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ORIGINAL PAPER
Integrative systematics of the genus Limacia O. F. Müller, 1781
(Gastropoda, Heterobranchia, Nudibranchia, Polyceridae)
in the Eastern Pacific
Roberto A. Uribe
1
&Fabiola Sepúlveda
2
&Jeffrey H. R. Goddard
3
&Ángel Valdés
4
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-
ses of specimens of the genus Limacia collected in the
Eastern Pacific Ocean indicate that four species of Limacia
occur in the region. Limacia cockerelli,previouslyconsidered
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 spe-
cies 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 genet-
ically 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 tax-
onomy, systematics and phylogeny, allowing researchers
to assess whether morphological variations correspond to
different species or merely represent intra-specific pheno-
typic expression due to environmental variation (Hebert
et al. 2004;Radulovicietal.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 nudi-
branchs (Hoover et al. 2015;LindsayandValdés2016;
Kienberger et al. 2016; Lindsay et al. 2016). Some of
these newly described taxa resulted from splitting wide-
spread species in the Northeastern Pacific into two sister
species, mainly allopatric, with limited overlap near the
SanFranciscoBayArea(Krugetal.2007;Lindsayand
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 biogeo-
graphic barriers that could promote allopatric divergence.
Communicated by V. Urgorri
This article is registered in ZooBank under urn:lsid:zoobank.org:pub:
A97ACF68-C637-4507-AF42-6A0065014FE5
Electronic supplementary material The online version of this article
(doi:10.1007/s12526-017-0676-5) contains supplementary material,
which is available to authorized users.
*Ángel Valdés
aavaldes@cpp.edu
1
Laboratorio de Biodiversidad y Ecología Bentónica, Instituto del Mar
del Perú –IMARPE, ChimboteLos Pinos s/n. Urb. Nueva Caleta,
Áncash, Perú
2
Laboratorio de Ecología Parasitaria y Epidemiología Marina
LEPyEM, Facultad de Ciencias del Mar y Recursos Biológicos,
Universidad de Antofagasta, Antofagasta, Chile
3
Marine Science Institute, University of California, Santa
Barbara, CA 93106, USA
4
Department of Biological Sciences, California State Polytechnic
University, 3801 West Temple Avenue, Pomona, CA 91768, USA
Mar Biodiv
DOI 10.1007/s12526-017-0676-5
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 morpho-
logically 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 sug-
gested 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 pub-
lished molecular study of a species found in both hemispheres
confirms that southern records of Aeolidia papillosa
(Linnaeus, 1761) constitute a distinct, cryptic, endemic spe-
cies 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 tem-
perate, tropical, and newly discovered southern temperate pop-
ulations. The genus Limacia is a group of polycerid dorid nu-
dibranchs characterized by having a unique body plan, with one
to several rows of elongate dorso-lateral appendages surround-
ing 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;Goslineretal.2015) but is rare. In the Eastern
Pacific, L. cockerelli has a broad geographic range from
Alaska to Baja California and displays considerable color var-
iation (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 ofthe 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, north-
ern Chile). The specimens from Chile were collected on a
barren ground system at 5 m depth. Additionally, two speci-
mens of Limacia janssi from San Carlos and Bahía
Magdalena, Mexico, were collected, examined and se-
quenced. 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 unsuit-
able 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 coxidase 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 anal-
yses, 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 cen-
trifugation 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 po-
lymerase (Promega, Madison, USA), 7 μl of 5 × PCR buffer,
5.6 μlofMgCl
2
(25 mM), 2.1 μl ofBSA (10 mg/ml), 0.7 μlof
deoxynucleotide triphosphate (dNTP) (10 mM), 1.4 μlofeach
primer (10 pM) and 5 μl of template DNA and 11.625 μlof
nuclease-free water. PCR conditions were as follows: 1) for
COI –initial denaturation 94 °C, 5 min; 35 cycles of denatur-
ation 94 °C, 1 min, annealing 44 °C, 30 s; extension 72 °C,
1 min; final extension 72 °C, 7 min; 2) for 16S –initial dena-
turation 94 °C, 2 min; 35 cycles of denaturation 94 °C, 30 s,
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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.
