To better illustrate the differences between the zse.pensoft.net species described in this study, we adapted and updated Camacho-Garcia and Gosliner’s (2008) comparative table of the Indo-Pacific Jorunna species, including recent distribution and morphological data observed by us, as well as the species J. /iviae sp. nov. and J. labialia (Table 2). This latter species is found in the western Indian Ocean and Red Sea but was under ‘Mediterranean and Western Atlantic’ species in Camacho-Garcia and Gosliner’s (2008) comparative tables. The Indo-Pacific species that most resembles J. /iviae sp. nov. 1s J. ramicola; a species first described from New Zealand and likely occurring in Mozambique (Tibiri¢a et al. 2017a). Jorunna ramicola is dull in color and bears long, slender outermost teeth which may be described as pectinate. However, these teeth bear much shorter and less numerous denticles, which do not form the distinct bunch as it does in J. Jiviae sp. nov. Moreover, the innermost Zoosyst. Evol. 99 (1) 2023, 63-75 fie Table 2. Comparative morphology of valid Jorunna species from the Indo-Pacific Ocean. J. funebris J. pantherina | J. rubescens J. parva J. hartleyi J. alisonae J. ramicola J. labialis J. liviae (Kelaart, 1859) | (Angas, 1864) | (Bergh, 1876) | (Baba, 1938) | (Burn, 1958) | Marcus, 1976 | Miller, 1996 sp. nov. Geographic | Indo-Pacific inc. | Australia and | Indo-Pacific inc. | Indo-Pacific inc. Southern Western and} Indo-Pacific inc. Red Sea & Mozambique range western Indian New Zealand western Indian | western Indian Australia Central Pacific | western Indian | western Indian Ocean Ocean Ocean Ocean Ocean Dorsal color | White to yellow- | Purplish brown | Cream-grey to Dark orange Pale pink, large | Pale grey, dark Pale grey to |White to dark dull) Pinkish grey, cream, dark to pale brown | yellow with oval | to dark brown, | brown patches | grey spots, line light brown grey with light | darker spots brown rings of with dark yellow spots, caryophyllidia encircled with of dark spots | with patches of brown spots sometimes different sizes patches rings, horizontal dark brown white or dark |fromrhinophores| — similar color present black stripes purple spots to gill Rhinophores | 14-20 lamellae 16 lamellae, 23-25 lamellae | 13-15 lamellae | 7-8 lamellae = 10 lamellae short, up to wide, 8-11 short, 6-8 terminal knob 13 lamellae, lamellae lamellae, terminal knob terminal knob Rhinophore | White with black | Base colorless, | Base black or | Base light yellow Cream-brown Pale-grey, Same than —_| White to pinkish color apexes uppermost cream-yellow, | with dark clubs, with pale grey- | speckled with mantle lamellae club black- or dark brown brown club dark brown, speckled in spotted with rachises with white on the club white white or cream- | light yellow clubs colored spots Branchial 6 tripinnate up to 11, bi 6-7, tripinnate 7, bipinnate 10, bipinnate 12, tripinnate 10, bipinnate 11, bipinnate | 6-9, bipinnate leaves tripinnate Gill color White with Same than Base white, dark} — Light yellow, Grey to brown Dark grey White to pinkish dark black mantle brown rachises base dark with cream delineations brown, tips glandular spots light-yellow, dark brown rachises Foot sole Dark spots Margins sparsely | Black stripes on Dark spots Pale gray Whitish-pink color around the speckled, sole | sole and laterals, | around margin, margin, sole | translucent white} sole white or brown line translucent white cream dorsally, translucent yellow sole Upper lip White to yellow Cream yellow, Yellowish, Cream in White color cream speckled with | sometimes with preserved black two brown spots animals on each side of the lip Oral tentacles) White to cream, Speckled Light white to Yellowish Pale gray White-pinkish color with brown yellow spots in some specimens Caryophyllidia =~ 220um = 250um = 133um = 220um = 140um size Mantle White, White, large, Absent White, Absent Glands distributed distributed distributed around the around the around the mantle edge mantle edge mantle edge Foot Dorsally visible | Dorsally visible | Dorsally visible | Dorsally visible Dorsally visible | No prolongation Rarely visible when the animal | when the animal | when the animal | when the animal when the animal present dorsally is in motion is in motion is in motion is in motion is in motion Radula 21 x (21.0.21) | 20 x (28.0.28) | 26 x (18.0.18) | 20 x (15.0.15) | 21 x (23.0.23) | 16 x (17.0.17) | 14 x (18.0.18) | 19 x (17.0.17) 38 x (6- in 20mnHong no more in 18mm in 6mm-long in 18mnHong in 20mnyong in 5bmmong in 10mm 7.26.0.26.6-7) preserved information specimen preserved preserved preserved preserved preserved in 20mm specimen specimen specimen specimen specimen specimen specimen Innermost Hamate, shorter | Hamate, small, | Hamate, blunt, Hamate, Hamate, lacking | Hamate, pointed, Hamate, hamate, single | Hamate, lacking teeth and thinner than | lacking denticles | lacking denticles elongated, denticles shorter than the | elongate, with cusp Close to denticles midlateral teeth, lacking denticles midlateral teeth, | up to 3 denticles base lacking denticles lacking denticles | near the cusp Midlateral Hamate, lacking | Hamate, lacking | Elongated, blunt, |} Hamate, lacking | Hamate, lacking | Hamate, pointed, | Hamate, pointed, | Hamate, lacking | Hamate, lacking teeth denticles