Proc. R. Soc. B (2012) 279, 1061–1067
doi:10.1098/rspb.2011.1599
Published online 14 September 2011
Flying shells: historical dispersal of marine
snails across Central America
Osamu Miura1,2,*, Mark E. Torchin1, Eldredge Bermingham1,
David K. Jacobs3 and Ryan F. Hechinger4
1
Smithsonian Tropical Research Institute, Apartado 0843-03092, Balboa, Ancon,
Republic of Panama
2
Division of Marine Biotechnology, Oceanography Section, Science Research Centre,
Kochi University, 200 Monobe, Nankoku, Kochi 783-8502, Japan
3
Department of Ecology and Evolutionary Biology, University of California, Los Angeles,
CA 90095, USA
4
Marine Science Institute and Department of Ecology, Evolution and
Marine Biology University of California, Santa Barbara, CA 93106, USA
The geological rise of the Central American Isthmus separated the Pacific and the Atlantic oceans about
3 Ma, creating a formidable barrier to dispersal for marine species. However, similar to Simpson’s proposal that terrestrial species can ‘win sweepstakes routes’—whereby highly improbable dispersal events
result in colonization across geographical barriers—marine species may also breach land barriers given
enough time. To test this hypothesis, we asked whether intertidal marine snails have crossed Central
America to successfully establish in new ocean basins. We used a mitochondrial DNA genetic comparison
of sister snails (Cerithideopsis spp.) separated by the rise of the Isthmus. Genetic variation in these snails
revealed evidence of at least two successful dispersal events between the Pacific and the Atlantic after the
final closure of the Isthmus. A combination of ancestral area analyses and molecular dating techniques
indicated that dispersal from the Pacific to the Atlantic occurred about 750 000 years ago and that dispersal in the opposite direction occurred about 72 000 years ago. The geographical distribution of
haplotypes and published field evidence further suggest that migratory shorebirds transported the
snails across Central America at the Isthmus of Tehuantepec in southern Mexico. Migratory birds
could disperse other intertidal invertebrates this way, suggesting the Central American Isthmus may
not be as impassable for marine species as previously assumed.
Keywords: geminate species; Cerithideopsis; Cerithidea; Isthmus of Panama; Central America
1. INTRODUCTION
The separation of populations by geographical barriers
and dispersal across those barriers are two major opposing
forces determining species formation and distribution.
The final closure of the Central American Seaway by
the rise of the Isthmus of Panama divided two tropical
oceans [1] and had profound consequences for marine
biota [2,3]. Marine organisms were separated by a land
barrier and adapted to very different environments, promoting genetic divergence and allopatric speciation [3].
Although several studies have considered the divergence
of populations separated by the Isthmus [2,3], natural dispersal of marine organisms across Central America has
received little attention.
In 1940, Simpson [4] proposed that terrestrial species
can win highly improbable ‘sweepstakes’ and disperse
across ocean barriers. He later noted that ‘a possible dispersal event, however improbable. . .becomes probable if
enough time elapses’ [5, p. 171]. We hypothesized that
some marine species may be particularly prone to winning
such sweepstakes dispersal across Central America by
* Author for correspondence ([email protected]).
Electronic supplementary material is available at http://dx.doi.org/
10.1098/rspb.2011.1599 or via http://rspb.royalsocietypublishing.org.
Received 29 July 2011
Accepted 25 August 2011
hitchhiking on birds. Some intertidal snails are abundant
in migratory shorebird feeding habitats, can adhere to
feathers, bills and legs of birds [6–9], and can survive
after being ingested [10,11]. Because many species of
shorebirds regularly cross Central America at both the Isthmus of Panama and the Isthmus of Tehuantepec [12–16],
we suspected that such snails could be transported across
Central America by this route.
The Pacific horn snail, Cerithideopsis californica
(¼Cerithideopsis mazatlanica and Cerithideopsis valida)
and the Atlantic horn snail, Cerithideopsis pliculosa constitute a pair of ‘geminate species [17]’ formed following
separation by the geological formation of the Isthmus of
Panama [18]. These species (formerly in the genus
Cerithidea [19]) are common and often very abundant
in intertidal mangrove and mudflat habitats [19], which
are major foraging areas for migratory shorebirds.
Further, the snails are known to survive ingestion by
birds [10], suggesting that they may be particularly
prone to avian transport. When documenting that
C. californica and C. pliculosa were geminate species,
Miura et al. [18] provided preliminary evidence that
these snails may have historically dispersed between the
Pacific and the Atlantic coasts. Here, we provide a more
rigorous testing of this possibility, broadly sampling
snails along both coasts and evaluating genetic evidence
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Snails flew across Central America
which indicates that these marine snails have indeed
crossed the Central American land barrier.
2. MATERIAL AND METHODS
(a) Sample collections
We collected C. californica and C. pliculosa from 29 populations across 308 of latitude along both coasts of North
and Central America (electronic supplementary material,
table S1). Snails were either frozen or preserved in
95 per cent EtOH. All samples were stored at 2208C in
the laboratory for molecular analyses.
