Advertisement

Marine Biodiversity

, Volume 48, Issue 4, pp 1815–1832 | Cite as

Integrative systematics of the genus Limacia O. F. Müller, 1781 (Gastropoda, Heterobranchia, Nudibranchia, Polyceridae) in the Eastern Pacific

  • Roberto A. Uribe
  • Fabiola Sepúlveda
  • Jeffrey H. R. Goddard
  • Ángel Valdés
Original Paper

Abstract

Morphological examination and molecular analyses of specimens of the genus Limacia collected in the Eastern Pacific Ocean indicate that four species of Limacia occur in the region. Limacia cockerelli, previously considered to range from Alaska to Baja California, is common only in the northern part of its former range. An undescribed pseudocryptic species, previously included as L. cockerelli, 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 species similar to L. cockerelli is described from Antofagasta, Chile and constitutes the first record of the genus Limacia in the Southeastern Pacific Ocean. These two new species are formally described herein. Finally, Limacia janssi is a genetically and morphologically distinct tropical species ranging from Baja California to Panama. Species delimitation analyses based on molecular data and unique morphological traits from the dorsum, radula, and reproductive systems are useful in distinguishing these species

Keywords

Mollusca New species Molecular taxonomy Pseudocryptic species 

Introduction

Molecular markers have become a powerful tool in taxonomy, systematics and phylogeny, allowing researchers to assess whether morphological variations correspond to different species or merely represent intra-specific phenotypic expression due to environmental variation (Hebert et al. 2004; Radulovici et al. 2010). Recent use of these markers has helped reveal high levels of cryptic species diversity in heterobranch sea slugs from the Eastern Pacific Ocean, including sacoglossans (Krug et al. 2007), cephalaspideans (Cooke et al. 2014), and nudibranchs (Hoover et al. 2015; Lindsay and Valdés 2016; Kienberger et al. 2016; Lindsay et al. 2016). Some of these newly described taxa resulted from splitting widespread species in the Northeastern Pacific into two sister species, mainly allopatric, with limited overlap near the San Francisco Bay Area (Krug et al. 2007; Lindsay and Valdés 2016) or further north (Kienberger et al. 2016). However, less often, newly discovered cryptic species pairs are sympatric along most of their ranges (Hoover et al. 2015; Lindsay et al. 2016). These differences raise intriguing questions about the mechanisms of speciation (allopatric vs. ecological) involved in producing those species pairs, as well as the possible existence of biogeographic barriers that could promote allopatric divergence.

Most of these newly discovered cryptic species pairs are restricted to the Northeastern Pacific; very little is known about the molecular systematics and biogeography of Southeastern Pacific taxa, or their relationships with morphologically similar northern species. Some Southern Hemisphere temperate species display resemblances with northern taxa (e.g., Polycera alabe Collier & Farmer, 1964, Rostanga pulchra MacFarland, 1905), and have been suggested to belong to the same species (e.g., Schrödl 2003; Schrödl and Grau 2006). However, temperate northern and southern regions are separated by the Panamic Biogeographic Province, a 4,000 km-long stretch of tropical waters from the mouth of the Gulf of California to the Gulf of Guayaquil (Briggs and Bowen 2012). In fact, the only published molecular study of a species found in both hemispheres confirms that southern records of Aeolidia papillosa (Linnaeus, 1761) constitute a distinct, cryptic, endemic species from Chile (Kienberger et al. 2016).

In this paper we examine the Eastern Pacific species of the genus Limacia O. F. Müller, 1781, which include northern temperate, tropical, and newly discovered southern temperate populations. The genus Limacia is a group of polycerid dorid nudibranchs characterized by having a unique body plan, with one to several rows of elongate dorso-lateral appendages surrounding the entire notum. Species of the genus Limacia occur in a handful of mainly temperate but also tropical disjunct areas, including Western and Southern Africa [Limacia lucida (Stimpson, 1854), Limacia annulata Vallès, Valdés & Ortea, 2000], the Eastern Pacific [Limacia cockerelli (MacFarland, 1905), Limacia janssi (Bertsch & Ferreira, 1974)], the Eastern Atlantic and Mediterranean [Limacia clavigera (O. F. Müller, 1776), Limacia iberica Caballer, Almón & Pérez, 2016] and the Western Pacific [Limacia ornata (Baba, 1937)]. A possibly undescribed species appears to be widespread in the tropical Indian Ocean, from Eastern Australia to Tanzania (Gosliner et al. 2008; Gosliner et al. 2015) but is rare. In the Eastern Pacific, L. cockerelli has a broad geographic range from Alaska to Baja California and displays considerable color variation (McDonald & Nybakken 1980; Behrens and Hermosillo 2005), making it a potential candidate for a species complex. In contrast, L. janssi is restricted to the Panamic tropical region and is less variable (Behrens and Hermosillo 2005). Newly collected specimens from Chile are morphologically similar to L. cockerelli and appear to belong to the same species, but this needs anatomical and molecular confirmation.

In order to better understand the biological diversity and phylogeny of the Eastern Pacific species of the genus Limacia we examined specimens belonging to both L. cockerelli and L. janssi, covering most of their ranges and including the newly collected specimens from Chile. We used an integrative approach (molecular, morphological and ecological data when available) in an attempt to unravel the systematics of this group and detect possible cryptic diversity.

Materials and methods

Source of specimens

From 2014 to 2016, nine specimens of Limacia cockerelli were collected from rocky intertidal and subtidal sites on the Eastern Pacific coast: seven from the Northeast Pacific coast (four from Oregon and three from California) and two from the Southeast Pacific coast (Bolsico Cave, Antofagasta, northern Chile). The specimens from Chile were collected on a barren ground system at 5 m depth. Additionally, two specimens of Limacia janssi from San Carlos and Bahía Magdalena, Mexico, were collected, examined and sequenced. Samples were preserved in 95–99% ethanol and deposited at the California State Polytechnic Invertebrate Collection (CPIC), the Natural History Museum of Los Angeles County (LACM) and the Sala de Colecciones Biológicas, Universidad Católica del Norte, Chile (SCBUCN). Details on collection localities and dates as well as museum registration numbers are provided in the material examined section for each species. Additional specimens from the LACM were examined morphologically but were unsuitable for molecular work. Photographs of the type material of Limacia cockerelli were obtained from the National Museum of Natural History, Smithsonian Institution (USNM).

DNA extraction and amplification

For molecular analyses, partial sequences of the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S were amplified using pairs LCO and HCO (Folmer et al. 1994) and 16Sar-L and 16Sbr-H (Palumbi et al. 1991). The 16S rRNA region is more conserved, and it is used for phylogenetic analyses, instead. COI mtDNA has a higher mutation accumulation rate and is commonly used in species delimitation analyses (Hebert et al. 2004). The DNA of each individual was isolated following a modified protocol based on Miller et al. (1988) involving treatment with sodium dodecyl sulfate, digestion with Proteinase K, NaCl protein precipitation, and subsequent ethanol precipitation of the DNA. Modifications included centrifugation at 10 °C, maintaining ethanol at −20 °C and adding to the last step in the protocol a wash with 70% ethanol.

