Biological Journal of the Linnean Society, 2008, 95, 409–424. With 4 figures
A peninsula as an island: multiple forms of evidence for
overwater colonization of Baja California by the
gartersnake Thamnophis validus
ALAN DE QUEIROZ1* and ROBIN LAWSON2
1
826 Delmar Way, Reno NV 89509, USA
Center for Comparative Genomics and Department of Herpetology, California Academy of Sciences,
55 Music Concourse Drive, San Francisco, CA 94118, USA
2
Received 11 May 2007; accepted for publication 18 January 2008
The recent shift toward dispersal rather than vicariant explanations of disjunct distributions has been driven by
the use of molecular data to estimate divergence dates between lineages. However, other kinds of evidence can also
be critical in evaluating such biogeographic hypotheses. In the present study, we used a multifaceted approach
employing diverse analyses of mitochondrial DNA sequences to assess explanations for the disjunct distribution of
the gartersnake Thamnophis validus. The occurrence of this species in the Cape Region of the Baja California
peninsula, isolated from mainland populations that occur along the west coast of Mexico, might be explained by:
(1) separation of the peninsula from mainland Mexico through rifting 4–8 Mya (tectonic vicariance); (2) fragmentation of the range of this semi-aquatic species because of post-Pleistocene aridification (vicariance by aridification);
(3) natural overwater dispersal across the Gulf of California; or (4) human introduction. Divergence dating
indicates that peninsular and mainland T. validus separated from each other within the last 0.5 Myr, thus rejecting
tectonic vicariance. In addition, the estimated closest mainland relatives of peninsular snakes are found farther
north than expected under this hypothesis. Three findings argue against vicariance by aridification: (1) peninsular
snakes and their closest mainland relatives are more genetically similar than predicted; (2) the location of closest
mainland relatives is farther south than predicted; and (3) the species is absent from areas where one might expect
to find relict populations. Taken together, refutation of the vicariance hypotheses and the fact that the estimated
closest mainland relatives are found almost directly across the Gulf from the Cape Region supports some form of
overwater colonization. Various additional arguments suggest that natural dispersal is more likely than human
introduction. The present study emphasizes the need for multiple kinds of evidence, beyond divergence dates, to
discriminate among hypotheses and to provide independent sources of corroboration or refutation in historical
biogeography. © 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95,
409–424.
ADDITIONAL KEYWORDS: aridification – difference-by-distance – dispersal – divergence dating – human
introduction – Mexico – mitochondrial DNA – phylogeography – tectonics – vicariance.
INTRODUCTION
Historical biogeography is currently in a state
of upheaval, largely driven by the ease of obtaining molecular sequences and the development of
methods for estimating lineage divergences from such
data. In particular, the use of molecular dating
methods to infer whether the ages of particular
*Corresponding author. E-mail: [email protected]
lineage divergences are consistent with vicariance
hypotheses has revolutionized the study of disjunct
distributions. The results of such analyses often have
strongly refuted previously accepted vicariance
hypotheses and, consequently, have led to a widespread shift toward a more dispersalist view (Hedges,
1996; Winkworth et al., 2002; Lavin et al., 2004;
McDowall, 2004; McGlone, 2005; de Queiroz, 2005;
Yoder & Nowak, 2006; Measey et al., 2007).
Some of the above studies may give the impression
that the estimation of divergence dates is a panacea
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
409
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A. DE QUEIROZ and R. LAWSON
for dispersal–vicariance debates. However, most of
the cited cases compared just two hypotheses (usually
an old tectonic vicariance event versus more recent
dispersal) and supported a dispersal event estimated
to be several to many times younger than the hypothesized vicariance event. Many other cases will not be
so easily discriminated by divergence dating and may
require a more diverse approach. Furthermore, even
if an estimated divergence date favors one hypothesis,
it would be wise to seek corroboration from other
kinds of evidence given the continuing debate over
the accuracy of divergence date estimates (Graur &
Martin, 2004; Heads, 2005).
In the present study, we evaluated a difficult case
concerning the Mexican Pacific Lowlands Gartersnake (Thamnophis validus), a species that occurs on
the coastal plain of western Mexico and disjunctly in
the Cape Region of the Baja California peninsula
(Conant, 1969; Rossman, Ford & Seigel, 1996)
(Fig. 1); hereafter referred to as ‘Baja California’.
Recent discussions of the biogeographic history of
Baja California reptiles have explained such disjunct
distributions by tectonic vicariance (Murphy, 1983;
Grismer, 1994b; Murphy & Aguirre-Léon, 2002). Specifically, these taxa are thought to have become isolated on the peninsula when the Gulf of California
formed through rifting several million years ago
(see below). However, other plausible hypotheses
(i.e. vicariance by aridification, natural overwater dispersal, and human introduction) might explain the
distributions of some of these taxa, and there have
been few rigorous tests of the various explanations
(for an exception, see Macey et al., 2004).
We make use of several kinds of evidence to evaluate the case of T. validus. In particular, we use
divergence date estimates, an analysis of the relationship between sequence difference and geographic distance (hereafter, ‘difference by distance’), the location
of the closest mainland relatives of Baja California
T. validus, and the presence/absence of relict populations as primary forms of evidence to discriminate
among the various hypotheses. We also consider other
information to evaluate the hypotheses not refuted by
the primary evidence. This multifaceted approach
enables stronger discrimination among hypotheses
than would divergence age estimates alone. We
discuss our findings with respect to previous studies
of Baja California disjunctions and to the development of a more expansive historical biogeography.
HYPOTHESES
AND PREDICTIONS
We present the four hypotheses to explain the
disjunct distribution of T. validus along with the predictions made by these hypotheses concerning
divergence dates, difference-by-distance results, and
the location of the closest mainland relatives of peninsular snakes. We tested these predictions using
mitochondrial (mt)DNA sequences of specimens from
Baja California and several mainland localities.
Tectonic vicariance
Murphy (1983) and Grismer (1994b) suggested that
the formation of the Gulf of California by tectonic
processes separated Baja California populations of
many species of reptiles and amphibians, including T.
validus, from mainland relatives (Fig. 1A). Baja California apparently began rifting away from mainland
Mexico in the late Miocene or early Pliocene, creating
the Gulf of California (= Sea of Cortés) in the process
(Curray & Moore, 1984; Lonsdale, 1989; Stock &
Hodges, 1989; Oskin & Stock, 2003). Although many
aspects of this process continue to be debated, there is
general agreement that the Gulf had reached at least
its current north-south extent by 4 Mya (Grismer,
1994b; Gans, 1997; Carreño & Helenes, 2002; Murphy
& Aguirre-Léon, 2002). The various geological reconstructions indicate a maximum age for separation of
the Cape Region from the mainland ranging from
5.5 Mya (Curray & Moore, 1984) to 8.2 Mya (Oskin &
Stock, 2003).
