Integrative Systematics of the Genus Limacia in the Eastern Pacific

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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 morphologically 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; 35 cycles of denaturation 94 °C, 30 s, Mar Biodiv annealing 50 °C, 30 s; extension, 72 °C, 1 min; final extension package MrBayes (Huelsenbeck and Ronquist 2001) with 6 72 °C, 7 min. substitution types for 16S (nst = 6) and 2 substitution types PCR products were cleaned using Ultra clean kit (Mobio), for COI (nst = 2), and considering gamma distributed rate var- and both DNA strands were sequenced directly at Macrogen iation as well as the proportion of invariable positions, accord- (Seoul, Korea; http://www.macrogen.com). Complementary ing to the evolutionary model determined by jModeltest for sequences were assembled and edited using ProSeq v2.9 each gene (GTR + I + G and HKY + I + G respectively). The (Filatov 2002). The fragments obtained were aligned with BI concatenated analysis was partitioned by genes. All BI anal- sequences of species of the genus Limacia obtained from yses included two runs of six chains for 10 million generations, GenBank using the Clustal 2 software package (Larkin et al. sampling every 1,000 generations and a burn-in of 25%. ML 2007). All new DNA sequences have been deposited in analyses were performed using the software package RaXML GenBank, and the accession numbers are given in Table 1. (Stamatakis 2006) with the GTR + G evolution model, which was the best fit for the entire alignment. To determine the nodal support in ML a 10,000 bootstrap analysis was implemented. Phylogenetic analyses

COI and 16S sequences were trimmed to 603 and 458 base Species delimitation analyses pairs, respectively. Gene concatenation (COI + 16S = 1061 bp) was performed in Mesquite v.2.75 (Maddison and Maddison In order to compare the genetic distances among specimens of 2011). The software jModelTest 0.1 (Posada 2008)was Limacia, we calculated the pairwise p-distances (between in- employed to determine the best-fit nucleotide substitution mod- dividual sequences and in average by species) for 16S and el for each gene and for the entire alignment accompanying COI using MEGA 6 (Tamura et al. 2013). evolutionary parameter values for the data under the Akaike The COI gene was used to aid in determining the number information criterion (Akaike 1974). Bayesian inference (BI) of species present in the L. cockerelli species complex, using and maximum likelihood (ML) analyses were conducted for the approximation of delineation of species boundaries in the the concatenated data set as well as for individual genes (COI Automatic Barcode Gap Discovery method (ABGD) and 16S). Triopha catalinae (Cooper, 1863) and T. maculata (Puillandre et al. 2012). This method estimates the distribution MacFarland, 1905 were selected as the outgroups based on of pairwise genetic distances between the aligned sequences their close phylogenetic relationship with the genus Limacia. and then it statistically infers multiple potential barcode gaps Sequences of the outgroup taxa were obtained from GenBank as minima in the distribution of pairwise distances, thereby (Table 1). BI analyses were performed using the software partitioning the sequences such that the distance between

Table 1 Specimens used for molecular analyses, including locality data, GenBank accession numbers and museum voucher numbers

Species Locality Voucher GenBank Accession No. Source

COI 16S

Limacia cockerelli Middle Cove, Cape Arago, Oregon, USA SCBUCN 4606 KX673492 KX673501 This study Limacia cockerelli Whiskey Creek, Oregon, USA SCBUCN 4607 KX673491 KX673502 This study Limacia mcdonaldi sp. nov. Carpinteria, California, USA SCBUCN 4608 KX673494 KX673500 This study Limacia mcdonaldi sp. nov. Carpinteria, California, USA SCBUCN 4609 KX673495 KX673499 This study Limacia mcdonaldi sp. nov. Carpinteria, California, USA - KX673493 - This study Limacia mcdonaldi sp. nov. Carmel Point, California, USA CPIC 01885 KY622051 KY622049 This study Limacia antofagastensis sp. nov. Antofagasta, Chile LACM 3356 KX673496 - This study Limacia antofagastensis sp. nov. Antofagasta, Chile LACM 3357 KX673497 KX673498 This study Limacia janssi Bahía Magdalena, Mexico - - KX673503 This study Limacia janssi San Carlos, Mexico CPIC 01503 KY622050 KY622048 This study Limacia sp. 1 False Bay, South Africa CASIZ 176312 HM162692 HM162602 Pola and Gosliner 2010 Limacia sp. 2 Cape Province, South Africa CASIZ 176276 HM162693 HM162603 Pola and Gosliner 2010 Limacia clavigera Cadiz, Spain MNCN EF142906 EF142952 Pola et al. 2007 15.05/46736 Limacia clavigera Kristineberg, Bohuslän, Sweden - AJ223268 AJ225192 Thollesson 2000 Triopha catalinae San Francisco, California, USA CASIZ 170648 HM162690 HM162600 Pola and Gosliner 2010 Triopha maculata Marin County, California, USA CASIZ 181556 HM162691 HM162601 Pola and Gosliner 2010 Mar Biodiv two sequences taken from distinct clusters will be larger than morphology were used as the main internal traits for spe- the barcode gap (Puillandre et al. 2012). For this analysis cies identification and characterization. The reproductive seven sequences of specimens initially identified as organs were examined by removing them from the animal L. cockerelli were used: two from Chile, three from through a dorsal incision and drawn under a Nikon SMZ- California and two from Oregon. 100 dissecting microscope with a camera lucida attach- COI alignments were uploaded at http://wwwabi.snv. ment. The penises were removed, mounted on a micro- jussieu.fr/public/abgd/abgdweb.html and ABGD was run scope slide and drawn while viewed with an Olympus with the default settings (Pmin = 0.001, Pmax = 0.1, Steps = CX31 compound microscope utilizing a camera lucida 10, X (relative gap width) = 1.5, Nb bins = 20) and with K2P attachment. The buccal mass of each specimen examined distances. was removed and the tissue surrounding the jaws and radula was dissolved using 10% sodium hydroxide Morphological examination (NaOH). Jaws and radulae were rinsed in water, dried, mounted, and sputter coated for examination under a At least two specimens of each putative species were dis- Jeol JSM-6010 variable pressure SEM at the California sected. The reproductive anatomy and radular State Polytechnic University.

