doi: 10.1111/j.1420-9101.2005.00906.x
The Isthmus of Panama: a major physical barrier to gene flow in a
highly mobile pantropical seabird
T. E. STEEVES,* D. J. ANDERSON & V. L. FRIESEN*
*Department of Biology, Queen’s University, Kingston, ON, Canada
Department of Biology, Wake Forest University, Winston-Salem, NC, USA
Keywords:
Abstract
gene divergence;
masked booby;
MDIV;
mitochondrial control region;
phylogeography;
population divergence.
To further test the hypothesis that the Isthmus of Panama is a major barrier to
gene flow in pantropical seabirds, we applied phylogeographic methods to
mitochondrial control sequence variation in masked booby (Sula dactylatra)
populations on either side of the Isthmus of Panama and the southern tip of
Africa. In accord with Steeves et al. (2003), we found that all Caribbean
masked boobies with the ‘secondary contact’ cytochrome b haplotype (m-B)
shared a control region haplotype (Sd_100), which grouped with Indian–
Pacific haplotypes and not Caribbean–Atlantic haplotypes. In addition, Sd_100
was more closely related to control region haplotypes in the Indian Ocean
than in the Pacific. We also found that the ‘secondary contact’ birds diverged
more recently from extant populations in the Indian Ocean than in the Pacific.
Thus, it appears that these masked boobies did not breach the Isthmus of
Panama. Rather, birds likely dispersed around the southern tip of Africa during
favourable oceanographic conditions in the Pleistocene.
Introduction
The emergence of the Isthmus of Panama approximately
3 million years ago (Coates & Obando, 1996) isolated
Pacific and Atlantic populations of many tropical marine
taxa (e.g. sea urchins – Lessios et al., 1999; McCartney
et al., 2000; Lessios et al., 2001; fishes – Bowen et al.,
2001; Muss et al., 2001; sea turtles – Bowen et al.,
1992,1998). Despite the great dispersal potential of many
seabirds (e.g. Anderson, 1993; Schreiber et al., 2002),
recent phylogeographic studies indicate that the Isthmus
of Panama is also an effective barrier to gene flow in
several pantropical species (i.e. terns – Avise et al., 2000;
boobies – Steeves et al., 2003). However, the divergence
of extant Pacific and Atlantic populations does not
correspond to the closure of the Panamanian Seaway.
Rather, populations appear to have diverged within the
last c. 640 000 years (Avise et al., 2000; Steeves et al.,
2003). These results suggest that the Isthmus of Panama
has restricted gene flow between Pacific and Atlantic
Correspondence: T. E. Steeves, School of Biological Sciences, University of
Canterbury, Private Bag 4800, Christchurch, New Zealand.
Tel.: +64-3-364-2987 ext. 7019; fax: +64-3-364-2590;
e-mail: tammy.steeves@canterbury.ac.nz
1000
pantropical seabird populations in one of two ways: it has
either never been breached (e.g. species arose following
the emergence of the Isthmus of Panama and subsequently spread between the Pacific and Atlantic via the
Indian Ocean), or it has rarely been breached (e.g. species
spread between the Pacific and Atlantic via dispersal over
the Isthmus of Panama; Steeves et al., 2003).
There are several reasons to suspect that the Isthmus of
Panama has never been breached. Despite their high
mobility, pantropical seabirds avoid flight over land.
Although the Isthmus of Panama is only 35 km at its
narrowest, its interior is dominated by steep, rugged
mountains and upland plains. In addition, the present
width of the landbridge represents an interglacial minimum: during the glacial periods that dominated the
Pleistocene, when global sea levels dropped an average of
120 m below present levels (Siddall et al., 2003), the
narrowest section of the Isthmus of Panama would have
been c. 200 km wide. Global sea level was higher than
present during some interglacial periods but it rarely
exceeded 10 m above current levels (Karner et al., 2002).
Thus, although the minimum width of the Isthmus of
Panama may have decreased slightly during some of
these periods, the topography of its interior terrain would
have remained relatively unchanged. For these reasons,
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY OF EVOLUTIONARY BIOLOGY
Physical barriers to gene flow in masked boobies
any dispersal over the Isthmus of Panama would likely be
passive and occur only during stochastic events such as
severe storms.
In contrast, fluctuating oceanographic conditions during the Pleistocene suggest that active dispersal around
the southern tip of Africa is more plausible. For many
tropical marine taxa, the cold Benguela Current off
southwestern Africa has restricted gene flow between
Atlantic and Indian Ocean populations since its appearance in the Late Pliocene (e.g. Bowen et al., 1992; Lessios
et al., 1999; McCartney et al., 2000; Bowen et al., 2001,
but see also Lessios et al., 2001). However, recent
paleoceanographic studies indicate that whereas the
Benguela Current had a tendency to weaken and/or
shift northwards during major transitions from glacial to
interglacial conditions in the Middle – Late Pleistocene,
the warm Agulhas Current off southeastern Africa had a
tendency to strengthen and intrude into the Atlantic
(Flores et al., 1999; Pierre et al., 2001; Chen et al., 2002).