Given the above ages, the tectonic vicariance
hypothesis predicts that the divergence for Baja California snakes and their closest mainland relatives
should be between 4 and 8.2 Mya (Fig. 1A). The
mtDNA divergence could be older because the mtDNA
lineage separation could predate the vicariance event.
However, a date clearly younger than 4 Mya would
argue against the hypothesis. Tectonic vicariance also
predicts that Baja California animals should be most
closely related to those from the region of western
Mexico where the Cape Region is thought to have
been attached, namely, the states of Nayarit or Jalisco
(Gastil, Minch & Phillips, 1983; Curray & Moore,
1984) (Fig. 1A) This second prediction is weaker than
the first because the predicted intraspecific relationship could have been erased by several million years
of gene flow among and lineage extinction within
mainland populations.
Vicariance by aridification
Thamnophis validus is a semi-aquatic species that is
rarely found far from water (Conant, 1969; de
Queiroz, Henke & Smith, 2001). Known habitats for
the species include rivers, streams, irrigation canals,
marshes, and lagoons (Conant, 1969). With this in
mind, Conant (1946, 1969) suggested that, during
more mesic times in the late Pleistocene, the species
was continuously distributed from the Cape Region
north through Baja California around the head of the
Gulf of California and southward into mainland
Mexico (Fig. 1B). Beginning at approximately the end
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
GARTERSNAKE COLONIZATION OF BAJA CALIFORNIA
411
A
Rio Yaqui
Rio Yaqui
Rio Yaqui
El Dorado
El Dorado
El Dorado
San Blas
San Blas
8.2 million years ago
San Blas
4 million years ago
Present
B
Rio Yaqui
Rio Yaqui
Rio Yaqui
El Dorado
El Dorado
El Dorado
San Blas
13,000 years ago
San Blas
San Blas
9,000 years ago
Present
C
Rio Yaqui
Rio Yaqui
El Dorado
El Dorado
San Blas
After 8.2 million years ago
San Blas
Present
Figure 1. Hypotheses to explain the disjunct occurrence of Thamnophis validus in the Cape Region of Baja California.
A, tectonic vicariance (figure modified from Gans, 1997). B, vicariance by aridification. C, overwater dispersal either
naturally or by human transport. Under some geological reconstructions, the 8.2 Mya figure in (A) and (C) for the initial
opening of the Gulf of California would be replaced by a figure of 5.5 Mya; none of our conclusions would be affected by
this change. Ranges of T. validus are indicated by shading. Past coastlines have been depicted with the shape of the
modern coastline for ease of interpretation. For details, see text.
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
412
A. DE QUEIROZ and R. LAWSON
of the Pleistocene, increased aridity (Van Devender,
1990; Thompson & Anderson, 2000; Lozano-García,
Ortega-Guerrero & Sosa-Najéra, 2002) could have
eliminated habitats suitable for T. validus in the
northern parts of its range, thus separating Baja
California and mainland populations from each other
(Fig. 1B). Aridification in this region probably began
approximately 13 000 to 10 000 years ago, depending
on the location (Cole, 1990; Van Devender, 1990;
Thompson et al., 1993; McAuliffe & Van Devender,
1998), and habitats were close to their present positions by approximately 7000 years ago (Thompson &
Anderson, 2000). Radiocarbon dates are translated
to calendar year dates, when necessary, following
Reimer et al. (2004).
According to this aridification hypothesis, Cape
Region snakes became isolated from other conspecific
populations between 7000 and 13 000 years ago
(Fig. 1B). However, the closest relatives of Cape
Region populations would likely have been peninsular
populations that failed to survive the aridification
episode; the divergence of Cape Region snakes from
their nearest mainland relatives would have occurred
at some unspecified earlier time. More helpfully, the
hypothesis predicts that, given a positive relationship
between sequence difference and the geographic distance between populations, Baja California snakes
should be differentiated from mainland snakes at
least to the degree expected by the overland distance
between them. We evaluated this prediction by
comparing Baja California-mainland sequence differentiation to that among mainland populations
(difference-by-distance analyses). The hypothesis also
weakly predicts that Baja California snakes should be
most closely related to snakes in the northern part of
the species’ mainland distribution (Fig. 1B).
Natural overwater dispersal
Thamnophis validus could have colonized Baja
California by dispersal across the Gulf of California
(Conant, 1946, 1969), either directly in the water or
on natural rafts (Fig. 1C). Conant rejected this
hypothesis in favor of his aridification hypothesis.
This hypothesis predicts that the divergence date
for Baja California snakes and their closest mainland
relatives should be younger than 8.2 Mya (Fig. 1C),
although, again, the mtDNA lineage separation could
predate the dispersal event. If the divergence date is
very recent, the hypothesis weakly predicts that the
closest mainland relatives of Baja California snakes
should be from an area that is close to the Cape
Region by water, namely, from the vicinity of El
Dorado in the central region of the state of Sinaloa
(Fig. 1C), because this would require the shortest
voyage.
Human introduction
Thamnophis validus could have been introduced to
Baja California (Fig. 1C) by watercraft in either prehistoric or historic times (Conant, 1969; although,
again, Conant rejected this idea in favor of the aridification hypothesis). Although there are no verified
early American human remains from the vicinity of
the Cape Region on either side of the Gulf of California, human specimens from both coastal California
and south-central Mexico have been dated to 12 800–
12 900 years ago (González et al., 2003). Such early
occupation of the general area is also indicated by the
presence of Clovis-type projectile points in the state of
Sonora (Sánchez, 2001). We take these values as the
approximate maximum age for human introduction of
T. validus. In historic times, snakes could have been
transported across the Gulf in the ships of Spanish
conquistadors as early as the 1530s (Miller, 1974).
This hypothesis predicts that the divergence date
for Baja California snakes and their closest mainland
relatives should be within the last 13 000 years
although, again, the mtDNA lineage separation could
be older. As for natural dispersal, the human introduction hypothesis weakly predicts that the closest
mainland relatives of Baja California snakes should
be from central Sinaloa (Fig. 1C).