We isolated DNA using a modified CTAB procedure [20].
A small piece of tissue from the foot of each snail was homogenized in a solution of 300 ml 2x CTAB and 10 mg ml21
proteinase K, and incubated at 608C for approximately 1 h,
extracted once with phenol/chloroform (v : v, 1 : 1) and precipitated with two volumes of ethanol. The DNA pellets
were briefly washed in 75 per cent ethanol, air-dried for
approximately 30 min and dissolved in 50 ml of H2O.
(b) DNA sequencing
We analysed mitochondrial DNA encoding the cytochrome
oxidase c subunit I (CO1) gene to identify major mitochondrial clades and their phylogeographic patterns. We also
analysed the 16S ribosomal RNA gene (16S), 12S ribosomal
RNA gene (12S), cytochrome b gene (Cytb) and NADH
dehydrogenase subunit 6 gene (ND6) for a single individual
from each major clade to estimate times of divergence events.
The primer pairs used in this study are listed in electronic
supplementary material, table S2. PCR reactions ran for 35
cycles under the following conditions: denaturing at 948C
for 60 s, annealing at 508C for 60 s (458C for CO1) and
extension at 728C for 90 s. The 35 cycles were preceded by
an initial denaturing at 948C for 1 min, followed by a final
extension of 728C for 7 min. The PCR products of the
samples were purified and sequenced using an automated
sequencer (ABI 3130xl). Sequences analysed in this study
were deposited in GenBank (accession nos HQ724852–
HQ725015).
(c) Estimation of phylogenetic relationships and
genetic parameters
Sequences were aligned by CLUSTALW, implemented in
BIOEDIT [21] for further phylogenetic analyses. Phylogenetic
trees based on the CO1 sequences were constructed using
maximum-likelihood analysis (ML) and Bayesian inference
(BI). We used the Akaike Information Criterion to determine
the best model using MODELTEST [22] (the sequences were
not partitioned for codon positions). The GTR þ I þ G
model was selected for the CO1 gene. The most closely
related congener, Cerithideopsis pulchra, was selected for an
outgroup based on a higher level phylogeny of Cerithideopsis
[18]. The ML analysis was conducted using PHYML [23]
using a BioNJ starting tree and rate parameter optimization.
Node robustness was assessed using non-parametric bootstrapping and 1000 replicates. The BI tree was obtained
using MRBAYES [24]. The dataset was run for four million
generations with a sample frequency of 100. The first 25
per cent of trees were discarded, such that only 30 001
trees were accepted. Because the software automatically analysed the data in two independent runs, a total of 60 002 trees
were analysed to estimate phylogenetic relationships and posterior probability value of each clade. Convergence between
Proc. R. Soc. B (2012)
the two runs was tested by examining the potential scale
reduction factors.
We used a molecular clock on the five mitochondrial genes
(CO1, 16S, 12S, CytB and ND6; approx. 3000 bp total) to
estimate divergence time of the major genetic clades. We calibrated the molecular clocks with the final closure of the
Panamanian Isthmus at 3.1–2.8 Myr ago [3]. We analysed
only mitochondrial DNA variation because the available
nuclear genes are generally too conserved to allow comparison between the geminate snails [18]. While amplified
fragment length polymorphism (AFLP) markers would be
ideal to evaluate shallow divergences [25], mitochondrial
DNA provided sufficient resolution for tracking potential dispersal events across the Isthmus owing to its rapid rate of
sequence divergence [26] and absence of recombination
[27]. A likelihood ratio test rejected (p , 0.01) the hypothesis of a molecular clock [28]. Thus, dates of divergence
were inferred using a relaxed molecular clock, following
the uncorrelated relaxed lognormal clock implemented in
BEAST v. 1.5.4 [29]. A uniform prior distribution was
used for the split of the clades assumed to be separated by
the rise of the Isthmus. The Yule speciation model was
used as a tree prior. The best evolutionary model was determined using MODELTEST [22], which selected HKY for the
12S gene, HKY þ I for 16S gene, K3Puf þ I for ND6
gene and HKY þ I þ G for the CO1 and Cytb genes. The
combined dataset was partitioned among genes. We used
gene-specific models and unlinked all parameters among
genes. The analysis was run for 10 million generations,
sampled every thousand steps and the first thousand samples
were discarded as burn-in. To check for convergence and to
visualize the results, we used TRACER v. 1.4.1 and FIGTREE
v. 1.2.3 [29]. Ideally, fossil calibration would provide another
independent measure for our dating analysis [30], but fossil
records for Cerithideopsis are not available.
We obtained CO1 haplotypes network trees using maximum
parsimony and TCS 1.21 [31].
We tested the monophyly of snails from the Pacific or
snails from the Atlantic using both the Shimodaira–
Hasegawa (SH) test [32] and the approximately unbiased
(AU) test [33] in CONSEL [34]. We constrained the Pacific
and the Atlantic snails to be monophyletic, respectively, and
tested it against the unconstrained (best) tree.
