INTRODUCTIONTop

Runcinids are small heterobranch sea slugs with an average size of about 4 mm. The largest species known is Runcinida elioti (Baba, 1937) from Amakusa (Japan) which reaches a maximum length of 8 mm (Burn 1963Burn R. 1963. Australian Runcinacea (Mollusca, Gastropoda). Aust. Zool. 13: 9-22.). These slugs inhabit intertidal and shallow rocky shores and are specialized herbivores, feeding on macrophytic algae (Burn 1963Burn R. 1963. Australian Runcinacea (Mollusca, Gastropoda). Aust. Zool. 13: 9-22., Thompson and Brodie 1988Thompson T.E., Brodie G. 1988. Eastern Mediterranean Opisthobranchia: Runcinidae (Runcinacea), with a review of Runcinid classification and a description of a new species from Fiji. J. Moll. Stud. 54: 339-346., Schmekel and Cappellato 2001Schmekel L., Capellato D. 2001. Contributions to the Runcinidae. I. Six new species of the genus Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 51: 141-160.). They are characterized by having an undivided dorsum, a foot lacking parapodial lobes, and an anus located next to the gill under the right posterior side of the mantle. An external or internal vestigial shell may be present, but it is absent in most species (Thompson 1976Thompson T.E. 1976. Biology of Opisthobranch Molluscs. Vol I. The Ray Society, London, 207 pp., Burn and Thompson 1998Burn R., Thompson T.E. 1998. Order Cephalaspidea. In: Beesley P.L, Ross G.J.B., Wells A. (eds), Mollusca: The Southern Synthesis. Fauna of Australia. Vol. 5. Part B, CSIRO Publishing, Melbourne, pp. 565-1234., Schmekel and Cappellato 2001Schmekel L., Capellato D. 2001. Contributions to the Runcinidae. I. Six new species of the genus Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 51: 141-160.).

The runcinids have traditionally been included in the order Cephalaspidea based on anatomical features such as nervous and reproductive systems (Ghiselin 1963Ghiselin T. 1963. On the functional and comparative anatomy of Runcina setoensis Baba, an opisthobranch gastropod. Publ. Seto Mar. Biol. Lab. 11: 389-398., Kress 1977Kress A. 1977. Runcina ferruginea n. sp. (Opisthobranchia: Cephalaspidea), a new runcinid from Great Britain. J. Mar. Biol. Assoc. UK 57: 201-211., Schmekel 1985Schmekel L. 1985. Aspects of evolution within the opisthobranchs. In Trueman E.R., Clarke M.R. (eds) The Mollusca, Evolution, 10. Academic Press, Orlando: New York. pp. 221-267.). However, Malaquias et al. (2009)Malaquias M.A.E., Mackenzie-Dodds J., Bouchet P., et al. 2009. A molecular phylogeny of the Cephalaspidea sensu lato (Gastropoda: Euthyneura): Architectibranchia redefined and Runcinacea reinstated. Zool. Scr. 38: 23-41., based on molecular phylogenetic analyses, demonstrated that runcinids were not part of the Cephalaspidea radiation but warrant their own ordinal assignment, a suggestion first proposed by Odhner (1968)Odhner N.H. 1968. Sous-Classe des Opisthobranches. In: Franc A., Grassé P.P. (ed.) Traité de Zoologie 5: 608-893, Masson, Paris. and later corroborated by Jörger et al. (2010)Jörger K.M., Stöger I., Kano Y., et al. 2010. On the origin of Acochlidia and other enigmatic euthyneuran gastropods, with implications for the systematics of Heterobranchia. BMC Evol. Biol. 10: 323., Wägele et al. (2014)Wägele H., Klussmann-Kolb A., Verbeek E., et al. 2014. Flashback and foreshadowing - a review of the taxon Opisthobranchia. Org. Divers. Evol. 14: 133-149. and Oskars et al. (2015)Oskars T.R., Bouchet P., Malaquias, M.A.E. 2015. A new phylogeny of the Cephalaspidea (Gastropoda: Heterobranchia) based on expanded taxon sampling and gene markers. Mol. Phylog. Evol. 89: 130-150..

The order Runcinida (Burn 1963Burn R. 1963. Australian Runcinacea (Mollusca, Gastropoda). Aust. Zool. 13: 9-22.) comprises two families, Runcinidae H. Adams and A. Adams, 1854 and Ilbiidae Burn, 1963 with nine and two genera, respectively. Within the family Runcinidae, Runcina is the most species-rich genus, with 38 valid species, of which 29 occur in European waters (Cervera et al. 2004Cervera J.L., Calado G., Gavaia C., et al. 2004. An annotated and updated checklist of the opisthobranchs (Mollusca: Gastropoda) from Spain and Portugal (including islands and archipelagos). Bol. Inst. Esp. Oceanogr. 20: 1-122., Schmekel and Cappellato 2002Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102., Ortea et al. 2015Ortea J., Moro L., Bacallado J.J. 2015. Babosas Marinas Canarias. Turquesa Ed., Tenerife, 144 pp.). The small size of these animals and the fact that most species have dark, dull cryptic colour patterns render the runcinids difficult to detect and identify.

