Bulletin of the Southern California Academy of Sciences
Volume 112 | Issue 3
Article 2
2013
Genetic Structure of Polycera alabe and P. atra
(Mollusca: Opistobranchia: Nudibranchia) in the
Pacific Coast of North America
Monica Santander
California State Polytechnic University, masantander@csupomona.edu
Angel Valdes
California State Polytechnic University - Pomona, aavaldes@csupomona.edu
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Recommended Citation
Santander, Monica and Valdes, Angel (2013) "Genetic Structure of Polycera alabe and P. atra (Mollusca: Opistobranchia:
Nudibranchia) in the Pacific Coast of North America," Bulletin of the Southern California Academy of Sciences: Vol. 112: Iss. 3.
Available at: http://scholar.oxy.edu/scas/vol112/iss3/2
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Santander and Valdes: Genetic structure in Polycera alabe and P. atra
Bull. Southern California Acad. Sci.
112(3), 2013, pp. 176–184
E Southern California Academy of Sciences, 2013
Genetic structure of Polycera alabe and P. atra
(Mollusca: Opistobranchia: Nudibranchia) in
the Pacific coast of North America
Monica Santander and Ángel Valdés
Department of Biological Sciences, California State Polytechnic University, 3801 West
Temple Avenue, Pomona, California 91768, USA, aavaldes@csupomona.edu
Abstract.—Polycera alabe and Polycera atra are closely related opisthobranch sea
slugs found in coastal habitats along the eastern Pacific. Both species are extremely
variable in external coloration and some of this variation appears to be correlated
with geographic range. To determine the phylogenetic relationships and genetic
structure of P. alabe and P. atra molecular phylogenies were generated using two
genes: H3 (nuclear) and 16S (mitochondrial). Sequence data indicate that
populations of P. atra are genetically homogeneous and lack geographic structure
along the range of the species. In contrast, Polycera alabe consists of three previously
unrecognized, distinct clades with overlapping ranges. The northernmost clade of
P. alabe is sister to P. atra, thus the current definition of P. alabe constitutes a
paraphyletic assemblage. The southernmost clade presents morphological differences in the radula compared to the other two clades. These data suggest that P. alabe is
most likely a species complex.
The opisthobranch mollusks Polycera alabe Collier & Farmer, 1964 and Polycera atra
MacFarland, 1905 (family Polyceridae Alder & Hancock, 1845) are benthic marine
invertebrates found in coastal habitats along the eastern Pacific. Anecdotal evidence
suggests that El Niño Southern Oscillation (ENSO) plays significant roles in range
expansions and contractions of both species (Kerstitch and Bertsch 2007), which
otherwise have allopatric ranges, with the boundary at Punta Eugenia and Isla Cedros,
Mexico (Angulo Campillo 2003, 2005). The known geographic range of P. atra spans
from Cape Arago, Oregon (Goddard 1984) to La Paz, Mexico (Angulo Campillo 2003,
2005). However, the northernmost record of P. atra in Oregon was during the intense El
Niño of 1982–1983 (Goddard 1984), and reports of this species south of Punta Eugenia
are very rare (Angulo Campillo 2003, 2005). Polycera alabe is found from the Channel
Islands in Southern California to Panama (Behrens and Hermosillo 2005), with an
isolated record from northern Chile (Schrödl 2003), but the only record of P. alabe north
of Punta Eugenia, in Southern California, was during the strong El Niño of 1997–1998
(Engle and Richards 2001).
Both P. atra and P. alabe are extremely variable in external coloration. Behrens and
Hermosillo (2005) recognized four distinct color forms in P. alabe that they classified as
variations A–D, each with slightly different geographic ranges. Variation A is found
throughout the Gulf of California, the Pacific coast of Baja California and Southern
California; variation B is found in the Pacific coast of Mexico; variation C is present from
Mexico to Costa Rica and Panama; and variation D is only found in Central America.
Based on these data, Behrens and Hermosillo (2005) suggested the possibility of the
existence of several species. Animals of P. atra are also found to vary in coloration from
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GENETIC STRUCTURE OF POLYCERA ALABE AND P. ATRA
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Table 1. List of specimens used in the study; including locality, voucher number, and GenBank
Accession numbers. Specimens for which the radula was studied are marked with an asterisk (*).