We used three types of ancestral area analyses to identify
ancestral areas for the major clades: (i) dispersal –vicariance
analysis [35], (ii) ancestral area analysis (AAA [36]), and
(iii) weighted ancestral area analysis [37]. All AAAs used a
cladogram based on the phylogeny shown in figure 1.
We compared CO1 haplotype variation within and among
populations using statistics from analysis of molecular
variance using ARLEQUIN v. 3.0 [38].
3. RESULTS AND DISCUSSION
We found high mitochondrial DNA CO1 genetic variation along both coasts (figure 1). Snails were separated
into two major phylogenetic clades (clades A and B),
both composed of several genetically well-separated subgroups. First, we identified the genetic divergence
associated with the closure of the Central American
Seaway [3]. The two major clades A and B are characterized by 8.5 per cent K2P distance and 29.7 per cent Ks
(silent site) distance (point 1, figure 1). This distance corresponds with the CO1 genetic distances that Lessios [3]
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O. Miura et al.
Snails flew across Central America
1063
H
17
90/100
G
19
A-1
point 2
clade A
100/100
O
E
20
N
19
100/100
100/100
81/100
F
20
4
A-2
A-3
33
D
4
L
19
M
18
K
C
point 1
19
19
78/77
B-1
clade B
B
J
24
I
19
A
94/100
100/100
87
B-2
C. pulchra
0.01
Figure 1. Molecular phylogeny and geographical distribution of the intertidal snails, Cerithideopsis californica and C. pliculosa. A
maximum-likelihood tree was constructed based on 873 bp of the CO1 gene. The major clades are categorized as clades A, B
and the detailed subclades are within. Numbers near nodes are the support values for the clade from the different analyses
(ML/BI). The scale bar represents the phylogenetic distances expressed as units of expected nucleotide substitutions per
site. Phylogenetic relationships within the clades are shown in electronic supplementary material, figure S1. The distributions
of the major genetic clades are shown at the right side of the figure. Numbers near the geographical points indicate sample size.
Letters indicate sampling sites (see electronic supplementary material, table S1).
reported for four other gastropod geminate species pairs
separated by the closure of the Seaway (average K2P distance 7.4–9.2% and average Ks distance 23.5–30.3%)
and with what Miura et al. [18] report for this geminate
pair. Interestingly, both clades A and B contain individuals
from both coasts. This is obvious in figure 1 and, further,
monophyly of the snails from the Pacific or from the
Atlantic is rejected by both the SH and AU tests (p ,
0.01). A divergence within clade A (point 2) separates
Pacific and Atlantic individuals (subclades A-1 and A-2;
figure 1). Genetic divergence (K2P ¼ 4.0%, Ks ¼ 12.6%)
at this point was less than half the divergence characterizing
point 1 or the distances separating other geminate gastropods [3]. Similarly, the Pacific and the Atlantic snails in
subclade B-1 exhibited a small divergence (K2P ¼ 0.9%,
Ks ¼ 2.4%; see electronic supplementary material, figure
S1). These results suggest that separation by the rise of
the Isthmus of Panama produced the major genetic
clades, A and B, and that after the final closure of the
Seaway there were at least two dispersal events between
the Pacific and the Atlantic.
We used the phylogenetic patterns of each major clade
to reconstruct ancestral distributions and infer directionality of the cross land barrier dispersals. All three
historical biogeographic analyses identified the Pacific as
the ancestral area of clade A, and the Atlantic as the
ancestral area of clade B (table 1). These methods
implicitly employ phylogenetic diversity to infer the
ancestral area, as ancestral populations probably have
higher diversity than more recently colonized sites.
Indeed, clade A has two highly diverged subclades in
the Pacific (A-1 and A-3), while only a single subclade
was found in the Atlantic (A-2). Similarly, clade B has
two genetically distinct subclades in the Atlantic (B-1
and B-2), while only one of those (B-1) appears in the
Proc. R. Soc. B (2012)
Table 1. The results of the ancestral area analyses. (Ancestral
areas were estimated by dispersal–vicariance analysis
(DIVA), the ancestral area analysis (AAA), and the weighted
ancestral area analysis (WAAA). All results indicate the
Pacific origin of clade A and the Atlantic origin of clade B.)
clade
ocean
DIVA
AAA (G/L)
WAAA (PI)
clade A
Pacific
Atlantic
O
—
2.00
0.50
3.00
0.33
clade B
Pacific
Atlantic
—
O
0.50
2.00
0.33
3.00
Pacific. Importantly, snails from the upper Gulf of
California form a unique subclade (A-3, figure 1), owing
to the isolating influence driven by the Baja California
Peninsula [39], and the presence of this unique and old
lineage in the Pacific further indicates a Pacific origin of
clade A. Similarly, the basal subclade B-2 was found only
along the Atlantic Coast of Nicaragua (figure 1), suggesting
the Atlantic origin of clade B. These results further support
that the rise of the Isthmus of Panama isolated clade A
in the Pacific and clade B in the Atlantic. These groups
diversified to several subclades within each ocean after
the Isthmus formed. Subsequently, individuals from clade
A dispersed from the Pacific to the Atlantic, forming subclade A-2, and individuals of subclade B-1 dispersed
more recently from the Atlantic to the Pacific, creating its
current distribution.
