Zoosyst. Evol. 100 (1) 2024, 49-59 | DOI 10.3897/zse.100.113707 > PENSUFT. yee BERLIN First occurrence of the genus Pleurobranchaea Leue, 1813 (Pleurobranchida, Nudipleura, Heterobranchia) in British waters, with the description of a new species Martina Turani', Leila Carmona**, Peter J. Barry*, Hayden L. Close*, Ross Bullimore’, Juan Lucas Cervera?? 1 Department of Natural History, University Museum, University of Bergen, Bergen, Norway 2 Departamento de Biologia, Facultad de Ciencias del Mar y Ambientales, Campus de Excelencia Internacional del Mar (CEI* MAR), Universidad de Cadiz, Av. Republica Saharaui, s/n, 11510 Puerto Real, Spain 3 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, Spain 4 Centre for Environment Fisheries and Aquaculture Science (CEFAS), Lowestoft, Suffolk, UK https://zoobank. org/579E D8AF-40E7-4F E4-ASAF-ED8&7567D5DCC Corresponding author: Leila Carmona (leila.carmona@uca.es) Academic editor: M. Glaubrecht # Received 5 October 2023 # Accepted 28 November 2023 @ Published 26 January 2024 Abstract In the north-eastern Atlantic and Mediterranean Sea, the pleurobranchid genus Pleurobranchaea Leue, 1813 is represented by two species, Pleurobranchaea meckeli (Blainville, 1825) and Pleurobranchaea morosa (Bergh, 1892). The former is a well-known species distributed from northern Spain to Senegal and the Mediterranean Sea, while the second is a poorly-described species. In this contribution, species delimitation analyses (ABGD and COI/16S p-distances) identified a third undescribed Pleurobranchaea species from samples collected in south-western UK waters and the Gulf of Cadiz (SW Spain). This new species, Pleurobranchaea britannica sp. nov., is also supported by several morphological synapomorphies. The British specimens constitute the first occur- rence of the genus Pleurobranchaea in UK waters. Key Words Atlantic Ocean, Gulf of Cadiz, Mediterranean Sea, molluscan diversity, Pleurobranchaea britannica, Pleurobranchaeidae, southwest UK, systematics Introduction The family Pleurobranchaeidae Pilsbry, 1896 was estab- lished for heterobranchs, characterised by an oval body, broad oral veil, rolled rhinophores and variable colours. Members of this family have a bipinnate gill in the mid- dle of the right side, which may or may not be covered by the mantle (Garcia-Gomez and Cervera 2011). The latter character led to their inclusion together with the Umbrac- ulida (Order Notaspidea). However, phylogenetic analy- ses, based on morphological and molecular data (Marty- nov and Schrodl 2009; Goébbeller and Klussmann-Kolb 2010), demonstrated that the Pleurobranchaeidae should be considered as a separate group. Under an alternative classification scheme, Wagele and Willan (2000) intro- duced the Nudipleura to unite Pleurobranchoidea and Nu- dibranchia. Some characteristics the Nudipleura have in common are: the loss of the shell, the presence of papil- lae on the notum, a hermaphroditic reproductive system with simultaneous maturation of the gametes and obligate cross-fertilisation involving copulation. Members of the Pleurobranchaeidae are active hunt- ers of invertebrates and typically inhabit sedimentary substrates. The family is ubiquitous and they can be Copyright Turani, M. 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. 50 found from the intertidal zone to the circalittoral. The family is considered monophyletic and comprises three genera: Pleurobranchaea Leue, 1813, Euselenops Pils- bry, 1896 and Pleurobranchella Thiele, 1925. The genus Pleurobranchaea constitutes 15 valid species (Alvim et al. 2014; MolluscaBase 2023a) that inhabit temperate or tropical waters across a wide geographical range (Mu- niain et al. 2007; Alvim et al. 2014). The type species of the genus, Pleurobranchaea meckeli (Blainville, 1825), is the only well-known species from European waters (Bergh 1897; Vayssiere 1901; Marcus and Gosliner 1984). This species has been recorded in many locali- ties from the Mediterranean Sea, as well as in several localities around the Atlantic Iberian coasts, Madeira, Canary Islands, Azores (Cervera et al. 2004) and Cape Verde Archipelago (Vayssiere 1901). Marcus and Gos- liner (1984) described two additional species of Pleuro- branchaea, based on preserved material collected from Turkey, Israel (P. notmec) and Algiers (P. vayssierei). However, Cervera and Garcia-Gomez (1988), follow- ing Willan (pers. comm.), considered both names to be junior synonyms of P. meckeli. Bergh (1892) described P. morosa from a