MATERIAL AND METHODS
SAMPLING
We collected specimens of T. validus from seven mainland and three Baja California localities (Fig. 2;
Table 1). Outgroups were specimens of T. melanogaster, T. errans, T. cyrtopsis, and T. chrysocephalus
(de Queiroz, Lawson & Lemos-Espinal, 2002). We also
used specimens of several other species of Thamnophis and other natricine genera for the divergence
dating analyses (Fig. 3).
DNA
ISOLATION, AMPLIFICATION, AND SEQUENCING
We used liver tissue or shed skins to obtain total
genomic DNA. We digested tissues for 3–4 h at 65 °C
with constant motion in 2 mL of lysis buffer (Tris HCl
100 mM at pH 8.0, EDTA 50 mM at pH 8.0, NaCl
10 mM, SDS 0.5%) containing 60 mg mL-1 of proteinase K. Digestion was followed by two extractions with
phenol/CHCl3 and one with CHCl3 alone. DNA was
precipitated from the aqueous layer with 2.5 volumes
of pure ethanol and then washed in 80% ethanol,
dried, and redissolved in TE buffer (Tris 10 mM,
EDTA 1 mM, pH 8.0).
Template DNA for the polymerase chain reaction
(PCR) was prepared by diluting stock DNA with TE
buffer to give spectrophotometric optical density readings in the range 0.2–0.5 at A260. We amplified mito-
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
GARTERSNAKE COLONIZATION OF BAJA CALIFORNIA
413
Table 1. List of specimens of Thamnophis validus used in the present study
Taxon
Thamnophis validus
Thamnophis validus
Thamnophis validus
Thamnophis validus
Thamnophis validus
Thamnophis validus
thamnophisoides
Thamnophis validus
Thamnophis validus
Thamnophis validus
Thamnophis validus
Locality, State
#Specimens
Voucher numbers
validus
validus
validus
validus
validus
Río Yaquí, Sonora
Río Sinaloa, Sinaloa
El Dorado, Sinaloa
Río Panuco, Sinaloa
San Blas, Nayarit
Tepic, Nayarit
3
3
2
1
2
2
UCM 61507-61509
UCM 61510-61512
UCM 61513, 61514
UCM 61515
LSUMZ 37964, CAS 224079
CAS 224080, 224081
isabellae
celaeno
celaeno
celaeno
Boca de Apiza, Michoacan
El Chorro, Baja California Sur
Boca de la Sierra, Baja California Sur
San José del Cabo, Baja California Sur
3
2
1
1
CAS 224074-224076
UCM 61296, no voucher
no voucher
UCM 61899
Localities are listed from north to south on the mainland and then from north to south in Baja California.
CAS, California Academy of Sciences; LSUMZ, Louisiana State University Museum of Natural Science; UCM, University
of Colorado Museum of Natural History.
chondrial DNA from template DNA in 100-mL
reactions using a hot-start method in a thermal cycler
with a 7-min denaturing step at 94 °C followed by 40
cycles of denaturing for 40 s at 94 °C, primer annealing for 30 s at 46 °C, and elongation for 1 min at
72 °C. PCR products were purified using the Promega
Wizard PCR Preps DNA Purification System.
We performed cycle sequencing on the PCR products
using the Big Dye (Perkin-Elmer) reaction premix for
50 cycles of 96 °C for 10 s, 45 °C for 5 s, and 60 °C for
4 min. Nucleotide sequences were determined using an
ABI 310 Genetic Analyzer (Applied Biosystems). Oligonucleotide primers for amplification and sequencing
of the cytochrome b (cyt b) and NADH dehydrogenase
genes (ND1, ND2, and ND4) were as described in de
Queiroz et al. (2002). Primers for cytochrome oxidase I
(CO I) were as described in Schätti & Utiger (2001).
For each specimen, we obtained complete sequences of
cyt b (1117 nucleotides), ND1 (964 nucleotides), and
ND2 (1032 nucleotides), and partial sequences of ND4
(696 of 1338 nucleotides) and CO I (607 of 1602
nucleotides). The tRNA sequences amplified with the
ND4 gene were not used because of ambiguous alignments and lack of informative variation. Sequences
have been deposited in GenBank (accession numbers
EF417342–417460 and EF460849). The Appendix
gives GenBank numbers for sequences obtained from
previous studies.
PHYLOGENETIC
ANALYSES
We estimated genealogical relationships among
mtDNA lineages using maximum parsimony (MP)
and maximum likelihood (ML) as implemented in
PAUP*, version 4.0b10 (Swofford, 2002) and statistical parsimony (Templeton, Crandall & Sing, 1992)
as implemented in TCS, version 1.13 (Clement,
Posada & Crandall, 2000). Incongruence length difference tests (Farris et al., 1994) for all possible
pairs of the five genes indicated no significant heterogeneity among genes (results not shown). These
results and the fact that the mitochondrial genome
is inherited as a single locus led us to combine the
data for all genes. We used the default settings in
PAUP* and TCS in all analyses unless otherwise
indicated. For MP and ML analyses, trees were
rooted using the four outgroup species noted
above.
For the MP analyses, we used the heuristic search
method with tree bisection and reconnection (TBR)
branch swapping and starting trees obtained by
random stepwise addition (100 replicates). All character state changes were weighted equally. Bootstrap
analyses (2000 replicates) used the same method
except that only a single stepwise addition was used
in each replicate.
For the ML analyses, we chose a model of sequence
evolution using MODELTEST, version 3.06 (Posada &
Crandall, 1998). The specific model chosen was the
HKY + G model with the following parameter values:
transition/transversion
ratio = 8.56247
(kappa =
18.920149); nucleotide frequencies: A = 0.34016,
C = 0.30751, G = 0.10474, T = 0.24759; shape parameter a for G distribution = 0.0834345. ML analyses
were conducted using the heuristic search mode with
‘as is’ addition and TBR branch-swapping. Bootstrap
analyses (500 replicates) used the same model and
search options.
Unlike MP and ML, statistical parsimony is specifically designed to estimate relationships among
haplotypes within a species. The method explicitly
distinguishes between haplotypes that occur at the
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
414
A. DE QUEIROZ and R. LAWSON
tips of branches and those at interior nodes, with the
probability that a haplotype is interior (i.e. ancestral
to other haplotypes) being directly related to its frequency (Crandall, Templeton & Sing, 1994). Although
it would be useful to identify a haplotype ancestral to
those from Baja California, all haplotypes in our
dataset had frequencies of either one or two, giving
little reason to assign haplotypes to tip versus interior positions. Thus, we used statistical parsimony
only as a third method to identify the closest mainland relatives of Baja California T. validus.