Fig. 1 Bayesian consensus phylogenetic tree based on the concatenated (Bayesian) are included. The blue clade represents samples from Chile, molecular data (COI + 16S) for species of the genus Limacia.Numbers the red clade represents samples from California and the green clade on the branches represent bootstrap support values for ML and posterior represents samples from Oregon probabilities of Bayesian inference, only values > 50 (ML) and > 0.5 Mar Biodiv 79) 76) Results

Phylogenetic analyses 13.2– (78 12.7– (75 – – 9.5 (57) 9.9 (59) 13.2 (79) A total of 8 sequences of the 16S rRNA gene were obtained in this study: two of L. cockerelli from Oregon, 77) 13.1 76) 12.4 three of L. cockerelli from California, one of L. cockerelli from Chile and two of L. janssi.Forthe 12.8– (74 12.6– (75 COI gene, 9 sequences 602 bp long were obtained: two – – of L. cockerelli from Oregon, four of L. cockerelli from 10.5 (63) 11.4 (68) 1.0 (4) 14 (84) L. clavigera* L. clavigera** California, and two of L. cockerelli from Chile. The BI 75) 12.3 83) 14.5 (87) 14.6 (87) 73) 12.5 and ML analysis of the concatenated dataset produced

entheses. The number of sequences per gene (16S/ trees with the same topology but varying BI posterior Limacia

sp. 2 probabilities (pp) and ML bootstrap values (mlb). Both 12.5– (73 13.8– (82 12.1– (71 – – – trees recovered four clades for Eastern Pacific species of 4.5 (20) 2.7 (11) 10.8 (65) 4.3 (26) Limacia the genus Limacia (Fig. 1); one clade included only specimens from Chile (pp = 0.96; mlb = 100), another clade 77) 12.1 81) 11.8 included only specimens from California (pp = 0.97;

fferences between par mlb = 86), another clade included only specimens from sp. 1 Oregon (pp = 1; mlb = 100) and the last clade included 12.8– (75 13.5– (80 – – specimens of L. janssi (pp = 1; mlb = 100). The individual

13.5 (81) 14 (84) gene analyses (Fig. S1) also produced the same topology, although support values in the 16S tree were generally 85) 13.6 (82) 13.6 71) 13.3 – – 66) 12.5

– lower. pper triangular) among species of the genus 11 (64 – ge number of bp-pairwise di Species delimitation analyses 9.6 (42) 3.6 (16) 8.3 (34) 0.5 (2) 9.5 (42) 6.5 (29) L. janssi Limacia 10.6

The genetic divergence among species in the genus Limacia is 49) 13.6-14.1 (82 27) 11.5-11.8 (69 as high as 10.7% (e.g., L. cockerelli from California and Limacia sp. 1) (Table 2). For the COI gene, a total of 68 8.1 (46 – 4.5 (22 – polymorphic sites were identified among sequences of speci- 3.7 – L. antofagastensis sp. nov. mens initially identified as L. cockerelli. Intraspecific genetic variability was <1% in each species: 0.8% (n = 2 sequences) 50) 7.6 – 41) 8.9 (39) – 8) for L. cockerelli from Oregon [now L. cockerelli], 0.2–0.7% – (n = 4 sequences) for L. cockerelli from California [now 8.3– (47

– L. mcdonaldi sp. nov.], and 0.5% (n = 2 sequences) for

sp. nov. L. cockerelli from Chile [now L. antofagastensis sp. nov.]. 16S rRNA (lower triangular) and COI mtDNA (u The smallest genetic distance among two clades was observed 9) –

and is shown as an average percentage with the avera between L. cockerelli from California and from Chile 2(8

– [L. mcdonaldi sp.nov.andL. antofagastensis sp.nov.respec-

L. cockerelli L. mcdonaldi tively] (3.7–4.5%) and the largest genetic distance was observed between L. cockerelli from Oregon [now L. cockerelli]andL. clavigera (14.5–14.6%) (Table 2). The ABGD analysis showed a tri-modal pairwise genetic 1/1 8.9 (39) 9.1 (40) 8.9 (39) 1/1 7.1 (29) 7.8 (32) 7.8 (32) 1/1 9.3 (41) 9.0-9.3 (40 2/1 6.6 (29) 6.1 (27) 6.4 (28) 1/1 10 (44) 10.7 (47) 10.5 (46) 10.3 (45) No. of sequences 2/2 7.8 distance (K2P) distribution with a clear gap located between 2 and 4% of genetic distance and a second gap located between 6

sp. nov. 1/2 2.3 (10) 1.4-1.8 (6 and 7% of genetic distance (Fig. 2a). The method used detected three stable candidate species with estimated prior maximum sp. nov. 3/4 1.8

Mean pairwise sequence divergences for the divergences of intra-specific diversity (P) as large as 3.59% sp. 2 sp. 1 (one-tail 95% confidence interval) (Fig. 2b). Notably, the three species recovered correspond to the three clades for L. cockerelli = Spain; ** = Norway COI) is shown. * L. clavigera** The average p-distance was calculated by species L. clavigera* Limacia L. janssi Limacia L. antofagastensis L. mcdonaldi Table 2 L. cockerelli recovered in the phylogenetic analysis (Fig. 1,Fig.S1). Mar Biodiv

species Limacia janssi is also morphologically and genetically distinct (Figs. 1 and 3g). No consistent differences were observed in penial morphology and therefore the penial spines are not illustrated. Other anatomical differences are summa- rized in the descriptions and remarks for each species below. A review of the literature and available type material indicates that the name Limacia cockerelli should be retained for the northern species, whereas the southern species and the Southern Hemisphere species are undescribed. We provide an updated taxonomy for the Limacia cockerelli species complex below. Mollusca Gastropoda Cuvier, 1795 Heterobranchia Burmeister, 1837 Nudibranchia Cuvier, 1817 Polyceridae Alder & Hancock, 1845 Limacia O. F. Müller, 1781 Limacia cockerelli (MacFarland, 1905) (Figs. 3a–c, 4a, 5) Laila cockerelli MacFarland 1905: 47.