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 mod-
el 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 var-
iation as well as the proportion of invariable positions, accord-
ing 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 anal-
yses 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 in-
dividual 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
Tabl e 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 Polaand 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
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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 dis-
sected. The reproductive anatomy and radular
morphology were used as the main internal traits for spe-
cies 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 attach-
ment. The penises were removed, mounted on a micro-
scope 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.
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
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Results
Phylogenetic analyses
A total of 8 sequences of the 16S rRNA gene were ob-
tained 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.Forthe
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 spec-
imens 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.
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 speci-
mens 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 smallestgenetic distance among two clades was observed
between L. cockerelli from California and from Chile
[L. mcdonaldi sp.nov.andL. antofagastensis sp.nov.respec-
tively] (3.7–4.5%) and the largest genetic distance was ob-
served 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
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).
Tab l e 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
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Morphological examination
Based on the molecular evidence above, as well as morpho-
logical 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 cov-
ered with numerous, small tubercles (Fig. 3a–c)andasouth-
ern 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. 1and 3g). No consistent differences were ob-
served 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 indi-
cates 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 com-
plex 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
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 2–3rows 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 poste-
rior ends. The dorsum bears numerous small tubercles, ar-
ranged 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 retrac-
tile, with short stalks and large clubs, bearing 10–12 lamellae.
Posterior end of the foot projecting beyond the dorsum,
forming a nearly triangular tail.
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
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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
tipinothers.
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 nar-
row, elongate, widening into the muscular deferent duct
distally. Vagina elongate, as wide as the deferent duct,
joining the seminal receptacle-connecting duct before en-
tering the bursa copulatrix. Bursa copulatrix inflated, thin-
walled, about 10 times larger in volume than the seminal
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
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receptacle. Seminal receptacle oval, muscular, connected
to the female gland complex by a short uterine duct,
which emerges from the duct connecting the seminal re-
ceptacle 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 ofa rectangular plate with an irregular
surface (Fig. 5). Innermost lateral tooth very narrow and del-
icate, hook-shaped, with a single curved cusp. Second inner-
most 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)toPointLoma,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 rib-
bons 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;Goddard1984).
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
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
Mar Biodiv
describing the large pallial papillae, he described the Bmedian
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 Bmuch smaller individuals of the
same species^collected from San Pedro by T. D. A.
Cockerell, Bfor 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’s1905 and 1906
papers, as well as his acknowledgment of Anna Nash, Bartist
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 col-
lected by Cockerell in San Pedro, but this time mentions
receiving these specimens, along with Cockerell’sBnotes up-
on 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 descrip-
tion of L. cockerelli. Near the end of the second paragraph (p.
134) he adds to the description of the low dorsal tubercles the
phrase, Bthe 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 spe-
cies 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.
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
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
Mar Biodiv
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)
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 7–8mmpreserved
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 pre-
served 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), inter-
tidal, 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 poste-
rior ends. Dorsum with a single medial row of tubercles run-
ning from the area anterior to the rhinophores to in front of the
gill. Gill composed of 5–6 bipinnate branchial leaves, ar-
ranged in a circle surrounding the anus. Rhinophores retrac-
tile, 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, elon-
gate, 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 ofa rectangular plate with an irregular
surface (Fig. 7). Innermost lateral tooth very narrow and
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)
Mar Biodiv
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, person-
al communication to JG, 15 May 2016) to Cabo San Lucas, Baja
California Sur (Lance 1961) and into the Gulf of California to
BahiadeLosAngeles(Keen1971; Angulo-Campillo 2003,2005).
Diet Limacia mcdonaldi sp. nov. has been photographed in
California on unidentified encrusting anascan bryozoans (e.g.,
Clark 2006;Green2007).
Reproduction and Development: Nothing is known about re-
production and development in this species.
