Mar Biol
DOI 10.1007/s00227-014-2444-4
Original Paper
Multiscale patterns of genetic structure in a marine snail
(Solenosteira macrospira) without pelagic dispersal
Stephanie J. Kamel · Richard K. Grosberg ·
Jason A. Addison
Received: 25 July 2013 / Accepted: 5 April 2014
© Springer-Verlag Berlin Heidelberg 2014
Abstract The northern Gulf of California (NGC) is one
of the most dynamic and productive marine ecosystems
in the world, yet knowledge about population connectivity and dispersal patterns is lacking for many of its resident species. Using nuclear and mitochondrial markers,
we investigated the effects of open water, geographical distance and suitable habitat on patterns of genetic structure
of Solenosteira macrospira, a benthic buccinid whelk with
direct development. We collected samples in April 2004,
2005 and May 2007 from the upper NGC (31°34.39″N,
114°44.45″W). Phylogenetic analyses, hierarchical analyses of variance and Bayesian assignment tests substantiated
a break between the east and west coasts. Genetic distance
between population pairs increased with geographical distance, but only when assuming a U-shaped dispersal pathway over the open water of the NGC. Given S. macrospira’s
association with rocky intertidal habitats, and its limited
Communicated by S. Uthicke.
Electronic supplementary material The online version of this
article (doi:10.1007/s00227-014-2444-4) contains supplementary
material, which is available to authorized users.
S. J. Kamel (*)
Department of Biology and Marine Biology, Center for Marine
Science, University of North Carolina Wilmington, 601 S.
College Road, Wilmington, NC 29403, USA
e-mail: kamels@uncw.edu
R. K. Grosberg
Center for Population Biology, Department of Evolution
and Ecology, College of Biological Sciences, University
of California, 1 Shields Ave, Davis, CA 95616, USA
J. A. Addison
Biology Department, University of New Brunswick,
P.O. Box 4400, Fredericton, NB E3B 5A3, Canada
dispersal potential, we assumed that the geographical distribution of rocky habitat would play a significant role in
genetic differentiation of S. macrospira. Nevertheless,
populations separated by sand were more similar than populations separated by rocks. The influence of open water,
geographical distance and suitable habitat (rocks vs. sand)
also varied significantly across different genetic markers
that presumably evolve at different rates. Specifically, the
more rapidly evolving nuclear microsatellites suggested
that physical transport processes strongly influence genetic
differentiation on contemporary time scales, even in a species with direct benthic development. This underscores the
strong, and potentially homogenizing, effect of present-day
ocean circulation patterns in the NGC.
Introduction
Sessile and sedentary marine invertebrates exhibit a range
of dispersal potentials, with some species having long-lived
larvae that may drift and feed for months in the ocean, and
others entirely lacking a pelagic larval stage. In general,
marine species with long-lived larvae and extensive dispersal potential tend to exhibit genetic structure over greater
geographical ranges than species with short-lived larval
phases and limited dispersal potential. Although many
marine species conform to this expectation (reviewed in
Bohonak 1999; Kinlan and Gaines 2003; Dawson et al.
2006; Cowen and Sponaugle 2009), a surprisingly large
number of species display patterns of genetic structure
that contradict predictions based primarily on estimates of
pelagic larval duration (Kyle and Boulding 2000; Thorrold
et al. 2002; Palumbi 2003; Marko 2004; Lester et al. 2007;
Hellberg 2009; Shanks 2009; Weersing and Toonen 2009;
Winston 2012). The paradox of Rockall embodies this
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Mar Biol
Fig. 1 Map of the NGC. Thick
coastlines represent rocky
shore and sampling locations
are indicated by blue circles.
Biogeographic regions are
separated by the dotted lines:
red line boundary between the
east and west coasts, green line
boundary between the northern
and central Gulf regions. Islands
within the Gulf are as follows:
(1) Isla Angel de la Guarda and
(2) Isla Tiburon. Redrawn from
Riginos and Nachman (2001)
33
32
Latitude (°N)
31
30
29
28
117
116
115
114
113
112
111
Longitude (°W)
contradiction: Littorina saxatilis, a brooding gastropod (as
well as several other invertebrate species lacking a planktonic stage) has successfully colonized isolated island sites,
whereas Littorina littorea, with planktotrophic larvae, has
not (Johannesson 1988).
These inconsistencies highlight the fact that dispersal
and gene flow in marine systems often reflect complex
interactions involving multiple life-history traits and larval
behaviors, patterns of water circulation, historical and contemporary barriers to dispersal, and the spatial distribution
of suitable habitat (Perrin and Borsa 2001; Marko 2004;
Lourie et al. 2005; Ayre et al. 2009; Cowen and Sponaugle
2009; Morgan et al. 2009; Pinsky et al. 2012). For example,
dispersal by gametes (Grosberg 1991; Yund 1995; Bishop
1998), detached larvae (Martel 1993), and adults (Hellberg
1994, 1996; Worcester 1994), may lead to more extensive
gene flow and larger ranges than would be expected based
on larval dispersal potential alone (Winston 2012). Disentangling the contributions of each of these processes to contemporary genetic structure is essential for understanding
13
present-day dispersal routes, scales of connectivity, and
population dynamics.