One of the taxonomically difficult species of the European fauna is Runcina brenkoae Thompson, 1980, which, together with Runcina adriatica Thompson, 1980 and Runcina zavodniki Thompson, 1980, has been described from the Adriatic Sea. Runcina brenkoae is characterized by an elongated body with a characteristic pattern of anastomosing black blotches, a red-brown ground colour, clusters of chalk-white spots on both sides of the head behind the eyes, and presence of two gills. However, Thompson and Brodie (1988)Thompson T.E., Brodie G. 1988. Eastern Mediterranean Opisthobranchia: Runcinidae (Runcinacea), with a review of Runcinid classification and a description of a new species from Fiji. J. Moll. Stud. 54: 339-346. referred to specimens of R. brenkoae collected near Rovinj (Croatia), the type locality, which depicted several differences in respect to the original description: the presence of a developed crest, a pale fawn ground colour and the absence of white spots. Nevertheless, the specimens possessed key features of the species: the anastomosing black blotches and presence of only two gills. Schmekel and Cappellato (2002)Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102. reported the species outside the Adriatic Sea for the first time in Banuyls-sur-Mer (French Mediterranean coast) and Ballesteros et al. (2016)Ballesteros M., Madrenas E., Pontes M. 2016. Actualización del catálogo de los moluscos opistobranquios (Gastropoda: Heterobranchia) de las costas catalanas. Spira 6: 1-28. reported R. brenkoae in Catalonia (Spanish northeastern coast).

The use of integrative taxonomic approaches, and in particular of molecular phylogenetics, has revealed the existence of numerous species complexes and contributed to the discovery of unknown species among heterobranch sea slugs (Padula et al. 2014Padula V., Araújo A.K., Matthews-Cascon H., et al. 2014. Is the Mediterranean nudibranch Cratena peregrina (Gmelin, 1791) present on the Brazilian coast? Integrative species delimination and description of Cratena minor n. sp. J. Moll. Stud. 80: 575-584., Austin et al. 2018Austin J., Gosliner T., Malaquias M.A.E. 2018. Systematic revision, diversity patterns, and trophic ecology of the tropical Indo-West Pacific sea slug genus Phanerophthalmus A. Adams, 1850 (Cephalaspidea, Haminoeidae). Invertebr. Syst. 32: 1336-1387., Krug et al. 2018Krug P.J., Berriman J.S., Valdés Á. 2018. Phylogenetic systematics of the shelled sea slug genus Oxynoe Rafinesque, 1814 (Heterobranchia: Sacoglossa), with integrative descriptions of seven new species. Invertebr. Syst. 32: 950-1003., among others). The variable chromatic patterns described for R. brenkoae hint at yet another possible example of cryptic diversity masked under a single species name, but to date the taxonomy of this elusive species has only been studied on the basis of morphology.

Here we investigate for the first time the taxonomic status of the taxonomically difficult species Runcina brenkoae following an integrative approach combining multi-locus molecular phylogenetics and morpho-anatomical characters, based on specimens from the central and western Mediterranean Sea and the southern Iberian coastline of Portugal and Spain.

MATERIALS AND METHODSTop

Taxon sampling

Specimens identified as Runcina brenkoae were collected by the authors and colleagues from algae and seagrass or were obtained on loan from the Zoologische Staatssammlung München, Germany (ZSM). Specimens were photographed alive and preserved in 96% EtOH. The newly collected material was deposited at the Museo Nacional de Ciencias Naturales (MNCN), Madrid, Spain.

For the molecular analyses we also obtained sequences of Ilbia ilbi Burn, 1963 and additional Runcina species, namely R. adriatica Thompson, 1980, R. ferruginea Kress, 1977, R. hornae Schmekel and Cappellato, 2001 and R. coronata (Quatrefages, 1844), plus two specimens previously identified as Runcina cf. bahiensis Cervera, Garcia-Gomez and Garcia, 1991 and Runcina cf. hansbechi Schmekel and Cappellato, 2001. Furthermore, sequences of the runcinid Lapinura divae (Ev. Marcus and Er. Marcus, 1963), the acteonoid Micromelo undatus (Bruguière, 1792) and the aplysiid Aplysia dactylomela Rang, 1828 were obtained from GenBank and included in the analyses (Table 1).

DNA extraction, amplification and sequencing

Tissue samples were taken from the foot and DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA). Partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI), and 16S rRNA(16S), and nuclear histone H3 (H3) genes were amplified by polymerase chain reaction (PCR) using the universal primers LCO1490 and HCO2198 (Folmer et al. 1994Folmer R.O., Black M., Hoeh W., et al. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294-299. for COI); 16S ar-L and 16S br-H (Palumbi et al.1991Palumbi S.R., Martin A., Roman S., et al. 1991. The Simple Fools’ Guide to PCR. Dept. Zoology & Kewalo Laboratory, Univ. Hawaii, 46 pp. for 16S); and H3aF and H3aR (Colgan et al. 1998Colgan D., McLauchlan A., Wilson G.D.F., et al. 1998. Histone H3 and U2 snRNA DNA sequences and arthropod molecular evolution. Aust. J. Zool. 46: 419-437. for H3). PCRs were conducted in a 25 µl reaction volume containing 1 µl of both forward and reverse primers (10 µM), 2.5 µl of dNTP (2 mM), a gene-dependent amount of magnesium chloride (25 mM), 0.25 µl of Qiagen DNA polymerase (5 units/µl), 5 µl of “Q-solution” (5x), 2.5 µl of Qiagen buffer (10x) (Qiagen Taq PCR Core Kit) and 2 µl of genomic DNA. Amplification of COI was performed with an initial denaturation for 5 min at 94°C, followed by 35-36 cycles of 1 min at 94ºC, 30s at 45°C (annealing temperature) and 1 min at 72ºC, with a final extension of 10 min at 72°C. Amplification of 16S began with an initial denaturation for 5 min at 94°C, followed by 35–36 cycles of 1 min at 94°C, 30s at 42 and 49°C (annealing temperatures) and 1 min at 72°C, with a final extension of 10 min at 72ºC. Amplification of H3 was performed with an initial denaturation for 5 min at 94ºC, followed by 35 cycles of 1 min at 94°C, 30s at 52°C (annealing temperature) and 1 min at 72°C, with a final extension of 10 min at 72°C. Successful PCR products were sent to Macrogen, Inc for purification and sequencing on a 3730XL DNA sequencer (Applied Biosystems). All new DNA sequences have been deposited in GenBank (Table 1).