denticles lacking denticles denticles denticles lacking denticles | lacking denticles denticles denticles Outermost Hamate, lacking | Hamate, lacking | Shorther than Smaller than Elongated, Small, elongated, first | shorter, hamate, | pectinate, 5-8 teeth denticles denticles midlateral teeth, | midlateral teeth. | lacking denticles elongated, three outermost pointed long fibrous curved pointed, | The 5 outermost smooth or teeth with up to denticles lacking denticles | have up to 5 sometimes with 3 denticles denticles a single denticle near the cusp Labial Smooth With jaw Smooth Smooth With jaw With jaw With jaw With jaw Smooth cuticule elements elements elements elements elements Accessory | Present, curved | Present, long | Present, curved | Present, spine ~ | Present, spine ~ | Present, curved | Present, curved | Present, curved Present, gland and spine ~ 717um | pointed spine | spine = 3.7mm 477um long 198um long | spine = 1.03mm | spine ~ 2.25mm | spine ~ 600um | curved spine = spine long long long long long 1.25mm long teeth in J. ramicola are denticulated, while in J. /iviae sp. nov. they are simple hamate. In addition, the labial cuticle in J. ramicola bears jaw elements (Camacho-Garcia and Gosliner 2008), while in J. /iviae sp. nov. it is smooth. Externally, they can be easily separated by the color of the rhinophores, which in J. ramicola is dark pigmented and in J. /iviae sp. nov. whitish pink. Additional differ- ences are provided in the comparative Table 2. zse.pensoft.net 74 Conclusions Based on morphological and genetic data there is no doubt that J. /iviae sp. nov. 1s a newly discovered species. Here we provide the first sequence of Jorunna species to the WIO. We recommend further efforts to sequence other Jorunna species in order to clarify the monophyly of the genus and phylogenetic relationships. In addition, J. funebris specimens from different geographic regions should be morphologically and genetically examined as they may represent a species complex. Acknowledgements We are grateful to Miguel Gongales (Park Warden) for assisting with the collection permit. We thank Daniela de Abreu and Alvaro A. Vetina for helping with the de- positing of specimens at Museu de Historia Natural de Maputo (MHNM). We are in great debt to Rupert Cor- nelius from Back to Basics Adventures for always going out of his way to support research in Ponta do Ouro. We also would like to express our gratitude to Elena Ortega Jimenez and Enrique Gonzales Ortegon (CSIC-ICMAN) for kindly allowing us to use the Nikon SMZ18 for the dissection. Finally, we thank the following technicians for their valuable work: Juan Gonzales (Servicios Cen- trales de la Ciencia y Tecnologia de la UCA—EB), Cristi- na Paradela Guerrero (Servicio de Técnicas No Destruc- tivas, MNCN-CSIC) and Francisco Javier Domingues Marchan (Servicios Periféricos de Biologia Molecular, INMAR-UCA). Molecular, SEM and micro-tomography were supported by funds received by the research group “Marine Biology and Fisheries” PAIDI RNM-213. References Alfaro ME, Zoller S, Lutzoni F (2003) Bayes or bootstrap? 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Northern France: La Rochelle Norway: Fraya Northern Ireland: Ballyhenry Island Spain: Pontevedra, Galicia Spain: Huelva Mozambique: Ponta do Ouro Mozambique: Ponta do Ouro Mozambique: Ponta do Ouro Mozambique: Ponta do Ouro Species Jorunna artsdatabankia Jorunna artsdatabankia Jorunna artsdatabankia Jorunna funebris Jorunna funebris Jorunna funebris Jorunna tomentosa lineage A Jorunna tomentosa lineage A Jorunna tomentosa lineage A Jorunna tomentosa lineage B Jorunna tomentosa lineage B Jorunna tomentosa lineage B Jorunna onubensis Jorunna liviae sp. nov. Jorunna liviae sp. nov. Jorunna liviae sp. nov. Jorunna liviae sp. nov. Supplementary material 1 Bayesian maximum credibility tree of the COI and 16S sequence alignments for Jorunna liviae sp. nov. Authors: Yara Tibirica, Jenny Stromvoll, Juan Lucas Cervera Data type: phylogenetic Explanation note: Bayesian maximum credibility tree of the COI and 16S sequence alignments. Posteri- or probabilities (PP) are indicated above each and bootstrap values (BS) are indicated below each branch. Branch lengths indicates the proportion of substitu- tions. PP< 0.5 and BS< 50 are not shown. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/zse.99.95222 suppl Voucher Col H3 NTNU-VM-58891 MW784174 MW810589 ZMBN 125946 MW784173 MW810590 ZMBN 127749 MW784172 - CPICO0633 KP871645 KP871669 IPOO11 MN690306 - IPO0302 MN690307 - ZMBN 127710 MW784177 MW810611 ZMBN 127711 MW784180 MW810603 ZMBN 125512 MW784175 MW810597 NTNU-VM-58888 MW784204 MW810600 ZMBN 127709 MW784190 MW810594 ZMBN 132446 MW784193 MW810599 ZMBN 125474 MW784171 MW810587 MNCN15.05/200189 OP948384 OP939409 MNCN15.05/200187 OP948382 0P939411 MNCN15.05/200188 O0P948383 0P939410 MNCN15.05/94693 OP948385 0P939412 Supplementary material 2 Table of locality Authors: Yara Tibirica, Jenny Stromvoll, Juan Lucas Cervera Data type: occurences Explanation note: Table of locality of collected specimens of Jorunna liviae sp. nov. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons. org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow us- ers to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://do1.org/10.3897/zse.99.95222 suppl2 zse.pensoft.net