The northern Gulf of California (NGC) is a region of
primary importance to Mexican fisheries and has been
identified as a conservation priority, currently encompassing three marine protected areas (MPAs), including two
biosphere reserves (Cisneros-Mata 2004; IUCN 2005).
The NGC has a complex geologic and oceanographic history and is one of the most productive and diverse marine
ecosystems in the world (Ezcurra et al. 2002). Its waters
exhibit extreme seasonal temperature variation, tidal amplitudes that may exceed 10 m, and a diversity of substrates
and habitats (Briggs 1974). The Baja California coast of
the NGC consists largely of rocky shores interspersed
with short stretches of sand and sandy mud (Fig. 1). All
of the mainland coastline of the NGC lies within the state
of Sonora. The intertidal and shallow subtidal zones consist of sand and mud forming long beaches and estuaries,
only occasionally interrupted by rocky outcrops and boulder fields (Fig. 1; Brusca 2004). In addition, the Colorado
Mar Biol
River forms a large delta, emptying into the NGC between
Baja California and Sonora, potentially creating a boundary
between the two coasts.
Despite the biological and economic importance of the
NGC, little is known about population connectivity and
dispersal patterns for most of its resident species. Previous studies of Gulf fauna have typically concerned regional
scale patterns (>100–1,000 km) of genetic differentiation
(e.g., Riginos and Nachman 2001; Bernardi et al. 2003;
Riginos 2005; Hurtado et al. 2007; Deng and Hazel 2010),
focusing less on the effects of smaller-scale oceanographic
or habitat features on population genetic structure. Organisms restricted to patchy habitats and with low dispersal
potential represent promising systems in which to study
fine-scale patterns and processes of divergence. On the one
hand, high levels of population subdivision are expected in
animals restricted to a single patchy habitat type, because
surrounding unsuitable habitats can constitute effective
dispersal barriers (Hurtado et al. 2010). Gene flow should
be limited to proximate populations, with genetic differentiation increasing between increasingly distant populations (i.e, isolation-by-distance; Wright 1943). On the other
hand, whether limited larval dispersal translates into a geographical pattern of gene flow consistent with an isolationby-distance model remains unclear because mechanisms
other than the movement of larvae may influence population connectivity in marine species (Hellberg 1994).
In this paper, we characterize patterns of genetic structure in the buccinid whelk, S. macrospira, a gonochoric,
direct-developing intertidal gastropod which produces
benthic egg capsules and is restricted to rocky outcrops
on mudflats in the upper NGC. Its widespread distribution
on both coasts of the upper Gulf coupled with its habitat
specificity presents an opportunity to simultaneously evaluate the importance of several potential mechanisms on patterns of population differentiation. Specifically, we test
these hypotheses relating to patterns of genetic structure in
S. macrospira: (1) The open waters of the NGC present a
significant barrier to dispersal, leading to deep intraspecific
divergence between Sonoran and Baja Californian populations; (2) direct development and limited dispersal should
lead to a strong positive relationship between geographical
and genetic distances between populations; and (3) populations separated by unsuitable habitat (sandy substrate)
should be more differentiated than populations separated
by suitable habitat (rocks).
We use three molecular markers (nDNA, mtDNA, and
microsatellites) to infer patterns of divergence among populations across most of the documented range of S. mac‑
rospira. We utilize this approach because the most complete
explanation for both past and present levels of population
connectivity can only be achieved by exploiting the variable mutation rates present among the different markers.
For example, rapidly evolving loci such as microsatellites
are suited to measuring contemporary gene flow, whereas
slower evolving markers, like mtDNA, reveal patterns of
divergence that have accrued over hundreds to thousands
of generations (Wares and Cunningham 2001; Marko and
Hart 2011; Doellman et al. 2011; Panova et al. 2011). Past
events can thus be examined over wider time scales, allowing for the separation of different aspects of a species’ evolutionary history (Crandall et al. 2000; Sunnucks 2000;
Garrick et al. 2009). We conclude by assessing whether the
relative importance of the mechanisms outlined above (i.e.,
open water, geographical distance, and discontinuities in
suitable habitat) varies across historical and contemporary
time scales. Population genetic patterns offer clues to past
geological and environmental events, which, in turn, can
improve our understanding of the complex relationships
among life histories, biogeography, oceanography, and
genetic structure in the northern Gulf region.