single specimen collected from the Pico-Faial Channel (Azores) at 130 m depth. No fur- ther records are attributed to this second species, since Marcus and Gosliner (1984) did not include P. morosa in their review of the Pleurobranchaeidae due to its in- sufficient description. The northernmost record of Pleurobranchaea in the eastern Atlantic had been from northern Spain (Cervera et al. 2004), but recent bottom trawls conducted along the English Channel in 2018 and 2019, as well as in the Gulf of Cadiz (SW Iberian Peninsula) in 2020 collected specimens of an unknown pleurobranchid with an exter- nal appearance slightly different from Pleurobranchaea meckeli. During the surveys, the collected specimens were initially considered as a different morphotype of P. meckeli, but a detailed examination of the external and internal anatomy revealed marked differences from all 10.00°O 6.00°O 4.00°O _ 2.00°O 0.00° | a 8.00°O 52,00°N ee 50.00°N 48,00°N 46.00°N Turani, M. et al.: New species of Pleurobranchaea in the SW of UK other species of the genus. Subsequently, a phylogenetic analysis, based on two mitochondrial (cytochrome-oxi- dase subunit I and 16SrRNA) and one nuclear (Histone 3) marker, in conjunction with species delimitation analy- ses, Supported the status of this morphotype as a new and previously undescribed species, which is formally de- scribed in the present paper. Materials and methods Taxon sampling and molecular data Fourteen specimens of an undescribed species of Pleuro- branchaea were collected during two different campaigns in southern England (Fig. 1A) and one in the Gulf of Cadiz (Fig. 1B). The surveys in the Western Channel and Celtic Sea occurred during the 2018 and 2019 Centre for En- vironment, Fisheries and Aquaculture Science (CEFAS) Quarter One South West EcoSystem (QISWECOS) sur- veys. This survey series used two commercially rigged 4 m beam trawls with 80 mm cod-end mesh, with one of the trawls fitted with a 40 mm liner to facilitate the col- lection of epibenthic species. The bathymetric range cov- ered by the trawls was approximately 20—200 m. The sur- vey conducted by the Instituto Espafiol de Oceanografia (IEO-CSIC) in the Gulf of Cadiz, offshore between Faro and Cadiz was collected by beam trawl of 190 cm and the effective height was 60 cm above the bottom with a duration of 15 min. The material collected in these campaigns was de- posited either in the Natural History Museum of London (NHM) or in the National Museum of Natural Sciences of Madrid (MNCN) (see Table 1). Twenty-four specimens of three different species (11 Pleurobranchaea meckeli, 4 P. maculata and 14 of the new species) were sequenced in this study to obtain partial sequences of two mitochon- drial (cytochrome-oxidase subunit I or COI and 16SrR- NA or 16S) and one nuclear (Histone 3 or H3) markers. 12.00°O 10.00°O 8.00°O 6.00°O 4.00 40.00°N 38.00°N 36.00°N 34.00°N Figure 1. Sampling stations where Pleurobranchaea britannica sp. nov. material was collected. A. The map on the left shows the south of England: the red dots refer to the 2018 campaign and the green dots to the 2019 campaign; B. The map on the right shows part of Spain and the orange dot is where samples were collected in 2019. zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 49-59 Additionally, sequences of these genes from ten close- ly-related pleurobranchid taxa were added from Gen- Bank, including two additional species of Pleurobran- chaea (P. californica and P. inconspicua) and Bathydoris aioca (Er. Marcus and Ev. Marcus, 1962), Bathydoris clavigera (Thiele, 1912) and Berthella plumula (Mon- tagu, 1803) as outgroups (Table 1). Morphological analysis The samples were stored in 95%—100% ethanol to al- low DNA extraction. Two specimens were dissected by dorsal incision. The internal morphology of the re- productive system was examined and drawn using a Leica Wild M8 dissection microscope. The buccal bulb was removed and placed in a 10% sodium hydroxide (NaOH) solution for three days until the radulae and jaws were cleaned of the surrounding tissue. The rad- ula and jaws were then rinsed in demineralised water 51 and rinsed at least twice with 96% ethanol before dry- ing. Clean radulae and jaws were mounted on SEM stubs with dissection pins and coated with gold for examination with a Nova NanoSEM scanning electron microscope (SEM) at the Central Services of Scientif- ic and Technological Research (SC-ICYT) unit of the University of Cadiz. DNA extraction and amplification Genomic