Species
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
P.
cf. capensis
hedgpethi
atra
atra
atra
atra *
atra
alabe
alabe *
alabe *
alabe
alabe
alabe *
alabe *
alabe
Locality
Date
Voucher
16S
H3
South Africa
San Francisco, CA
San Francisco, CA
Monterey Bay, CA
Santa Barbara, CA
Long Beach, CA
La Paz, MX
Sonora, MX
Sonora, MX
Sonora, MX
La Paz, MX
Jalisco, MX
Jalisco, MX
Colima, MX
Guerrero, MX
11 Sep 2009
12 Sep 2009
19 Aug 2010
18 Aug 2010
17 Jul 2010
2 Apr 1974
Mar 1975
Nov 1975
May 1976
2 Apr 1974
15 Feb 2008
18 Feb 2008
28 Jan 1976
18 Mar 2004
CASIZ 176907
CPIC 00805
CPIC 00806
CPIC 00425
CPIC 00807
CPIC 00808
LACM 140740
LACM 140737
LACM 140736
LACM 76-5
LACM 140738
LACM 174944
LACM 174953
LACM 140739
CPIC 00809
HM162597
KF425278
KF425277
KF425276
KF425275
KF425272
KF425273
KF425274
KF425267
KF425271
KF425270
KF425268
KF425269
HM162503
KF425292
KF425291
KF425290
KF425289
KF425288
KF425287
KF425284
KF425285
KF425286
KF425279
KF425283
KF425282
KF425280
KF425281
very dark to light; MacFarland (1966) indicated that southern animals tend to be lighter,
suggesting some geographic variation. However, both our observations and unpublished
data (H. Bertsch, pers. comm.) indicate a possible relationship between coloration and
diet. Both species feed commonly on bryozoans of the genus Bugula and Membranipora
(McDonald 1983), and P. atra collected on Membranipora are consistently lighter than
those found on Bugula.
The objective of this paper is to examine the genetic structure of P. alabe and P. atra in
the Pacific coast of North America, including specimens from northern California to the
southern Mexican state of Guerrero. A mitochondrial (16S) and a nuclear (H3) gene were
sequenced.
Material and Methods
Source of Specimens
Eight specimens of P. alabe, five specimens of P. atra, and one specimen of Polycera
hedgpethi Marcus, 1964 were sequenced (Table 1). Specimens were obtained from the
collections of the Natural History Museum of Los Angeles County (LACM) and the Cal
Poly Pomona research collections (CPIC). Sequences for one specimen of Polycera cf.
capensis Quoy & Gaimard, 1824 deposited at the California Academy of Sciences
(CASIZ) were obtained from GenBank.
Morphological Examination
Four specimens of Polycera alabe were dissected, and the radula of each was removed.
Surrounding tissue on the radula was removed manually and then placed in low
concentration [0.1] sodium hydroxide for approximately 24 hours to remove any
remaining tissue. The radula was then dried, mounted with electrically conductive
double-sided adhesive tape, and sputter coated with gold/palladium alloy for examination
with a scanning electron microscope (SEM) Hitachi S-3000N at the Natural History
Museum of Los Angeles County.
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Table 2. Forward (F) and reverse (R) PCR primers used to amplify regions of the nuclear H3 gene and
mitochondrial 16S and COI genes.
Name
Sequence 59–39
Source
H3
HexAF (F)
HexAR (R)
ATG GCT CGT ACC AAG CAG ACG GC
ATA TCC TTG GGC ATG ATG GTG AC
Colgan et al. (1998)
Colgan et al. (1998)
CGC CTG TTT ATC AAA AAC AT
CCG GTC TGA ACT CAG ATC ACG T
AAA GAC GAG AAG ACC CTT AGA GTT TT
AAA ACT CTA AGG GTC TTC TCG TCT TT
Palumbi et al. (1996)
Palumbi et al. (1996)
Ornelas-Gatdula et al. 2011
Ornelas-Gatdula et al. 2011
16S rRNA
16Sar-L (F)
16Sbr-H (R)
16Sar-FAP (F)
16Sbr-FAP (R)
DNA Extraction
DNA extraction was performed using either a hot ChelexH protocol or the DNeasyH
Blood and Tissue Kit (Qiagen). Approximately 1–3 mg of tissue was taken from the foot
or tail of the animals and cut into fine pieces for extraction for each of the two protocols.