Materials and methods
Specimen collection and sampling
Solenosteira macrospira is an intertidal buccinid whelk,
native to the NGC, always found associated with rocks
and boulders on mudflats and sandy shores. Its range
extends south to Puertecitos (30°35.06″N, 114°63.89″W)
on the Baja California side, and to Guaymas (27°91.83″N,
110°89.89″W) on the Sonoran side (Brusca 2004; Houston 1978). During the spawning season (March–May),
females attach egg capsules to the shells of male conspecifics (Kamel and Grosberg 2012). Each egg capsule contains
≈200–300 eggs; on average, 3–20 hatchlings per capsule
emerge as crawl-away juveniles, about 1 month after oviposition. We sampled a total of 588 individual S. macrospira
from seven locations in the upper NGC (Fig. 1). We collected snails from the Puerto Peñasco (Sonora) (n = 144)
and San Felipe (Baja California) (n = 139) sites in April
2004, and again from Puerto Peñasco (n = 143) in April
2005, as part of another project. In May 2007, we collected
individuals from all other locations (n = 19–38 snails per
site). Upon collection, we removed the snails from their
shells and preserved them in 95 % ethanol.
Genetic data collection
Mitochondrial and nuclear genes
We extracted genomic DNA from ethanol preserved muscle tissue following the cetyltrimethyl ammonium bromide (CTAB) protocol described in Grosberg et al. (1996).
We used PCR to amplify fragments of two mitochondrial
13
Mar Biol
genes (cytochrome c oxidase subunit I, COI; cytochrome
b, Cyt b), and an intron from the single-copy nuclear
gene Calmodulin (Cal). We used the universal primers
LCO1490 and HCO2198 to amplify COI (Folmer et al.
1994), and we used the complete mtDNA genome of Ilya‑
nassa (=Nassarius obsoleta) to design primers to amplify
880 base pairs of Cyt b (Io7908L 5′- WCACTATACKG
CHCATGTAG-3′ and Io8794R 5′-TGYTCATAHGGWG
YTTCWACVGG-3′). We identified the nuclear cal gene
through BLAST searches of I. obsoleta expressed sequence
tags (ESTs) deposited in GenBank (GenBank accession no.
EV826015). Using custom primers anchored in the coding region (IoCalL 5′-CTGAGATCAAGGAGGCGTTG-3′
and IoCalR 5′-CACCATCAAGGTCAGCC TCT-3′), we
identified and sequenced a combination of coding and noncoding (intron) DNA. From these preliminary sequences,
we designed species-specific exon-primed intron crossing (EPIC) primers for the cal gene in Solenosteira
(SmCalL 5′-ATCCATCTGTGCCAGA AACC-3′; SmCalR
5′-AACAGCCCTCAACACAGCTT-3′).
We performed all PCRs in 15 µL volumes using an
Applied Biosystem GenAmp®9600 thermal cycler. PCR
mixes consisted of 15–30 ng of template DNA, 1× PCR
Buffer, 2.5 mM MgCl2, 0.2 mM dNTP (Promega), 0.1 mg/
mL bovine serum albumin (BSA, NEB), 0.25 µM forward
and reverse primers, 0.6 U of AmpliTaq® DNA polymerase
(Applied Biosystems). The cycling protocol consisted of an
initial denaturation step of 3 min at 94 °C, followed by 35
cycles of 94 °C denaturation for 30 s, annealing at 49 °C for
30 s, and extension at 72 °C for 50 s for COI and Cytb, and
a 94 °C denaturation for 30 s, annealing at 56 °C for 30 s,
and extension at 70 °C for 1 min for cal. All PCR protocols
included a final extension step at 72 °C for 3 min. We treated
PCR products with Exonuclease 1 (USB) and Shrimp Alkaline Phosphatase (USB) and direct sequenced with one or
both primers using an Applied Biosystems 3730 Capillary
Electrophoresis Genetic Analyzer. Sequences were edited
and aligned using the SEQUENCE NAVIGATOR and
AUTO ASSEMBLER programs (Applied Biosystems). We
inferred the allelic phases of individuals heterozygous at the
cal gene using the program PHASE version 2.1.1 (Stephens
et al. 2001; Stephens and Scheet 2005). This program uses
a Bayesian method to reconstruct haplotypes from a population sample and to estimate the uncertainty associated with
each phase call. We ran the program using the default model
allowing for recombination (-MR0 flag) for 1,000 iterations
with a burn-in of 1,000 and a thinning interval of five. We
repeated the analysis five times and examined the haplotype
frequency estimates and goodness-of-fit measures to ensure
consistency across runs. We excluded individuals whose
haplotype reconstructions were ambiguous (P < 0.95) from
the data analysis. All sequences were deposited in GenBank
(Accession numbers KF956938-KF957436).
13
Microsatellites
Ecogenics GmbH (Zurich, Switzerland) constructed a
library from size-selected genomic DNA ligated into
SAULA/SAULB-linker (Armour et al. 1994) and enriched
by magnetic bead selection with biotin-labeled (AAG)10
and (GCGT)7 oligonucleotide repeats (Gautschi et al.