Each PCR reaction included 0.175 μl of GoTaq DNA polymerase (Promega, Madison, USA), 7 μl of 5 × PCR buffer, 5.6 μl of MgCl2 (25 mM), 2.1 μl of BSA (10 mg/ml), 0.7 μl of deoxynucleotide triphosphate (dNTP) (10 mM), 1.4 μl of each primer (10 pM) and 5 μl of template DNA and 11.625 μl of nuclease-free water. PCR conditions were as follows: 1) for COI – initial denaturation 94 °C, 5 min; 35 cycles of denaturation 94 °C, 1 min, annealing 44 °C, 30 s; extension 72 °C, 1 min; final extension 72 °C, 7 min; 2) for 16S – initial denaturation 94 °C, 2 min; 35 cycles of denaturation 94 °C, 30 s, annealing 50 °C, 30 s; extension, 72 °C, 1 min; final extension 72 °C, 7 min.

PCR products were cleaned using Ultra clean kit (Mobio), and both DNA strands were sequenced directly at Macrogen (Seoul, Korea; http://www.macrogen.com). Complementary sequences were assembled and edited using ProSeq v2.9 (Filatov 2002). The fragments obtained were aligned with sequences of species of the genus Limacia obtained from GenBank using the Clustal 2 software package (Larkin et al. 2007). All new DNA sequences have been deposited in GenBank, and the accession numbers are given in Table 1.
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 15.05/46736

EF142906

EF142952

Pola et al. 2007

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

Phylogenetic analyses

COI and 16S sequences were trimmed to 603 and 458 base pairs, respectively. Gene concatenation (COI + 16S = 1061 bp) was performed in Mesquite v.2.75 (Maddison and Maddison 2011). The software jModelTest 0.1 (Posada 2008) was employed to determine the best-fit nucleotide substitution model for each gene and for the entire alignment accompanying evolutionary parameter values for the data under the Akaike information criterion (Akaike 1974). Bayesian inference (BI) and maximum likelihood (ML) analyses were conducted for the concatenated data set as well as for individual genes (COI and 16S). Triopha catalinae (Cooper, 1863) and T. maculata MacFarland, 1905 were selected as the outgroups based on their close phylogenetic relationship with the genus Limacia. Sequences of the outgroup taxa were obtained from GenBank (Table 1). BI analyses were performed using the software package MrBayes (Huelsenbeck and Ronquist 2001) with 6 substitution types for 16S (nst = 6) and 2 substitution types for COI (nst = 2), and considering gamma distributed rate variation as well as the proportion of invariable positions, according to the evolutionary model determined by jModeltest for each gene (GTR + I + G and HKY + I + G respectively). The BI concatenated analysis was partitioned by genes. All BI analyses included two runs of six chains for 10 million generations, sampling every 1,000 generations and a burn-in of 25%. ML analyses were performed using the software package RaXML (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.

Species delimitation analyses

In order to compare the genetic distances among specimens of Limacia, we calculated the pairwise p-distances (between individual sequences and in average by species) for 16S and COI using MEGA 6 (Tamura et al. 2013).

The COI gene was used to aid in determining the number of species present in the L. cockerelli species complex, using the approximation of delineation of species boundaries in the Automatic Barcode Gap Discovery method (ABGD) (Puillandre et al. 2012). This method estimates the distribution of pairwise genetic distances between the aligned sequences and then it statistically infers multiple potential barcode gaps as minima in the distribution of pairwise distances, thereby partitioning the sequences such that the distance between two sequences taken from distinct clusters will be larger than the barcode gap (Puillandre et al. 2012). For this analysis seven sequences of specimens initially identified as L. cockerelli were used: two from Chile, three from California and two from Oregon.

COI alignments were uploaded at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html and ABGD was run with the default settings (Pmin = 0.001, Pmax = 0.1, Steps = 10, X (relative gap width) = 1.5, Nb bins = 20) and with K2P distances.

Morphological examination

At least two specimens of each putative species were dissected. The reproductive anatomy and radular morphology were used as the main internal traits for species identification and characterization. The reproductive organs were examined by removing them from the animal through a dorsal incision and drawn under a Nikon SMZ-100 dissecting microscope with a camera lucida attachment. The penises were removed, mounted on a microscope slide and drawn while viewed with an Olympus CX31 compound microscope utilizing a camera lucida attachment. The buccal mass of each specimen examined was removed and the tissue surrounding the jaws and radula was dissolved using 10% sodium hydroxide (NaOH). Jaws and radulae were rinsed in water, dried, mounted, and sputter coated for examination under a Jeol JSM-6010 variable pressure SEM at the California State Polytechnic University.

Results

Phylogenetic analyses

A total of 8 sequences of the 16S rRNA gene were obtained in this study: two of L. cockerelli from Oregon, three of L. cockerelli from California, one of L. cockerelli from Chile and two of L. janssi. For the COI gene, 9 sequences 602 bp long were obtained: two of L. cockerelli from Oregon, four of L. cockerelli from California, and two of L. cockerelli from Chile. The BI and ML analysis of the concatenated dataset produced trees with the same topology but varying BI posterior probabilities (pp) and ML bootstrap values (mlb). Both trees recovered four clades for Eastern Pacific species of the genus Limacia (Fig. 1); one clade included only specimens from Chile (pp = 0.96; mlb = 100), another clade included only specimens from California (pp = 0.97; mlb = 86), another clade included only specimens from Oregon (pp = 1; mlb = 100) and the last clade included specimens of L. janssi (pp = 1; mlb = 100). The individual gene analyses (Fig. S1) also produced the same topology, although support values in the 16S tree were generally lower.
Fig. 1

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

Species delimitation analyses

The genetic divergence among species in the genus Limacia is 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 polymorphic sites were identified among sequences of specimens initially identified as L. cockerelli. Intraspecific genetic variability was <1% in each species: 0.8% (n = 2 sequences) for L. cockerelli from Oregon [now L. cockerelli], 0.2–0.7% (n = 4 sequences) for L. cockerelli from California [now L. mcdonaldi sp. nov.], and 0.5% (n = 2 sequences) for L. cockerelli from Chile [now L. antofagastensis sp. nov.]. The smallest genetic distance among two clades was observed between L. cockerelli from California and from Chile [L. mcdonaldi sp. nov. and L. antofagastensis sp. nov. respectively] (3.7–4.5%) and the largest genetic distance was observed between L. cockerelli from Oregon [now L. cockerelli] and L. clavigera (14.5–14.6%) (Table 2).
Table 2

Mean pairwise sequence divergences for the 16S rRNA (lower triangular) and COI mtDNA (upper triangular) among species of the genus Limacia

 

No. of sequences

L. cockerelli

L. mcdonaldi sp. nov.

L. antofagastensis sp. nov.

L. janssi

Limacia sp. 1

Limacia sp. 2

L. clavigera*

L. clavigera**

L. cockerelli

2/2

 

7.8–8.3 (47–50)

7.6–8.1 (46–49)

13.6-14.1 (82–85)

13.6 (82)

13.6–13.8 (82–83)

14.5 (87)

14.6 (87)

L. mcdonaldi sp. nov.

3/4

1.8–2 (8–9)

 

3.7–4.5 (22–27)

11.5-11.8 (69–71)

13.3–13.5 (80–81)

11.8–12.1 (71–73)

12.5–12.6 (75–76)

12.4–12.7 (75–76)

L. antofagastensis sp. nov.

1/2

2.3 (10)

1.4-1.8 (6–8)

 

10.6–11 (64–66)

12.5–12.8 (75–77)

12.1–12.5 (73–75)

12.3–12.8 (74–77)

13.1–13.2 (78–79)

L. janssi

2/1

6.6 (29)

6.1 (27)

6.4 (28)

 

13.5 (81)

14 (84)

14 (84)

13.2 (79)

Limacia sp. 1

1/1

10 (44)

10.7 (47)

10.5 (46)

10.3 (45)

 

10.8 (65)

4.3 (26)

9.9 (59)

Limacia sp. 2

1/1

9.3 (41)

9.0-9.3 (40–41)

8.9 (39)

9.5 (42)

6.5 (29)

 

10.5 (63)

11.4 (68)

L. clavigera*

1/1

7.1 (29)

7.8 (32)

7.8 (32)

8.3 (34)

0.5 (2)

2.7 (11)

 

9.5 (57)

L. clavigera**

1/1

8.9 (39)

9.1 (40)

8.9 (39)

9.6 (42)

3.6 (16)

4.5 (20)

1.0 (4)

 

The average p-distance was calculated by species and is shown as an average percentage with the average number of bp-pairwise differences between parentheses. The number of sequences per gene (16S/COI) is shown.