DNA was extracted from foot tissue of speci- mens using the DNeasy Blood & Tissue Kits of Qiagen (Qiagen, Inc., Valencia, Ca., USA) and stored in ex- traction buffer at —20 °C prior to amplification. Partial sequences of H3 were amplified by polymerase chain re- action (PCR) using the universal primers H3F and H3R (Colgan et al. 2000), while universal and specific prim- ers or their combination were used for the COI and 16S (Table 2). Table 1. List of species used for this study including sample locality, voucher numbers and GenBank accession numbers. Species Locality Voucher numbers GenBank accession numbers col 16S H3 Berthella plumula (Montagu, 1803) Ballyhenry Is., Northern Ireland, UK CASIZ 193034 MK542770 MK542742 MK542803 Prodoris clavigera Thiele, 1912 South Shetland |., Elephant |., Antartica CASIZ 167553 JX274106 JX274067 KP940463 Bathydoris aioca California CPIC 01053 KP153283° ~KP153249" + KP153316 Er. Marcus & Ev. Marcus, 1962 Pleurobranchaea maculata Auckland, New Zealand - JN675223 - - Quoy & Gaimard, 1832 Pleurobranchaea maculata Auckland, New Zealand - JN675222 - - Pleurobranchaea maculata Auckland, New Zealand - JN675221 - - Pleurobranchaea maculata Auckland, New Zealand - JN675220 - - Pleurobranchaea californica California - - FJ917440 - MacFarland, 1966 Pleurobranchaea inconspicua Bergh, 1897 Patagonia, Argentine, Atlantic Ocean MNCN 15.05/94846 - OR723442 OR715121 Pleurobranchaea meckeli (Blainville, 1825) Blanes, Spain, Mediterranean Sea - AY345026 - - Pleurobranchaea meckeli Blanes, Spain, Mediterranean Sea - - - EF133470 Pleurobranchae meckeli 005 Castellaneta Marina, Italy, MNCN 15.05/94841 - OR723434 OR715119 Mediterranean Sea Pleurobranchaea meckeli 006 Castellaneta Marina, Italy, MNCN 15.05/94842 OR687335 OR7/723435 OR715118 Mediterranean Sea Pleurobranchaea meckeli 008 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/94843 OR687340 OR7/23441 OR715116 Pleurobranchaea meckeli 010 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/94844 OR687336 OR7/23436 OR715117 Pleurobranchaea meckeli 016 Laayoun, Morocco, Atlantic Ocean MNCN 15.05/94845 OR687341 OR723437 - Pleurobranchaea meckeli 21.1 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/94837 OR687337 OR7/23438 OR715120 Pleurobranchaea meckeli 21.2 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/94838 OR687339 OR7/23440 £OR715115 Pleurobranchaea meckeli 21.3 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/94839 OR687338 OR723439 OR715114 Pleurobranchaea meckeli 22.1 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/94840 OR807509 - - Pleurobranchaea britannica sp. nov. 17.1 Southwest of England NHMUK 20230086 OR687348 OR723452 OR715123 Pleurobranchaea britannica sp. nov. 17.2 Southwest of England NHMUK 20230087 OR687349 = OR723446 =O0R715122 Pleurobranchaea britannica sp. nov. 17.3 Southwest of England NHMUK 20230085 OR687351 OR723453 = =OR715124 Pleurobranchaea britannica sp. nov. 17.4 Southwest of England NHMUK 20230088/1 OR687347 OR7/23444 OR715125 Pleurobranchaea britannica sp. nov. 18.1 Southwest of England MNCN 15.05/200180 - OR723447 - Pleurobranchaea britannica sp. nov. 18.2 Southwest of England NHMUK 20230089 - - OR715126 Pleurobranchaea britannica sp. nov. 18.3 Southwest of England NHMUK 20230090 - - OR715127 Pleurobranchaea britannica sp. nov. 18.4 Southwest of England NHMUK 20230091 - - OR715128 Pleurobranchaea britannica sp. nov. 20.1 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/200181 OR687345 OR723448 OR715132 Pleurobranchaea britannica sp. nov. 20.2 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/200182 OR687343 OR723449 OR715133 Pleurobranchaea britannica sp. nov. 20.3 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/200183 OR687346 OR723450 OR715129 Pleurobranchaea britannica sp. nov. 20.4 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/200184 OR687342 OR/23451 OR715130 Pleurobranchaea britannica sp. nov. 20.5 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/200185 OR687344 OR7/723445 OR715134 Pleurobranchaea britannica sp. nov. 20.6 Gulf of Cadiz, Spain, Atlantic Ocean MNCN 15.05/200186 OR687350 OR/23443 OR715131 zse.pensoft.net oy Table 2. List of primers used for this study. Turani, M. et al.: New species of Pleurobranchaea in the SW of UK Gene Primer ID Sequence (5’to-3’) Annealing Temperature (°C) Source COl LCO1490 (F) GGTCAACAAATCATAAAGATATTGG 50 Folmer et al. (1994) HCO2198 (R) TAAACTTCAGGGTGACCAAAAATCA 50 Folmer et al. (1994) Pluero26F (F) GAGTTGGGGACTTCAGGAGC 46 This study