Type material

Holotype: USNM 1811290, Monterey Bay, California

Other material examined

Middle Cove, Cape Arago State Park, Oregon (43°18′N, 124°24′W), intertidal, 10 Aug 1972, 2 specimens 13–20 mm Fig. 2 Distribution of pairwise distances for the COI gene and automatic preserved length (LACM 1972–106.14). South Cove, Cape barcode gap discovery (ABGD). a. Frequency distribution of K2P Arago State Park, Oregon (43°18′N, 124°24′W), intertidal, 8 distances among COI sequences. b. ABGD results showing the number Aug 1971, 1 specimen 15 mm preserved length (LACM of groups obtained for a range of prior maximum divergences of – intraspecific diversity. Dashed line indicates the upper bound of 1971 88.16). Fort Ross Cove, Sonoma County, California estimated maximum limits for intraspecific genetic divergences that (38°30.8′N, 123°14.7′W), 22 Oct 1976, 1 specimen 11 mm resulted in three stable candidate species preserved length (LACM 1976–8.26). Cortez Bank, Los Angeles County, California, 21–27 m depth, 22 Sep 1971, 1 Morphological examination specimen 6 mm preserved length (LACM 140729).

Based on the molecular evidence above, as well as morpho- External anatomy logical data, we concluded that Limacia cockerelli is a species complex that includes two Northern Hemisphere species: a Live animals up to 26 mm long. Body oval to elongate, northern species (based on specimens from Oregon and completely surrounded by 2–3 rows of elongate, club-shaped, Northern California) characterized by having the dorsum cov- dorso-lateral papillae (Fig. 3a–c). Papillae vary in length and ered with numerous, small tubercles (Fig. 3a–c)andasouth- width considerably, typically larger towards the center of the ern species (based on specimens from California) with a single body and smaller and thinner towards the anterior and poste- row of orange-red tubercles on the dorsum (Fig. 3d–f). A rior ends. The dorsum bears numerous small tubercles, ar- Southern Hemisphere species (based on specimens from ranged irregularly from anterior to the rhinophores to behind Northern Chile) is also distinct and characterized by having the gill. Gill composed of 6–8 bipinnate branchial leaves, a single row of orange-red tubercles on the dorsum, but the arranged in a circle surrounding the anus. Rhinophores retrac- rhinophoral clubs are half white with the apical half orange- tile, with short stalks and large clubs, bearing 10–12 lamellae. red (Fig. 3h), instead of being almost completely red as in the Posterior end of the foot projecting beyond the dorsum, Northern Hemisphere species. Additionally, the tropical forming a nearly triangular tail. Mar Biodiv

Fig. 3 Photographs of living animals. a–c, Limacia cockerelli (MacFarland, 1905), specimens from Asilomar, California (a), Pecho, California (b), Shell Beach, California (c); d–f, Limacia mcdonaldi sp. nov., specimens from Carmel Point, California (d), Point Loma, California (e), La Jolla, California (f); g, Limacia janssi (Bertsch & Ferreira, 1974), specimen from Puerto Vallarta, Mexico; h, Limacia antofagastensis sp. nov., specimen from Antofagasta, Chile

Background color opaque white, viscera visible in Internal anatomy some specimens as a pinkish area. Dorso-lateral papillae translucent white, with an elongate opaque white core Reproductive system triaulic (Fig. 4a). Ampulla with one visible through the surface, and bright orange distally. fold, connecting directly into the female gland complex, Dorsal tubercles white, with small apical orange dots in near the proximal opening of the prostate. Prostate nar- some specimens. Gill either completely white or with red row, elongate, widening into the muscular deferent duct blotches on the apical region of the branchial leaves. distally. Vagina elongate, as wide as the deferent duct, Rhinophores with white stalks and bright red clubs. Tail joining the seminal receptacle-connecting duct before en- uniformly white in some specimens or with an orange-red tering the bursa copulatrix. Bursa copulatrix inflated, thin- tipinothers. walled, about 10 times larger in volume than the seminal Mar Biodiv

Fig. 4 Diagrams of the reproductive anatomy of the species studied. a, Limacia cockerelli (MacFarland, 1905), specimen from Oregon (LACM 72–104), Limacia mcdonaldi sp. nov., specimen from Southern California (CPIC 00889); c, Limacia antofagastensis sp. nov., holotype, Antofagasta, Chile (LACM 3356); d, Limacia janssi (Bertsch & Ferreira, 1974), specimen from San Carlos, Mexico (CPIC 1503). Abbreviations: am, ampula; bc, bursa copulatrix; dd, deferent duct; fgc, female gland complex; pr,prostate;sr,seminal receptacle; vg, vagina