Phylogenetic analyses

Sequences were edited in Genious v10.2.3 (Drummond et al. 2009) and aligned using MAFFT (Katoh et al. 2009Katoh K., Asimenos G., Toh H. 2009. Multiple alignment of DNA sequences with MAFFT. Methods Mol. Biol. 537: 39-64.) implemented in Geneious v10.2.3 (Drummond et al. 2009Drummond A.J., Ashton B., Cheung M., et al. 2009. GENEIOUS v4.6. Available from: http://www.geneious.com/) with the default settings (Auto [FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i; depends on data size]). Sequences from the protein-coding genes COI and H3 were translated into amino acids to check for stop-codons. Hypervariable regions of the 16S alignment where homology could not be confidently established were removed using Gblocks under relaxed settings (Talavera and Castresana 2007Talavera G., Castresana J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Syst. Biol. 56: 564-577.). Nevertheless, analyses including and excluding these regions provided similar results. Therefore, final analyses were performed including all bases. Sequences of the COI, 16S and H3 genes were trimmed to 658, 457 and 328 nucleotides, respectively. All three genes were concatenated using Mesquite v3.2 (Maddison and Maddison 2018Maddison W.P., Maddison D.R. 2018. Mesquite: a modular system for evolutionary analysis.), resulting in a final dataset of 1443 base pairs. Single gene and concatenated (H3+COI+16S) analyses were performed. Saturation for the first, second and third codon positions of the COI and H3 genes were calculated in MEGA v7.0 (Kumar et al. 2016Kumar S., Stecher G., Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.).

The best-fit evolutionary model for each gene was determined in jModeltest v2.1.6 (Guindon and Gascuel 2003Guindon S., Gascuel O. 2003. A simple, fast, and accurate method to estimate large phylogenies by maximum-liklihood. Syst. Biol. 52: 696-704., Darriba et al. 2012Darriba D., Taboada G.L., Doallo R., et al. 2012. jModelTest2: more models, new heuristics and parallel computing. Nat. Methods 9: 696-704.) under the Akaike information criterion (Akaike 1974Akaike H. 1974. A new look at the statistical model identification. IEEE Transactions on Automatic Control 19: 716-723.). The GTR + G + I model was selected for the COI and H3 genes, and GTR + G for the 16S gene. Bayesian inference (BI) analyses were performed in MrBayes v. 3.2.1 (Ronquist and Huelsenbeck 2003Ronquist F., Huelsenbeck J.P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574.) with a random starting tree and two parallel runs of 107 generations. Convergence was checked in TRACER v1.7.1 (Rambaut et al. 2018Rambaut A., Drummond A.J., Xie D., et al. 2018. Posterior Summarization in Bayesian Phylogenetics Using Tracer 1.7. Syst. Biol. 67: 901-904.) with a burn-in of 25%. Nodes with a posterior probability (PP) ≥0.95 (Alfaro et al. 2003Alfaro M.E., Zoller S., Lutzoni F. 2003. Bayes or bootstraps? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Mol. Biol. Evol. 20: 255-266.) were considered well supported and discussed. Maximum likelihood (ML) analysis was executed using RAxML v8 (Stamatakis 2014Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic anaslysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313.) and node support was assessed with nonparametric bootstrapping (BS) with 5000 replicates. Nodes with bootstrap values (BS)≥70 (Hillis and Bull 1993Hillis D.M., Bull J.J. 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42: 182-192.) were considered significant and were discussed. Both BI and ML trees were visualized in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/). Minimum and maximum pairwise uncorrected p-distances of COI within and between species were calculated in MEGA v7.0 using all sequences available. (Kumar et al. 2016Kumar S., Stecher G., Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets.).

Species delimitation analyses

The Automatic Barcode Gap Discovery (ABGD) (Puillandre et al. 2012Puillandre N., Lambert A., Brouillet S., et al. 2012. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol. Ecol. 21: 1864-1877.) and Bayesian Poisson tree processes (bPTP) (Zhang et al. 2013Zhang J., Kapli P., Pavlidis P., et al. 2013. A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29: 2869-2876.) were used to aid delimitation of species. For the ABGD we used the alignment from the fast-evolving COI gene with default settings (Pmin=0.001, Pmax=0.1, Steps=10, X=1.2, Nb bins=20) under the three models of evolution available, namely Jukes-Cantor (JC69), Kimura (K80) and Simple Distance. The bPTP analysis is an updated version of the original maximum likelihood PTP (modelling speciation in terms of the number of substitutions), which adds Bayesian support values to delimit species. The bPTP analyses were run with the COI and 16S trees using the webserver (https://species.h-its.org/ptp/) (Zhang et al. 2013Zhang J., Kapli P., Pavlidis P., et al. 2013. A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29: 2869-2876.).