Pluro526R (R) AATAGCCCCCGCCAATACTG 46 This study jgLCO1490 (F) TITCIACIAAYCAYAARGAYATT GG 50 This study jgHCO2198 (R) TAIACYTCIGGRTGICCRAARAAYCA 50 This study 16S Sar-L (F) CGCCTGTTTATCAAAAACAT 52 Palumbi (1996) Sbr-H (R) CCGGTCTGAACTCAGATCACGT 52 Palumbi (1996) F52 (F) ATAGCCGCGGTACTTTGACC 55 This study R384 (R) AGTCCAACATCGAGGTCACA 55 This study H3 H3F (F) ATGGCTCGTACCAAGCAGAGVGC 58 Colgan et al. (1998) H3R (R) ATATCCTTRGGCATRATRGTGAC 58 Colgan et al. (1998) Reactions took place in a total volume of 25 ul including 2 wl of template DNA, 1 ul of both forward and reverse primers (10 uM), 2.5 ul of dNTP (2 mM), a gene- dependent amount of magnesium chloride (25 mM), 0.25 ul of Qiagen DNA polymerase (5 u/ul), 5 wl of “Q-solution” (5x) and 2.5 ul of Qiagen buffer (10x; Qiagen Taq PCR Core Kit cat. no. 201225). Magnesium chloride (MgCl2) amounts were 4.5 ul for COI and 16S and 3 ul for H3. The COI fragment was amplified with initial denaturation step for 1 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, an annealing step for 30 s at 52 °C (universal primer) or 50-46 °C (specific primer) and 30 s at 72 °C. A final extension step for 3 min at 72 °C was added to ensure extension. For 16S, the thermal cycle profile began with denaturation for 1 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 16s at 52 °C (universal primer) or 16s at 55 °C (specific primer) and 30 s at 72 °C, with a final extension step for 3 min at 72 °C. Finally, the H3 amplification was performed with an initial denaturation for 2 min at 95 °C, followed by 40 cycles of 30 s at 94 °C, an annealing step for 30 s at 58 °C and 1 min at 72 °C, with a final extension step at 72 °C for 7 min. A negative control (no template) was included in each reaction. PCR products were visualised by electrophoresis on a 2% agarose gel and those viable were purified and amplified in both directions by Macrogen Inc. All new sequences obtained were deposited in GenBank. Nucleotide sequence alignment and phylogenetic reconstruction All DNA chromatograms were assembled and edited using Geneious version 10.0.9 (http://www.geneious. com, Kearse et al. 2012). The sequences were aligned with MAFFT v.7.402 server (Katon and Standley 2013) using the L-INS-1 iterative refinement algorithm via the CIPRES Portal Science Gateway (Miller et al. 2010). The alignments were further optimised by eye using AliView (Larsson 2014) and trimmed to 328 bp (H3), 658 bp (COI) and 450 bp (16S). These partitions were subsequently concatenated with FASconCAT-G zse.pensoft.net v.1.0, resulting in 1436 bp alignment. Pairwise uncor- rected p-distances between each available taxon were conducted for the COI and 16S gene using PAUP v.4.0 (Swofford 2002). The best model of evolution was determined with jModelTest 2.1.10 (Darriba et al. 2012). The model identified with the Akaike Information Criterion (AIC) (Akaike 1998) was the Tamura-Nei model (TrN+G) for H3 and the general time-reversible model (GTR+G) for COI and 16S. Phylogenetic analyses were conducted un- der two optimal criteria: Maximum Likelihood (ML) and Bayesian Inference (BI). The ML phylogenetic trees were inferred using RAXML v.8.2.12 (Stamatakis 2014) under the GTRGAMMA model. Node support was assessed with rapid bootstrap analysis with 1000 replicates. Anal- ysis stopped the search for bootstrap after 100 replicates with the autoMRE-based bootstopping criterion. Values > 70% were interpreted as significant nodal support (Hillis and Bull 1993). Bayesian Inference analyses were conducted using MrBayes v.3.2.7 (Ronquist et al. 2012). Two runs were performed in parallel with four independent MCMC chains (one heated, three cold) and default priors. The analyses were run for 5,000,000 generations, saving a tree every 1000 generations and discarding the first 1250 trees of each analysis as “burn-in.” Nodal support was estimated as posterior probabilities (PP), with values > 90% taken as significant (Huelsenbeck and Rannala 2004). Species delimitation analyses Species delimitation analyses involved two methods: (a) the Automatic Barcode Gap Discovery (ABGD; Puillan- dre et al. 2012), through a simple distance matrix, based on the COI and 16S genes (generated in MEGA v.7.0.18) as input file under default intra- and interspecific priors (pmin = 0.001; pmax = 0.10) in 10 steps and with a rela- tive gap width of 1.5; (b) a pairwise genetic distance ma- trix, based on the COI sequences generated in PAUP v.4.0 (Swofford 2002). Zoosyst. Evol. 100 (1) 2024, 49-59 Results