receptacle. Seminal receptacle oval, muscular, connected Diet Throughout its range Limacia cockerelli specializes on to the female gland complex by a short uterine duct, the encrusting anascan bryozoan Hincksina velata (Hincks, which emerges from the duct connecting the seminal re- 1882) (McDonald and Nybakken 1978; Goddard 1984, ceptacle to the bursa copulatrix. 1998; personal observations). Radular formula 62 × 12.1.1.1.1.1.12 (LACM 14076) to 80 × 14.1.1.1.1.1.14 (LACM 72–106). In each half-row the Reproduction Limacia cockerelli deposits pale pink egg rib- rachidian tooth consists of a rectangular plate with an irregular bons in flat, tightly coiled spirals up to 15 mm in total diameter surface (Fig. 5). Innermost lateral tooth very narrow and del- and with up to 4.5 turns (O’Donoghue and O’Donoghue icate, hook-shaped, with a single curved cusp. Second inner- 1922;Goddard1984). most tooth wide and robust, with an elongate, blunt main cusp pointing outwards and an even smaller secondary cusp located Development Limacia cockerelli from Cape Arago, Oregon next to it; tooth base with a transverse thick fold crossing the hatched as planktotrophic veligers with clear shells averaging tooth from the inner (higher, thicker) to outer (lower) side. 141.8 ± 2.8 μm (n = 10) after an embryonic period of 17 days Outer teeth are simple plates, innermost with short bases and at 10–13 °C from eggs averaging 95.4 ± 2.4 μm (n = 10) in inconspicuous cusps on their inner lower corners and apical diameter (Goddard 1984). depressions, becoming nearly square towards the center, with no distinct cusps, and oval towards the outer end of the half- Remarks row. Laila cockerelli MacFarland, 1905 is the type species of the Biology genus Laila MacFarland, 1905, which was synonymized with the genus Limacia by Ortea et al. (1989). MacFarland (1905) Range Ketchikan, Alaska (Behrens 2004)toPointLoma,San originally described Laila cockerelli from Monterey Bay, in a Diego, California (Vitsky 2008). one page, preliminary description lacking illustrations. After Mar Biodiv

Fig. 5 Limacia cockerelli (MacFarland, 1905), scanning electron specimen; c, View of several complete rows, specimen from Oregon micrographs of the radula. A, View of several complete rows, specimen (LACM 14076); d, Detail of the innermost teeth, same specimen from Oregon (LACM 72–106); b, Detail of the innermost teeth, same describing the large pallial papillae, he described the Bmedian receiving these specimens, along with Cockerell’s Bnotes up- portion of dorsum with numerous low scattered tubercles of on the same.^ MacFarland (1906) includes one detail not varying size^ (MacFarland 1905, p. 47), consistent with the found in his 1905 description suggesting that he incorporated characteristics of the northern species described herein. At the Cockerell’s material from San Pedro in his expanded descrip- end of his description MacFarland (1905, p. 47) mentions, tion of L. cockerelli. Near the end of the second paragraph (p. with no further detail, the Bmuch smaller individuals of the 134) he adds to the description of the low dorsal tubercles the same species^ collected from San Pedro by T. D. A. phrase, Bthe largest near the median line.^ This is consistent Cockerell, Bfor whom the species is named.^ MacFarland with the southern species of L. cockerelli delineated above and (1906) slightly expanded the description of L. cockerelli and described below. However, there is no evidence that this spe- included illustrations of a living specimen, radular teeth and cies was included in MacFarland’s original 1905 description penial armature. Based on the size and distribution of the of L. cockerelli, and the Holotype (USNM 181290) has a dorsal tubercles, the specimen illustrated by MacFarland dorsum covered by scattered low tubercles (Fig. 6a). 1906: pl. 27, fig. 15) and here reproduced in (Fig. 6b), is Therefore we confidently apply the name L. cockerelli to the clearly the northern species. Its collection location was not northern species. precisely specified, but was almost certainly the Monterey Limacia cockerelli is clearly distinguishable from other Peninsula given the focus of MacFarland’s 1905 and 1906 Eastern Pacific species of Limacia because the dorsum, from papers, as well as his acknowledgment of Anna Nash, Bartist anterior to the rhinophores to behind the gill, bears scattered of the Hopkins Seaside Laboratory^ for the illustration of the small tubercles. All other species have a smooth dorsum with living specimen (MacFarland 1906, note preceding plate 22). either no tubercles (as in some specimens of Limacia janssi), MacFarland (1906, p. 135) again refers to the specimens col- or tubercles forming a medial row. Additionally, L. cockerelli lected by Cockerell in San Pedro, but this time mentions is recovered as a distinct species in the species delimitation Mar Biodiv

7 mm preserved length (CPIC 01018). Portuguese Bend, Palos Verdes, California (33°44′20″N, 118°22′20″W), intertidal, 10 Nov 1939, 2 specimens 14 mm preserved length (LACM 1939–117.16). Santa Catalina Island, California, 27 Aug 1968, 1 specimen 6 mm preserved length (LACM 140730). Catalina Harbor, Santa Catalina Island, California (33°26′N, 118°30′W), intertidal, 7 Mar 1970, 4 specimens 5–8 mm preserved length (LACM 1970–8.8). Fisherman’s Cove, Santa Catalina Island, California, 1–2.5 m depth, 14 Aug 1970, 3 specimens 5–9 mm preserved length (LACM 140733). Cabrillo Tidepools, San Pedro, California, 1 Feb 2014, 1 specimen 10 mm preserved length (CPIC 00889).