For the ChelexH extraction, the tissue was rinsed and rehydrated using 1.0 mL TE buffer
(10 mM Tris, 1 mM EDTA, pH 8.0) for 15–20 minutes. A 10% (w/v) ChelexH 100 (100–
200 mesh, sodium form, Bio-Rad) was prepared using TE buffer. After rehydration, the
mixture was then centrifuged, 975.00 mL of the supernatant was removed, and 175.00 mL
of the ChelexH solution was added. Samples were then heated in a 56uC water bath for
20 minutes, heated in a 100uC heating block for 8 minutes, and the supernatant was used
for PCR. The DNeasyH protocol supplied by the manufacturer was followed, with some
modifications. The elution step was modified such that the first elution was collected
using 100.00 mL of Buffer AE and was allowed to incubate at room temperature for
5 minutes. In a new test tube, a second elution step was conducted using 200.00 mL of
Buffer AE and was also allowed to incubate at room temperature for 5 minutes. The first
elution was used for PCR. Such a variety of extraction protocols was implemented
because not all tissue samples could be amplified when using the same extraction method.
The use of the DNeasyH extraction protocol was only used as an alternative to the
ChelexH extraction technique when it became apparent that the DNA could not be
successfully extracted using that technique.
Primers
Universal H3 (Colgan et al. 1998) and 16S primers (Palumbi et al. 1996) were used to
amplify the regions of interest for all specimens (Table 2). Internal primers for 16S
designed for another group of opisthobranchs (Ornelas-Gatdula et al. 2011) were used
resulting in additional partial sequences for some specimens. Multiple attempts to obtain
CO1 sequences using different primers (LCO1490/HCO2198, developed by Folmer et al.
1994 and NAF-COI/NAR-COI, developed by Ornelas-Gatdula et al. 2011) were
unsuccessful.
PCR Amplification and Sequencing
The master mix was prepared using 34.75 mL H2O, 5.00 mL of either Buffer B
(ExACTGene, Fisher Scientific) or Taq Buffer, 5.00 mL 25 mM MgCl2, 1.00 mL 20mM
dNTPs, 1.00 mL 10mM primer 1, 1.00 mL 10mM primer 2, 0.25 mL 5 mg/mL Taq, and
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2.00 mL extracted DNA. Some master mixes included 2 mL 20mg/ml bovine serum
albumin (Fermentas) and therefore 32.75 mL of H2O in order to maximize enzymatic
activity. Reaction conditions for H3 and 16S rRNA were as follows: an initial
denaturation for 2 min at 94uC, 35 cycles of 1) denaturation for 30 sec at 94uC,
2) annealing for 30 sec at 50uC, and 3) elongation for 1 min at 72uC, and a final
elongation for 7 min at 72uC. PCR products yielding bands of appropriate size
(approximately 375 bp for H3 and 195 bp for the 16S fragments) were purified using the
Montage PCR Cleanup Kit (Millipore). Cleaned PCR samples were quantified using a
NanoDrop 1000 Spectrophotometer (Thermo Scientific). PCR products were diluted to
6.0 ng/mL, and a sample of each primer was diluted to 2.0 pmol/mL. These samples were
sequenced at the City of Hope DNA Sequencing Laboratory (Duarte, CA) using
chemistry type BigDye V1.1 for fragments less than 500 bp.
Phylogenetic Analyses
Sequences for each gene were assembled and edited using Geneious Pro 4.7.4
(Biomatters Ltd.). Geneious was also used to extract the consensus sequence between the
primer regions and to construct the alignment for each gene using the default parameters.