Morphology

To complete and compare the results obtained by molecular phylogenetics and species delimitation analyses, specimens previously identified as Runcina brenkoae and Runcina sp. from Croatia (Adriatic) (3), Catalonia (Mediterranean, Spain) (6), Cádiz (Atlantic, Spain) (1) and Algarve (Atlantic, Portugal) (5), and one specimen early identified as R. adriatica from Banyuls-sur-Mer (France) were studied for their morpho-anatomy. Animals were dorsally dissected and the buccal bulbs were extracted and dissolved in a solution of 10% sodium hydroxide to expose the radula. The radulae and gizzard plates were then immersed in water, dried and mounted for scanning electron microscopy (SEM) with a Nova NanoSEM 450 available at the University of Cádiz (Cádiz, Spain). The reproductive system was examined and drawn using a dissecting microscope with the aid of a camera lucida.

RESULTSTop

Phylogenetic analyses

The concatenated (H3+COI+16S) tree provided better resolution than the individual gene analyses (Fig. 1, and Supplementary material Figs S1, S2 and S3). No saturation was observed, even in the third codon position. Both BI and ML analyses supported the monophyly of the genus Runcina (PP=1; BS=100) and showed L. divae to be its sister lineage (PP=0.98; BS=86). The species Ilbia ilbi was rendered sister to the Lapinura + Runcina clade (PP=1; BS=100). In the Runcina clade the species R. ferruginea was rendered sister to a sub-clade containing all remaining species (PP=1; BS=80). The specimens previously identified as R. brenkoae split into three subclades all with maximum support (PP=1; BS=100). The first clade (Group A) includes specimens from Portugal; the second clade (Group B) includes one specimen previously identified as Runcina adriatica from France (Mediterranean) and specimens from Spain (Atlantic and Mediterranean); and the third clade (Group C) includes specimens from Croatia and Spain (Mediterranean) (Fig. 1).

Species delimitation analyses

The ABGD analysis of the COI sequences with all three models of evolution resulted in 11 groups with three of them corresponding to the same R. brenkoae groups, A, B and C, recovered in the BI and ML analyses (Fig 1A). However, the recursive partition, at lower values of prior intraspecific divergence (P), recovered seven groups for the “R. brenkoae complex”, separating specimens from Group A and C into two distinct groups each, and specimens from Group B into three distinct groups (not shown).

Regarding the COI uncorrected p-distances, the minimum distance was 11.7% between Groups A and B; 9.6% between Groups A and C; and 10.4% between Groups B and C. The maximum distance was 0% within specimens of Group A, 4% within Group B, and 4.6% within Group C (Table 2). Between species in the genus Runcina the COI uncorrected p-distances ranged from 9.3% to 15.1%, while between the genera Runcina and Lapinura they ranged from 16.3% to 20.7%. No COI gene sequences from Ilbia ilbi were available for this analysis. The results obtained with the bPTP analysis were congruent with the ABGD output in suggesting the same three groups of Runcina brenkoae (Fig 1B).

The molecular results support the occurrence of three species under the name Runcina brenkoae, and this hypothesis is backed by morphological differences across specimens from the three R. brenkoae clades (see Systematic description section). Therefore, we present below a redescription of R. brenkoae and we describe two new species.

Systematic descriptionTop

Family RUNCINIDAE H. Adams and A. Adams, 1854
Genus Runcina Forbes in Forbes and Hanley, 1851
Runcina brenkoae Thompson, 1980
(Figs 2, 5A-C, 6A, D)

Runcina brenkoae Thompson 1980Thompson T.E. 1980. New species of the bullomorph genus Runcina from the northern Adriatic Sea. J. Moll. Stud. 46: 154-157.: 156, fig. 1C. Thompson and Brodie 1988Thompson T.E., Brodie G. 1988. Eastern Mediterranean Opisthobranchia: Runcinidae (Runcinacea), with a review of Runcinid classification and a description of a new species from Fiji. J. Moll. Stud. 54: 339-346.: 340, fig. 1D. Schmekel and Capellato 2001Schmekel L., Capellato D. 2001. Contributions to the Runcinidae. I. Six new species of the genus Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 51: 141-160.: 144, Pl. I g; 145, Pl. II k; and 148, Pl. III a, b; Schmekel and Capellato 2002Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102.: 98, Pl. VI a-c. Ballesteros et al. 2016Ballesteros M., Madrenas E., Pontes M. 2016. Actualización del catálogo de los moluscos opistobranquios (Gastropoda: Heterobranchia) de las costas catalanas. Spira 6: 1-28.: 4, fig. 7A.

Type material. Holotype (NHMUK 197913W) Natural History Museum, London, UK (not studied because the material is only available as micro-slide preparations).

Type locality. Rovinj, Croatia.