External anatomy

Live animals up to 26 mm long. Body oval to elongate, completely surrounded by 2–3 rows of elongate, club-shaped, dorso-lateral papillae (Fig. 3d–f). Papillae vary in length and width considerably, typically larger towards the center of the body and smaller and thinner towards the anterior and posterior ends. Dorsum with a single medial row of tubercles running from the area anterior to the rhinophores to in front of the gill. Gill composed of 5–6 bipinnate branchial leaves, ar- Fig. 6 Limacia cockerelli (MacFarland, 1905). a, Photograph of the ranged in a circle surrounding the anus. Rhinophores retrac- preserved holotype (USNM 1811290), from Monterey Bay, California, tile, with short stalks and large clubs, bearing 12–15 lamellae. scale bar = 1 mm (photo Yolanda Villacampa); b, Original drawing Posterior end of the foot projecting beyond the dorsum, published by MacFarland (1906: pl. 27, fig. 15) forming a nearly triangular tail. Background color opaque white, viscera visible in some analyses and specimens of this species form a distinct clade in specimens as a pinkish area. Dorso-lateral papillae translucent the phylogenetic analyses (Fig. 1). white, with an elongate opaque white core visible through the Limacia mcdonaldi sp. nov. ZooBank registration:urn: surface, and orange-red spherical to oval apical structures. lsid:zoobank.org:act:5C99B6E2-C62C-462B-935E- Dorsal tubercles orange red, in some specimens the orange- DC7B88CDD718 red pigment spreads into the dorsal surface around the tubercles. (Figs. 3d–f, 4b, 7) Gill white with red blotches on the apical ends of the branchial leaves. Rhinophores with translucent white stalks and orange- Type material red clubs. Tail white with a large orange spot at the distal end. ’ Holotype: LACM 3359, White s Point, Palos Verdes, Internal anatomy California, 15 mm preserved length. Reproductive system triaulic (Fig. 4b). Ampulla with two Other material examined folds, connecting directly into the female gland complex, near the proximal opening of the prostate. Prostate narrow, elon- Naples Reef, Santa Barbara County, California (34°28′N, gate, convoluted, widening into the muscular deferent duct 120°13′W), 13–18 m depth, 2 specimens 7–8mmpreserved distally. Vagina short, much narrower than the deferent duct, length (LACM 1970–74.2). Point Vicente, Palos Verdes, joining the seminal receptacle-connecting duct before entering California, 29 Feb 1924, 2 specimens 5–7 mm preserved the bursa copulatrix. Bursa copulatrix inflated, thin-walled, length (LACM 140731). White’s Point, Palos Verdes, about 20 times larger in volume than the seminal receptacle. California (33°43′N, 118°18.5′W), intertidal, 9 Dec 1969, 6 Seminal receptacle oval, muscular, connected to the female specimens 4–14 mm preserved length (LACM 1969–37.21); gland complex by a short uterine duct. 8 Jan 1971, 12 specimens 5–15 mm preserved length (LACM Radular formula 69 × 12.1.1.1.1.1.12 (CPIC 01018) to 1971–1.4); 27 Jan 1971, 1 specimen 10 mm preserved length 72 × 12.1.1.1.1.1.12 (CPIC 00889). In each half-row the (LACM 1971–35.2); 3 Nov 1971, 2 specimens 8–12 mm pre- rachidian tooth consists of a rectangular plate with an irregular served length (LACM 140732); 19 Apr 2014, 1 specimen surface (Fig. 7). Innermost lateral tooth very narrow and Mar Biodiv

Fig. 7 Limacia mcdonaldi sp. nov., scanning electron micrographs of the specimen; c, View of several complete rows, specimen from Southern radula. a, View of several complete rows, specimen from Southern California (CPIC 01018); d, Detail of the innermost teeth, same specimen California (CPIC 00889); b, Detail of the innermost teeth, same delicate, with a single curved cusp. Second innermost tooth Reproduction and Development: Nothing is known about re- wide and robust, with an elongate, blunt main cusp pointing production and development in this species. outwards and a smaller secondary cusp located next to it; tooth Derivatio nominis base with a transverse thick fold crossing the tooth from the Named in honor of Gary McDonald, who has spent de- inner (higher, thicker) to outer (lower) side. Outer teeth are cades studying and documenting California nudibranchs with simple plates, innermost with short bases and inconspicuous exemplary care, precision, and generosity. His review of the cusps on their inner lower corners and apical depressions, nudibranchs of California (McDonald 1983), compilations of becoming nearly square towards the center, with less distinct the literature, collection of specimens (housed at the cusps, and oval towards the outer end of the half-row. California Academy of Sciences), and publicly available dig- ital images have been exhaustive and continue to facilitate our Biology research on nudibranchs from the Eastern Pacific.

Range Salt Point, Sonoma County, California (D. Mason, person- Remarks al communication to JG, 15 May 2016) to Cabo San Lucas, Baja California Sur (Lance 1961) and into the Gulf of California to Limacia mcdonaldi sp. nov. is clearly distinguishable from BahiadeLosAngeles(Keen1971; Angulo-Campillo 2003, 2005). L. cockerelli by having the dorsal tubercles arranged in a single line, running from the area anterior to the rhinophores to in Diet Limacia mcdonaldi sp. nov. has been photographed in front of the gill. These tubercles are always orange-red, where- California on unidentified encrusting anascan bryozoans (e.g., as in L. cockerelli the dorsal tubercles are smaller, never form a Clark 2006;Green2007). line and are either white or have a central orange-red spot. Mar Biodiv

Fig. 8 Limacia antofagastensis sp. nov., scanning electron micrographs of the radula. a, View of several complete rows, holotype, Antofagasta, Chile (LACM 3356); b, Detail of the innermost teeth, same specimen