DIVERGENCE
DATING
To estimate the divergence age between Baja California T. validus and the most closely-related mainland
specimen, we used the penalized likelihood method of
Sanderson (2002) as implemented in the software r8s,
version 1.71 (Sanderson, 2006). This method allows
different substitution rates on each branch of the tree
but, in the final choice of a substitution model, a
penalty is incorporated that imposes a greater cost for
models with substitution rates that change more
quickly from branch to branch. A smoothing factor
determines the level of the penalty. We report results
using a wide range of smoothing factor values that
produce models ranging from ones in which the
changes in substitution rate from branch to branch
are essentially unconstrained to ones in which substitution rates on all branches are virtually identical
(a molecular clock).
The tree topology used in these analyses was a
composite of the intraspecific ML tree for T. validus
(present study), the Thamnophis tree of de Queiroz
et al. (2002), the thamnophiine tree of Alfaro &
Arnold (2001), the Natrix tree of Guicking et al.
(2006), and analyses indicating the outgroup status of
Afronatrix anoscopus (R. Lawson, unpubl. data).
We constructed this composite tree following the
protocol described in de Queiroz & Rodríguez-Robles
(2006). Branch lengths were obtained from a ML
analysis of all the genes except CO I (because data
from this gene were not available for all the taxa) for
all the included taxa using the model chosen by
Modeltest for these data. The specific model used was
the TVM + I + G model with the following parameter
values: rate matrix - AC = 0.49202, AG = 8.40996,
AT = 1.00661, CG = 0.30448, CT = 8.40996, GT =
1.00000;
nucleotide
frequencies - A = 0.359135,
C = 0.347652, G = 0.077262, T = 0.215952; proportion
of invariant sites = 0.485274; shape parameter a for G
distribution = 1.111108. Seven specimens of T. validus
that contributed zero-length branches were excluded
from these analyses.
We calibrated the divergence age analyses with four
points: (1) the first fossil appearance of Thamnophis
(Egelhoff Quarry and Hottell Ranch Rhino Quarries,
Nebraska; Holman, 2000) at 13.6–14.0 Mya (Carrasco
et al., 2005) as an estimate of the deepest split among
extant members of the genus; (2) the first fossil
appearance of the tribe Thamnophiini (Black Bear
Quarry II, South Dakota; Holman, 2000) at 18.8–
19.5 Mya (Carrasco et al., 2005) as an estimate of the
deepest split among extant thamnophiines; (3) the
split between Natrix natrix and Natrix tessellata at
13–22 Mya (Guicking et al., 2006); and (4) the split
between these two Natrix species and Natrix maura
at 18–27 Mya (Guicking et al., 2006). The two Natrix
calibration points were estimated from amino acid
sequences of the same four genes used in the present
study; however, these points were derived from fossil
calibration points other than those we used (Guicking
et al., 2006).
We used a bootstrapping method to estimate the
sampling variation associated with the divergence
date estimates. We generated 100 bootstrap replicates
of the dataset (minus the CO I gene) using PHYLIP,
version 3.6 (Felsenstein, 2005) and used PAUP* to
estimate the phylogeny for each replicate by ML with
the same model and parameter values as for the
previous penalized likelihood analyses. To speed the
searches, we constrained interspecific relationships to
match those in the composite tree and used a fast
heuristic search method with no branch-swapping.
From the tree for each replicate, we obtained the
divergence date between Baja California snakes and
their closest mainland relatives by penalized likelihood using the oldest calibration points (which makes
the analysis conservative; see below), a smoothing
factor of 0.1, which was the optimal value for the
original data, and other settings as above. This set of
divergence dates reflects sampling error in the estimation of both the branch lengths and the relationships within T. validus.
Divergence dates obtained from the above methods
are for gene lineage divergences rather than population divergences. Coalescent methods can be used to
obtain estimates of the age of population divergences
(Nielsen & Wakeley, 2001; Rannala & Yang, 2003). We
did not use such population-based methods because
we sampled few individuals from each population and
because we did not have a robust estimate of the
mutation rate, which these methods require. Our
interpretations of the results consider the fact that
our age estimates are of gene lineage rather than
population divergences.
RELATIONSHIP
BETWEEN SEQUENCE DIFFERENCE
AND OVERLAND DISTANCE
To test the prediction of the aridification hypothesis
that the sequence difference between peninsular and
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
GARTERSNAKE COLONIZATION OF BAJA CALIFORNIA
mainland snakes should reflect the large overland
distance separating them, we first evaluated the relationship between percent sequence difference and
geographic distance for all mainland populations. For
this analysis, individuals from the same drainage
system into the ocean were considered members of
the same population and those from different drainages were considered members of different populations. The one exception to this rule was that
individuals collected near sea level in the area of San
Blas, Nayarit, were considered members of a different
population than those from approximately 1000 m
elevation in the area of Tepic, Nayarit (Fig. 2),
although both areas are part of the same drainage. In
addition to the elevational difference, snakes from
these two areas are morphologically distinct (Conant,
1969). In all cases, members of the same population
were collected within 5 km of each other whereas
members of different populations were collected at
least 50 km from each other. We used the mean
percent sequence difference for all possible pairwise
comparisons of individuals as the measure of
sequence difference between two populations. Geographic distance was measured as the mean straightline distance for all possible pairwise comparisons of
individuals from the two populations compared.
We used a Mantel test as implemented in IBD
(Isolation by Distance), version 1.52 (Bohonak, 2002)
to determine whether there is a significant relationship between percent sequence difference and geographic distance. This test uses populations as the
units for randomization in creating a null distribution, thus avoiding the pseudoreplication inherent in
using each pairwise comparison between two populations as the data points. We performed this test using
10 000 randomizations, and used both the raw data
and log-transformed data, as suggested by Slatkin
(1993).
An obvious next step would be to determine
whether the percent sequence difference between
Baja California snakes and their closest mainland
relatives falls within a 95% prediction interval
derived from a regression for mainland populations,
assuming that the relevant geographic distance
between the peninsular and mainland populations is
the overland distance between them. However, the
Mantel test only indicates whether a significant positive or negative relationship between genetic and
geographic distances exists; it does not estimate an
equation for the relationship. Available regression
methods are not helpful in this case because they
either use non-independent population pairs as data
points or else drastically reduce the sample size (e.g.
by using each population only once, which would give
N = 3 in this case). Thus, we evaluated the aridification hypothesis only through a qualitative consider-
415
ation of how its prediction fits with all the percent
sequence difference comparisons among mainland
populations. To make this evaluation, we calculated
the mean percent sequence difference for all pairwise
comparisons between Baja California specimens and
the specimens from the mainland population estimated to be most closely related to the Baja California individuals. The overland distance between Baja
California populations and this mainland population
was calculated as the straight-line distance from the
northernmost Baja California locality to the northern
apex of the Gulf of California plus the straight-line
distance from this latter point to the locality of the
mainland population in question. The point we used
for the northern apex of the Gulf of California reflects
the lowered sea level at the last glacial maximum,
when the peninsular and mainland ranges of T.
validus are hypothesized to have been contiguous.