Examined material. (MNCN 15.05/88086): Split, Croatia, 03 Aug 2014, 1.5 mm in length preserved, depth 1 m. Found washing Posidonia (dissected and sequenced). (MNCN 15.05/88087): Roses, Catalonia, Spain, coll. Marina Poddubetskaia, 08 Aug 2016, 1 mm in length preserved, depth 8 mm. Found on Posidonia (dissected, sequenced). (MNCN 15.05/88088): Nin, Croatia, coll. Alen Petani, 04 Apr 2017, 3.5 mm in length preserved, depth 0.5-1 m (dissected and sequenced). (MNCN 15.05/88090): Roses, Catalonia, Spain, coll. Marina Poddubetskaia, 19 Jul 2017, 1 mm in length preserved, depth 9 m (sequenced). (MNCN 15.05/88089): Nin, Croatia, coll. Alen Petani, 26 Dic 2017, 1.5 mm in length preserved, depth 0-1 m (sequenced).

External morphology (Fig. 2). Body moderately elongated and tapered. Notum smooth. Foot as wide as notum, showing a developed median pallial crest. Ground colour of body red-brown, sometimes translucent pale fawn bearing a pattern of anastomosing dark blotches on notum, margin and sole of foot. Eyes difficult to discern. Chalk-white spots all over body, more concentrated on margin of tail, both sides of head behind eyes and on metapodium in front of dark band. Some specimens with small red spots on margin of tail and surface of metapodium. The slugs have a longitudinal band of dark brown or wine-red colour on the surface of the metapodium. Two equal-sized translucent gills with white spots bearing pinnules on right posterior side of body. Anal pore situated beneath gills.

Internal anatomy (Figs 5A-C, 6A, D). Radular formulae 20 × 1.1.1 (MNCN 15.05/88086, MNCN 15.05/88088). Rachidian tooth boomerang-shaped with long, smooth lateral wings on each side. Central part of rachidian tooth bilobed; masticatory edge contains a pair of cockle-shaped rounded pads, each pad with 8-10 denticles. Median deep and broad depression is present between the pads; a small denticle may be present (Fig. 5A). Lateral teeth smooth, elongate and curved like a swan neck (Fig. 5B). Triangular jaws present. Four gizzard plates with 5-7 lamellae (Fig. 5C). Shell absent. Reproductive system monaulic. Female gland mass slightly divided into two lobes. Common genital duct connecting the female gland to the exterior on right posterior side of the body. Bursa copulatrix absent. Female gland placed on right posterior side of digestive gland (Fig. 6A). Male copulatory organ opens to the right of the mouth. Short and unarmed penial papilla projects into the atrium. Prostate gland long and cylindrical. Slender seminal vesicle with half size of prostate gland (Fig. 6D).

Runcina lusitanica n. sp.
(Figs 3, 5D-F, 6B, E)
http://zoobank.org/FAECCA78-B65B-47E6-8081-B2ABA0020F70

Examined material. Holotype (MNCN 15.05/200065): Near Faro, Algarve, Portugal, coll. Jorge Antonio Domínguez Godino, May 2015, 4 mm in length preserved (dissected and sequenced). Paratypes (MNCN 15.05/88091): Near Faro, Algarve, Portugal, coll. Jorge Antonio Domínguez Godino, May 2015, 5 mm in length preserved (dissected and sequenced). (MNCN 15.05/88092): Near Faro, Algarve, Portugal, coll. Jorge Antonio Domínguez Godino, May 2015, 5 mm in length preserved (dissected and sequenced). (MNCN 15.05/88093): Near Faro, Algarve, Portugal, coll. Jorge Antonio Domínguez Godino, May 2015, 4.5 mm in length preserved (dissected and sequenced). (MNCN 15.05/88094): Near Faro, Algarve, Portugal, coll. Jorge Antonio Domínguez Godino, May 2015, 4.5 mm in length preserved (dissected and sequenced).

Etymology. Lusitania was the name of a Roman province in the west of the Iberian Peninsula that occupied much of what now is Portugal.

External morphology (Fig. 3). Body elongated and moderately broad. Notum smooth. Foot as wide as notum. Posterior part of notum rounded without pallial crest. Ground colour of body brown and translucent yellowish bearing a pattern of anastomosing dark blotches on notum and margin of foot. Some specimens have a large pale fawn patch on the posterior part of head and notum. Eyes not visible. White spots on some specimens. Longitudinal band, sometimes wide, of dark brown colour on surface of metapodium. Two large, yellowish gills with dark spots bearing irregular pinnules on right posterior side of body. Upper gill unipinnate and the most ventral bipinnate. Anal pore situated beneath gills.

Internal anatomy (Figs 5D-F, 6B, E). Radular formulae 25 × 1.1.1 (MNCN 15.05/88092) and 29 × 1.1.1 (MNCN 15.05/88093). Rachidian tooth boomerang shaped with one long and smooth lateral wing on each side. Central part of rachidian tooth bilobed; masticatory edge contains a pair of flat, comb-shaped pads, each one possessing 10-12 denticles. Median deep and broad depression is present between the pads; a small denticle present (Fig. 5D). Lateral teeth smooth, elongate and curved like a swan neck (Fig. 5E). Triangular jaws present. Four gizzard plates with 10-11 lamellae (Fig. 5F). Shell absent. Reproductive system monaulic. Female gland mass divided into two lobes, located on right side and behind the digestive gland. Bursa copulatrix absent. Common genital duct opening to exterior on right posterior side of body (Fig. 6B). Male copulatory organ comprises a relatively large atrium, which opens on right side next to mouth. Short, unarmed, conical penial papilla projects inside atrium. Long and cylindrical prostate gland. Elongated and convoluted seminal vesicle (Fig. 6E).