2000a, b). The microsatellite PCR amplification mixture
contained 15–30 ng of template DNA, 1× PCR Buffer,
2.5 mM MgCl2, 0.2 mM dNTP (Promega), 0.1 mg/mL
bovine serum albumin (BSA, NEB), 0.25 µM forward
and reverse primers (Table S1), 0.6 U of AmpliTaq® DNA
polymerase (Applied Biosystems). The cycling protocol
consisted of an initial denaturation step of 3 min at 94 °C,
followed by 35 cycles of 94 °C denaturation for 30 s,
annealing at 49 °C for 30 s, and extension at 72 °C for 30 s.
One microliter of the amplification products was added to
9 µL of formamide and ABI Gene Scan 500 size standard
and loaded onto a 96-well plate for analysis on an ABI
Prism 3100 Capillary Electrophoresis Genetic Analyzer.
We visualized and scored fragment data using STRand
(Version 2.3.69; Toonen and Hughes 2001).
Analyses
Genetic diversity
For the microsatellite loci, we used GENEPOP 3.4 (Raymond and Rousset 1995) to estimate expected heterozygosity (HE) and to test for linkage disequilibrium and
conformity to Hardy–Weinberg expectations (HWE). We
calculated allelic richness (RS) using FSTAT 2.9.3.2 (Goudet 1995), by standardizing to the smallest sample size
(n = 19). For the DNA sequence data, we used DNASP
4.90 (Rozas et al. 2003) to calculate haplotype diversity
(h), and nucleotide diversity (π).
Genetic differentiation
We calculated Weir and Cockerham’s F statistics (FIS,
FIT, FST) for both the microsatellite markers and DNA
sequences using ARLEQUIN (Excoffier et al. 2005) and
tested for significance by 10,100 permutations of the data.
We tested for pairwise differences between populations
using FSTAT (Goudet 1995) for the microsatellites and
ARLEQUIN for the DNA sequences.
Open water as a barrier to gene flow
We used an analysis of molecular variance (AMOVA) to
test whether there was regional genetic subdivision between
the east and west coasts of the Gulf. We ran the AMOVAs
using F statistics for the microsatellites and Tamura and
Mar Biol
Nei (1993) distances for the DNA sequences and tested for
significance with 10,100 permutations of the data. To visualize the phylogenetic relationships among mtDNA and cal
sequences, we constructed 95 % statistical parsimony networks using TCS 1.18 (Clement et al. 2000).
We used the Bayesian model-based clustering algorithm
implemented by the program structure 2.2.3 (Pritchard et al.
2000) to characterize the underlying genetic structure in the
microsatellite data. We assessed the number of genetic clusters (K) among our seven S. macrospira samples for values
of K ranging from 1 to 10 using the admixture model, with
allele frequencies correlated among populations and ignoring
prior population information. We ran 10 Bayesian Markov
Chain Monte Carlo (MCMC) searches of between 150,000
and 1,000,000 steps with a burn-in of 10 %, and used the
method of (Evanno et al. 2005) to find the best-fit value of K.
To estimate the magnitude of gene flow (m) and divergence times (t) between populations on the east and west
sides of the NGC, we ran a coalescent-based analysis of the
COI data in IMa2 (Hey and Nielsen 2007), with a MCMC
approach under an isolation with migration (IM) model
of divergence. We conducted preliminary runs using very
large priors to determine the maximum values of the model
parameter estimates. Our final analyses were run with a
burn-in until a flat trend-line appeared, after which we sampled 200,000 genealogies. We used a conservative mutation
rate for COI of 2.3 % per million years (Knowlton et al.
1993; Hellberg and Vacquier 1999).
Dispersal routes and isolation‑by‑distance in S. macrospira
Since little is known about dispersal routes in the NGC, especially for species that lack pelagic larvae, we investigated
several dispersal scenarios for S. macrospira. We constructed
our geographical distance matrices such that sampled
populations were joined in a stepping-stone manner, and we
tested four connectivity hypotheses. Geographical distances
between populations were measured using several dispersal routes: (1) an inverted U pattern, assuming no dispersal
across the southern waters of the NGC; (2) a U-shaped pattern, assuming dispersal across the southern waters of the
NGC; (3) the shortest shoreline distance from either of the
above directions; or (4) great circle distances (i.e., the shortest distances between points along the surface of the Earth).
To characterize the relationship between inferred levels of
gene flow and geographical distance between populations,
we conducted Mantel tests for nonrandom associations
between matrices of geographical and genetic distances
using the program IBDWS (Jensen et al. 2005). We calculated genetic distances using Slatkin’s (1993) similarity
, based on allelic frequencies for the microsatellites
index, M
and Kimura 2-parameter distances for the DNA sequences.
We used an AIC model selection procedure to identify which
of the geographical distance matrices best explained the patterns of genetic distance (Burnham and Anderson 2002).