Another difference observed in the animals here examined is External anatomy that the rhinophoral clubs of L. cockerelli are typically bright red, contrasting with the orange-red pigment on the rest of the Live animals up to 11 mm long. Body oval to elongate, body, whereas in L. mcdonaldi sp. nov. the rhinophoral clubs completely surrounded by 2–3 rows of elongate, club-shaped, are the same orange-red as the tips of the dorso-lateral papillae dorso-lateral papillae (Fig. 3H). Papillae vary in length and and dorsal tubercles. width considerably, typically larger towards the posterior Anatomically, L. mcdonaldi sp. nov. has a proportionally end of the body. Dorsum with a single row of spherical tuber- smaller seminal receptacle (about 20 times smaller in volume cles running from the area anterior to the rhinophores to in than the bursa copulatrix) than L. cockerelli (about 10 times front of the gill. Gill composed of 5 bipinnate branchial leaves, smaller). More importantly, the oviduct connects directly into arranged in a circle surrounding the anus. Rhinophores retrac- the seminal receptacle of L. mcdonaldi sp. nov., whereas it tile, with short stalks and large clubs, bearing 11 lamellae. emerges from the duct connecting the seminal receptacle and Background color opaque white, viscera visible as a pink- the bursa copulatrix in L. cockerelli. No consistent differences ish area. Dorso-lateral papillae translucent white, with an elon- were observed in the radular or penial morphology. Limacia gate opaque white core visible through the surface, and mcdonaldi sp. nov. is also recovered as a distinct species in the orange-red spherical to oval apical structures. Dorsal tubercles species delimitation analyses and specimens of this species orange red. Gill white with some orange-red apical pigment. form a distinct clade in the phylogenetic analyses (Fig. 1). Rhinophores with translucent white stalks, clubs orange-red Guernsey (1912, fig. 39a) provided the earliest known il- on the apical half and creamy-white on the basal half. lustration of L. mcdonaldi sp. nov. (as L. cockerelli) and clearly shows the dorsal tubercles aligned along the mid-line of the dorsum in a specimen collected intertidally at Laguna Beach, Internal anatomy California. The specimen illustrated in color by Johnson and Snook (1927, pl. 9, fig. 3) is also clearly L. mcdonaldi sp. nov., Reproductive system triaulic (Fig. 4c). Ampulla with one fold, although no collection locality was specified. connecting directly into the female gland complex, next to the Limacia antofagastensis sp. nov. ZooBank registration: proximal opening of the prostate. Prostate narrow, elongate, urn:lsid:zoobank.org:act:BE4CB683-4695-42FE-8058- convoluted, widening into the muscular deferent duct distally. BEEC640240FB Vagina short, much narrower than the deferent duct, (Figs. 3h, 4c, 8) connecting directly into the bursa copulatrix. Bursa copulatrix inflated, thin-walled, about 15 times larger in volume than the seminal receptacle. Seminal receptacle oval, muscular, con- Type material nected to the female gland complex by a short uterine duct. Radular formula 72 × 12.1.1.1.1.1.12 (LACM 3356). In Holotype: LACM 3356, Antofagasta, Chile (23°38′39″S, each half-row the rachidian tooth consists of a rectangular 70°24′39″W),11mmpreservedlength,2014.Paratype: plate with an irregular surface (Fig. 8). Innermost lateral tooth LACM 3357, Antofagasta, Chile (same coordinates as holo- very narrow and delicate, with a single curved cusp. Second type), 9 mm preserved length, 2014. innermost tooth wide and robust, with an elongate, hook- Mar Biodiv shaped main cusp pointing outwards and a smaller, blunt sec- Limacia janssi (Bertsch & Ferreira, 1974) ondary cusp located next to it; tooth base with a transverse (Figs. 3g, 4d, 9) thick fold crossing the tooth from the inner (higher, thicker) to outer (lower) side. Outer teeth are simple plates, innermost with short bases and inconspicuous cusps on their inner lower Type material corners and apical depressions, becoming narrower with less distinct cusps towards the outer end of the half-row. Holotype: CASIZ 19044, Bahía Santa Elena, Guanacaste, Costa Rica (not examined). Biology Other material examined Range Only known from Bolsico, Peninsula de Mejillones, Antofagasta, northern Chile. Smith Island, Bahía de los Ángeles, Mexico, intertidal, May1976,1specimen8mmpreservedlength(LACM Diet The specimens of Limacia antofagastensis sp.nov.were 140734). Honeymoon Island, San Carlos Bay, Mexico, 2 m observed and photographed on small rocky platforms cover depth, May 1977, 2 specimens 6–7 mm preserved length by encrusting and filamentous red and green algae. However (LACM 25080). North end of the BTurtle Pen^,Isla underneath the rocks were small orange bryozoan colonies Coronado, Bahía de los Ángeles, Mexico (29°05.0′N, that probably constitute their diet. 113°31.3′W), intertidal, 15–17 May 1976, 1 specimen 4 mm preserved length (LACM 1976–5.7). West side of Isla Reproduction and development of this species are unknown. Coronado, Bahía de los Ángeles, Mexico (29°03.7′N, Derivatio nominis 113°31.0′W), intertidal, 11–14 May 1976, 1 specimen 5 mm The name Limacia antofagastensis is dedicated to preserved length (LACM 1976–3.1). San Carlos, Baja Antofagasta, the type locality, where the specimens were California Sur, Mexico, 3 m depth, 1 Sep 2015, 1 specimen collected. 5 mm preserved length (CPIC 01503). Los Arcos, Puerto Vallarta, Jalisco, Mexico, 16 Feb 2008, 2 specimens 5– Remarks 9 mm preserved length (LACM 174940). SE of Isla Canal de Afuera, Panama (7°41.48′N, 81°38.38′W), 5–15 m depth, Limacia antofagastensis sp. nov. is externally very similar to 21 May 2003, 1 specimen 5 mm preserved length (LACM Limacia mcdonaldi sp. nov. as both species share the presence 153339). of a single line of orange-red tubercles on the dorsum. The only obvious difference between the two species is the pig- mentation of the rhinophores, which have an almost complete- External anatomy ly orange-red club in L. mcdonaldi sp.nov.whereasin L. antofagastensis sp. nov. only the apical half is orange-red Live animals up to 8 mm long. Body oval, completely and the other half is white. surrounded by 2 rows of elongate to oval, often globose, Anatomically, L. antofagastensis sp. nov. is character- dorso-lateral papillae (Fig. 3h). Papillae vary in length and ized by having a proportionally large seminal receptacle width considerably, typically larger and more globose towards in comparison to the size of the bursa copulatrix (about 15 the posterior end of the body and smaller and thinner towards times smaller in volume than the bursa copulatrix), which the anterior ends. Dorsum either completely smooth or with a is different from L. mcdonaldi sp.nov.inwhichthesem- single medial row of tubercles running from the area anterior inal receptacle is about 20 times smaller in volume than to the rhinophores to in front of the gill. Gill composed of 3 the bursa copulatrix. Limacia cockerelli also has a larger bipinnate branchial leaves, arranged in a circle surrounding seminal receptacle than L. mcdonaldi sp. nov., about 10 the anus. Rhinophores retractile, with short stalks and large times smaller than the bursa copulatrix, but it is distin- clubs, bearing 10–13 lamellae. Posterior end of the foot guishable from that of L. antofagastensis sp. nov. because projecting beyond the dorsum, forming a nearly triangular tail. the uterine duct does not connect to the seminal receptacle Background color opaque white, viscera visible as a pink- directly. The radula of L. antofagastensis sp. nov. is vir- ish area. Dorso-lateral papillae translucent white, with an elon- tually indistinguishable from those of L. cockerelli and gate opaque white core visible through the surface, becoming L. mcdonaldi sp. nov. (Fig. 8). orange red towards the apex; larger papillae each with a sub- Genetically, Limacia antofagastensis sp. nov. is recovered apical opaque white sphere. Dorsum with numerous irregular as a distinct species in the species delimitation analyses and orange patches. Gill completely off-white to cream. specimens of this species form a distinct clade in the phyloge- Rhinophores with white stalks and orange clubs. Tail uniform- netic analyses (Fig. 1). ly white. Mar Biodiv