* = Spain; ** = Norway

The ABGD analysis showed a tri-modal pairwise genetic distance (K2P) distribution with a clear gap located between 2 and 4% of genetic distance and a second gap located between 6 and 7% of genetic distance (Fig. 2a). The method used detected three stable candidate species with estimated prior maximum divergences of intra-specific diversity (P) as large as 3.59% (one-tail 95% confidence interval) (Fig. 2b). Notably, the three species recovered correspond to the three clades for L. cockerelli recovered in the phylogenetic analysis (Fig. 1, Fig. S1).
Fig. 2

Distribution of pairwise distances for the COI gene and automatic barcode gap discovery (ABGD). a. Frequency distribution of K2P distances among COI sequences. b. ABGD results showing the number of groups obtained for a range of prior maximum divergences of intraspecific diversity. Dashed line indicates the upper bound of estimated maximum limits for intraspecific genetic divergences that resulted in three stable candidate species

Morphological examination

Based on the molecular evidence above, as well as morphological data, we concluded that Limacia cockerelli is a species complex that includes two Northern Hemisphere species: a northern species (based on specimens from Oregon and Northern California) characterized by having the dorsum covered with numerous, small tubercles (Fig. 3a–c) and a southern species (based on specimens from California) with a single row of orange-red tubercles on the dorsum (Fig. 3d–f). A Southern Hemisphere species (based on specimens from Northern Chile) is also distinct and characterized by having a single row of orange-red tubercles on the dorsum, but the rhinophoral clubs are half white with the apical half orange-red (Fig. 3h), instead of being almost completely red as in the Northern Hemisphere species. Additionally, the tropical 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 summarized in the descriptions and remarks for each species below.
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

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)
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

Fig. 5

Limacia cockerelli (MacFarland, 1905), scanning electron micrographs of the radula. A, View of several complete rows, specimen from Oregon (LACM 72–106); b, Detail of the innermost teeth, same specimen; c, View of several complete rows, specimen from Oregon (LACM 14076); d, Detail of the innermost teeth, same specimen

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 preserved length (LACM 1972–106.14). South Cove, Cape Arago State Park, Oregon (43°18′N, 124°24′W), intertidal, 8 Aug 1971, 1 specimen 15 mm preserved length (LACM 1971–88.16). Fort Ross Cove, Sonoma County, California (38°30.8′N, 123°14.7′W), 22 Oct 1976, 1 specimen 11 mm preserved length (LACM 1976–8.26). Cortez Bank, Los Angeles County, California, 21–27 m depth, 22 Sep 1971, 1 specimen 6 mm preserved length (LACM 140729).

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. 3a–c). 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. The dorsum bears numerous small tubercles, arranged irregularly from anterior to the rhinophores to behind the gill. Gill composed of 6–8 bipinnate branchial leaves, arranged in a circle surrounding the anus. Rhinophores retractile, with short stalks and large clubs, bearing 10–12 lamellae. Posterior end of the foot projecting beyond the dorsum, forming a nearly triangular tail.

Background color opaque white, viscera visible in some specimens as a pinkish area. Dorso-lateral papillae translucent white, with an elongate opaque white core visible through the surface, and bright orange distally. Dorsal tubercles white, with small apical orange dots in some specimens. Gill either completely white or with red blotches on the apical region of the branchial leaves. Rhinophores with white stalks and bright red clubs. Tail uniformly white in some specimens or with an orange-red tip in others.

Internal anatomy

Reproductive system triaulic (Fig. 4a). Ampulla with one fold, connecting directly into the female gland complex, near the proximal opening of the prostate. Prostate narrow, elongate, widening into the muscular deferent duct distally. Vagina elongate, as wide as the deferent duct, joining the seminal receptacle-connecting duct before entering the bursa copulatrix. Bursa copulatrix inflated, thin-walled, about 10 times larger in volume than the seminal receptacle. Seminal receptacle oval, muscular, connected to the female gland complex by a short uterine duct, which emerges from the duct connecting the seminal receptacle to the bursa copulatrix.

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 rachidian tooth consists of a rectangular plate with an irregular surface (Fig. 5). Innermost lateral tooth very narrow and delicate, hook-shaped, with a single curved cusp. Second innermost tooth wide and robust, with an elongate, blunt main cusp pointing outwards and an even smaller secondary cusp located next to it; tooth base with a transverse 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 corners and apical depressions, becoming nearly square towards the center, with no distinct cusps, and oval towards the outer end of the half-row.

Biology

Range

Ketchikan, Alaska (Behrens 2004) to Point Loma, San Diego, California (Vitsky 2008).

Diet

Throughout its range Limacia cockerelli specializes on the encrusting anascan bryozoan Hincksina velata (Hincks, 1882) (McDonald and Nybakken 1978; Goddard 1984, 1998; personal observations).

Reproduction

Limacia cockerelli deposits pale pink egg ribbons in flat, tightly coiled spirals up to 15 mm in total diameter and with up to 4.5 turns (O’Donoghue and O’Donoghue 1922; Goddard 1984).

Development

Limacia cockerelli from Cape Arago, Oregon hatched as planktotrophic veligers with clear shells averaging 141.8 ± 2.8 μm (n = 10) after an embryonic period of 17 days at 10–13 °C from eggs averaging 95.4 ± 2.4 μm (n = 10) in diameter (Goddard 1984).

Remarks

Laila cockerelli MacFarland, 1905 is the type species of the genus Laila MacFarland, 1905, which was synonymized with the genus Limacia by Ortea et al. (1989). MacFarland (1905) originally described Laila cockerelli from Monterey Bay, in a one page, preliminary description lacking illustrations. After describing the large pallial papillae, he described the “median portion of dorsum with numerous low scattered tubercles of varying size” (MacFarland 1905, p. 47), consistent with the characteristics of the northern species described herein. At the end of his description MacFarland (1905, p. 47) mentions, with no further detail, the “much smaller individuals of the same species” collected from San Pedro by T. D. A. Cockerell, “for whom the species is named.” MacFarland (1906) slightly expanded the description of L. cockerelli and included illustrations of a living specimen, radular teeth and penial armature. Based on the size and distribution of the dorsal tubercles, the specimen illustrated by MacFarland 1906: pl. 27, fig. 15) and here reproduced in (Fig. 6b), is clearly the northern species. Its collection location was not precisely specified, but was almost certainly the Monterey Peninsula given the focus of MacFarland’s 1905 and 1906 papers, as well as his acknowledgment of Anna Nash, “artist of the Hopkins Seaside Laboratory” for the illustration of the living specimen (MacFarland 1906, note preceding plate 22). MacFarland (1906, p. 135) again refers to the specimens collected by Cockerell in San Pedro, but this time mentions receiving these specimens, along with Cockerell’s “notes upon the same.” MacFarland (1906) includes one detail not found in his 1905 description suggesting that he incorporated Cockerell’s material from San Pedro in his expanded description of L. cockerelli. Near the end of the second paragraph (p. 134) he adds to the description of the low dorsal tubercles the phrase, “the largest near the median line.” This is consistent with the southern species of L. cockerelli delineated above and described below. However, there is no evidence that this species was included in MacFarland’s original 1905 description of L. cockerelli, and the Holotype (USNM 181290) has a dorsum covered by scattered low tubercles (Fig. 6a). Therefore we confidently apply the name L. cockerelli to the northern species.
Fig. 6