Runcina marcosi n. sp.
(Figs 4, 5G-I, 6C, F, G)
http://zoobank.org/1E0B605C-C403-41F4-881B-3439F2D9C41C

Examined material. Holotype (MNCN 15.05/200066): La Caleta (Cádiz), Andalusia, southwestern Spain, coll. Josep Romà, 17 May 2015, 2.5 mm in length preserved, depth 0.5 – 1 m. Found on samples of the brown algae Halopteris scoparia (dissected and sequenced). Paratypes (ZSM MOL 201442089): Banyuls-sur-Mer, France (Mediterranean), coll. Bastian Brenzinger and Timea Neusser, 02 Jul 2014, 1.3 mm in length preserved (sequenced). (MNCN 15.05/88095): Mataró, Catalonia, northeastern Spain, coll. Manuel Ballesteros, 22 Sep 2015, 1 mm in length preserved. Found on green algae (dissected and sequenced). (MNCN 15.05/88096): Roses, Catalonia, northeastern Spain, coll. Carles Galià, 29 May 2017, 1 mm in length preserved. Found on roots of seagrass Posidonia oceanica (dissected and sequenced). (MNCN 15.05/88098): Roses, Catalonia, northeastern Spain, coll. Marina Poddubetskaia, 30 Aug 2017, 1.5 mm in length preserved (dissected and sequenced). (MNCN 15.05/88097): Roses, Catalonia, northeastern Spain, coll. Marina Poddubetskaia, 19 Jul 2018, 2 mm in length preserved, depth 4 m (sequenced).

Etymology. This species is dedicated to Marcos Martínez Vazquez, husband of the first author, for all his help, enthusiasm and support during the course of this work.

External morphology (Fig. 4). Body moderately elongated. Notum smooth. Foot as wide as notum. Some specimens show developed median pallial crest. Ground colour of body red-brown or translucent pale fawn bearing a pattern of anastomosing dark or reddish blotches on notum, margin of foot and metapodium. Eyes difficult to discern. White spots all over the body. Longitudinal band of dark brown or wine-red colour on surface of metapodium. Two translucent gills bearing regular pinnules on right posterior side of body. Upper gill unipinnate and the most ventral bipinnate. Anal pore situated beneath gills.

Internal anatomy (Fig. 5G-I, 6C, F, G). Radular formulae 10 x 1.1.1 (MNCN 15.05/88097) and 13 × 1.1.1 (MNCN 15.05/88095). Rachidian tooth boomerang-shaped with long and smooth lateral wings on each side. Central part of rachidian tooth bilobed; masticatory edge contains a pair of flat, comb-shaped pads, each one with 10-11 denticles. Median deep and broad depression present between the pads; small denticle absent (Fig. 5G). Lateral teeth smooth, elongate and curved like a swan neck (Fig. 5H). Triangular jaws present. Four gizzard plates with 7-8 lamellae (Fig. 5I). Shell absent. Reproductive system monaulic. Female gland mass placed on right side and behind the digestive gland. Divided into two lobes, perhaps albumen and mucous glands. Long common genital duct connects the female gland to exterior on right posterior side of body. Bursa copulatrix absent (Fig. 6C). Elongated and cylindrical male copulatory organ. Atrium opens to right side of mouth. Short and unarmed penial papilla projects into the atrium. Cylindrical prostate gland. Slender seminal vesicle with half size of prostate gland (Fig. 6F, G).

DISCUSSIONTop

Recent molecular studies on heterobranch sea slugs, mostly nudibranchs, have demonstrated the existence of many complexes of cryptic species (Austin et al. 2018Austin J., Gosliner T., Malaquias M.A.E. 2018. Systematic revision, diversity patterns, and trophic ecology of the tropical Indo-West Pacific sea slug genus Phanerophthalmus A. Adams, 1850 (Cephalaspidea, Haminoeidae). Invertebr. Syst. 32: 1336-1387., Layton et al. 2018Layton K.K.S., Gosliner T.M., Wilson N.G. 2018. Flexible colour patterns obscure identification and mimicry in Indo-Pacific Chromodoris nudibranchs (Gastropoda: Chromodorididae). Mol. Phylog. Evol. 124: 27-36., Korshunova et al. 2019Korshunova T.A., Picton B., Furfaro G., et al. 2019. Multilevel fine-scale diversity challenges the ‘cryptic species’ concept. Sci. Rep. 9: 6732., among many others). Up to now, most studies related to the order Runcinida have focused only on morphological aspects in order to identify and describe new species and genera (Cervera et al. 1991Cervera J.L., García-Gómez J.L., García F.J. 1991. The genus Runcina Forbes and Haley, 1851 (Opisthobranchia: Cephalaspidea) in the Strait of Gibraltar, with the description of a new species from the bay of Algeciras. J. Moll. Stud. 57: 199-208., Chernyshev 2006Chernyshev A.V. 2006. New data on mollusks of the family Runcinidae (Gastropoda: Opisthobranchia) from Russian Far Eastern seas. Bull. Russian 10: 122-125., Moro and Ortea 2015Moro L., Ortea J. 2015. Nuevos taxones de babosas marinas de las islas Canarias y de Cabo Verde (Mollusca: Heterobranchia). Vieraea 43: 21-86.). Our contribution is the first to use molecular phylogenetics combined with morphology to test the status of taxonomically difficult European runcinids, with a focus on the Runcina brenkoae species complex. Our study recognized three distinct species within this complex, namely R. brenkoae Thompson, 1980 proper and two new species described here as R. marcosi n. sp. and R. lusitanica n. sp. (Table 3).