Systematics Superorder Nudipleura Wagele & Willan, 2000 Order Pleurobranchida Gray, 1827 Superfamily Pleurobranchoidea Gray, 1827 Family Pleurobranchaeidae Pilsbry, 1896 Genus Pleurobranchaea Leue, 1813 Pleurobranchaea britannica sp. nov. https://zoobank. org/81238088-87DA-4163-81F8-06AEB3ADDCAA Material examined. Holotype: NHMUK 20230085, 18 mm preserved length, (49°54'5.306"N, 6°45'7.056"W), southern England, 103 m depth, Apr 2019. Paratypes: NHMUK 20230087, 19 mm _ preserved length, (49°35'59.389"N, 4°39'48.485"W) southwest England, 91.98 m depth, Mar 2018; NHMUK 20230086, 18 mm preserved length, (49°42'13.429"N, 4°6'28.514"W) southwest England, 81.12 m depth, Mar 2018; NHMUK 20230091, 22 mm preserved length, (50°5'10.929"N, 3°41'34.436"W) southwest England, 68.94 m depth, Mar 2018; MNCN 15.05/200180, 24 mm preserved length, (50°2'41.978"N, 4°3'33.805"W) southwest England, 75.69 m depth, Mar 2018, dissected specimen; NHMUK 20230090, 18 mm preserved length, (49°54'5.306"N, 6°45'7.056"W), southwest England, 103 m depth, Apr 2019; NHMUK 20230089, 19 mm preserved length, (49°54'5.306"N, 6°45'7.056"W), southwest England, 103 m depth, Apr 2019; NHMUK 20230088/1, 20 mm preserved length, (49°54'5.306"N, 6°45'7.056"W), south- west England, 103 m depth, Apr 2019, dissected spec- imen; MNCN 15.05/200181, 7 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Feb 2020; MNCN 15.05/200182, 8 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Feb 2020; MNCN 15.05/200183, 11 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Feb 2020; MNCN 15.05/200184, 10 mm pre- served length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Ca- diz, 555 m depth, Feb 2020; MNCN 15.05/200185, 11 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Feb 2020; MNCN 15.05/200186, 9 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Feb 2020. Additional ma- terial: MNCN 15.05/94837, 41 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Mar 2020; MNCN 15.05/94838, 43 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Mar 2020; MNCN 15.05/94839, 48 mm preserved length, (36°16'19.56"N, 7°32'52.8"W) Gulf of Cadiz, 555 m depth, Mar 2020; MNCN 15.05/94840, 42 mm preserved length, (36°16'19.56"N, 7°32'5S2.8"W) Gulf of Cadiz, 555 m depth, Mar 2020. Diagnosis. Body oval, large, translucent with a min- ute cream/ochre pigmentation. Some specimens with 53 opaque white specks irregularly spread all over mantle, oral veil, gill and posterior region of the foot not covered by the mantle. Rhinophores with dark spots on the front and white ones on the back. Gill bipinnate, with 15-18 pairs of pinnules and smooth rachis. Caudal spur absent. Outermost radular teeth bicuspid. Seminal receptacle short; bursa copulatrix at the end of the vagina and di- rectly fused to it. Description. External morphology (Fig. 2). Body oval and large, with a rough mantle forming irregu- lar polygons delimited by shallow grooves (Fig. 2A, B). Base colour translucent with a minute cream to ochre pigmentation, which may not always be present. Opaque white specks might appear irregularly spread all over mantle, oral veil, gill and posterior region of the foot not covered by the mantle. Speckles density vari- able. Viscera partially visible through mantle in lighter individuals. Posterior part of foot round with no caudal spur (Fig. 2A). Moreover, no pedal gland was observed. Sole patterned. Anterior part of rhinophores brown and posterior covered with close white dots (Fig. 2A). Oral veil with trapezoid shape and fused with mantle where rhinophores are inserted. Veil front edge not smooth, but slightly irregular. Some specimens with series of white specks at veil corners. Gill located on the right side of the body, clearly visible and not covered by the mantle. Gill bipinnate, with 15-18 pairs of pinnules and smooth rachis. Gill with same base colour, white grains almost always present and variable in density, being visible on rachis and pinnules. Genital openings in front of gill and nephropore, clearly visible since it is covered by a circular fleshy papilla, which may have white dots. Anus opens above the 6" and 7" pinnule of the gill. Internal anatomy (Figs 3, 4) Radula almost rectan- gular with no rachidian teeth. Radular formulae are: 35 x (53—50).0.(53-50) (NHMUK 20230088/2); 33 x (54— 56).0.