Limacia cockerelli (MacFarland, 1905). a, Photograph of the preserved holotype (USNM 1811290), from Monterey Bay, California, scale bar = 1 mm (photo Yolanda Villacampa); b, Original drawing published by MacFarland (1906: pl. 27, fig. 15)

Limacia cockerelli is clearly distinguishable from other Eastern Pacific species of Limacia because the dorsum, from anterior to the rhinophores to behind the gill, bears scattered small tubercles. All other species have a smooth dorsum with either no tubercles (as in some specimens of Limacia janssi), or tubercles forming a medial row. Additionally, L. cockerelli is recovered as a distinct species in the species delimitation analyses and specimens of this species form a distinct clade in the phylogenetic analyses (Fig. 1).

Limacia mcdonaldi sp. nov. ZooBank registration:urn:lsid:zoobank.org:act:5C99B6E2-C62C-462B-935E-DC7B88CDD718

(Figs. 3d–f, 4b, 7)
Fig. 7

Limacia mcdonaldi sp. nov., scanning electron micrographs of the radula. a, View of several complete rows, specimen from Southern California (CPIC 00889); b, Detail of the innermost teeth, same specimen; c, View of several complete rows, specimen from Southern California (CPIC 01018); d, Detail of the innermost teeth, same specimen

Type material

Holotype: LACM 3359, White’s Point, Palos Verdes, California, 15 mm preserved length.

Other material examined

Naples Reef, Santa Barbara County, California (34°28′N, 120°13′W), 13–18 m depth, 2 specimens 7–8 mm preserved length (LACM 1970–74.2). Point Vicente, Palos Verdes, California, 29 Feb 1924, 2 specimens 5–7 mm preserved length (LACM 140731). White’s Point, Palos Verdes, California (33°43′N, 118°18.5′W), intertidal, 9 Dec 1969, 6 specimens 4–14 mm preserved length (LACM 1969–37.21); 8 Jan 1971, 12 specimens 5–15 mm preserved length (LACM 1971–1.4); 27 Jan 1971, 1 specimen 10 mm preserved length (LACM 1971–35.2); 3 Nov 1971, 2 specimens 8–12 mm preserved length (LACM 140732); 19 Apr 2014, 1 specimen 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, arranged in a circle surrounding the anus. Rhinophores retractile, with short stalks and large clubs, bearing 12–15 lamellae. Posterior end of the foot projecting beyond the dorsum, forming a nearly triangular tail.

Background color opaque white, viscera visible in some specimens as a pinkish area. Dorso-lateral papillae translucent white, with an elongate opaque white core visible through the surface, and orange-red spherical to oval apical structures. Dorsal tubercles orange red, in some specimens the orange-red pigment spreads into the dorsal surface around the tubercles. Gill white with red blotches on the apical ends of the branchial leaves. Rhinophores with translucent white stalks and orange-red clubs. Tail white with a large orange spot at the distal end.

Internal anatomy

Reproductive system triaulic (Fig. 4b). Ampulla with two folds, connecting directly into the female gland complex, near the proximal opening of the prostate. Prostate narrow, elongate, convoluted, widening into the muscular deferent duct distally. Vagina short, much narrower than the deferent duct, joining the seminal receptacle-connecting duct before entering the bursa copulatrix. Bursa copulatrix inflated, thin-walled, about 20 times larger in volume than the seminal receptacle. Seminal receptacle oval, muscular, connected to the female gland complex by a short uterine duct.

Radular formula 69 × 12.1.1.1.1.1.12 (CPIC 01018) to 72 × 12.1.1.1.1.1.12 (CPIC 00889). In each half-row the rachidian tooth consists of a rectangular plate with an irregular surface (Fig. 7). Innermost lateral tooth very narrow and delicate, with a single curved cusp. Second innermost tooth wide and robust, with an elongate, blunt main cusp pointing outwards and a smaller secondary cusp located next to it; tooth base with a transverse 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 corners and apical depressions, becoming nearly square towards the center, with less distinct cusps, and oval towards the outer end of the half-row.

Biology

Range

Salt Point, Sonoma County, California (D. Mason, personal communication to JG, 15 May 2016) to Cabo San Lucas, Baja California Sur (Lance 1961) and into the Gulf of California to Bahia de Los Angeles (Keen 1971; Angulo-Campillo 2003, 2005).

Diet

Limacia mcdonaldi sp. nov. has been photographed in California on unidentified encrusting anascan bryozoans (e.g., Clark 2006; Green 2007).

Reproduction and Development: Nothing is known about reproduction and development in this species.

Derivatio nominis

Named in honor of Gary McDonald, who has spent decades studying and documenting California nudibranchs with exemplary care, precision, and generosity. His review of the nudibranchs of California (McDonald 1983), compilations of the literature, collection of specimens (housed at the California Academy of Sciences), and publicly available digital images have been exhaustive and continue to facilitate our research on nudibranchs from the Eastern Pacific.

Remarks

Limacia mcdonaldi sp. nov. is clearly distinguishable from L. cockerelli by having the dorsal tubercles arranged in a single line, running from the area anterior to the rhinophores to in front of the gill. These tubercles are always orange-red, whereas in L. cockerelli the dorsal tubercles are smaller, never form a line and are either white or have a central orange-red spot. Another difference observed in the animals here examined is that the rhinophoral clubs of L. cockerelli are typically bright red, contrasting with the orange-red pigment on the rest of the body, whereas in L. mcdonaldi sp. nov. the rhinophoral clubs are the same orange-red as the tips of the dorso-lateral papillae and dorsal tubercles.

Anatomically, L. mcdonaldi sp. nov. has a proportionally smaller seminal receptacle (about 20 times smaller in volume than the bursa copulatrix) than L. cockerelli (about 10 times smaller). More importantly, the oviduct connects directly into the seminal receptacle of L. mcdonaldi sp. nov., whereas it emerges from the duct connecting the seminal receptacle and the bursa copulatrix in L. cockerelli. No consistent differences were observed in the radular or penial morphology. Limacia mcdonaldi sp. nov. is also recovered as a distinct species in the species delimitation analyses and specimens of this species form a distinct clade in the phylogenetic analyses (Fig. 1).

Guernsey (1912, fig. 39a) provided the earliest known illustration 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, California. The specimen illustrated in color by Johnson and Snook (1927, pl. 9, fig. 3) is also clearly L. mcdonaldi sp. nov., although no collection locality was specified.