We calibrated the molecular clock for the five mitochondrial genes assuming that the divergence at point 1
corresponds to the final closure of the Central American
Seaway at 3.1 – 2.8 Myr ago [3]. Analyses indicated that
dispersal from the Pacific to the Atlantic occurred about
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Snails flew across Central America
dispersal from
the Pacific to the Atlantic
A-1 (Pacific)
A-2 (Atlantic)
the rise of the Isthmus
A-3 (Pacific)
dispersal from
the Atlantic to the Pacific
B-1 (Atlantic)
B-1 (Pacific)
B-2 (Atlantic)
C. pulchra
4.0
3.5
3.0
2.5
2.0
Ma
1.5
1.0
0.5
0
Figure 2. Divergence time estimates for the major clades in C. californica and C. pliculosa based on the CO1, 16S, 12S, Cytb
and ND6 genes (totally 2952 bp). Horizontal bars represent the upper and lower interval bounds for 95% of the highest
posterior densities (HPDs). Each gene tree is shown in electronic supplementary material, figure S2.
750 000 years ago (430 000–1 100 000 years ago: 95%
highest posterior density (HPD); figure 2) and that dispersal
from the Atlantic to the Pacific occurred about 72 000 years
ago (13 000–147 000 years ago: 95% HPD, figure 2).
Until the 1914 breach of the Panama Isthmus by the
Panama Canal, coastal organisms were completely separated by land. Dispersal over the Central American land
barrier is the most likely explanation for our results. It is
implausible that the snails dispersed around the North
American arctic or the southern tip of South America
(approx. 20 000 km) because the geographical range of
Cerithideopsis does not extend beyond warm temperate
regions [40] and the snails are either direct developers
or have short-lived larvae [41]. Additionally, the absence
of identical mitochondrial haplotypes on both sides of the
Isthmus of Panama despite extensive sampling (electronic
supplementary material, table S1) suggests that Cerithideopsis has not dispersed through the approx. 100 year
old Panama Canal, as have some other marine organisms
[42,43]. Temporary connections between the Pacific and
the Atlantic Oceans about 1.9 Ma [44] are thought to
have facilitated the post-Isthmian dispersal of some
marine species [45]. However, our dates (0.02 – 0.14
and 0.43 –1.1 Myr ago) for dispersal events are too late
for this and much earlier than would be predicted for
human-mediated dispersal.
We posit that transport by migrating shorebirds is the
most parsimonious explanation for the bidirectional dispersal of Cerithideopsis across Central America. Cerithideopsis
snails live in habitats used by millions of shorebirds.
These shorebirds regularly cross Central America over
two migration flyways: the Isthmus of Tehuantepec
in southern Mexico [12–14,16], and the Isthmus of
Panama [15]. Further, birds can transport small invertebrates [7]. Small, juvenile Cerithideopsis snails could
become attached to bird feathers or appendages via their
Proc. R. Soc. B (2012)
sticky mucus. Additionally, several species of shorebirds
ingest Cerithideopsis and the snails can survive. For
example, Sousa [10] found approximately 30 per cent survivorship of Cerithideopsis snails in regurgitation pellets of
the willet (Tringa semipalmata). Regurgitation can occur
days to weeks after feeding [46], which is sufficient time
to cross either the Tehuantepec or Panama trans-Isthmian
flyways (200 and 70 km wide, respectively).
To identify the most likely locations of the over-land dispersals, we constructed haplotype networks. Concerning
the dispersal of clade A from the Pacific to the Atlantic,
all encountered members of subclade A2 were in the Gulf
of Mexico (figure 1). Further, the network tree shows
that A2 haplotypes in Galveston, USA, are most closely
related to Pacific A1 haplotypes (figure 3a). This suggests dispersal occurred in the Gulf of Mexico, across
the Isthmus of Tehuantepec rather than over the narrower
Isthmus of Panama. The lack of geographical structure for
Pacific A1 precludes elucidating a specific Pacific source
location for the colonization (table 2). Concerning the
dispersal from the Atlantic to the Pacific, the network tree
of the subclade B1 shows that all Pacific haplotypes were
derived from a haplotype currently in Mandinga, Mexico
(figure 3b). This suggests that snails dispersed south
across the Isthmus of Tehuantepec from the Gulf of
Mexico to the Pacific. Here, the data suggest a source for
the Atlantic to the Pacific dispersal (Mandinga) but the
lack of genetic structure in Pacific B1 populations precludes
pinpointing a colonization location (table 2). The relative
lack of genetic structure in Pacific Cerithideopsis populations
(both A1 and B1) may be owing to greater planktonic
dispersal ability of Pacific snails at low latitudes [41].
Nevertheless, the available data suggest that both dispersal
events occurred across the Isthmus of Tehuantepec.