(54—-56) (MNCN15.05/200180) (Fig. 3A). All teeth with two long and blade-shaped cusps. The outermost cusp is larger, while the one facing the centre of the rad- ula is smaller and sometimes covered by the next tooth. Innermost teeth slightly more elongated and with finer tips, while outermost teeth with rounder tip (Fig. 3B). Jaws elongated. Anterior part of jaw elements hexag- onal, hand-shaped, with 4 to 9 denticles along anterior edge. The jaw elements have a depression in the middle (Fig. 3C, D). Reproductive system (Fig. 4) begins with the her- maphroditic duct which first widens into the ampulla and thereafter narrows and divides into two parts: one entering the prostate gland and the other continuing to oviduct. The prostate gland is composed of small and pyramidal-shaped papillae. Exiting from prostate, vas deferens entering the penial sac, anchored by a retrac- tor muscle to the inner body wall. Inside the sac, pe- nis relatively straight, with a couple of twists, but not coiled and apparently not cuticularised. The oviduct zse.pensoft.net 54 Turani, M. et al.: New species of Pleurobranchaea in the SW of UK Figure 2. A. Living specimens of Pleurobranchaea britannica sp. nov. collected on Survey CEND 0518, southwest England. Photo by Ross Bullimore (NHMUK 20230085); B. Two young individuals of P. britannica sp. nov. from the Gulf of Cadiz, Spain (MNCN 15.05/200181; MNCN 15.05/200182); C. Specimen of P. meckeli from Morocco, Mediterranean Sea (MNCN 15.05/94845). A - ss y . . 30 ym 40 ym Figure 3. Pleurobranchaea britannica sp. nov. Scanning electron micrographs of radula and jaw. A. Complete radula (MNCN15.05/200180); B. Lateral teeth of radula (MNCN15.05/200180); C. View from above of the anterior part of the jaw (MNCN15.05/200180); D. Lateral view of the anterior part of the jaw (MNCN15.05/200180). zse.pensoft.net Zoosyst. Evol. 100 (1) 2024, 49-59 widens slightly forming a bilobed seminal receptacle, narrowing before entering laterally into the muscular vagina. Copulatory bursa spherical, placed at the distal end of vagina. Copulatory bursa not very muscular, its wall being delicate and thinner than the vagina’s. Fe- male gland and vagina join laterally, very close to the female orifice. There are two different genital openings: the opening closest to the nephropore is the female one, the further one is male. Etymology. The species name in Latin refers to the British waters where this species was initially found. Distribution. The species has been found in a num- ber of locations in the southwest of UK waters and the Gulf of Cadiz, see Fig. 1, but we hypothesise that it could probably be distributed throughout the Atlantic coast of Spain, Portugal and France up to the southwest approach- es to the English Channel. Type locality and habitat. South-western England (see Fig. 1A). Collected from a range of depths (70—110 m) and a range of substrates that include areas of mosaic rock and mixed sediments and areas of muddier sediments. a Figure 4. Pleurobranchaea britannica sp. nov. Reproductive system (NHMUK 20230088/5). Abbreviations: a — atrium; am — ampulla; bc — bursa copulatrix; dd — deferent duct; fg — fe- male gland; hd — hermaphroditic duct; ov — oviduct; p — penis; pr — prostate; r — retractor muscle; ps — penis sac; sr — seminal receptacle; v — vagina. Phylogenetic analysis We obtained 17 sequences for COI, 20 for 16S and 21 for H3 genes. The combined data set (COI+H3+16S) provided better resolution than the COI, 16S and H3 separately. Fig. 5 shows the phylogenetic hypothesis based on the combined dataset (COI+H3+16S) con- structed by Bayesian Inference (BI). The topology of the Maximum Likelihood (ML) tree was very similar to that obtained by BI (former not shown). All the sequenc- es of the new species clustered in a single, well-sup- ported clade (PP = 1; BS = 88), included in a broader well-supported clade (PP =1; BS = 80) together with 55 Pleurobranchaea inconspicua, P. maculata and P. cali- fornica. All the specimens of Pleurobranchaea meckeli clustered in a different and maximum supported clade (PP =1; BS = 100) including the remaining species of Pleurobranchaea we analysed. Uncorrected p-distances (%) between P. britannica sp. nov. and the species P. maculata and P. meckeli ranged from 12.7% to 18.3%, respectively (Table 3). The ABGD species delimitation analysis recovered three putative species, based on the COI gene with both Jukes-Cantor (JC69) and Kimura (K80) parame- ters, while for the 16S alignment retrieved four putative species. These differences are due to the fact that se- quences of P. inconspicua and P. californica were not available for COI, whereas for 16S, there were no data for P. maculata. Table 3. Maximum