Limacia antofagastensis sp. nov. ZooBank registration:urn:lsid:zoobank.org:act:BE4CB683-4695-42FE-8058-BEEC640240FB

(Figs. 3h, 4c, 8)
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

Type material

Holotype: LACM 3356, Antofagasta, Chile (23°38′39″S, 70°24′39″W), 11 mm preserved length, 2014. Paratype: LACM 3357, Antofagasta, Chile (same coordinates as holotype), 9 mm preserved length, 2014.

External anatomy

Live animals up to 11 mm long. Body oval to elongate, completely surrounded by 2–3 rows of elongate, club-shaped, dorso-lateral papillae (Fig. 3H). Papillae vary in length and width considerably, typically larger towards the posterior end of the body. Dorsum with a single row of spherical tubercles running from the area anterior to the rhinophores to in front of the gill. Gill composed of 5 bipinnate branchial leaves, arranged in a circle surrounding the anus. Rhinophores retractile, with short stalks and large clubs, bearing 11 lamellae.

Background color opaque white, viscera visible as a pinkish area. Dorso-lateral papillae translucent white, with an elongate opaque white core visible through the surface, and orange-red spherical to oval apical structures. Dorsal tubercles orange red. Gill white with some orange-red apical pigment. Rhinophores with translucent white stalks, clubs orange-red on the apical half and creamy-white on the basal half.

Internal anatomy

Reproductive system triaulic (Fig. 4c). Ampulla with one fold, connecting directly into the female gland complex, next to the proximal opening of the prostate. Prostate narrow, elongate, convoluted, widening into the muscular deferent duct distally. Vagina short, much narrower than the deferent duct, 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, connected to the female gland complex by a short uterine duct.

Radular formula 72 × 12.1.1.1.1.1.12 (LACM 3356). In each half-row the rachidian tooth consists of a rectangular plate with an irregular surface (Fig. 8). Innermost lateral tooth very narrow and delicate, with a single curved cusp. Second innermost tooth wide and robust, with an elongate, hook-shaped main cusp pointing outwards and a smaller, blunt secondary cusp located next to it; tooth base with a transverse 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 corners and apical depressions, becoming narrower with less distinct cusps towards the outer end of the half-row.

Biology

Range

Only known from Bolsico, Peninsula de Mejillones, Antofagasta, northern Chile.

Diet

The specimens of Limacia antofagastensis sp. nov. were observed and photographed on small rocky platforms cover by encrusting and filamentous red and green algae. However underneath the rocks were small orange bryozoan colonies that probably constitute their diet.

Reproduction and development of this species are unknown.

Derivatio nominis

The name Limacia antofagastensis is dedicated to Antofagasta, the type locality, where the specimens were collected.

Remarks

Limacia antofagastensis sp. nov. is externally very similar to Limacia mcdonaldi sp. nov. as both species share the presence of a single line of orange-red tubercles on the dorsum. The only obvious difference between the two species is the pigmentation of the rhinophores, which have an almost completely orange-red club in L. mcdonaldi sp. nov. whereas in L. antofagastensis sp. nov. only the apical half is orange-red and the other half is white.

Anatomically, L. antofagastensis sp. nov. is characterized by having a proportionally large seminal receptacle in comparison to the size of the bursa copulatrix (about 15 times smaller in volume than the bursa copulatrix), which is different from L. mcdonaldi sp. nov. in which the seminal receptacle is about 20 times smaller in volume than the bursa copulatrix. Limacia cockerelli also has a larger seminal receptacle than L. mcdonaldi sp. nov., about 10 times smaller than the bursa copulatrix, but it is distinguishable from that of L. antofagastensis sp. nov. because the uterine duct does not connect to the seminal receptacle directly. The radula of L. antofagastensis sp. nov. is virtually indistinguishable from those of L. cockerelli and L. mcdonaldi sp. nov. (Fig. 8).

Genetically, Limacia antofagastensis sp. nov. is recovered as a distinct species in the species delimitation analyses and specimens of this species form a distinct clade in the phylogenetic analyses (Fig. 1).

Limacia janssi (Bertsch & Ferreira, 1974)

(Figs. 3g, 4d, 9)
Fig. 9

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

Type material

Holotype: CASIZ 19044, Bahía Santa Elena, Guanacaste, Costa Rica (not examined).

Other material examined

Smith Island, Bahía de los Ángeles, Mexico, intertidal, May 1976, 1 specimen 8 mm preserved length (LACM 140734). Honeymoon Island, San Carlos Bay, Mexico, 2 m depth, May 1977, 2 specimens 6–7 mm preserved length (LACM 25080). North end of the “Turtle Pen”, Isla Coronado, Bahía de los Ángeles, Mexico (29°05.0′N, 113°31.3′W), intertidal, 15–17 May 1976, 1 specimen 4 mm preserved length (LACM 1976–5.7). West side of Isla Coronado, Bahía de los Ángeles, Mexico (29°03.7′N, 113°31.0′W), intertidal, 11–14 May 1976, 1 specimen 5 mm preserved length (LACM 1976–3.1). San Carlos, Baja California Sur, Mexico, 3 m depth, 1 Sep 2015, 1 specimen 5 mm preserved length (CPIC 01503). Los Arcos, Puerto Vallarta, Jalisco, Mexico, 16 Feb 2008, 2 specimens 5–9 mm preserved length (LACM 174940). SE of Isla Canal de Afuera, Panama (7°41.48′N, 81°38.38′W), 5–15 m depth, 21 May 2003, 1 specimen 5 mm preserved length (LACM 153339).

External anatomy

Live animals up to 8 mm long. Body oval, completely surrounded by 2 rows of elongate to oval, often globose, dorso-lateral papillae (Fig. 3h). Papillae vary in length and width considerably, typically larger and more globose towards the posterior end of the body and smaller and thinner towards the anterior ends. Dorsum either completely smooth or with a single medial row of tubercles running from the area anterior to the rhinophores to in front of the gill. Gill composed of 3 bipinnate branchial leaves, arranged in a circle surrounding the anus. Rhinophores retractile, with short stalks and large clubs, bearing 10–13 lamellae. Posterior end of the foot projecting beyond the dorsum, forming a nearly triangular tail.

Background color opaque white, viscera visible as a pinkish area. Dorso-lateral papillae translucent white, with an elongate opaque white core visible through the surface, becoming orange red towards the apex; larger papillae each with a subapical opaque white sphere. Dorsum with numerous irregular orange patches. Gill completely off-white to cream. Rhinophores with white stalks and orange clubs. Tail uniformly white.

Internal anatomy

Reproductive system triaulic (Fig. 4d). Ampulla with one fold, connecting directly into the female gland complex, next to the proximal opening of the prostate. Prostate narrow, elongate, widening into the muscular deferent duct distally. Vagina short, much narrower than the deferent duct, connecting directly into the bursa copulatrix. Bursa copulatrix oval, inflated, thin-walled, about 4 times larger than the seminal receptacle. Seminal receptacle oval, muscular, connected to the female gland complex by a short uterine duct.

Radular formula 62 × 10.1.1.1.1.1.10 (CPIC 01503) to 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 surface (Fig. 9). Innermost lateral tooth very narrow and delicate, with a single curved cusp. Second innermost tooth wide and robust, with an elongate, hook-shaped main cusp pointing outwards and a smaller, blunt secondary cusp located next to it; tooth base with a transverse 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 corners and apical depressions, becoming narrower with less distinct cusps towards the outer end of the half-row.

Biology

Range

Northern Gulf of California (Bertsch 2014) to Panama (Hermosillo 2004).