Open water, distance, and unsuitable habitat as barriers
to gene flow
Several factors, acting singly or in combination, could
account for the observed patterns of genetic differentiation
in S. macrospira. To explore the collective effects of open
water, geographical distance and the distribution of suitable
habitat on genetic distance, we conducted multiple regression analyses on all markers. First, we defined three prediction vectors reflecting distances between population pairs
based on (a) open water (0 = same coast, 1 = different coast;
WATER); (b) geographical distance (km separating the populations; SDIST); (c) discontinuous habitat due to sandy shores
(0 = population pairs separated by rocky shore or open water,
1 = population pairs separated by sandy shore; SAND).
We used the geographical distance vector corresponding to
the U-shaped pattern with dispersal across the south NGC
(SDIST), since this matrix had the lowest AIC value (see
“Results”). Although there were three types of habitats separating the population pairs (water, sand, and rocks), the effects
of these habitats could not be estimated independently but
could only be estimated relative to each other. This is because
assigning three presence/absence binary codes for each habitat type resulted in a design matrix that was not invertible and
could not be solved for a least squares solution. Therefore, the
predictor variables WATER and SAND represent the effect of
open water minus the effect of rocky shore and the effect of
sandy shore minus the effect of rocky shore, respectively. This
makes it possible to test whether discontinuous habitat (water
or sand) increases genetic distance relative to continuous
habitat (rock). In both cases, a positive correlation coefficient
would indicate that the populations separated by discontinuous
habitat are more genetically distant than populations separated
by continuous habitat (see Riginos and Nachman (2001) for a
similar approach). We standardized all vectors by subtracting
the mean and dividing by one SD (Selkoe et al. 2010). The
sums of squares and associated statistics for the whole model
were calculated following standard regression procedures. We
estimated significances for the whole model and partial regression coefficients by permuting the order of values in the vector
of genetic distances and recalculating the regression 10,000
times using the program RT version 2.1 (Manly 1997).
Results
Genetic diversity
We genotyped 588 specimens of S. macrospira from seven
locations in the upper NGC. Our sequence alignments
13
Mar Biol
Table 1 Summary of sample sizes and genetic diversity for the microsatellites, the combined mtDNA sequences (COI + CytB), and the Calmo‑
dulin intron
Sample site
mtDNA—1,390 bp
N
nH
Microsatellites—6 loci
Calmodulin—557 bp
h
Hd
π
nD
h
Hd
π
nA
Ho
He
Rs
West NGC
Punta Estrella (PE)
San Felipe (SF)
Playa Bonita (PB)
Campo los Compadres (CC)
East NGC
Los Pinitos (LP)
Puerto Penasco (PP)
Almejas (AS)
38
138
33
37
25
26
20
21
6
10
8
6
0.4267
0.7385
0.7000
0.5571
0.0003
0.0008
0.0007
0.0007
46
58
42
40
3
2
1
7
0.4050
0.1000
0.0000
0.4590
0.0020
0.0005
0.0000
0.0027
38
138
33
37
0.58
0.67
0.65
0.68
0.69
0.69
0.71
0.78
7.3
6.5
7.4
8.4
19
287
36
19
29
23
5
5
10
0.7310
0.7414
0.6403
0.0010
0.0012
0.0011
34
60
32
5
6
6
0.8220
0.6430
0.5080
0.0036
0.0027
0.0026
19
287
36
0.73
0.71
0.63
0.73
0.72
0.76
5.6
6.2
6.2
Total
588
163
38
0.7139
0.0010
312
14
0.5720
0.0029
588
0.66
0.73
6.8
nH number of haplotypes sampled, nA number of alleles sampled, nD number of diploid genotypes, HO observed heterozygosity, HE expected heterozygosity, RS average allelic richness, h number of unique haplotypes, Hd haplotype diversity, π nucleotide diversity
included 609 bp of COI, 781 bp of CytB, and 557 bp of the
nuclear intron Cal. We detected 13 unique haplotypes for
COI and 32 haplotypes for CytB; nucleotide polymorphism
(π) was more than twice as high for CytB (0.0013) than for
COI (0.0005). Since many populations of S. macrospira
had only one or two unique COI haplotypes, we combined
both COI and CytB into a single 1,390-bp haplotype in
the individuals for which we sequenced both fragments
(nH = 163; h = 38; Table 1).
We reconstructed the nuclear haplotypes using the maximum-likelihood methods implemented by PHASE, which
successfully identified allelic states with >95 % certainty in
156 of the 163 (>96 %) individuals we directly sequenced
for the Cal intron. We detected 14 unique haplotypes, and
haplotype diversity (Hd) ranged considerably from 0 at
Playa Bonita (PB) in the western gulf (Baja California) to
0.822 at Los Pinitos (LP) on the eastern shore of Sonora
(Table 1).