The sequences were not trimmed after alignment. A total of 395 bp for H3, and 495 bp
for 16S were used for the phylogenetic analyses. Two other species of Polycera, P. cf.
capensis and P. hedgpethi, were selected as outgroups. Because of the lack of
comprehensive phylogenies for Polycera, outgroup choice was based on available
sequences and specimens. An analysis was conducted for both genes concatenated. To
assess whether H3 and 16S have significantly conflicting signals, the incongruence length
difference (ILD) test (Mickevich and Farris 1981, Farris et al. 1994), implemented in
PAUP*4.0 (Swofford 2002) as the partition homogeneity test (Swofford 2002), was
conducted for all genes combined. The levels of saturation for each gene and for the first
and second versus third codon positions of H3 were investigated using the substitution
saturation test developed by Xia et al. (2003) and Xia and Lemey (2009) implemented in
the program DAMBE (Xia and Xie 2001). The Akaike information criterion (Akaike
1974) was executed in jModelTest (Posada 2008) to determine the best-fit model of
evolution. Maximum likelihood analyses were conducted using GARLI v0.96b8 (Zwickl
2006). Model parameters determined from jModelTest were entered into GARLI and the
default parameters were used at 100 replicates each. Robustness of each clade was
assessed by bootstrap support (Felsenstein 1985) based on 2000 replicates with heuristic
search, TBR branch-swapping algorithm, multrees option, and 100 random additions.
Bayesian analyses were executed in MrBayes 3.1.2 (Huelsenbeck and Ronquist 2001),
partitioned by gene (unlinked). The Markov chain Monte Carlo analysis was run with
two runs of six chains for five million generations, with sampling every 1000 generations.
Convergence of runs was assessed using Tracer 1.5 (Rambaut and Drummond 2009). The
default 25% burn-in was applied before constructing majority-rule consensus tree/s.
Diagnostic nucleotides for each clade were identified visually in the alignments after
collapsing identical haplotypes using the program Collapse 1.2 (Posada 2004).
Results
Phylogenetic Analyses
The phylogenetic analyses produced a resolved and well-supported phylogenetic
hypothesis for the specimens examined (Figure 1). There is very little genetic diversity
and no phylogenetic structure in P. atra. All specimens examined from San Francisco
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Fig. 1. Bayesian consensus tree of the specimens examined based on H3 gene and 16 gene sequences.
Posterior probabilities and bootstrap values (from the maximum likelihood analysis) are indicated for each
branch. Sequenced specimens for which photographs were available are illustrated. Photographs by David
Behrens (CPIC 00809), Leroy Poorman (LACM 140738), and Ángel Valdés (all others).
Bay, California to La Paz, Mexico are identical for H3 and almost identical for 16S (only
one nucleotide substitution was found in a specimen from Monterey Bay, California).
The clade containing all the specimens identified as P. atra is well supported (posterior
probability 5 1). In contrast, P. alabe exhibits a large degree of variation in both genes,
providing a strong phylogenetic signal. The phylogenetic analyses have recovered three
distinct and well-supported clades including specimens identified as P. alabe. One of the
clades (named the Northern Clade herein) includes specimens collected in Sonora and La
Paz, Mexico (both in the Gulf of California) and Jalisco, Mexico. This clade is well
supported (posterior probability 5 0.95) and it is sister to P. atra. Within this Northern
Clade three specimens from the Gulf of California form a well-supported subclade
(posterior probability 5 0.97). The clade containing the Northern Clade of P. alabe and
P. atra is also well supported (posterior probability 5 0.98). The two other clades of P.
alabe are called Southern Clade 1 and 2 herein and they appear to be sister, but this is
weakly supported (posterior probability 5 0.8). Southern Clade 1 includes specimens
collected in Sonora and Jalisco and is well supported (posterior probability 5 0.96),
whereas Southern Clade 2 includes animals collected in southern Mexico (Colima and
Guerrero) and is also well supported (posterior probability 5 0.97). The ranges of the
Northern Clade and Southern Clade 1 overlap in the central Gulf of California
(Figure 2). However, the specimen of Southern Clade 1 from Sonora was collected during
the El Niño of 1977 and it could be the result of unusual oceanographic conditions. Based
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Fig. 2. Phylogeny of Polycera alabe and P. atra with a map showing the geographic location where the
specimens were collected. Records representing large range shifts during ENSO events are labeled with
bold font. In yellow is a record of P. alabe from Southern California not sequenced in this study.
on the information available, it appears that P. atra + the Northern Clade and the
Southern Clade 2 are completely allopatric.