Diet

Unidentified encrusting anascan bryozoan (see images in Hermosillo et al. 2005).

Reproduction

Limacia janssi deposits flat, spiral, peach-colored egg masses (Gonsalves-Jackson 2004).

Development

Gonsalves-Jackson (2004) reported that Limacia janssi from Panama hatched as planktonic veligers 110 um long after 7 days at 10 °C from eggs averaging 70.2 ± 5.1 μm in diameter (n = 10). Eggs and hatching larvae of these sizes are indicative of planktotrophic development in nudibranchs (Goddard 2004).

Remarks

Limacia janssi is the only tropical member of the genus Limacia in the Eastern Pacific and is very different morphologically from the other temperate species. For example, the dorso-lateral papillae of L. janssi are elongate to oval, often globose, whereas in the other species they are elongate, club-shaped. The color pattern of L. janssi is also distinct. Whereas temperate Eastern Pacific species are white with orange-red pigment on the rhinophores, tips of dorso-lateral papillae and gill, L. janssi has numerous irregular orange patches on the dorsum and the dorso-lateral papillae are translucent white, with an elongate opaque white core visible through the surface, becoming orange red towards the apex. Also, the larger papillae of L. janssi have a subapical opaque white sphere, and these structures are absent in the other species. Finally, Limacia janssi is recovered as a distinct species in the species delimitation analyses and specimens of this species form a distinct clade in the phylogenetic analyses (Fig. 1).

Limacia janssi is superficially similar to Limacia ornata (Baba, 1937), originally described from Japan (Baba 1937). Both species have globose dorso-lateral appendages and smooth dorsums. However, L. ornata is completely covered by conspicuous orange to orange-red spots (including the dorso-lateral appendages), which are absent in L. janssi.

Discussion

The present paper adds two additional taxa to the known diversity in the genus Limacia, a relatively species-poor group, until now containing only seven described species. A recent study in Europe (Caballer et al. 2016) revealed the existence of another cryptic species very similar to Limacia clavigera and although that particular study did not include molecular data, there are consistent morphological and external differences between the two species examined. Further research may reveal additional diversity, particularly in less studied areas such as Western and Southern Africa, where two poorly known species appear to coexist, and the tropical Indo-Pacific where several specimens belonging to one or more undescribed species have been reported (Caballer et al. 2016; Coleman 2008; Gosliner et al. 2008; Gosliner et al. 2015).

A third color form of L. cockerelli with distinctive red patches on the dorsum has been reported in the Northeastern Pacific literature (e.g., Behrens and Hermosillo 2005) and could constitute an additional pseudocryptic species. However, specimens of this red color form share the same habitat and range as typical L. cockerelli (see Wakeling 2001; Klug 2014) and feed on the same bryozoan prey, Hincksina velata (see Zade 2008; Hershman 2016). Also, the dorsal papillae are similar in size and distribution to those on L. cockerelli. We did not have access to specimens of this color form for this study, but based on the available evidence we consider it as a color variant of L. cockerelli until sequence data becomes available.

The present study constitutes another example of previously undetected diversity of heterobranch sea slugs along the Eastern Pacific Ocean, and supplements a series of recent studies providing evidence of the existence of numerous cryptic and pseudocryptic species of heterobranch sea slugs in this region (Krug et al. 2007; Cooke et al. 2014; Hoover et al. 2015; Lindsay and Valdés 2016; Kienberger et al. 2016; Lindsay et al. 2016). As in the cases of the Diaulula sandiegensis (Cooper, 1863) and the Doriopsilla albopunctata (Cooper, 1863) species complexes, Limacia cockerelli and the new species Limacia mcdonaldi sp. nov. show a substantial range overlap; in this particular case the overlap region stretches from San Diego, California to Salt Point in Sonoma County, an expanse of nearly 850 kilometers of coastline. Based on the examination of over 600 images of specimens of Limacia available on Flickr (https://www.flickr.com/search/?text=Limacia%20cockerelli&view_all=1&sort=date-taken-desc) and iNaturalist (http://www.inaturalist.org/taxa/50059-Limacia-cockerelli), it appears that L. mcdonaldi sp. nov. has been common in the Monterey and San Francisco 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 seven years prior to this warming event, only a few of the images of specimens of Limacia available from Central and Northern California were L. mcdonaldi sp. nov., suggesting this species was much less prevalent in the northern range overlap region when oceanographic conditions were more normal. Also, examination of photographic evidence suggests that L. mcdonaldi sp. nov. is more abundant in the southern portion of the range overlap region, where L. cockerelli is rare. Limacia mcdonaldi sp. nov. appears to specialize on different bryozoan than L. cockerelli, but until this is confirmed it is difficult to speculate on how the range overlap of the two species is maintained.

Another intriguing question is what process of speciation led to the formation of the partially sympatric Limacia cockerelli and Limacia mcdonaldi sp. nov. as well as the allopatric Limacia antofagastensis sp. nov., which is restricted to the southern hemisphere. Based on the molecular phylogenies here presented, these three species share a common ancestor and therefore their recent evolution was probably confined to the Eastern Pacific. There are no two identical documented cases of cryptic and pseudocryptic speciation for the Eastern Pacific in which sister species pairs display similar ranges and/or range overlaps. Thus, it is difficult to compare the biogeographic pattern observed in Eastern Pacific species of Limacia to other cases. In fact most documented cases for cryptic and pseudocryptic species pairs of heterobranch sea slugs have very limited range overlaps in the Eastern Pacific. As mentioned above only the Diaulula sandiegensis and the Doriopsilla albopunctata species complexes display substantial 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 fulva (MacFarland, 1905), as there are no obvious barriers to dispersal, past or present, their ranges overlap completely, and they show some differences in reproductive anatomy that could derive from differential sexual selection. On the other hand, Lindsay et al. (2016) suggested that glaciation driven vicariance may have resulted in the allopatric speciation of Diaulula sandiegensis and Diaulula odonoghuei (Steinberg, 1963) as the latter maintains a transpacific range, and the split between the two species coincides with major cooling events. With the limited available information it is difficult to provide hypotheses for the causes of the speciation between the Eastern Pacific species of Limacia. Further research on the habitat use, possible reproductive barriers between sympatric species, and divergence times may provide insights into the causes of speciation. However, the split between L. antofagastensis sp. nov. and its closest northern hemisphere relative L. mcdonaldi sp. nov. may have an obvious explanation. The range of L. antofagastensis sp. nov. is separated from that of L. mcdonaldi sp. nov. by the Panamic Biogeographic Province, a 4,000 km-long stretch of tropical waters from the mouth of the Gulf of California to the Gulf of Guayaquil (Briggs and Bowen 2012). The ocean temperature in the Panamic Province varied considerably in the past, for example, cooling events during the Pleistocene greatly reduced average temperatures (Lawrence et al. 2006). Although the Panamic Province remained tropical (Stanley 1984), oceanographic changes resulted in faunal migrations and reshuffling of benthic molluscan communities (Roy et al. 1995) potentially allowing migration of temperate species of Limacia across this stretch of tropical water. Subsequent vicariance caused by the return to more tropical conditions, such as those occurring presently, would have resulted in the separation of Northern and Southern Hemisphere lineages. Again, additional data on divergence times may shed light on the evolution and speciation within Eastern Pacific species of Limacia. However, the lack of obvious calibration points prevents further research on this topic at this time.