Specifically, we assumed that sea level at the last
glacial maximum was approximately 135 m lower
than it is today (Clark & Mix, 2002) and used a sea
depth chart (Dauphin & Ness, 1991) to determine the
approximate location of the northern apex of the Gulf
at that time. All distances were obtained using a
program that calculates the distance between two
points from latitude and longitude coordinates (http://
jan.ucc.nau.edu/~cvm/latlongdist.html).
RESULTS
PHYLOGENETIC
ANALYSES
The 50% majority-rule ML bootstrap tree for relationships among mtDNA lineages of T. validus is shown
in Figure 2 superimposed on a map of the collecting
localities. The MP bootstrap tree is identical to the
ML tree, except that the El Dorado lineage that is
sister to the clade consisting of the other El Dorado
lineage and the Baja California lineages in the ML
tree collapses to the next more basal node in the MP
tree. The MP bootstrap percentages are also very
similar to those in the ML tree, except for the
collapsed branch just mentioned.
Statistical parsimony creates three separate haplotype networks: the first consisting of haplotypes from
the northernmost locality (Río Yaquí), the second
those from the southernmost locality (Boca de Apiza),
and the third containing all the remaining haplotypes. This separation into three networks is consistent with the MP and ML trees (Fig. 2). In the third
network, two haplotypes are placed at interior nodes
and the position of one lineage that is part of a
polytomy in the ML bootstrap tree is resolved. In all
other respects, including the placement of the two El
Dorado lineages as the closest relatives of the Baja
California haplotypes, this third network is identical
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
416
A. DE QUEIROZ and R. LAWSON
Sonora
100
100
A
74
96
60
B
Baja California Sur
99
58
99
C
Sinaloa
D
H
I
61
99
100
J
Nayarit
E
62
F
Jalisco
0
200
400
km
Colima
G
Michoacan
Figure 2. 50% majority-rule maximum likelihood bootstrap tree of mtDNA lineages of Thamnophis validus superimposed on a map of specimen localities. Mainland localities correspond to populations as defined in the text. Baja California
localities represent all three peninsular localities. For cases in which mitochondrial DNAs from multiple individuals from
a single locality formed a clade, only a single lineage is indicated (i.e. this is why bootstrap percentages are given in some
cases for what appear to be single lineages). A, Río Yaquí, Sonora; B, Río Sinaloa, Sinaloa; C, El Dorado, Sinaloa; D, Río
Panuco, Sinaloa; E, San Blas, Nayarit; F, Tepic, Nayarit; G, Boca de Apiza, Michoacán; H, El Chorro, Baja California Sur;
I, Boca de la Sierra, Baja California Sur; J, San José del Cabo, Baja California Sur.
Table 2. Divergence ages (Mya) from penalized likelihood analyses for the separation of Baja California Thamnophis
validus mtDNA lineages from the closest mainland lineage
Smoothing factor
Calibration
0.001
0.01
0.1
1
10
100
1000
10 000
100 000
Oldest
Midpoint
Youngest
Oldest w/min ages
0.33
0.32
0.32
0.33
0.33
0.32*
0.32
0.33
0.33*
0.32
0.32*
0.33
0.33
0.32
0.32
0.33
0.30
0.29
0.29
0.30
0.28
0.28
0.28
0.28
0.30
0.29
0.29
0.30*
0.30
0.28
0.26
0.30
0.29
0.27
0.24
0.29
The first three calibrations refer to the use of the oldest date, the midpoint, and the youngest date in the range of values
for each fixed calibration point. Oldest w/min ages refers to the use of the oldest dates as fixed points for the two Natrix
calibrations and the oldest dates as minimum ages for the Thamnophis and Thamnophiini calibrations. The smoothing
factor varies the penalty for among-branch rate heterogeneity, with larger values producing more clock-like models
(see text). With the smoothing factor set to 100 000 the model is essentially a clock model.
*The result under the optimal smoothing factor for that set of calibrations.
to the corresponding part of the ML tree if one
assumes the same placement of the root.
DIVERGENCE
DATING
Penalized likelihood analyses gave divergence ages
between Baja California T. validus and the closest
mainland specimen of 0.24–0.33 Mya, depending on
smoothing factor values and whether the youngest,
midpoint, or oldest age for each calibration point was
used (Fig. 3; Table 2). To evaluate the consistency of
the results across calibration points, we also performed analyses using all possible combinations of
the four calibration points with the oldest age in the
range used for each point. These analyses gave divergence ages between 0.29 and 0.33 Mya.
The bootstrap analysis gave a mean divergence age
of 0.30 Mya with a 95% confidence interval of 0.13–
0.47 Mya. The greatest divergence age from the 100
bootstrap replicates was 0.67 Mya.
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
GARTERSNAKE COLONIZATION OF BAJA CALIFORNIA
417
Baja Calif - Mainland
T. validus El Chorro
Tectonic vicariance
T. validus El Dorado
T. validus Rio Yaqui
T. melanogaster
T. errans
T. nigronuchalis
T. cyrtopsis
T. atratus
*
T. chrysocephalus
T. sauritus
T. sirtalis
*
Nerodia fasciata
Storeria dekayi
Natrix maura
*
45
40
35
30
Natrix natrix
*
25
Natrix tessellata
20
15
10
5
0
Millions of Years BP
Figure 3. Chronogram of Thamnophis validus and related taxa from penalized likelihood analysis. The arrow indicates
the estimated divergence of Baja California from mainland T. validus at 0.33 Mya, which does not coincide with the
postulated time of formation of the Gulf of California (4–8.2 Mya) indicated by the bar. The nodes marked by asterisks
were used as calibration points. In this particular analysis, the points used were the oldest dates given in the original
references. The Río Yaquí sequence represents the population of T. validus estimated to be most distantly related to the
Baja California T. validus. Most of the T. validus sequences were left out of the figure to reduce clutter, but were used
in the analysis producing the shown result.