Derivatio nominis
Named in honor of Gary McDonald, who has spent de-
cades 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 dig-
ital 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 sin-
gle line, running from the area anteriorto the rhinophores to in
front of the gill. These tubercles are always orange-red, where-
as in L. cockerelli the dorsal tubercles are smaller, never form a
line and are either white or have a central orange-red spot.
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
Mar Biodiv
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 il-
lustration of L. mcdonaldi sp. nov. (as L. cockerelli) and clear-
ly 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)
Type material
Holotype: LACM 3356, Antofagasta, Chile (23°38′39″S,
70°24′39″W),11mmpreservedlength,2014.Paratype:
LACM 3357, Antofagasta, Chile (same coordinates as holo-
type), 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 tuber-
cles 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 retrac-
tile, with short stalks and large clubs, bearing 11 lamellae.
Background color opaque white, viscera visible as a pink-
ish area. Dorso-lateral papillae translucent white, with an elon-
gate 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,
connectingdirectly into the bursa copulatrix. Bursa copulatrix
inflated, thin-walled, about 15 times larger in volume than the
seminal receptacle. Seminal receptacle oval, muscular, con-
nected 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-
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
Mar Biodiv
shaped main cusp pointing outwards and a smaller, blunt sec-
ondary 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 pig-
mentation of the rhinophores, which have an almost complete-
ly orange-red club in L. mcdonaldi sp.nov.whereasin
L. antofagastensis sp. nov. only the apical half is orange-red
and the other half is white.
Anatomically, L. antofagastensis sp. nov. is character-
ized 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.inwhichthesem-
inal 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 distin-
guishable 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 vir-
tually 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 phyloge-
netic analyses (Fig. 1).
Limacia janssi (Bertsch & Ferreira, 1974)
(Figs. 3g,4d,9)
Type material
Holotype: CASIZ 19044, Bahía Santa Elena, Guanacaste,
Costa Rica (not examined).
Other material examined
Smith Island, Bahía de los Ángeles, Mexico, intertidal,
May1976,1specimen8mmpreservedlength(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 BTurtle 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 pink-
ish area. Dorso-lateral papillae translucent white, with an elon-
gate opaque white core visible through the surface, becoming
orange red towards the apex; larger papillae each with a sub-
apical opaque white sphere. Dorsum with numerous irregular
orange patches. Gill completely off-white to cream.
Rhinophores with white stalks and orange clubs. Tail uniform-
ly white.
Mar Biodiv
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 di-
rectly into the bursa copulatrix. Bursa copulatrix oval, inflat-
ed, thin-walled, about 4 times larger than the seminal recepta-
cle. Seminal receptacle oval, muscular, connected to the fe-
male 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 ofa rectangular plate with an irregular
surface (Fig. 9). Innermost lateral tooth very narrow and del-
icate, 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 incon-
spicuous cusps on their inner lower corners and apical depres-
sions, 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
110umlongafter7daysat10°Cfromeggsaveraging70.2±
5.1 μm in diameter (n = 10). Eggs and hatching larvae of these
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
Mar Biodiv
sizes are indicative of planktotrophic development in nudi-
branchs (Goddard 2004).
Remarks
Limacia janssi is the only tropical member of the genus
Limacia in the Eastern Pacific and is very different morpho-
logically 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 sur-
face, 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 di-
versity 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 differ-
ences 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;Klug2014) 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 previous-
ly 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 cryp-
tic 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 allo-
patric Limacia antofagastensis sp. nov., which is restricted to
the southernhemisphere. 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
Mar Biodiv
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 substan-
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
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 explana-
tion. 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 exam-
ple, cooling events during the Pleistocene greatly reduced av-
erage temperatures (Lawrence et al. 2006). Although the
Panamic Province remained tropical (Stanley 1984), oceano-
graphic changes resulted in faunal migrations and reshuffling
of benthic molluscan communities (Roy et al. 1995) potential-
ly 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 specia-
tion within Eastern Pacific species of Limacia.However,the
lack of obvious calibration points prevents further research on
this topic at this time.
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.
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