Notes

Acknowledgements

We thank Alejandro Ramírez, Julissa Rassa, Eduardo Nahualhuen, and Pedro Coronado for their assistance during diving activities as well as the crew of Santa Maria S.A. for providing logistic support in Northern Chile. We also thank Zambra López for her help with software support and Craig Hoover and Sandra Millen for providing several specimens for this study. Ellen Strong facilitated obtaining photographs of the Holotype of L. cockerelli taken by Yolanda Villacampa. The SEM work was conducted at the California State Polytechnic University SEM laboratory supported by the US National Science Foundation (NSF) grant DMR-1429674. Lindsey Groves (LACM) assisted with the curation of specimens and access to the LACM collection. Financial support was provided by project 5303 and Laboratorio de Modelamiento de Sistemas Ecológicos Complejos (LAMSEC) of the Universidad de Antofagasta, Chile.

Supplementary material

12526_2017_676_MOESM1_ESM.docx (588 kb)
ESM 1 (DOCX 588 kb)

References

  1. Akaike H (1974) A new look at the statistical model identification. IEEE Trans Autom Control 19:716–722CrossRefGoogle Scholar
  2. Angulo-Campillo OJ (2003) Variación espacio-temporal de las poblaciones de opistobranquios (Mollusca: Opisthobranchia) en tres localidades de B.C.S., Mexico. Master of Science Thesis, Departamento de Pesquerías y Biología Marina, Centro Interdisciplinario de Ciencias Marinas: La Paz, Baja California Sur, Mexico (unpublished).Google Scholar
  3. Angulo-Campillo O (2005) A four year survey of the opisthobranch fauna (Gastropoda, Opisthobranchia) from Baja California Sur, Mexico. Vita Malacologica 3:43–50Google Scholar
  4. Baba K (1937) Opisthobranchia of Japan (II). Jour. Dept. Agr. Kyushu Imp. Univ. 5:289–344, pls.1–2.Google Scholar
  5. Behrens (2004) Pacific Coast Nudibranchs, Supplement II New Species to the Pacific Coast and New Information on the Oldies. Proc Calif Acad Sci 55(2):11–54Google Scholar
  6. Behrens DW, Hermosillo A (2005) Eastern Pacific nudibranchs, a guide to the opisthobranchs from Alaska to Central America. Sea Challengers, Monterey, California. 137 pp., 314 photos.Google Scholar
  7. Bertsch H (2014) Biodiversity in La Reserva de la Biósfera Bahía de los Ángeles y Canales de Ballenas y Salsipuedes: Naming of a new genus, range extensions and new records, and species list of Heterobranchia (Mollusca: Gastropoda), with comments on biodiversity conservation within marine reserves. The Festivus 46:158–177Google Scholar
  8. Bertsch H, Ferreira AJ (1974) Four new species of nudibranchs from tropical West America. The Veliger 16:343–353Google Scholar
  9. Briggs JC, Bowen BW (2012) A realignment of marine biogeographic provinces with particular reference to fish distributions. J Biogeogr 39:12–30CrossRefGoogle Scholar
  10. Caballer M, Almón B, Pérez J (2016) The sea slug genus Limacia Müller, 1781 (Mollusca: Gastropoda: Heterobranchia) in Europe. Cah Biol Mar 57:35–42Google Scholar
  11. Clark T (2006) ‘Limacia cockerelli.’ Available at http://week.divebums.com/2006/Jun12-2006/index.html [7th image from top]. Accessed 15 July 2016.
  12. Coleman N (2008) Nudibranchs Encyclopedia. Catalogue of Asia/Indo-Pacific sea slugs. Neville Coleman’s Underwater Geographic Pty: Springwood Queensland, Australia, 416 ppGoogle Scholar
  13. Cooke S, Hanson D, Hirano Y, Ornelas-Gatdula E, Gosliner TM, Chernyshev AV, Valdés A (2014) Cryptic diversity of Melanochlamys sea slugs (Gastropoda, Aglajidae) in the North Pacific. Zool Scripta 43:351–369CrossRefGoogle Scholar
  14. Di Lorenzo E, Mantua NJ (2016) Multi-year persistence of the 2014/15 North Pacific marine heatwave. Nat Clim Chang. doi: 10.1038/nclimate3082 CrossRefGoogle Scholar
  15. Filatov D (2002) Proseq: a software for preparation and evolutionary analysis of DNA sequence data sets. Mol Ecol Not 2:621–624CrossRefGoogle Scholar
  16. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3:294–299PubMedGoogle Scholar
  17. Goddard JHR (1984) The opisthobranchs of Cape Arago, Oregon, with notes on their biology and a summary of benthic opisthobranchs known from Oregon. The Veliger 27:143–163Google Scholar
  18. Goddard JHR (1998) A summary of the prey of nudibranch molluscs from Cape Arago, Oregon. Opisthobranch Newsletter 24:11–14Google Scholar
  19. Goddard JHR (2004) Developmental mode in benthic opisthobranch molluscs from the northeast Pacific Ocean: feeding in a sea of plenty. Can J Zool 82:1954–1968CrossRefGoogle Scholar
  20. Gonsalves-Jackson DC (2004) Opisthobranch mollusks across the Isthmus of Panama: systematics and biogeographic distribution of developmental types. Dissertation, Florida Institute of Technology (unpublished).Google Scholar
  21. Gosliner TM, Valdés A, Behrens DW (2015) Nudibranch and sea slug identification, Indo-Pacific. New World Publication: Jacksonville, Florida 408 pp.Google Scholar
  22. Gosliner TM, Behrens DW, Valdés Á (2008) Indo-Pacific Nudibranchs and Sea Slugs. A field guide to the World’s most diverse fauna. Sea Challengers Natural History Books and California Academy of Sciences: California 426 pp.Google Scholar
  23. Green B (2007) Sea scallop and Cockerell’s dorid (Limacia cockerelli). Available at https://www.flickr.com/photos/lemurdillo/1369606503 Accessed 15 July 2016
  24. Guernsey M (1912) Some of the Mollusca of Laguna Beach. In: First Annual Report of the Laguna Marine Laboratory pp. 68–82. Department of Biology, Pomona College: Claremont, CaliforniaGoogle Scholar
  25. Hebert P, Penton E, Burns J, Janzen D, Hallwachs W (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci 101:14812–14817CrossRefGoogle Scholar
  26. Hermosillo A (2004) Opisthobranch mollusks of Parque Nacional de Coiba, Panamá (tropical Eastern Pacific). The Festivus 36:105–117Google Scholar
  27. Hermosillo A, Behrens DW, Ríos-Jara E (2005) Opistobranquios de México. CONABIO: Guadalajara, Mexico, 143 ppGoogle Scholar
  28. Hershman D (2016) Limacia cockerelli. Available at: https://www.flickr.com/photos/hershman/26068755511 Accessed 17 February 2017
  29. Hoover C, Lindsay T, Goddard JHR, Valdés Á (2015) Seeing double: pseudocryptic diversity in the Doriopsilla albopunctataDoriopsilla gemela species complex of the north‐eastern Pacific. Zool Scripta 44:612–631CrossRefGoogle Scholar
  30. Huelsenbeck J, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755CrossRefGoogle Scholar
  31. Johnson ME, Snook HJ (1927) Seashore animals of the Pacific coast. Macmillan Company: New York; reprinted 1967 by Dover Publications: New York, 633 ppGoogle Scholar