and minimum COI gene pairwise uncorrect- ed p-distances (%). Species P. meckeli P. maculata P. meckeli P. maculata 16.2-17.5 P. britannica sp. nov. 16.3-18.3 12.7-13.9 Discussion Our analyses support, from both the molecular and morphological approaches, the existence of Pleuro- branchaea britannica sp. nov. as a distinct species. The geographical range of this species and that of P. meck- eli partially overlap, but they can be distinguished by both external and internal characters. Externally, the new species lacks a caudal spur and has a transparent cream-coloured base with variation in the density of white spots and the mottled or “net” pattern which can be darker or lighter. Pleurobranchaea meckeli exhibits a conspicuous caudal spur and has a brown net pattern all over the body (Fig. 2A). Furthermore, the colour pat- tern of the rhinophores of P. britannica is characteristic with dark spots on the front and white spots on the back, respectively. Other differences between P. meckeli and P. britannica are summarised in the Appendix 1. This Appendix also includes the main differences between the known species from the Atlantic Ocean and Medi- terranean Sea. For example, we also observed differenc- es in the radula formula and the outermost teeth which are bicuspid in P. britannica, while they are unicuspid in P. meckeli. In the reproductive system, we can find several clear differences: the seminal receptacle of P. meckeii is larger than in P. britannica and, therefore, easier to observe. The bursa copulatrix in P. meckeli is not directly connected to the vagina, but with a small tube that connects the two structures. In the new species, the bursa copulatrix is located at the end of the vagina, directly connected with it. Another difference can be ob- served in the structure of the penis since, in P. meckeli, it performs various loops within the penal sac, while in P. britannica, it does not. zse.pensoft.net 56 0.84 52 Pleurobranchaea incospicua 13 Argentina i Pleurobranchaea californica FJ917440 California 98 100 Turani, M. et al.: New species of Pleurobranchaea in the SW of UK col 16S Pleurobranchaea britannica sp.nov. 20.3 Spain Pleurobranchaea britannica sp.nov. 20.1 Spain Pleurobranchaea brifannica sp.nov. 20.5 Spain Pleurobranchaea britannica sp.nov. 20.2 Spain Pleurobranchaea britannica sp.nov. 20.4 Spain Pleurobranchaea britannica sp.nov. 20.6 Spain Pleurobranchaea britannica sp.nov. 17.2 England Pleurobranchaea britannica sp.nov. 17.4 England Pleurobranchaea britannica sp.nov. 17.3 England Pleurobranchaea britannica sp.nov. 17.1 England Pleurobranchaea britannica sp.nov. 18.1 England i Pleurobranchaea britannica sp.nov. 18.4 England Pleurobranchaea britannica sp.nov. 18.3 England Pleurobranchaea britannica sp.nov. 18.2 England el ie ce Camano Ae Pleurobranchaea maculata JN6G75223 New Zealand Pleurobranchaea maculata JN6G75222 New Zealand Pleurobranchaea maculata JN675220 New Zealand ele RINT leurobranchaea maculata JN675221 New Zealand Pleurobranchaea meckeli 05 Italy Pleurobranchaea meckeli EF 133470 Spain a Se yal: gF Sat ee: sae aw el prt. Pleurobranchaea meckeli 22.1 Spain Pleurobranchaea meckeli 21.2 Spain Pleurobranchaea meckeli 21.3 Spain Pleurobranchaea meckeli 21.1 Spain Pleurobranchaea meckeli 08 Spain Pleurobranchaea meckeli 16 Morocco Pleurobranchaea meckeli 10 Spain Pleurobranchaea meckeli 06 Italy Pleurobranchaea meckeli AY345026 Spain Bathydoris clavigera 1 Bathydoris aioca Berthella plumula Figure 5. Phylogenetic hypothesis of Pleurobranchaea systematics, based on concatenated dataset (COI+16S+H3) inferred by Bayesian analysis. Significant support values are given as BI posterior probabilities (below branch) and ML bootstrap percentages (above branch). Rectangles are automatic barcode gap discovery for the COI and 16S dataset. White rectangles indicate the lack of those sequences in the alignment. Pleurobranchaea britannica sp. nov. does not corre- spond to the poor descriptions of other animals currently accepted as synonyms of P. meckeli (see MolluscaBase (2023b)). For example, Pleurobranchaea dellechiaii Véra- ny, 1846 has a minimal external description, but the mantle has red dots even after death, which are not present in the new species. Pleurobranchaea notmec Marcus, Ev. and Gosliner 1984 has a caudal spur, absent in P. britannica and the penis is much longer (as in P. meckeli) compared to the new species. Pleurobranchaea vayssierei Marcus, Ev. and Gosliner 1984 also has a caudal spur and an elongat- ed