We obtained multi-locus microsatellite genotypes for
588 individuals. Observed heterozygosities ranged from
0.58 to 0.73 among all seven S. macrospira populations
(Table 1), with between 7 and 16 alleles per locus averaged across seven populations (Table S1). We detected a
small but significant excess of homozygotes at three loci
(Soleno01, Soleno13, Soleno22), and the exact test of Raymond and Rousset (1995) indicated a small but significant
global departure from HWE (FIS = 0.014, P < 0.001, Table
S1). There was no signal of linkage disequilibrium between
microsatellite loci (data not shown).
Open water as a barrier to gene flow
The AMOVA analysis, comparing eastern versus western populations from the NGC, revealed a strong and
13
significant hierarchical structure for the mtDNA and
microsatellite loci, but not the Cal intron (Table 2). With
the exception of the microsatellites, for which pairwise differences among populations were less variable than those
using mtDNA, the proportion of genetic variation among
populations within groups was very high. This suggests
that the non-significant among-group comparison for Cal
might be due to the exceptionally high pairwise differentiation between populations (Table 2).
Parsimony networks constructed with both the mtDNA
and Cal DNA sequences revealed striking differences in the
frequency of unique haplotypes, supporting genetic structuring across the Gulf (Fig. 2). Both genes had common
and widespread haplotypes found at high frequency in all
populations, but they also had several low frequency haplotypes unique to individual locations on both the eastern and
western coasts of the Gulf.
Bayesian clustering analyses identified two significant
clusters of microsatellite genotypes which corresponded
to the four populations sampled from the western NGC
and the three populations sampled from the eastern NGC.
Although the variance in the posterior probability of the
estimate of the number of clusters in the microsatellite data
was high for values of k ≥ 4, the Δk method of Evanno
et al. (2005) showed a strong mode at k = 2 that was more
than 6× higher than at k = 3.
Overall, our data support a strong east–west structuring of S. macrospira in the NGC. We therefore used
IMa2 to infer t, the time since the populations on both
sides of the Gulf diverged. Using a divergence rate of
2.3 % mya for the COI data, our multi-locus mtDNA
analysis suggests that the eastern and western Gulf populations split approximately 22,071 years ago (95 % CI
8,614–58,157).
Mar Biol
Table 2 Solenosteira macrospira population structure from the
analysis of molecular variance (AMOVA) comparing the population
groups along the western (PE, SF, PB, CC) and eastern (AS, PP, LP)
coasts of the NGC
Source of variation
several scenarios in S. macrospira. Across all three marker
classes, the AIC scores were lowest for the models with
the U-shaped southern dispersal route as the predictor
of genetic distances (Table 3), suggesting this is the most
likely dispersal route connecting the east (Sonora) and
west (Baja California peninsular) coasts. The geographical
matrix based on this dispersal route explained 48 % of the
variation in the mtDNA divergence (P = 0.004) and 26 %
of the divergence at the microsatellite loci (P = 0.020). The
results for all other hypothesized routes were not significant. Significant values are indicated in bold.
Variance compo- Percentage
nents
of variation
mtDNA (COI + CytB)
Among groups (ΦCT)
0.07**
7.11
Populations within groups (ΦSC)
0.126**
11.67
Within populations (ΦIS)
0.188**
81.22
ΦST
0.163**
Cal
Among groups (ΦCT)
0.139
Open water, distance, and unsuitable habitat as barriers
to gene flow
13.87
Populations within groups (ΦSC)
0.304**
26.27
Within populations (ΦIS)
0.401**
59.86
The multiple regressions of the three predictor vectors on
genetic distance were highly significant, but differed across
each of the three marker classes (Table 4). For the mtDNA,
the partial regression coefficient of SDIST (i.e., U-shaped
dispersal across the southern part of the upper NGC) was
significantly greater than random (whole model: r2 = 0.89,
F1, 20 = 34.84, P < 0.001). For the Cal intron, the partial
regression coefficients of WATER and SAND explained a
significant proportion of the variance in genetic distance
(whole model: r2 = 0.39, F1, 20 = 3.92, P = 0.047). The significant positive coefficient for WATER suggests that populations separated by water are more distantly related to each
other than populations separated by either rocks or sand.