  32. Keen AM (1971) Seashells of Tropical West America: Marine Mollusks from Baja California to Peru. Stanford University Press, California, 1064 ppGoogle Scholar
  33. Kienberger K, Carmona L, Pola M, Padula V, Gosliner TM, Cervera JL (2016) Aeolidia papillosa (Linnaeus, 1761) (Mollusca: Heterobranchia: Nudibranchia), single species or a cryptic species complex? A morphological and molecular study. Zool J Linn Soc 177:481–506CrossRefGoogle Scholar
  34. Klug D (2014) Nudibranch5April18-14. Available at: https://www.flickr.com/photos/diverdoug/14344674700 Accessed 17 February 2017
  35. Krug PJ, Ellingson RA, Burton R, Valdés A (2007) A new poecilogonous species of sea slug (Opisthobranchia: Sacoglossa) from California: Comparison with the planktotrophic congener Alderia modesta (Lovén, 1844). J Molluscan Stud 73:29–38CrossRefGoogle Scholar
  36. Lance JR (1961) A distributional list of Southern California opisthobranchs. The Veliger 4:64–69Google Scholar
  37. Larkin M, Blackshields G, Brown N, Chenna R, Mcgettigan P, Mcwilliam H, Valentin F, Wallace I, Wilm A, Lopez R, Thompson et al. (2007). Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948Google Scholar
  38. Lawrence KT, Liu Z, Herbert TD (2006) Evolution of the eastern tropical Pacific through Plio-Pleistocene glaciation. Science 312:79–83CrossRefGoogle Scholar
  39. Lindsay T, Valdés A (2016) The model organism Hermissenda crassicornis (Gastropoda: Heterobranchia) is a species complex. PLoS One 11:e0154265CrossRefGoogle Scholar
  40. Lindsay T, Kelly J, Chichvarkhin A, Craig S, Kajihara H, Mackie J, Valdés Á (2016) Changing spots: pseudocryptic speciation in the North Pacific dorid nudibranch Diaulula sandiegensis (Cooper, 1862) (Gastropoda: Heterobranchia). J Molluscan Stud. doi: 10.1093/mollus/eyw026 CrossRefGoogle Scholar
  41. MacFarland FM (1905) A preliminary account of the Dorididae of Monterey Bay, California, and vicinity. Proc Biol Soc Wash 18:35–54Google Scholar
  42. MacFarland FM (1906) Opisthobranchiate Mollusca from Monterey Bay, California, and vicinity. Bull US Bur Fish 25:109–151, pls. 18–31.Google Scholar
  43. Maddison WP, Maddison DR (2011) Mesquite: a modular system for evolutionary analysis. Version 2.75 http://mesquiteproject.org Accessed 30 July 2016
  44. McDonald GR (1983) A review of the nudibranchs of the California coast. Malacologia 24:114–276Google Scholar
  45. McDonald GR, Nybakken JW (1978) Additional notes on the food of some California nudibranchs with a summary of known food habits of California species. The Veliger 21:110–118Google Scholar
  46. McDonald GR, Nybakken JW (1980) Guide to the nudibranchs of California. American Malacologists, inc., Melbourne, Florida, 72 ppGoogle Scholar
  47. Miller S, Dykes D, Polesky H (1988) A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215CrossRefGoogle Scholar
  48. O’Donoghue CS, O’Donoghue E (1922) Notes on the nudibranchiate Mollusca from the Vancouver Island region. II. The spawn of certain species. Trans R Can Inst 14:131–143Google Scholar
  49. Ortea J, Quero A, Rodríguez G, Valdés A (1989) Estudio de Limacia clavigera (Müller, 1776) (Mollusca: Nudibranchia) con nota sobre su distribución geográfica y la validez del género Laila MacFarland, 1905. Rev Biol Univ Oviedo 7:99–107Google Scholar
  50. Palumbi S, Martin A, Romano S, Owen MacMillan W, Stice L, Grabowski G (1991) The Simple Fool’s Guide to PCR. Department of Zoology, University of Hawaii, Honolulu, 45 ppGoogle Scholar
  51. Pola M, Cervera JL, Gosliner TM (2007) Phylogenetic relationships of Nembrothinae (Mollusca: Doridacea: Polyceridae) inferred from morphology and mitochondrial DNA. Mol Phylogenet Evol 43:726–742CrossRefGoogle Scholar
  52. Pola M, Gosliner TM (2010) The first molecular phylogeny of cladobranchian opisthobranchs (Mollusca, Gastropoda, Nudibranchia). Mol Phylogenet Evol 56:931–941CrossRefGoogle Scholar
  53. Posada D (2008) JModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256CrossRefGoogle Scholar
  54. Puillandre N, Lambert A, Brouillet S, Achaz G (2012) ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Mol Ecol 21:1864–1877CrossRefGoogle Scholar
  55. Radulovici A, Archambault P, Dufresne F (2010) DNA barcoding for marine biodiversity: moving fast forward? Diversity 2:450–472CrossRefGoogle Scholar
  56. Roy K, Jablonski D, Valentine JW (1995) Thermally anomalous assemblages revisited: patterns in the extraprovincial latitudinal range shifts of Pleistocene marine mollusks. Geology 23:1071–1074CrossRefGoogle Scholar
  57. Schrödl M, Grau JH (2006) Nudibranchia from the remote southern Chilean Guamblin and Ipún islands (Chonos Archipelago, 44–45°S), with re-description of Rostanga pulchra MacFarland, 1905. Rev Chil Hist Nat 79:3–12CrossRefGoogle Scholar
  58. Schrödl M (2003) Sea slugs of southern South America: Systematics, biogeography and biology of Chilean and Magellanic Nudipleura (Mollusca: Opisthobranchia). Conch-Books, Hackenheim, Germany, 165 ppGoogle Scholar
  59. Stamatakis A (2006) RAxML-VI-HPC: Maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22:2688–2690CrossRefGoogle Scholar
  60. Stanley SM (1984) Temperature and biotic crises in the marine realm. Geology 12:205–208CrossRefGoogle Scholar
  61. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mole Biol Evol 30:2725–2729CrossRefGoogle Scholar
  62. Thollesson M (2000) Increasing fidelity in parsimony analysis of dorid nudibranchs by differential weighting, or a tale of two genes. Mol Phylogenet Evol 16(2):161–172CrossRefGoogle Scholar
  63. Vitsky A (2008) Limacia cockerelli. Available at http://week.divebums.com/2008/Sep08-2008/index.html [2nd image from top] Accessed 15 July 2016.
  64. Wakeling M (2001) Laila cockerelli colour forms from Canada. Available at http://www.seaslugforum.net/find/5549Accessed 17 February 2017
  65. Zade R (2008) Limacia cockerelli and scaleworm. Available at http://www.seaslugforum.net/find/21852 Accessed 17 February 2017

Copyright information

© Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Roberto A. Uribe
    • 1
  • Fabiola Sepúlveda
    • 2
  • Jeffrey H. R. Goddard
    • 3
  • Ángel Valdés
    • 4
  1. 1.Laboratorio de Biodiversidad y Ecología BentónicaInstituto del Mar del Perú – IMARPEChimbotePerú
  2. 2.Laboratorio de Ecología Parasitaria y Epidemiología Marina LEPyEM, Facultad de Ciencias del Mar y Recursos BiológicosUniversidad de AntofagastaAntofagastaChile
  3. 3.Marine Science InstituteUniversity of CaliforniaSanta BarbaraUSA
  4. 4.Department of Biological SciencesCalifornia State Polytechnic UniversityPomonaUSA

Personalised recommendations