Mar Biodiv DOI 10.1007/s12526-017-0676-5 ORIGINAL PAPER Integrative systematics of the genus Limacia O. F. Müller, 1781 (Gastropoda, Heterobranchia, Nudibranchia, Polyceridae) in the Eastern Pacific Roberto A. Uribe1 & Fabiola Sepúlveda2 & Jeffrey H. R. Goddard3 & Ángel Valdés4 Received: 6 December 2016 /Revised: 22 February 2017 /Accepted: 27 February 2017 # Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2017 Abstract Morphological examination and molecular analy- from Baja California to Panama. Species delimitation analyses ses of specimens of the genus Limacia collected in the based on molecular data and unique morphological traits from Eastern Pacific Ocean indicate that four species of Limacia the dorsum, radula, and reproductive systems are useful in occur in the region. Limacia cockerelli,previouslyconsidered distinguishing these species to range from Alaska to Baja California, is common only in the northern part of its former range. An undescribed Keywords Mollusca . New species . Molecular taxonomy . pseudocryptic species, previously included as L. cockerelli, Pseudocryptic species occurs from Northern California to the Baja California Peninsula and is the most common species of Limacia in Southern California and Northern Mexico. Another new spe- Introduction cies similar to L. cockerelli is described from Antofagasta, Chile and constitutes the first record of the genus Limacia in Molecular markers have become a powerful tool in tax- the Southeastern Pacific Ocean. These two new species are onomy, systematics and phylogeny, allowing researchers formally described herein. Finally, Limacia janssi is a genet- to assess whether morphological variations correspond to ically and morphologically distinct tropical species ranging different species or merely represent intra-specific pheno- typic expression due to environmental variation (Hebert Communicated by V. Urgorri et al. 2004;Radulovicietal.2010). Recent use of these This article is registered in ZooBank under urn:lsid:zoobank.org:pub: markers has helped reveal high levels of cryptic species A97ACF68-C637-4507-AF42-6A0065014FE5 diversity in heterobranch sea slugs from the Eastern Electronic supplementary material The online version of this article Pacific Ocean, including sacoglossans (Krug et al. (doi:10.1007/s12526-017-0676-5) contains supplementary material, 2007), cephalaspideans (Cooke et al. 2014), and nudi- which is available to authorized users. branchs (Hoover et al. 2015;LindsayandValdés2016; Kienberger et al. 2016; Lindsay et al. 2016). Some of * Ángel Valdés these newly described taxa resulted from splitting wide- [email protected] spread species in the Northeastern Pacific into two sister species, mainly allopatric, with limited overlap near the 1 Laboratorio de Biodiversidad y Ecología Bentónica, Instituto del Mar del Perú – IMARPE, ChimboteLos Pinos s/n. Urb. Nueva Caleta, SanFranciscoBayArea(Krugetal.2007;Lindsayand Áncash, Perú Valdés 2016) or further north (Kienberger et al. 2016). 2 Laboratorio de Ecología Parasitaria y Epidemiología Marina However, less often, newly discovered cryptic species LEPyEM, Facultad de Ciencias del Mar y Recursos Biológicos, pairs are sympatric along most of their ranges (Hoover Universidad de Antofagasta, Antofagasta, Chile et al. 2015; Lindsay et al. 2016). These differences raise 3 Marine Science Institute, University of California, Santa intriguing questions about the mechanisms of speciation Barbara, CA 93106, USA (allopatric vs. ecological) involved in producing those 4 Department of Biological Sciences, California State Polytechnic species pairs, as well as the possible existence of biogeo- University, 3801 West Temple Avenue, Pomona, CA 91768, USA graphic barriers that could promote allopatric divergence. Mar Biodiv Most of these newly discovered cryptic species pairs are Materials and methods restricted to the Northeastern Pacific; very little is known about the molecular systematics and biogeography of Source of specimens Southeastern Pacific taxa, or their relationships with morpho- logically similar northern species. Some Southern From 2014 to 2016, nine specimens of Limacia cockerelli Hemisphere temperate species display resemblances with were collected from rocky intertidal and subtidal sites on the northern taxa (e.g., Polycera alabe Collier & Farmer, 1964, Eastern Pacific coast: seven from the Northeast Pacific coast Rostanga pulchra MacFarland, 1905), and have been sug- (four from Oregon and three from California) and two from gested to belong to the same species (e.g., Schrödl 2003; the Southeast Pacific coast (Bolsico Cave, Antofagasta, north- Schrödl and Grau 2006). However, temperate northern and ern Chile). The specimens from Chile were collected on a southern regions