Contemplating the distribution of species across the
continents, Simpson argued that given enough time,
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Snails flew across Central America
Pacific Atlantic
A
I
B
J
C
K
L
D
M
E
N
F
G
O
H
O. Miura et al.
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(a)
(b)
22 steps
Figure 3. Mitochondrial haplotype networks of Cerithideopsis californica and C. pliculosa, subclades A1 and A2 (a), and subclade
B1 (b) based on the CO1 gene. Circle sizes are proportional to the number of individuals observed for each haplotype. The
small black circles represent unobserved single-nucleotide substitutions. The pie chart coloration/shading indicates the regions
where haplotypes were collected (see electronic supplementary material, table S1).
Table 2. Analysis of molecular variance of C. californica in
the Pacific (subclades A1 and B1) and C. pliculosa in the
Atlantic (subclades A2 and B1).
ocean
clade
Pacific
subclade
A1
subclade
B1
Atlantic
subclade
A2
subclade
B1
source of
variation
d.f.
among
within
total
among
within
total
11
108
119
8
69
77
among
within
total
among
within
total
2
47
47
7
59
66
per cent of
variation
FST
24.82
75.18
0.25
47.47
52.53
0.47
82.13
17.87
0.82
90.41
9.59
0.90
improbable events become probable [5]. Our results indicate that, similar to the way terrestrial species can disperse
over inhospitable water barriers to colonize land masses
[47,48], marine species may disperse over land to colonize
new oceans. While thousands of marine species were separated by the closure of the Central American Seaway, we
show that marine species can occasionally breach this
barrier despite the low probability of doing so. Several
potential filters limit the probability of successful colonizations, similar to contemporaneous species invasions,
which also involve a sequence of stages with sometimes
independent probabilities of failure [49]. Natural dispersal
events over land barriers by marine species are unlikely
because individuals may not be picked up, survive the journey, survive in the novel environment, find mates or fail to
establish purely owing to demographic stochasticity [50].
Thus, despite the fact that there are many migrating birds
that regularly contact with snails, it seems improbable that
snails would successfully disperse across Central America.
Indeed, our genetic evidence supports this; although successful dispersal and establishment of these marine snails
have occurred, it has rarely happened—being detected
only two times within 3 Myr. We suspect that future molecular genetic data will reveal that other marine species,
Proc. R. Soc. B (2012)
particularly common intertidal species, have probably also
been dispersed across Central America by birds.
Reproductive incompatibility between geminate
species pairs is not always complete as demonstrated by
laboratory hybridization experiments between geminate
species of gobioid fishes [51] and sea urchins [52,53].
We postulate that introgressive hybridization with
‘native’ individuals facilitated the retention and spread
of the dispersed ‘non-native’ alleles that we detected.
Despite being able to morphologically distinguish Pacific
from Atlantic snails (O. Miura 2009, personal observation), there were no obvious morphological differences
between individuals with the Pacific or the Atlantic mitochondrial haplotypes when they occurred in the same
locality. Future analyses of highly variable nuclear markers (such as AFLP or microsatellites) could confirm
the occurrence of introgressive hybridization between
the Pacific and the Atlantic snails.
Charles Darwin first postulated that invertebrates,
including marine snails, could be dispersed long distances
by birds [7]. However, in contrast to terrestrial and freshwater invertebrates [8,54], there is little evidence for this
for marine animals. Our genetic evidence coupled with evidence from field studies provide a conservative estimate
that marine snails crossed Central America on two separate
occasions, established their alleles, which subsequently
spread along both coasts. This suggests that not only is
such passive dispersal possible for marine organisms, but
that it can occur across seemingly insurmountable barriers.
Consistent with the emerging paradigm for island biogeography in which both vicariance processes and dispersal
shape the distribution of terrestrial species [55], these
marine dispersal events, while rare, could profoundly
impact the ecology and evolution of marine species.
We thank Y. Kam, S. Jagadeeshan, E. Ochoa, A. Jaramillo,
V. Frankel, J. Johnston, A. Cornejo, J. Lorda,
R. Bastida-Zavala, R. Guerrero-Arenas, I. Jiménez-Garcı́a,
A. Armitage, A. Schulze, D. Aguirre, E. Mendoza,
J. Violante-González, E. Siu-Estrada and N. Aleman for
field assistance. We are especially indebted to C. Schlöder
for invaluable field, laboratory and logistical assistance. We
are grateful to G. Ruiz for providing us with additional
specimens. We thank H. Lessios, O. Sanjur, S. Chiba,
O. Puebla, A. Kuris and K. Lafferty and two anonymous
reviewers for providing useful comments on the
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Snails flew across Central America
manuscript. We thank the crew of the R. V. Urraca and the
staff of the STRI’s Naos Island Laboratories, Bocas del
Toro Marine Station, Galeta Marine Station, Smithsonian
Marine Station at Fort Pierce and Carrie Bow Cay Marine
Laboratory. Support was provided by the STRI and the
Smithsonian Marine Science Network including an MSN
Post-doctoral Fellowship and an STRI Hoch fellowship to
O.M.; the Japan Society for the Promotion of Science
(Grant-in-Aid for JSPS Fellows to O.M.); and the
California Sea Grant. This work was facilitated by the
Global Invasions Network NSF RCN DEB-0541673.