Fig. 9 Limacia janssi (Bertsch & Ferreira, 1974), scanning electron same specimen; c, View of several complete rows, specimen from San micrographs of the radula. a, View of several complete rows, specimen Carlos, Mexico (LACM 25080); d, Detail of the innermost teeth, same from San Carlos, Mexico (CPIC 01503); b, Detail of the innermost teeth, specimen

Internal anatomy teeth are simple plates, innermost with short bases and inconspicuous cusps on their inner lower corners and apical depres- Reproductive system triaulic (Fig. 4d). Ampulla with one fold, sions, becoming narrower with less distinct cusps towards the connecting directly into the female gland complex, next to the outer end of the half-row. proximal opening of the prostate. Prostate narrow, elongate, widening into the muscular deferent duct distally. Vagina Biology short, much narrower than the deferent duct, connecting directly into the bursa copulatrix. Bursa copulatrix oval, inflat- Range Northern Gulf of California (Bertsch 2014) to Panama ed, thin-walled, about 4 times larger than the seminal recepta- (Hermosillo 2004). cle. Seminal receptacle oval, muscular, connected to the female gland complex by a short uterine duct. Diet Unidentified encrusting anascan bryozoan (see images in Radular formula 62 × 10.1.1.1.1.1.10 (CPIC 01503) to Hermosillo et al. 2005). 70 × 12.1.1.1.1.1.12 (LACM 25080). In each half-row the rachidian tooth consists of a rectangular plate with an irregular Reproduction Limacia janssi deposits flat, spiral, peach- surface (Fig. 9). Innermost lateral tooth very narrow and del- colored egg masses (Gonsalves-Jackson 2004). icate, with a single curved cusp. Second innermost tooth wide and robust, with an elongate, hook-shaped main cusp pointing Development Gonsalves-Jackson (2004) reported that outwards and a smaller, blunt secondary cusp located next to Limacia janssi from Panama hatched as planktonic veligers it; tooth base with a transverse thick fold crossing the tooth 110umlongafter7daysat10°Cfromeggsaveraging70.2± from the inner (higher, thicker) to outer (lower) side. Outer 5.1 μm in diameter (n = 10). Eggs and hatching larvae of these Mar Biodiv sizes are indicative of planktotrophic development in nudi- 2001;Klug2014) and feed on the same bryozoan prey, branchs (Goddard 2004). Hincksina velata (see Zade 2008; Hershman 2016). Also, the dorsal papillae are similar in size and distribution to those Remarks on L. cockerelli. We did not have access to specimens of this color form for this study, but based on the available evidence Limacia janssi is the only tropical member of the genus we consider it as a color variant of L. cockerelli until sequence Limacia in the Eastern Pacific and is very different morpho- data becomes available. logically from the other temperate species. For example, the The present study constitutes another example of previous- dorso-lateral papillae of L. janssi are elongate to oval, often ly undetected diversity of heterobranch sea slugs along the globose, whereas in the other species they are elongate, club- Eastern Pacific Ocean, and supplements a series of recent shaped. The color pattern of L. janssi is also distinct. Whereas studies providing evidence of the existence of numerous cryp- temperate Eastern Pacific species are white with orange-red tic and pseudocryptic species of heterobranch sea slugs in this pigment on the rhinophores, tips of dorso-lateral papillae and region (Krug et al. 2007; Cooke et al. 2014; Hoover et al. gill, L. janssi has numerous irregular orange patches on the 2015; Lindsay and Valdés 2016; Kienberger et al. 2016; dorsum and the dorso-lateral papillae are translucent white, Lindsay et al. 2016). As in the cases of the Diaulula with an elongate opaque white core visible through the sur- sandiegensis (Cooper, 1863) and the Doriopsilla face, becoming orange red towards the apex. Also, the larger albopunctata (Cooper, 1863) species complexes, Limacia papillae of L. janssi have a subapical opaque white sphere, and cockerelli and the new species Limacia mcdonaldi sp. nov. these structures are absent in the other species. Finally, show a substantial range overlap; in this particular case the Limacia janssi is recovered as a distinct species in the species overlap region stretches from San Diego, California to Salt delimitation analyses and specimens of this species form a Point in Sonoma County, an expanse of nearly 850 kilometers distinct clade in the phylogenetic analyses (Fig. 1). of coastline. Based on the examination of over 600 images of Limacia janssi is superficially similar to Limacia ornata specimens of Limacia available on Flickr (https://www.flickr. (Baba, 1937), originally described from Japan (Baba 1937). com/search/?text=Limacia%20cockerelli&view_all=1&sort= Both species have globose dorso-lateral appendages and date-taken-desc) and iNaturalist (http://www.inaturalist.org/ smooth dorsums. However, L. ornata is completely covered taxa/50059-Limacia-cockerelli), it appears that L. mcdonaldi by conspicuous orange to orange-red spots (including the sp. nov. has been common in the Monterey and San Francisco dorso-lateral appendages), which are absent in L. janssi. bay areas from 2014 through 2016, coincident with the recent marine heat wave in the Northeastern Pacific Ocean starting in 2014 (Di Lorenzo and Mantua 2016). However, for about Discussion seven years prior to this warming event, only a few of the images of specimens of Limacia available from Central and The present paper adds two additional taxa to the known di- Northern California were L. mcdonaldi sp. nov., suggesting versity in the genus Limacia, a relatively species-poor group, this species was much less prevalent in the northern range until now containing only seven described species. A recent overlap region when oceanographic conditions were more study in Europe (Caballer et al. 2016) revealed the existence normal. Also, examination of photographic evidence suggests of another cryptic