However, the significant negative coefficient for SAND
indicates that populations separated by sand are, on average,
0.362**
ΦST
Microsatellites
Among groups (ΦCT)
0.052*
5.15
Populations within groups (ΦSC)
0.048**
4.56
Within populations (ΦIS)
0.097**
90.30
ΦST
0.083**
ΦST represents the magnitude of genetic subdivision among populations in the absence of hierarchical structure
* P < 0.01; ** P < 0.001
Dispersal routes and isolation‑by‑distance in S. macrospira
Since little is known about dispersal routes around the
NGC, we used an isolation-by-distance model to explore
(b)
(a)
PE
SF
PB
CC
Fig. 2 95 % plausible networks for a S. macrospira mtDNA
(COI + CytB n = 163) and b S. macrospira (n = 312) cal intron haplotypes from the NGC. Diameters of circles are proportional to the
frequency of the haplotype, lines connect haplotypes differing by
LP
PP
AS
one mutation, and small open circles indicate missing intermediate
haplotypes. Locations sampled from the western NGC are shades of
blue and locations from the eastern NGC are shades of red. Sampling
abbreviations are as in Table 1
13
Mar Biol
Table 3 Isolation by distance tests for correlations between genetic
differentiation (FST) and geographical distance between sampling
locations
Table 4 Partial regression coefficients of biogeography, geographical
distance, and habitat for a multiple linear regression on genetic distance with probabilities determined by a modified Mantel test
Marker
Distance measurement
AIC
R2
P value
Marker
mtDNA
(a) Inverted U dispersal
(b) U-shaped dispersal
(c) Shortest shoreline
route
(d) Great circle distance
(a) Inverted U dispersal
(b) U-shaped dispersal
(c) Shortest shoreline
route
(d) Great circle distance
(a) Inverted U dispersal
(b) U-shaped dispersal
(c) Shortest shoreline
route
−19.03
−32.27
−19.96
0.02
0.48
0.06
0.22
0.004
0.13
mtDNA
−18.69
0.09
−0.23
0.13
0.001
0.01
0.02
0.004
0.32
0.54
0.15
0.53
0.05
−80.33
−82.52
−80.45
0.01
0.19
0.26
0.19
0.71
0.07
0.02
0.06
(d) Great circle distance
−78.35
0.11
0.10
Cal
Microsatellites
Mantel tests (10,000 permutations) were conducted on distance
matrices that correspond to four different dispersal scenarios (see
text)
genetically more similar than populations separated by
rocks. The microsatellite data display a pattern consistent
with the Cal intron: the multiple regression explained 71 %
of the variation (r2 = 0.71, F1, 20 = 7.14, P = 0.001), and
the partial regression coefficients of WATER and SAND
significantly differed from zero (P < 0.05).
Discussion
As expected for a species with direct development and limited mobility of adults, we found substantial population
structure at both local (<20 km) and regional (~400 km)
scales in populations of S. macrospira sampled from
seven sites across its range in the upper NGC. However,
like in many other species with limited dispersal potential
(reviewed in Hellberg 2009), patterns of genetic structure
did not conform to a simple linear stepping-stone model,
where the magnitude of genetic differentiation was a monotonic function of geographical distance between sampled
populations. Instead, multiple factors, some historical,
some contemporary, appear to govern the present-day distribution of genetic variation.
Open water as a barrier to gene flow
At the regional scale, phylogenetic relationships among
mtDNA haplotypes, AMOVA analyses, and Bayesian
assignment tests all show a significant break between
13
Variable Partial regression
coefficient (β)
WATER
0.13
SAND
0.04
SDIST
0.21
WATER
1.42
Cal
SAND −1.44
SDIST
0.11
0.24
Microsatellites WATER
SAND −0.25
SDIST
0.03
t value P value R2
0.89
0.27
3.96
2.27
−2.24
0.48
3.38
−3.48
0.34
0.79
0.001
0.04
0.04
0.64
0.004
0.003
0.96 0.35
0.89
0.59
0.71
Significant values are indicated in bold
populations in the eastern and western NGC. The demographic reconstruction suggests that the split between
eastern and western populations occurred approximately
20,000 years ago, coinciding with a rise in sea levels in
excess of 20 m after the last glacial maximum (26,500–
20,000 years ago) (Fairbanks 1989; Weaver et al. 2003).
Dispersal routes and isolation‑by‑distance in S. macrospira
At a more local scale, a U-shaped southern dispersal route
best explains the relationship between genetic distance and
geographical distance. This pattern of isolation-by-distance
is evident from both mtDNA and microsatellite markers,
suggesting that while the open waters of the NGC prevent
extensive mixing between eastern and western populations,
snails have been able to traverse the southern part of the
NGC over both historical and contemporary time scales,
encompassing periods when sea levels were both low and
high.
How did S. macrospira successfully disperse across the
waters of the NGC? Given that the upper NGC is flat and
shallow, it likely contracted significantly during the low
sea level periods of the Pleistocene (Hurtado et al. 2010),
potentially enabling stepping-stone dispersal across rocky
outcrops along the southern portion of S. macrospira’s
range (e.g., Hellberg 1995). However, the southern dispersal route is also the best-fit model for the genetic distances reflected by the microsatellite loci, which—given
their rapid rates of evolution—most directly represent
contemporary gene flow. Given the post-Pleistocene
rises in sea level that occurred in the Gulf, with maximal heights estimated to be about 4–9 m above present
levels (Ortlieb 1981, 1984), this pattern of contemporary
connectivity might best be explained by the presence of
a strong cyclonic (e.g., anti-clockwise) gyre present in
Mar Biol
the NGC that could transport rafted adults and juveniles
across the open waters in the southern part of their range
(Marinone et al. 2008; see section below). In this case,
egg-carrying males that are advected to suitable habitats
and release their offspring could be successful colonizers of new environments (Thiel and Haye 2006), in part
because offspring will hatch within a restricted area and
potentially remain there until sexually mature, increasing
the encounter rates of mates (Johannesson 1988; Schulze
et al. 2000).