Externally, all species of this complex are similar in colour, but R. marcosi n. sp., despite its chromatic variability, has a characteristic concentration of white spots on the anterior part of the body forming a “necklace”. R. brenkoae is the only one among the three species of the complex with both gills unipinnate, whereas R. lusitanica n. sp. and R. marcosi n. sp. have one gill unipinnate and the other bipinnate. R. lusitanica n. sp. reaches comparatively larger sizes (up to 5 mm in length in preserved animals), but overlaps chromatically with R. brenkoae. R. marcosi n. sp. shows a considerable chromatic variation and, in fact, some individuals can be confused with R. adriatica, which has chalk-white spots on the pallial crest and behind the eyes forming a “necklace” (Thompson 1980Thompson T.E. 1980. New species of the bullomorph genus Runcina from the northern Adriatic Sea. J. Moll. Stud. 46: 154-157., Thompson and Brodie 1988Thompson T.E., Brodie G. 1988. Eastern Mediterranean Opisthobranchia: Runcinidae (Runcinacea), with a review of Runcinid classification and a description of a new species from Fiji. J. Moll. Stud. 54: 339-346.). However, R. adriatica has three gills (two bipinnate and one unipinnate) and a higher number of radular rows (21 × 1.1.1) (Thompson 1980Thompson T.E. 1980. New species of the bullomorph genus Runcina from the northern Adriatic Sea. J. Moll. Stud. 46: 154-157.).

Anatomically these species differ in subtle details of the radula and gizzard plates. The pads of the rachidian tooth are more oval in shape in R. brenkoae, as observed by Schmekel and Cappellato (2001Schmekel L., Capellato D. 2001. Contributions to the Runcinidae. I. Six new species of the genus Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 51: 141-160., 2002)Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102., whereas in R. marcosi n. sp. and R. lusitanica n. sp. these pads are more flattened. In R. lusitanica n. sp. and R. brenkoae, a small denticle is present in the depression between the two pads, but it may be absent in some rows. The gizzard plates of R. brenkoae have 5-6 lamellae, while in R. marcosi n. sp. and R. lusitanica n. sp. they have 7-8 and 10-11 lamellae, respectively.

The male copulatory organ of the runcinids consists of a penial papilla projecting into an atrium, a prostate gland, and a seminal vesicle (Vayssière 1883Vayssière A. 1883. Recherches anatomiques sur les genres Pelta (Runcina) et Tylodina. Ann. Sci. Nat. Zool. 15: 1-46., Kress 1977Kress A. 1977. Runcina ferruginea n. sp. (Opisthobranchia: Cephalaspidea), a new runcinid from Great Britain. J. Mar. Biol. Assoc. UK 57: 201-211., Burn and Thompson 1998Burn R., Thompson T.E. 1998. Order Cephalaspidea. In: Beesley P.L, Ross G.J.B., Wells A. (eds), Mollusca: The Southern Synthesis. Fauna of Australia. Vol. 5. Part B, CSIRO Publishing, Melbourne, pp. 565-1234.). The male copulatory organ does not differ much between R. brenkoae and R. marcosi n. sp. The prostate is more curved in R. brenkoae than in R. marcosi n. sp., and the seminal vesicle in R. brenkoae is more rounded on one of the sides. Thompson (1980)Thompson T.E. 1980. New species of the bullomorph genus Runcina from the northern Adriatic Sea. J. Moll. Stud. 46: 154-157. did not mention any aspect of the male organ of R. brenkoae, nor did Thompson and Brodie (1988)Thompson T.E., Brodie G. 1988. Eastern Mediterranean Opisthobranchia: Runcinidae (Runcinacea), with a review of Runcinid classification and a description of a new species from Fiji. J. Moll. Stud. 54: 339-346., and Schmekel and Cappellato (2002)Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102. only reported that the copulatory organ of R. brenkoae was similar to that of R. ferruginea, which has the same basic anatomical structure as the species described here. In R. lusitanica n. sp. the penial papilla is larger than in R. brenkoae and R. marcosi and the posterior end of the cylindrical prostate narrows slightly into a very long and twisted seminal vesicle, which is not present in R. brenkoae and R. marcosi n. sp.