RELATIONSHIP
BETWEEN SEQUENCE DIFFERENCE
AND OVERLAND DISTANCE
The Mantel test showed that there is a significant
positive correlation between percent sequence difference and straight-line geographic distance among
mainland populations of T. validus (Z = 9344.6083,
r = 0.6646, one-sided P = 0.0025). The results were
similar when both variables were log-transformed
(Z = –9.2682, r = 0.7291, one-sided P = 0.0024).
Figure 4 shows percent sequence difference plotted
against straight-line geographic distance for all possible comparisons among mainland T. validus populations (open circles). The filled circle represents the
mean percent sequence difference for all possible com-
parisons between Baja California sequences and the
two El Dorado sequences (representing the mainland
population most closely related to the specimens from
Baja California), with the distance between them
being the overland distance around the head of the
Gulf of California. The sequence difference between
Baja California and El Dorado populations stands out
as peculiarly small given this overland distance.
DISCUSSION
EVALUATION
OF THE HYPOTHESES
Tectonic vicariance
This hypothesis predicts, at least weakly, that
the closest mainland relatives of Baja California
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
418
A. DE QUEIROZ and R. LAWSON
Sequence difference (%)
1.50
1.25
1.00
0.75
0.50
0.25
2000
1500
1000
500
0
0.00
Overland distance (km)
Figure 4. Relationship between percent sequence difference and geographic distance among populations of Thamnophis validus. Open circles represent comparisons for all
possible pairs of mainland populations (as defined in the
text). The filled circle represents the Baja California
sequences compared with the mainland population (El
Dorado) estimated to be most closely related to the peninsular sequences. For this comparison, the distance is the
overland distance around the head of the Gulf between El
Dorado and the northernmost Baja California locality,
which is the relevant distance under the vicariance by
aridification hypothesis.
T. validus should be found in the states of Nayarit or
Jalisco, considerably south of the current position of
the Cape Region (Fig. 1). However, the phylogenetic
analyses indicate that the closest relatives of peninsular snakes are from central Sinaloa (Fig. 2).
The clearest prediction of the tectonic vicariance
hypothesis is that the genetic divergence of Baja
California and mainland specimens should reflect at
least 4 Myr of separation (Fig. 1). The divergence age
estimates strongly counter this prediction: All our
point estimates of the mtDNA lineage divergence age
are 0.33 Mya or less and even the upper 95% confidence limit from the bootstrap analysis (0.47 Mya) is
nearly one order of magnitude younger than the age
predicted by the hypothesis.
Divergence date analyses can be biased in several
ways. However, these biases are inherently conservative with respect to rejecting the tectonic vicariance
hypothesis or are unlikely to be substantial enough to
rescue the hypothesis. First, the divergence age for a
particular genetic locus can predate but is unlikely
to postdate the age of separation of populations
(Edwards & Beerli, 2000). This means that our estimate of population separation age is biased to be
high, making the estimate conservative. Second, longterm nucleotide substitution rates (i.e. for divergences
of approximately 1–2 Mya or more) can be much
lower than short-term substitution rates (i.e. for
divergences of approximately 1–2 Mya or less) and
this can affect divergence estimates when calibrations
represent the long-term rate and the estimated node
represents the short-term rate (Ho & Larson, 2006).
Again, however, this bias should only make us overestimate the divergence age between Baja California
and mainland populations. Third, the use of the first
fossil appearance as the actual first appearance of a
taxon can bias divergence age estimates toward
younger dates (Heads, 2005). Although this is a
potential problem in our analyses, the bias is not as
clearcut as one might imagine. In particular, we used
the first fossil appearances of Thamnophis and Thamnophiini to provide ages for the most recent common
ancestors of the extant members of these taxa, yet the
fossils might actually represent stem groups that
predate these crown-group ancestors. We also used a
range of ages for each calibration point, including the
oldest (and thus most conservative) age given in the
paleontological references. Furthermore, because
analyses using all possible combinations of the four
calibration points strongly refute the tectonic vicariance hypothesis, one would have to make the extreme
assumption that all the calibrations severely underestimated taxon ages to rescue this hypothesis.
Vicariance by aridification
Under this hypothesis, Baja California and mainland
T. validus separated at least 7000 years ago and
probably much earlier. The divergence date estimate
of approximately 0.3 Mya represents a maximum age
for population divergence (Edwards & Beerli, 2000)
but, even if taken as the actual age of divergence, this
value is consistent with the aridification hypothesis.
Other evidence, however, casts strong doubt on the
post-Pleistocene aridification hypothesis and also on
similar explanations invoking older episodes of aridification. mtDNA sequence differences among mainland populations of T. validus tend to increase with
increasing geographic distance (Fig. 4). Given this
observation, a prediction of the aridification hypothesis is that the sequence difference between Baja
California snakes and their closest mainland relatives
should reflect the large overland distance between
them (over 1900 km). However, the observed sequence
difference for this comparison is relatively slight
(only 0.222%). The only inter-population comparison
showing a smaller sequence difference is one between
mainland populations that are only 55 km apart. All
other comparisons between mainland populations
give sequence differences of at least 0.35%. In short,
the small sequence difference between Baja California
and mainland snakes strongly argues against the
aridification hypothesis.
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
GARTERSNAKE COLONIZATION OF BAJA CALIFORNIA
This hypothesis also predicts, albeit weakly, that
the closest relatives of Baja California snakes should
be found in the northern part of the mainland distribution. This is not the case; the estimated closest
relatives of the peninsular snakes are from central
Sinaloa, more than 400 km south of the northernmost
locality sampled (Fig. 2).
Finally, under the aridification hypothesis, one
might expect to find relict populations of T. validus in
suitable habitats between the Cape Region and the
relatively contiguous mainland range of the species
(i.e. north of its main peninsular and mainland distributions). This expectation is compromised by a lack
of knowledge of the exact characteristics of suitable
habitat for the species, which highlights the need for
a quantitative niche modelling approach (Weaver,
Anderson & Guralnick, 2006). However, another relatively aquatic gartersnake, T. hammondii, has a relict
distribution that includes numerous freshwater oases
in Baja California north of the Cape Region (McGuire
& Grismer, 1993), and a less aquatic gartersnake, T.
marcianus, occurs along the Colorado River just north
of the Gulf of California (Rossman et al., 1996).
Thamnophis validus might be able to survive in these
or other aquatic habitats in Baja California and
Sonora, yet it apparently is not found in them, suggesting that its distribution never included these
more northern regions.
Natural overwater dispersal
This hypothesis entails colonization of Baja California
some time after the formation of the Gulf of California, but otherwise places no time constraints on the
colonization event. Thus, the estimated divergence
age does not refute the hypothesis.