Morphological Analyses
A morphological examination of representative specimens from the three P. alabe
clades indicates some differences in radular morphology. All radulae examined have a
hook-shaped innermost lateral tooth in each half-row, with a short, triangular cusp and a
secondary cusp mid-length (Figure 3). The innermost tooth is followed by a larger tooth
with a similar morphology. However, the number and morphology of the outer lateral
teeth is different among clades. Northern Clade and Southern Clade 1 exhibit identical
radula morphology, with 2–3 vestigial outer teeth (Figure 3A-D). However, specimens of
Southern Clade 2 consistently have 7–8 large outer teeth (Figure 3E-F).
Discussion
The sequence data and phylogenetic analyses presented here show that populations of
P. atra from a broad geographic range (San Francisco Bay, California to La Paz,
Mexico) are genetically very similar for 16S and H3. The 16S gene is variable enough to
distinguish among species of opisthobranchs and is even informative in population
genetics studies (Krug et al. 2012). The lack of geographic structure in P. atra suggests
rampant gene flow across the range of the species. Alternatively, this pattern might have
been caused by rapid postglacial range expansion of P. atra from refugia at the southern
range of the species. This is a well-documented explanation for the lack of genetic
structure in several other species of marine invertebrates along the Pacific Northwest
(e.g., Marko 1998, Hellberg et al. 2001, Edmands and Harrison 2003). Although the
northern range limit of P. atra is well south of the last glacial maximum, colder ocean
temperatures during the Pleistocene in California (Bemis et al. 2002) might have restricted
the range of this species, which could have rapidly expanded to the north during the
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Fig. 3. Radula SEM photographs for representatives of each clade of P. alabe and P. atra. All
photographs taken with the same orientation (distal above, proximal below). A, P. atra (CPIC 00808).
B, P. alabe Southern Clade 1 (LACM 76-5). C, P. alabe Northern Clade (LACM 140736). D, P. alabe
Nortern Clade (LACM 174953). E-F, P. alabe Southern Clade 2 (LACM 140739).
transition to the Holocene (see Jacobs et al. 2004). The specimen of P. atra from La Paz,
Mexico (confirmed with both genetic markers) was collected in April 1974 during a
strong La Niña, suggesting that this species has a broad dispersal potential and that water
temperature may be an important factor limiting its range.
The same genetic markers demonstrate a completely different picture for P. alabe. In
this case we have identified three well-supported clades with several molecular
synapomorphies in the 16S and H3 genes. Southern Clade 2 is also supported by radula
morphological differences, indicating that it is likely a distinct species. The other
two clades could also possibly constitute different species based on their genetic
synapomorphies. One of the clades of P. alabe (Northern Clade) is sister to P. atra, thus
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the current definition of P. alabe constitutes a paraphyletic assemblage. However, there is
no available genetic information available for populations from the Pacific coast of
Central and South America – the known range of P. alabe includes Costa Rica, Panama,
and Chile – and the sample size and sequence data are relatively small. Therefore, we do
not think it is appropriate to suggest taxonomic changes at this point.
Another problem is to elucidate which of the two clades found in the Gulf of California
(Northern Clade or Southern Clade 1) is the original P. alabe, since the range of both
clades includes the type locality of P. alabe (Isla de Cedros, Baja California). These
taxonomic issues should be addressed in a comprehensive study including specimens from
the entire range of P. alabe and detailed morphological examinations including the
reproductive anatomy of all the species involved.
Acknowledgements
We are extremely grateful to Alicia Hermosillo, David Behrens, and Hans Bertsch for
providing specimens and/or information that were essential for the completion of this
project. Lindsey Groves from the Natural History Museum of Los Angeles County
helped with access to the collection and curation of specimens. Hans Bertsch, Jeff
Goddard, and an anonymous reviewer made constructive comments that greatly
improved the manuscript. Monica Santander was supported by a NIH MBRS Research
Initiative for Scientific Enhancement (RISE) grant to Cal Poly Pomona (2 R25
GM061190-05A2). Additional support was provided by the Science Education
Enhancement Services Research and Mentor Program (SEES-RaMP), and a Kellogg
FuTURE Mini-Grant to Ángel Valdés, both from the California State Polytechnic
University. The SEM work was conducted at the Natural History Museum of Los
Angeles County, supported by the National Science Foundation under MRI grant DBI0216506 to A. Valdés et al., with the assistance of Giar-Ann Kung.
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