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Electronic supplementary material for:
Flying shells: historical dispersal of marine snails across Central America
Osamu Miura, Mark E. Torchin, Eldredge Bermingham, David K. Jacobs, Ryan F.
Hechinger
This supplement contains:
Figure S1
Figure S2
Table S1
Table S2
Panama-Coiba
Panama-RioVenado
Panama-AguaDulce
Panama-Bique
Panama-RioVenado
Panama-LaPlaya
Panama-RioVenado
Panama-Bique
Nicaragua-Corinto
76/100
Nicaragua-Corinto
Panama-AguaDulce
Panama-RioVenado
74/91
Panama-Bique
Panama-RioVenado
81/100
Panama-Bique
Panama-Bique
Nicaragua-Corinto
Panama-Coiba
Panama-LosAmarillos
Mexico-ElSesteo
90/100
Mexico-ElSesteo
Nicaragua-Corinto
Panama-Coiba
Panama-Coiba
Panama-Coiba
Panama-Coiba
Panama-Coiba
Nicaragua-Corinto
Panama-LosAmarillos
Panama-LosAmarillos
Panama-LosAmarillos
Panama-LaPlaya
Panama-LosAmarillos
Panama-LosAmarillos
Nicaragua-Corinto
Panama-Coiba
Panama-AguaDulce
Panama-Coiba
Panama-LosAmarillos
Mexico-Chacaua
64/100
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
51/100
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Mexico-Chacaua
Panama-Bique
Panama-Bique
Panama-LosAmarillos
Panama-LosAmarillos
Panama-LosAmarillos
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
100/100
USA-SanFrancisco
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SanFrancisco
Mexico-ElSesteo
Mexico-ElSesteo
Mexico-ElSesteo
Mexico-ElSesteo
Panama-RioMar
Panama-RioMar
Panama-RioVenado
Mexico-ElSesteo
Panama-Bique
Mexico-ElSesteo
Mexico-ElSesteo
Mexico-ElSesteo
Panama-AguaDulce
Panama-LaPlaya
Mexico-ElSesteo
Panama-RioMar
Mexico-ElSesteo
Mexico-ElSesteo
Mexico-ElSesteo
Mexico-ElSesteo
USA-SanFrancisco
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
USA-SantaBarbara
Panama-AguaDulce
Panama-Bique
Panama-Bique
Nicaragua-Corinto
Nicaragua-Corinto
Mexico-ElSesteo
Mexico-ElSesteo
Mexico-ElSesteo
Nicaragua-Corinto
Mexico-ElSesteo
Nicaragua-Corinto
Nicaragua-Corinto
Nicaragua-Corinto
Panama-RioMar
64/100 Panama-RioMar
Panama-RioMar
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
84/100
USA-CorpusCristy
USA-CorpusCristy
USA-CorpusCristy
100/100
USA-CorpusCristy
USA-Galveston
USA-Galveston
100/100
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
57/USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
USA-Galveston
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
85/100
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
Mexico-LaMancha
62/93 Mexico-LaMancha
Mexico-LaMancha
Mexico-LaPinta
Mexico-SanFelipe
Mexico-LaPinta
Mexico-SanFelipe
Mexico-LaPinta
Mexico-LaPinta
Mexico-LaPinta
Mexico-SanFelipe
Mexico-SanFelipe
Mexico-LaPinta
Mexico-SanFelipe
Mexico-SanFelipe
Mexico-LaPinta
Mexico-SanFelipe
81/100
Mexico-SanFelipe
Mexico-LaPinta
Mexico-LaPinta
Mexico-SanFelipe
Mexico-LaPinta
Mexico-SanFelipe
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
80/100 Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Mexio-Yalku
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Belize-StannCreek
Panama-RioVenado
Panama-Thirdlock
Panama-PuertoMutis
Panama-PuertoMutis
Panama-AguaDulce
Panama-AguaDulce
Panama-PuertoMutis
Panama-AguaDulce
Panama-PuertoMutis
Panama-Thirdlock
Panama-LaPlaya
Panama-Thirdlock
Panama-Thirdlock
Panama-LaPlaya
Panama-SantaCatalina
Panama-RioVenado
Panama-LaPlaya
Panama-AguaDulce
Panama-RioVenado
Panama-PuertoMutis
Panama-Thirdlock
Panama-SantaCatalina