The natural overwater dispersal hypothesis does
not make a strong prediction about the location of the
closest mainland relatives of the Baja California
snakes, especially given that currents in the Gulf
run both north and south simultaneously (Velasco
Fuentes & Marinone, 1999; Álvarez-Borrego, 2002).
Nonetheless, under this hypothesis, a best guess for
the location of these mainland relatives is the area
closest to the Cape Region by water, namely central
Sinaloa. Our phylogenetic analyses indicate that this
is in fact the area where the closest relatives of the
peninsular snakes occur (Fig. 2).
Snakes have managed to colonize many islands
by natural overwater dispersal, including some, such
as Madagascar (Nagy et al., 2003), the Galapagos
(Kricher, 2006), and Mexico’s Isla Clarión
(Brattstrom, 1990), that are farther from source areas
than the Cape Region is from mainland Mexico.
Nonetheless, Conant (1946) thought that the possibility that T. validus colonized Baja California by such
dispersal was remote because ‘any aquatic [i.e. fresh-
419
water] snake making a landfall would be apt to come
ashore on a dry and inhospitable coast, where conditions would be unsuited for its survival’ (Conant,
1946: 270). However, although the coast of the Cape
Region today is generally inhospitable to T. validus,
this was probably less true during wetter periods in
the past, when more streams would have reached the
ocean. Also, compared to other snake species in the
region, the distribution and habits of T. validus make
it a likely candidate for dispersal across the Gulf. In
particular, these gartersnakes are abundant near the
coast, including in brackish water habitats (Conant,
1969), they are proficient swimmers, and individuals
in some populations are relatively resistant to the
desiccating effects of saltwater, at least compared to
closely related species (Dunson, 1980). It may not be
coincidental that the only prior well-established case
of colonization by a thamnophiine snake over a
similar expanse of ocean is that of another coastal
and saltwater-adapted species, the Saltmarsh Snake
(Nerodia clarkii), which apparently colonized Cuba
from Florida (Gibbons & Dorcas, 2004).
Human introduction
As noted above, the divergence date estimate of
0.3 Mya between Baja California and mainland
snakes represents a maximum age for population
separation; the actual population separation could be
much more recent. The sequence difference between
Baja California snakes and their closest mainland
relative is less than that between the latter specimen
and another snake from the same population. In other
words, the sequence difference between Baja California and mainland snakes is consistent with a population divergence age of zero and, thus, does not
refute the hypothesis of human introduction.
In historic times, ships could have transported
snakes across the Gulf beginning in the 1530s (Miller,
1974). Differentiation of peninsular from mainland T.
validus might appear to argue against such a recent
introduction. In particular, peninsular T. validus are
diurnal whereas conspecific mainland snakes are
nocturnal (Conant, 1969) and upland populations of
peninsular T. validus have evolved a distinctive melanistic colour pattern not seen in mainland populations (Conant, 1969). However, many examples of
evolution documented in contemporary populations
suggest that changes of this scale can occur very
rapidly (Stockwell, Hendry & Kinnison, 2003).
Prehistoric human introduction of T. validus to
Baja California from across the Gulf might initially
sound implausible, but several arguments suggest
that such an occurrence is not so far-fetched. First, in
historic times, Seri Indians made crossings of the
northern part of the Gulf in traditional reed-boats
(Felger & Moser, 1985; Bowen, 2000), indicating that
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
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A. DE QUEIROZ and R. LAWSON
prehistoric crossings were possible. Second, a large
collection of dolphin bones at an archaeological site in
the Cape Region suggests prehistoric dolphin-hunting
and, thus, perhaps the use of boats in that specific
area (Porcasi & Fujita, 2000). Finally, a variety of
observations or inferences, including the apparently
rapid colonization of the Americas, suggests that the
initial migration of people from Asia through North
America into South America might have occurred via
seafaring along the Pacific Coast (Dixon, 1999;
Erlandson, 2002). In short, it is plausible that prehistoric people crossed the Gulf from central Sinaloa
to the Cape Region and that such crossings might
have occurred starting from the earliest occupation of
these areas, some 13 000 years ago.
Conclusions
Our results strongly refute both tectonic vicariance
and vicariance by aridification as explanations for
the disjunct distribution of T. validus. Refutation of
the tectonic vicariance hypothesis is especially significant given the prevalence of this explanation for
disjunct occurrences of reptile taxa in Baja California (Seib, 1980; Murphy, 1983; Grismer, 1994b;
Murphy & Aguirre-Léon, 2002). Of the 16 reptile
lineages (including T. validus) that Grismer (1994b)
suggested had been isolated in Baja California
during the early stages of formation of the Gulf (his
‘southern Miocene vicariant complex’), the relevant
divergence date has now been estimated in two
cases: T. validus, in which the divergence is too
recent to be explained by the formation of the Gulf,
and the two-legged amphisbaenian genus Bipes, in
which the divergence is too old (55 Mya) to be
explained by this event (Macey et al., 2004). Vicariance associated with the Gulf’s formation remains a
likely hypothesis for many Baja California disjunctions, but the divergence date results for T. validus
and Bipes emphasize the need for focused analyses
of the various taxa to evaluate whether a general
vicariant pattern exists.
By contrast to the vicariance hypotheses for T.
validus, natural overwater dispersal and human
introduction cannot be rejected and are rendered
more likely by the finding that the estimated closest
relative of peninsular snakes was found almost
directly across the Gulf. Natural dispersal seems
more likely than human introduction for several
reasons: (1) the time window for natural dispersal is
much longer than that for human introduction; (2) T.
validus has characteristics that make it a reasonably
likely candidate for natural dispersal (see above); (3)
it is uncertain whether prehistoric people crossed the
Gulf in the appropriate region; and (4) T. validus does
not fall into the obvious categories of reptile taxa
prone to be introduced by humans, namely those used
for food (Felger & Moser, 1985; Grismer, 1994a;
Nabhan, 2002), those small enough even as adults to
be easily transported inadvertently (Austin, 1999), or
those kept as pets (Reed, 2005).
A
PENINSULA AS AN ISLAND
Historical explanations for the occurrence in Baja
California of nonflying tetrapod taxa that are also
found on the mainland have focused on two processes:
overland dispersal from the base of the peninsula
(Savage, 1960; Taylor & Regal, 1978; Murphy, 1983)
and vicariant events separating peninsular taxa or
populations from their mainland relatives (Savage,
1960; Conant, 1969; Seib, 1980; Murphy, 1983;
Grismer, 1994b; Riddle et al., 2000; Murphy &
Aguirre-Léon, 2002). In addition to our study of T.
validus, two general observations suggest that more
attention should be given to the possibility of overwater colonization of Baja California. First, the peninsula is approximately 1100 km long and is
separated from the mainland by the Gulf of California, which is less than 220 km across at its widest
point. In other words, Baja California is a large target
that, along its entire length, is separated by a relatively narrow saltwater barrier from a potential
source of colonists.