penis wrapped in the penal sac, but the vagina is long and quite narrow unlike P. britannica which has a wide vagina which connects the bursa copulatrix to the outside. Those last two names (P. notmac and P. vayssierei) were proposed from preserved material without any information provided of the living individuals’ colouration. All these morphological and anatomical differences which separate P. britannica sp. nov. as a standalone species are supported by our genetic data. In fact, individuals iden- tified as P. britannica form a single well-supported taxon (PP = 1; BS = 88), which 1s also separate from P. meckeli, zse.pensoft.net the geographically closest species. To date, the only Eu- ropean (excluding the Azores) species of the genus Pleu- robranchaea meckeli, has not been recorded further north than Iberian coasts. Therefore, P. britannica represents the first record of the genus Pleurobranchaea in British waters and the second valid species from European seas. Acknowledgements We thank the survey scientists and crew of “RV Cefas En- deavour” their help for the collection of the specimens of the new species from the English Channel. Likewise, Carlos Farias (Spanish Institute of Oceanography, IEO) donated the material of Pleurobranchaea britannica n. sp. and P. meckeli from the Gulf of Cadiz collected during several IEO ocean- ographic campaigns ARSA. 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Zoologial Journal of the Linnean Society 130: 83-181. https://doi. org/10.1111/j.1096-3642.2000.tb02196.x zse.pensoft.net New species of Pleurobranchaea in the SW of UK Turani, M. et al.: 58 (€202) Bunoalosd SINNOM S9LUI}AWOS SJOp ayIyM pue ‘(VIOZ) ‘|e 32 ‘feoupul|Ao ayjueW S9Ul] UMOIG 40 WIAY ‘(900Z) ‘Qs1e| SIUBd eUIseA aspa aU} YIM pasn} Wayed ayejnona BJBNaY ‘ell } eds ISA ‘SOW TI-OT | Jejnosnw pue JOUa\ue ay} SI} ‘suleys UUM ‘SUM pidsnoiun ‘Ulelun|/| ‘UeaUCLAYIPS|/| eyided SUIII0D ‘ayyM | xiUye|ndoo siyoes suge aeyjided | ysniym awos jugonsued} Jo dsno ‘(736 T) ‘onueny Jejnsuely juaon|sue.} esunq ayeuuadiun Asosuas JO YIM UMOJg ‘JOO} SuUaA0D =| Ayepuovas Auly J@ul|SO5 Waysam ‘|IZe1g | ULM plo} Aq ‘Ja\Ays papunol ‘sainuuid MO JeINBuUIs juagonsuet} jou ‘psonpal S 9WIaWOS (L681 ‘ysiag) pue snoe|| WauLON papunouwns Jeynonng adie] paqolg 92-02 Yum peog ‘UJOOLWUS uasalg| juasald SI ajuel| ‘pidsnaig v9'0'v9 X OE |enoidsuosu g Solu} XIS 0} OAY BUI|I09 ‘Ja|A\s jugon|sue.} (v10Z) ‘Ie 398 Jejnoyno xieindoo WIA (786 T) Suge jSauo esunq (QO6T ‘snoue|| JOUI|SO5) dey padojanap papunol paqojiy sainuuid 1Usi| UM SOLUIJBLUOS ‘AJ pue snoey| pue SNoJe || BOUIY {SoM [ESJOP UM 12M ‘ews 40 peqolla 9¢c-8I Si JU9SBJq 9JOS JOO} YOe|A ‘pidsnoig LGOLS X ¢cY ‘I9) e193 d s}op SYM UM esing P249A09 SI s}sIny Asoyejndoo 4O149}SO0d dy} sjods jouueyy jo ajdnoo e | jeouayds ospo pue umoig OVYM JeINSaAUI ysijsuy ouy UUM ‘WYsIe1}S ou} SI siyoel = quod einai) si dy ayy jo “Inojod eseq 0} dn 9oues4 eyjided AJOAnejos BUISeA YJOOLUS ‘anuew au} | Wed s0oLeque WIPa19 410 dsno pue jesnyiog Aysay Ss! slued 4ejnosnu ‘ayeuuidiq | yYwM pasny si | auQ ‘ayesedas queonjsue.} Asepuooas ‘uieds jo dejnouid & | pasuejnoyns jo pus pogojiq ‘sajnuuid pue sdeys | aie ‘pajuiod ‘J00j SULIBAOD | AU }SOWNNO | BG -GG'O'8S ‘aou “ds yseoo onuepy | Aq paran0d ON leqsip atin yy | —Apysiis 8I-GI plozedei, =| pue payjoy qesqy | jou apueW ‘pidsnoig -6G x 9€ eoiuUeq ‘d S98Y9}O|q YSHIYM Jejnsaui pue anbiquiezo|/| xujejndoo JUasid UMOIq (vIOZ) ‘le }9 “eEOeUU] 0} esung paqojiq yep Jo wiayed pidsnoaiun WIA ‘(~86T) ejnsuluad Japsoq puly epse} ‘q JO pue eulsen syed ‘SapIS yJOq UO | Yaa} JSOLWNO (786 ‘aul|Sos) JOUl|SO5) ade) joyseoo | uo dey yim | yeu} oj sejiis | seynpueys YJOOWS Sajqaqn} OT ueu} a0 JOO} a4} JAAO O€-0Z OOT'0'00T pue snoae|\) 7 SNOJE|A| onueny Woy | ‘sulpmyoidg | Si yajAjs aul su07 ‘sainuuld QZ | |JeEUS JO MOJ Ajauel p[-p | wasag} juasqy | Spuexa ayues| ‘pidsnaig X GE ejeqnq q ODIXA|/| (vIOZ) ‘Ie 38 | jo4iNd ‘yueg WIA “(y86T) | eweYyeg JealD pidsnoiun JQuUI|SO) —_ | “epuo|{ :onuepy 9]91ND JNOUUM JSOWU94NO / T (L681 ‘Ysieg) pue snouey| Wa]Sa/ JeinIsny\| aging ajsuls vy} sajnuuid v¢ Juasald jng ‘pidsnaig | 36°0'86 X ZE lIZISS@Be Y (syuaWeja xlyejndoo mef uo uj00} suluedo esing pue | aj9e}da001 sajoquap) ands 300} }SOWLI9}NO saouaiajoy | uonNgysig jewuar) BUIse/ jeulwias sas0ydoury mer jepney pue ajueyy pue J9UU| einpey saiseds ‘DEDYIUDAGOANA] JO SAIDads Jo soainyeay SIJSOUseIP Jo ATBUIWUNS “TY I[qQUL I xIpueddy zse.pensoft.net 59 Zoosyst. 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