The female part of the reproductive system in runcinids consists of an albumen and mucous gland opening to the outside through a common genital duct (Vayssière 1883Vayssière A. 1883. Recherches anatomiques sur les genres Pelta (Runcina) et Tylodina. Ann. Sci. Nat. Zool. 15: 1-46., Kress 1977Kress A. 1977. Runcina ferruginea n. sp. (Opisthobranchia: Cephalaspidea), a new runcinid from Great Britain. J. Mar. Biol. Assoc. UK 57: 201-211., Burn and Thompson 1998Burn R., Thompson T.E. 1998. Order Cephalaspidea. In: Beesley P.L, Ross G.J.B., Wells A. (eds), Mollusca: The Southern Synthesis. Fauna of Australia. Vol. 5. Part B, CSIRO Publishing, Melbourne, pp. 565-1234.). However, the presence of an ampulla and bursa copulatrix have been described for the species Runcina macfarlandi (Gosliner, 1991), R. coronate and Ilbia ilbi, among others (Vayssière 1883Vayssière A. 1883. Recherches anatomiques sur les genres Pelta (Runcina) et Tylodina. Ann. Sci. Nat. Zool. 15: 1-46., Burn 1963Burn R. 1963. Australian Runcinacea (Mollusca, Gastropoda). Aust. Zool. 13: 9-22., Gosliner 1991Gosliner T.M. 1991. Four new species and a new genus of opisthobranch gastropods from the Pacific coast of North America. The Veliger 34: 272-290.). All three species of the R. brenkoae complex have similar female glands and we were unable to recognize an ampulla and bursa copulatrix. In general, the female part of the reproductive system in runcinids is poorly studied and, for example, Thompson (1980)Thompson T.E. 1980. New species of the bullomorph genus Runcina from the northern Adriatic Sea. J. Moll. Stud. 46: 154-157., Thompson and Brodie (1988)Thompson T.E., Brodie G. 1988. Eastern Mediterranean Opisthobranchia: Runcinidae (Runcinacea), with a review of Runcinid classification and a description of a new species from Fiji. J. Moll. Stud. 54: 339-346. and Schmekel and Cappellato (2002)Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102. never referred to it.

Our study suggests that the geographical distribution of Runcina brenkoae proper is restricted to the Adriatic Sea (Croatia) and to the western Mediterranean (Spain and France), where it overlaps with the species R. marcosi n. sp., at least in northeastern Spain (Mediterranean Sea). Schmekel and Cappellato (2001Schmekel L., Capellato D. 2001. Contributions to the Runcinidae. I. Six new species of the genus Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 51: 141-160., 2002)Schmekel L., Capellato D. 2002. Contributions to the Runcinidae. II. Three new species and comparative studies on five stablished species of Runcina (Opisthobranchia Cephalaspidea) in the Mediterranean. Vie Milieu 52: 85-102. referred to its presence in Banyuls-sur-Mer (French Mediterranean coast) but their specimens were initially fixed in formalin (Ronald Janssen, pers. comm., Senckenberg Research Institute and Natural History Museum) and could not be tested for DNA. Thus, under the present taxonomic scenario the identity of these samples remains doubtful. The species R. lusitanica n. sp. is so far only known from the southern coast of Portugal. The distribution of R. marcosi n. sp. is restricted to southwestern Spain (Atlantic) and the western Mediterranean (Spain and France).

The present study is the first to evaluate the taxonomy of European species of runcinids using DNA data and to expose the occurrence of cryptic diversity among previously well-established species. Runcinids are small animals on average less than 5 mm in length, mostly with dull colour patterns, which complicates their identification and taxonomy. Runcinids clearly lack and will benefit from a DNA barcoding and molecular phylogenetics approach that could characterize the species molecularly, establishing a framework for understanding the value of colour patterns and morphological characters and their systematics.

ACKNOWLEDGEMENTSTop

We are grateful to all colleagues who provided specimens and photographs for this study, including Alen Petani, Jakov Prkić, Carles Galià, Marina Poddubetskaia, Josep Romà and Jorge Antonio Domíguez Godino. We thank Bastian Brenzinger and Melanie Mackenzie for lending specimens of the ZSM and Museum Victoria collections, respectively. We also thank Juan González (SC-ICYT, UCA) for assistance with SEM. This study was supported by two research projects, “La Caleta (Cádiz) e intermareales rocosos asociados: una ventana permanente al conocimiento de la biodiversidad marina” funded by Fundación Biodiversidad of the Ministry for Ecological Transition, and “Desentrañando la diversidad criptica en las regiones Lusitánica y Mediterránea: Heterobranquios marinos (Mollusca), Sílidos (Annelida) y Caprélidos (Arthropoda, Pancrustacea) como casos de estudio” funded by the University of Cádiz (PR2018-039) through a grant to J.L. Cervera. A. K. Araujo holds a PhD grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq/Brazil (Processo 205276/2014-8).

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SUPPLEMENTARY MATERIAL

The following supplementary material is available through the online version of this article and at the following link:
http://scimar.icm.csic.es/scimar/supplm/sm04907esm.pdf

Fig. S1. – Phylogenetic hypothesis based on BI of the H3 gene. Numbers on the left of the slash are posterior probabilities and those on the right bootstrap values derived from maximum likelihood. Unsupported branches not labelled.

Fig. S2. – Phylogenetic hypothesis based on BI of the COI gene. Numbers on the left of the slash are posterior probabilities and those on the right bootstrap values derived from maximum likelihood. Unsupported branches not labelled.

Fig. S3. – Phylogenetic hypothesis based on BI of the 16S gene. Numbers on the left of the slash are posterior probabilities and those on the right bootstrap values derived from maximum likelihood. Unsupported branches not labelled.