Second, nonflying tetrapods have apparently colonized islands by crossing most of the width of the Gulf
(i.e. mainland tetrapods have sometimes colonized
islands on the peninsular side and peninsular
tetrapods have sometimes colonized islands on the
mainland side). Examples include Tiger Whiptails
(Cnemidophorus tigris) from the mainland to Isla
Salsipuedes and Isla San Lorenzo Norte (Murphy &
Aguirre-Léon, 2002), Western Diamondback Rattlesnakes (Crotalus atrox) from the mainland to Isla
Tortuga and Isla Santa Cruz (Murphy & AguirreLéon, 2002; Castoe, Spencer & Parkinson, 2007), and
many cases involving deermice (Peromyscus; Hafner,
Riddle & Alvarez-Castañeda, 2001; Lawlor et al.,
2002; Carleton & Lawlor, 2005). If anything, one
would expect that crossings from the mainland to the
peninsula itself would be much more frequent than
these similarly distant crossings to the Gulf islands
because the peninsula is a much larger target than
the collection of islands.
These considerations suggest that many nonflying
tetrapod taxa may have colonized Baja California by
overwater dispersal, as if the peninsula were an
island. We suggest that future studies should sample
many peninsular and mainland populations to
increase the likelihood of detecting phylogeographic
relationships that would indicate such colonization
events (for an example involving cacti, see ClarkTapia & Molina-Freaner [2003]; for probable overwa-
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424
GARTERSNAKE COLONIZATION OF BAJA CALIFORNIA
ter dispersal of the Rosy Boa [Lichanura trivirgata]
from Baja California to the mainland, see Wood,
Fisher & Reeder [2008]).
BEYOND
CLADOGRAMS, BEYOND DIVERGENCE DATES
The historical biogeography that emerged in the
1970s was strongly focused on cladistic relationships
as the primary evidence to test hypotheses (Nelson &
Platnick, 1981; Wiley, 1988). In the last 15 years or
so, the field has come to use a greater diversity of
evidence, a general trend that may be typical of a
developing discipline (Donoghue & Moore, 2003).
One recent and promising approach is to incorporate diverse sources of information as parameters in
complex models. For example, dispersal and population persistence probabilities, divergence ages, and
phylogenetic relationships can be included in a single
model to estimate the likelihoods of different biogeographic scenarios (Ree et al., 2005); see also Knowles,
Carstens & Keat (2007) and Sanmartín, Wanntorp &
Winkworth (2007). This modelling approach can be
convincing if the sensitivity of the results to a wide
range of plausible parameter values is evaluated. If
the recent history of comparative biology in general is
any indication, such a complex, modelling approach is
likely to become an important aspect of historical
biogeography.
In the present study, we have taken a more primitive but more transparent approach, namely, employing diverse sources of evidence as multiple,
independent tests of hypotheses. Although our original data all came from a single mtDNA dataset, the
various analyses are independent in terms of their
ability to support or refute the hypotheses. For
example, the fact that difference-by-distance results
argue against vicariance by aridification does not
entail that the location of mainland relatives must
also do so. Compared with using only phylogenetic
relationships and divergence dating, this more
diverse approach allowed us to: (1) discriminate
among hypotheses that otherwise could not be separated and (2) add independent evidence to arguments
for or against particular hypotheses.
Other recent studies exemplify the use of multiple
independent tests to evaluate biogeographic hypotheses. For example, McDowall (2002) used independent tests involving intraspecific genetic variation,
parasitology, distribution patterns in relation to life
history, and known recent dispersal events, among
other evidence, to argue for widespread dispersal of
cool-temperate diadromous fishes among southern
continents. Similarly, the view that the biota of New
Zealand is derived primarily from oceanic dispersal
following the break-up of Gondwana has gained credence from independent arguments involving the
421
fossil record (Pole, 1994), geological evidence for the
Oligocene submergence of most or all of Zealandia
(Trewick, Paterson & Campbell, 2007), molecular
divergence dating (Winkworth et al., 2002), and the
conspicuous absence of taxa such as snakes and
terrestrial mammals (Trewick et al., 2007).
The use of multiple independent tests is obviously
desirable in science in general, but it is especially
critical in the historical sciences (Gould, 2002). Studying history, especially the history of quixotic entities
such as evolutionary lineages, typically involves
making assumptions that are difficult or impossible to
verify, with the result that conclusions from a single
test often are not convincing. In our view, this is a
major reason for historical biogeography to move
away from a narrow focus on cladistic relationships or
divergence dating. For that matter, it is a reason not
to rely entirely on the results of any single analysis,
no matter how sophisticated it is. A more integrative
historical biogeography should involve not only more
complex models, but also a greater attention to devising multiple, independent tests.
ACKNOWLEDGEMENTS
We thank Matthew Bealor, Tara Forbis, and Phil
Frank for collecting snakes; Carl Elliger and Eleanor
Visser for laboratory assistance; Lee Grismer for
information on collecting localities; Julio LemosEspinal for obtaining Mexican collecting and exportation permits; Chris Austin for answering questions
about specimens under his care; Dick Olmstead for
asking a key question about independent evidence;
and Tara Forbis, John Gatesy, Lee Grismer, Julio
Lemos-Espinal, Doug Rossman, and several anonymous reviewers for their helpful comments on the
manuscript.
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APPENDIX
GenBank accession numbers for sequences from previous studies (de Queiroz et al., 2002; Lawson et al.,
2005; Guicking et al., 2006) used in the present study.
Genes are given in the order cyt b, ND1, ND2, and
ND4: Afronatrix anoscopus – AF420073-420076;
Natrix maura – AY487695, AY873739, AY870613,
AY873706; N. natrix – AF471059, AY873757,
AY870630, AY873724; N. tessellata – AY866533,
AY873764, AY870635, AY873729; Nerodia fasciata –
AY866529,
AY873738,
AY870612,
AY873705;
Thamnophis atratus – AF420085-420088; T.
chrysocephalus – AF420108, AF420096-420098; T.
nigronuchalis – AF420153-420156; T. sauritus –
AF420177-420180; T. sirtalis – AF420193-420196
© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 95, 409–424