Panama-RioVenado
Panama-SantaCatalina
Panama-SantaCatalina
Panama-Thirdlock
Panama-LaPlaya
Panama-SantaCatalina
Panama-Thirdlock
Nicaragua-Corinto
Panama-SantaCatalina
Panama-PuertoMutis
Panama-SantaCatalina
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
64/85
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
-/61
63/98 USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
USA-SanFrancisco
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
85/100
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
68/71 Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-GuerreroNegro
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Mexico-Mandinga
Nicaragua-BlueFields
Nicaragua-BlueFields
Nicaragua-BlueFields
-/63
Nicaragua-BlueFields
87/100 Nicaragua-LagunadePerlas
Nicaragua-BlueFields
Nicaragua-LagunadePerlas
Nicaragua-BlueFields
Nicaragua-BlueFields
Nicaragua-LagunadePerlas
Panama-Colon
Panama-Colon
Panama-Colon
85/100
Panama-Colon
Panama-Colon
Panama-Colon
Panama-Colon
Panama-Colon
Panama-Colon
Panama-EscudodeVeraguas
Panama-EscudodeVeraguas
Panama-EscudodeVeraguas
Panama-EscudodeVeraguas
100/100 Panama-EscudodeVeraguas
Panama-EscudodeVeraguas
Panama-EscudodeVeraguas
78/77
Panama-EscudodeVeraguas
Panama-EscudodeVeraguas
82/100
Panama-EscudodeVeraguas
Panama-BocasdelToro
Panama-BocasdelToro
Panama-BocasdelToro
99/98
Panama-BocasdelToro
Panama-BocasdelToro
Nicaragua-LagunadePerlas
Nicaragua-LagunadePerlas
94/100
Mexio-Yalku
99/100 Mexio-Yalku
Mexio-Yalku
Nicaragua-Corinto
Nicaragua-Corinto
Panama-Thirdlock
Panama-LaPlaya
Nicaragua-Corinto
Nicaragua-Corinto
Panama-AguaDulce
Panama-PuertoMutis
Panama-SantaCatalina
Panama-LaPlaya
Nicaragua-Corinto
Nicaragua-Corinto
Nicaragua-LagunadePerlas
Nicaragua-LagunadePerlas
69/78
Nicaragua-LagunadePerlas
Nicaragua-LagunadePerlas
Nicaragua-LagunadePerlas
100/100
Nicaragua-BlueFields
Nicaragua-BlueFields
C. pulchra
92/99
90/100
A-1
A-2
A-3
B-1
94/100
B-2
0.01
Figure S1. Detailed maximum likelihood phylogenetic tree based on the COI gene.
Numbers near nodes are the support values for the clade (ML/BI). The samples from
the Pacific were indicated by blue and the samples from the Atlantic were indicated by red.
(a) CO1
(b) 16S
A-1(Pacific)
100/100
76/54
A-3 (Pacific)
100/89
91/97
A-1(Pacific)
62/53
A-2 (Atlantic)
96/98
A-2 (Atlantic)
A-3 (Pacific)
55/-
B-1 (Atlantic)
B-1 (Atlantic)
B-1 (Pacific)
B-1 (Pacific)
B-2 (Atlantic)
B-2 (Atlantic)
C. pulchra
C. pulchra
0.002
0.01
(c) 12S
(d) Cytb
A-1(Pacific)
97/100
79/81
100/100
A-2 (Atlantic)
63/93
A-3 (Pacific)
B-2 (Atlantic)
76/70
A-3 (Pacific)
89/83
B-1 (Atlantic)
70/94
A-1(Pacific)
A-2 (Atlantic)
B-1 (Atlantic)
B-1 (Pacific)
B-2 (Atlantic)
C. pulchra
B-1 (Pacific)
0.01
C. pulchra
0.002
(e) ND6
A-1(Pacific)
93/100
A-2 (Atlantic)
83/78
A-3 (Pacific)
B-1 (Atlantic)
100/100
B-1 (Pacific)
B-2 (Atlantic)
C. pulchra
0.02
Figure S2. Maximum likehihood phylogenetic trees based on the CO1 gene (a), 16S gene (b), 12S
gene (c), Cytb gene (d) and ND5 gene (e). Numbers near nodes are the support values for the
clade (ML/BI).
Table S1. Sampling sites of C. californica and C. pliculosa in the Americas. The N shows
total number of samples in each site, and the number of putative native individuals and the
invasive individuals are shown in the parenthesis. Genetic diversity (Pi) for all, putative
native, and putative invasive individuals in each sampling site are also estimated.