species very similar to Limacia clavigera that L. mcdonaldi sp. nov. is more abundant in the southern and although that particular study did not include molecular portion of the range overlap region, where L. cockerelli is rare. data, there are consistent morphological and external differ- Limacia mcdonaldi sp. nov. appears to specialize on different ences between the two species examined. Further research bryozoan than L. cockerelli, but until this is confirmed it is may reveal additional diversity, particularly in less studied difficult to speculate on how the range overlap of the two areas such as Western and Southern Africa, where two poorly species is maintained. known species appear to coexist, and the tropical Indo-Pacific Another intriguing question is what process of speciation where several specimens belonging to one or more led to the formation of the partially sympatric Limacia undescribed species have been reported (Caballer et al. cockerelli and Limacia mcdonaldi sp. nov. as well as the allo- 2016; Coleman 2008; Gosliner et al. 2008; Gosliner et al. patric Limacia antofagastensis sp. nov., which is restricted to 2015). the southern hemisphere. Based on the molecular phylogenies A third color form of L. cockerelli with distinctive red here presented, these three species share a common ancestor patches on the dorsum has been reported in the Northeastern and therefore their recent evolution was probably confined to Pacific literature (e.g., Behrens and Hermosillo 2005) and the Eastern Pacific. There are no two identical documented could constitute an additional pseudocryptic species. cases of cryptic and pseudocryptic speciation for the Eastern However, specimens of this red color form share the same Pacific in which sister species pairs display similar ranges and/ habitat and range as typical L. cockerelli (see Wakeling or range overlaps. Thus, it is difficult to compare the Mar Biodiv biogeographic pattern observed in Eastern Pacific species of Yolanda Villacampa. The SEM work was conducted at the California Limacia to other cases. In fact most documented cases for State Polytechnic University SEM laboratory supported by the US National Science Foundation (NSF) grant DMR-1429674. Lindsey cryptic and pseudocryptic species pairs of heterobranch sea Groves (LACM) assisted with the curation of specimens and access to slugs have very limited range overlaps in the Eastern Pacific. the LACM collection. Financial support was provided by project 5303 As mentioned above only the Diaulula sandiegensis and the and Laboratorio de Modelamiento de Sistemas Ecológicos Complejos Doriopsilla albopunctata species complexes display substan- (LAMSEC) of the Universidad de Antofagasta, Chile. tial range overlaps among sister taxa. Hoover et al. (2015) speculated that ecological speciation may be at the root of the formation of Doriopsilla albopunctata and Doriopsilla References fulva (MacFarland, 1905), as there are no obvious barriers to dispersal, past or present, their ranges overlap completely, and Akaike H (1974) A new look at the statistical model identification. IEEE – they show some differences in reproductive anatomy that Trans Autom Control 19:716 722 Angulo-Campillo OJ (2003) Variación espacio-temporal de las could derive from differential sexual selection. On the other poblaciones de opistobranquios (Mollusca: Opisthobranchia) en tres hand, Lindsay et al. (2016) suggested that glaciation driven localidades de B.C.S., Mexico. Master of Science Thesis, vicariance may have resulted in the allopatric speciation of Departamento de Pesquerías y Biología Marina, Centro Diaulula sandiegensis and Diaulula odonoghuei (Steinberg, Interdisciplinario de Ciencias Marinas: La Paz, Baja California 1963) as the latter maintains a transpacific range, and the split Sur, Mexico (unpublished). Angulo-Campillo O (2005) A four year survey of the opisthobranch between the two species coincides with major cooling events. fauna (Gastropoda, Opisthobranchia) from Baja California Sur, With the limited available information it is difficult to provide Mexico. Vita Malacologica 3:43–50 hypotheses for the causes of the speciation between the Baba K (1937) Opisthobranchia of Japan (II). Jour. Dept. Agr. Kyushu Eastern Pacific species of Limacia. Further research on the Imp. Univ. 5:289–344, pls.1–2. habitat use, possible reproductive barriers between sympatric Behrens (2004) Pacific Coast Nudibranchs, Supplement II New Species to the Pacific Coast and New Information on the Oldies. Proc Calif species, and divergence times may provide insights into the Acad Sci 55(2):11–54 causes of speciation. However, the split between Behrens DW, Hermosillo A (2005) Eastern Pacific nudibranchs, a guide L. antofagastensis sp. nov. and its closest northern hemisphere to the opisthobranchs from Alaska to Central America. Sea relative L. mcdonaldi sp. nov. may have an obvious explana- Challengers, Monterey, California. 137 pp., 314 photos. tion. The range of L. antofagastensis sp. nov. is separated from Bertsch H (2014) Biodiversity in La Reserva de la Biósfera Bahía de los Ángeles y Canales de Ballenas y Salsipuedes: Naming of a new that of L. mcdonaldi sp. nov. by the Panamic Biogeographic genus, range extensions and new records, and species list of Province, a 4,000 km-long stretch of tropical waters from the Heterobranchia (Mollusca: Gastropoda), with comments on biodi- mouth of the Gulf of California to the Gulf of Guayaquil versity conservation within marine reserves. The Festivus 46:158– (Briggs and Bowen 2012). The ocean temperature in the 177 Bertsch H, Ferreira AJ (1974) Four new species of nudibranchs from Panamic Province varied considerably in the past, for exam- tropical West America. The Veliger 16:343–353 ple, cooling events during the Pleistocene greatly reduced av- Briggs JC, Bowen BW (2012) A realignment of marine biogeographic erage temperatures (Lawrence et al. 2006). 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