are separated by the Panamic barren ground system at 5 m depth. Additionally, two speci- Biogeographic Province, a 4,000 km-long stretch of tropical mens of Limacia janssi from San Carlos and Bahía waters from the mouth of the Gulf of California to the Gulf of Magdalena, Mexico, were collected, examined and se- Guayaquil (Briggs and Bowen 2012). In fact, the only pub- quenced. Samples were preserved in 95–99% ethanol and lished molecular study of a species found in both hemispheres deposited at the California State Polytechnic Invertebrate confirms that southern records of Aeolidia papillosa Collection (CPIC), the Natural History Museum of Los (Linnaeus, 1761) constitute a distinct, cryptic, endemic spe- Angeles County (LACM) and the Sala de Colecciones cies from Chile (Kienberger et al. 2016). Biológicas, Universidad Católica del Norte, Chile In this paper we examine the Eastern Pacific species of the (SCBUCN). Details on collection localities and dates as well genus Limacia O. F. Müller, 1781, which include northern tem- as museum registration numbers are provided in the material perate, tropical, and newly discovered southern temperate pop- examined section for each species. Additional specimens from ulations. The genus Limacia is a group of polycerid dorid nu- the LACM were examined morphologically but were unsuit- dibranchs characterized by having a unique body plan, with one able for molecular work. Photographs of the type material of to several rows of elongate dorso-lateral appendages surround- Limacia cockerelli were obtained from the National Museum ing the entire notum. Species of the genus Limacia occur in a of Natural History, Smithsonian Institution (USNM). handful of mainly temperate but also tropical disjunct areas, including Western and Southern Africa [Limacia lucida (Stimpson, 1854), Limacia annulata Vallès, Valdés & Ortea, DNA extraction and amplification 2000], the Eastern Pacific [Limacia cockerelli (MacFarland, 1905), Limacia janssi (Bertsch & Ferreira, 1974)], the Eastern For molecular analyses, partial sequences of the mitochondrial Atlantic and Mediterranean [Limacia clavigera (O. F. Müller, genes cytochrome c oxidase subunit I (COI) and 16S were 1776), Limacia iberica Caballer, Almón & Pérez, 2016] and amplified using pairs LCO and HCO (Folmer et al. 1994)and the Western Pacific [Limacia ornata (Baba, 1937)]. A possibly 16Sar-L and 16Sbr-H (Palumbi et al. 1991). The 16S rRNA undescribed species appears to be widespread in the tropical region is more conserved, and it is used for phylogenetic anal- Indian Ocean, from Eastern Australia to Tanzania (Gosliner yses, instead. COI mtDNA has a higher mutation accumulation et al. 2008;Goslineretal.2015) but is rare. In the Eastern rate and is commonly used in species delimitation analyses Pacific, L. cockerelli has a broad geographic range from (Hebert et al. 2004). The DNA of each individual was isolated Alaska to Baja California and displays considerable color var- following a modified protocol based on Miller et al. (1988) iation (McDonald & Nybakken 1980; Behrens and Hermosillo involving treatment with sodium dodecyl sulfate, digestion 2005), making it a potential candidate for a species complex. In with Proteinase K, NaCl protein precipitation, and subsequent contrast, L. janssi is restricted to the Panamic tropical region ethanol precipitation of the DNA. Modifications included cen- and is less variable (Behrens and Hermosillo 2005). Newly trifugation at 10 °C, maintaining ethanol at −20 °C and adding collected specimens from Chile are morphologically similar to the last step in the protocol a wash with 70% ethanol. to L. cockerelli and appear to belong to the same species, but Each PCR reaction included 0.175 μl of GoTaq DNA po- this needs anatomical and molecular confirmation. lymerase (Promega, Madison, USA), 7 μl of 5 × PCR buffer, In order to better understand the biological diversity and 5.6 μlofMgCl2 (25 mM), 2.1 μl of BSA (10 mg/ml), 0.7 μlof phylogeny of the Eastern Pacific species of the genus Limacia deoxynucleotide triphosphate (dNTP) (10 mM), 1.4 μlofeach we examined specimens belonging to both L. cockerelli and primer (10 pM) and 5 μl of template DNA and 11.625 μlof L. janssi, covering most of their ranges and including the nuclease-free water. PCR conditions were as follows: 1) for newly collected specimens from Chile. We used an integrative COI – initial denaturation 94 °C, 5 min; 35 cycles of denatur- approach (molecular, morphological and ecological data when ation 94 °C, 1 min, annealing 44 °C, 30 s; extension 72 °C, available) in an attempt to unravel the systematics of this 1 min; final extension 72 °C, 7 min; 2) for 16S – initial dena- group and detect possible cryptic diversity. turation 94 °C, 2 min;