Ocean
Country ID
Local sites
Pacific Panama
A: Bique
Rio Venado
Third lock
Rio Mar
Agua Dulce
Puerto Mutis
La Playa
Los Amarillos
Santa Catalina
Coiba
Nicaragua B: Corinto
Mexico
C: Chacaua
D: El Sesteo
E: Guerrero Negro
F: San Felipe
La Pinta
USA
G: Santa Barbara
H: San Francisco
Atlantic Panama
I:
Nicaragua J:
Belize
Mexico
K:
L:
M:
USA
N:
O:
Colon
Escudo de Veraguas
Bocas del Drago
Blue fields
Laguna de Perlas
Stann Creek
Yal-ku
Mandinga
La Mancha
Corpus Cristy
Galveston
Coordinate
N 8°53'34.8" W 79°39'30.5"
N 8°53'39.7" W 79°35'38.0"
N 8°58'36.0" W 79°34'58.2"
N 8°27'17.5" W 79°58'10.7"
N 8°12'07.1 W 80°29'01.2"
N 7°55'26.4 W 81°03'13.8"
N 7°46'52.8" W 81°10'23.1"
N 7°42'45.6" W 81°14'26.2"
N 7°37'41.0" W 81°14'46.1"
N 7°20'30.0" W 81°41'37.2"
N 12°31'34.1" W 87°12'26.1"
N 15°58'13.1" W 97°41'24.7"
N 21°43'16.4" W 105°29'13.5"
N 28°0' 0" W 114°5'24.0"
N 30°58'58.4" W 114°49'15.7"
N 31°17'38.2" W 113°27'52.0"
N 34°24'01.5" W 119°32'07.5"
N 37°30'0" W 122°5"60.0"
Pacific Total
N 9°22'47.1" W 79°52'38.9"
N 9°05'59.4" W 81°33'34.0"
N 9°24'20.4" W 82°19'27.3"
N 11°52'17.6" W 83°43'14.2"
N 12°21'56.5" W 83°38'29.1"
N 16°46'22.0" W 88°18'10.7"
N 20°24'36.0" W 87°17'60.0"
N 19°02'56.6" W 96°04'38.1"
N 19°35'23.2" W 96°22'49.0"
N 27°49'14" W 97°05'04"
N 29°15'23.1" W 94°54'59.1"
Atlantic Total
N
10(10,0)
10(6,4)
8(0,8)
6(6,0)
10(5,5)
7(0,7)
9(3,6)
10(10,0)
8(0,8)
9(9,0)
19(12,7)
19(19,0)
19(19,0)
19(0,19)
10(10,0)
10(10,0)
19(19,0)
17(3,14)
219(141,78)
9(9,0)
10(10,0)
5(5,0)
9(9,0)
10(10,0)
18(18,0)
4(4,0)
9(0,9)
24(24,0)
4(0,4)
20(0,20)
122(78,74)
All
Pi
0.011
0.048
0.006
0.003
0.049
0.006
0.043
0.005
0.005
0.009
0.042
0.001
0.003
0.001
0.003
0.003
0.000
0.024
0.043
0.000
0.000
0.000
0.012
0.018
0.000
0.006
0.000
0.001
0.001
0.001
0.045
Native
Pi
0.011
0.014
n.d.
0.003
0.013
n.d.
0.011
0.005
n.d.
0.009
0.008
0.001
0.003
n.d.
0.003
0.003
0.000
0.000
0.016
0.000
0.000
0.000
0.012
0.018
0.000
0.006
0.000
n.d.
n.d.
n.d.
0.011
Invasive
Pi
n.d.
0.005
0.006
n.d.
0.008
0.006
0.005
n.d.
0.005
n.d.
0.003
n.d.
n.d.
0.001
n.d.
n.d.
n.d.
0.000
0.004
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.001
0.001
0.001
0.006
Table S2. The primers used for PCR reactions and sequencing.
Gene
COI
16S
12S
Cytb & ND6
Name
COI-bf
CalCOIF
CalCOISF
COI-6
CalCOIR
16Sar
16Sbr
12SaL
12SbH
ND6-F
UCYTB272R
Sequence
5'-GGGGCTCCTGATATAGCTTTTCC-3'
5'-GGAGCTCCAGATATGGCTTTYCC-3'
5'-ATTTTAATTCTYCCCGGRTTTGG-3'
5'-GGRTARTCNSWRTANCGNCGNGGYAT-3'
5'-TGAGCYTTAGTTCATCGAGAGTG-3'
5'-CGCCTGTTTATCAAAAACAT-3'
5'-CCGGTCTGAACTCAGATCACGT-3'
5'-AAACTGGGATTAGATACCCCACTAT-3'
5'-GAGGGTGACGGGCGGTGTGT-3'
5'-ATTAGCATTGGAAGCTAAA-3'
5'-GCRAANAGRAARTACCAYTC-3'
Direction
Forward
Forward
Forward
Reverse
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Reference
Miura et al., 2006
This study
This study
Shimayama et al., 1990
This study
Kessing et al., 1989
Kessing et al., 1989
Kocher et al., 1989
Kocher et al., 1989
This study
Merritt et al., 1998
References
Kessing, B., Croom, H., Martin, A., McIntosh, C., McMillan, W. & Palumbi, S. 1989 The
simple fool’s guide to PCR. Honolulu: University of Hawaii.
Kocher, T., Thomas, W., Meyer, A., Edwards, S., Pääbo, S., Villablanca, F. & Wilson, A.
1989 Dynamics of mitochondrial DNA evolution in animals: amplification and
sequencing with conserved primers. Proc. Natl. Acad. Sci. USA 86, 6196-6200.
Merritt, T., Shi, L., Chase, M., Rex, M., Etter, R. & Quattro, J. 1998 Universal cytochrome
b primers facilitate intraspecific studies in molluscan taxa. Mol. Mar. Biol.
Biotechnol. 7, 7-11.
Miura, O., Torchin, M. E., Kuris, A. M., Hechinger, R. F. & Chiba, S. 2006 Introduced
cryptic species of parasites exhibit different invasion pathways. Proc. Natl. Acad.
Sci. USA 103, 19818-23.
Shimayama, T., Himeno, H., Sasuga, J., Yokobori, S., Ueda, T. & Watanabe, K. 1990 The
genetic code of a squid mitochondrial gene. Nucleic Acids Symp. Ser. 22, 73-74.