The isolation-by-distance analyses also confirm that the
Colorado River delta represents a barrier to dispersal in S.
macrospira, since none of the models of dispersal which
included movement across it were significant. This result
is mirrored in the patterns of genetic differentiation among
populations: the two populations on either side of the delta
[(i.e., CC and LP) were highly significantly differentiated
across all markers; Table S2].
Open water, distance, and unsuitable habitat
When considering all variables simultaneously, mtDNA
markers revealed that geographical distance (based on a
southern dispersal route: SDIST) was the only significant
factor influencing patterns of population structure during
the Pleistocene (ca. 125 kya), suggesting that S. macrospira
could disperse across the southern portion of the upper
NGC. Dispersal was likely facilitated by the low sea level
periods of the Pleistocene (Hurtado et al. 2010). On the
other hand, the Cal intron and the microsatellites, reflecting more recent population processes, show that the open
waters of the NGC (WATER) now play a dominant role in
structuring populations and that geographical distance does
not contribute significantly to explaining patterns of contemporary population structure.
Contrary to expectations, unsuitable habitat (SAND) did
not present a significant barrier to gene flow: both nuclear
markers indicated that populations separated by sand were
more genetically similar than populations separated by
rocks. This is surprising given that areas lacking rocky
shore are typically strong barriers to gene flow in species
restricted to rocky intertidal habitats (Billot et al. 2003;
Johansson et al. 2008; Ayre et al. 2009). However, Johannesson and Warmoes (1990) found that the direct-developing gastropod L. saxatilis colonized breakwaters along
the Belgian coast that were often separated by kilometers
of unsuitable sandy habitats. They hypothesized that strong
currents running parallel to the shoreline transported snails
between breakwaters. Recent studies of other benthic invertebrates with direct development also suggest that longdistance dispersal via rafted transport on macroalgal mats
may extend connectivity beyond that expected from pelagic
larval duration alone (Helmuth et al. 1994; Waters and Roy
2004; Donald et al. 2005; Thiel and Haye 2006; Miranda
and Thiel 2008).
Moreover, studies of connectivity in the NGC using
numerical particle tracking have shown that strong regional
hydrodynamics produce a basin-wide cyclonic (anti-clockwise) gyre, which lasts throughout the summer months,
from June to August (Carrillo-Bribiezca et al. 2002;
Marinone et al. 2008, 2011). Flow patterns are strongly
northward on the eastern Sonoran coast but weakly southward on the peninsular side. Density distributions of juvenile rock scallops (Spondylus calcifer) and black murex
(Hexaplex nigritus), species whose larvae are considered
passive, are consistent with these flow patterns (CudneyBueno et al. 2009). Larval transport by this cyclonic gyre
has also been documented in larvae of the shrimp species
Litopenaeus stylirostris and Farfantepenaeus californ‑
iensis, and the main pathway of post-larval shrimp to the
Colorado River delta is via the Sonoran shoreline (GalindoBecti et al. 2010). On the western coast of the NGC, circulation patterns are significantly weaker and numerical
simulations reveal that particles do not travel far from their
origin in this region of the NGC (Marinone et al. 2011).
Thus, the combination of limited dispersal and weak circulation patterns on the west coast could explain the
higher level of population structure (i.e., populations less
than 20 km apart are significantly more differentiated than
their counterparts on the east coast), despite the presence
of largely continuous rocky habitat. Though adult S. mac‑
rospira are strongly associated with intertidal rocky substrate during the reproductive season, they may migrate
offshore into deeper waters outside of the mating season.
Given that sandy mud and deeper waters have been minimally sampled for this species, it may well be that rafting or currents transport adult snails substantial distances
across unsuitable reproductive habitat. To our knowledge,
there are no obvious barriers preventing S. macrospira
from crossing one rocky area to the next, and so it remains
an open question as to why this pattern of differentiation
exists.
Conclusions
Our range-wide study of genetic structure in S. macrospira
in the NGC suggests that on contemporary time scales
physical transport processes strongly influence local and
regional genetic differentiation, even in a species with
direct benthic development. This underscores the strong,
and potentially homogenizing, effect of present-day ocean
circulation patterns in the NGC (Cudney-Bueno et al.
2009; Aznar et al. 2010; Galindo-Becti et al. 2010). On
longer temporal and broader spatial scales, glacially driven
marine transgressions and regressions also appear to leave
13
Mar Biol
signatures on structure, evident in more slowly evolving
genetic markers. Given our meager understanding for most
species of the complex interactions among developmental
mode, habitat distribution, and circulation that determine
genetic structure, it remains a major challenge to understand patterns of connectivity in this ecologically and economically important region.
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