Such warm-water intrusions, albeit ephemeral, may
have facilitated active dispersal of highly mobile tropical
marine taxa between the Atlantic and the Indian Ocean.
Indeed, a recent phylogeographic study of olive ridley
turtles (Lepidochelys olivacea), a pantropical marine species, revealed that dispersal around the southern tip of
Africa did occur within the last c. 300 000 years (Bowen
et al., 1998).
Previous researchers were unable to test the hypothesis
that the Isthmus of Panama has not been breached by
pantropical seabirds due to restricted sampling (Steeves
et al., 2003) or limited genetic resolution (Avise et al.,
2000). Steeves et al. (2003) examined mitochondrial
cytochrome b sequence variation in red-footed (Sula
sula), brown (Sula leucogaster), and masked (Sula dactylatra) boobies from the central and eastern Pacific Ocean
and the Caribbean Sea. With the exception of one
haplotype in masked boobies (m-B), no haplotypes were
shared across the Isthmus of Panama in any of the three
species. However, due to the absence of samples from the
Indian Ocean, Steeves et al. (2003) could not determine
whether boobies initially dispersed between the Pacific
and the Caribbean around the southern tip of Africa or
over the Isthmus of Panama. For masked boobies,
because the most common Pacific haplotype (m-B) was
distantly related to the two unique Caribbean haplotypes
(m-C and m-D), Steeves et al. (2003) attributed the
presence of haplotype m-B in the Caribbean to secondary
contact, but were unable to determine whether secondary dispersal occurred via the Indian Ocean or over the
Isthmus of Panama. Subsequent analyses revealed that
masked boobies from the western Indian Ocean also
share cytochrome b haplotype m-B (n ¼ 6; TES, unpublished data). Thus, additional analyses that examine
sequence variation in a more rapidly evolving gene (e.g.
mitochondrial control region) are required to determine
whether masked boobies breached the Isthmus of Panama within the last c. 640 000 years.
1001
In this study, we expand on Steeves et al. (2003) and
examine mitochondrial control region sequence variation among masked booby populations on either side of
the Isthmus of Panama and the southern tip of Africa to
further test the hypothesis that the Isthmus of Panama is
a major barrier to gene flow in pantropical seabirds by
addressing the following predictions:
(a) If the presence of cytochrome b haplotype m-B in the
Caribbean is due to secondary contact then Caribbean
masked boobies with cytochrome b haplotype m-B
will have control region haplotypes that are more
closely related to haplotypes found in the Indian
Ocean and the Pacific than to haplotypes found in
the Caribbean. In addition, time since secondary
divergence will be more recent than time since initial
divergence of Caribbean masked boobies and masked
boobies in the Indian Ocean and the Pacific.
(b) If secondary dispersal occurred around the southern
tip of Africa (i.e. not over the Isthmus of Panama)
then Caribbean masked boobies with the ‘secondary
contact’ cytochrome b haplotype m-B will have
control region haplotypes that are more closely
related to haplotypes found in the Indian Ocean
than to haplotypes found in the Pacific. In addition,
the ‘secondary contact’ birds will have diverged more
recently from extant populations in the Indian Ocean
than populations in the Pacific.
Methods
Sampling and laboratory protocols
Blood or feather samples were collected from 120 masked
boobies in the Caribbean Sea (Isla Monito; Steeves
et al., 2003), the Atlantic Ocean (Ascension Island; this
study), the western Indian Ocean (Cosmoledo Atoll; this
study), and the eastern Pacific Ocean (Isla San Benedicto;
this study; Fig. 1). DNA was purified from samples using
standard phenol/chloroform extraction and isopropanol
precipitation (Sambrook et al., 1989). To avoid nuclear
copies of the mitochondrial control region, we amplified
and sequenced a fragment that included the 3¢ end of the
ND6 gene, the complete tRNAglu gene as well as all of
Domain I and most of Domain II of the control region in a
subset of the feather samples using a general light-strand
primer designed for birds (ND6L-end, O. Haddrath,
unpublished) and a more species-specific heavy-strand
primer designed for northern gannets (Morus bassanus,
SbMCR-H800, 5¢-CCAATACGTCAACCGTCTCAT-3¢). To
confirm the mitochondrial origin of these sequences, we
compared them to published ND6, tRNAglu, and control
region sequences and identified all of the avian conserved blocks in Domain II (Desjardin & Morais, 1990;
Baker & Marshall, 1997; Randi & Lucchini, 1998). We
then developed two species-specific primers (SdMCRL100B, 5¢-AATTCGTGGAAGCAGTCACA-3¢; SdMCRH750, 5¢-GGGAACCAAAAGAGGAAAACC-3¢) to amplify
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1002
T. E. STEEVES ET AL.
Fig. 1 Sampling locations for masked boobies. 1: Caribbean Sea (Isla Monito); 2:
Atlantic Ocean (Ascension Island); 3: western Indian Ocean (Cosmoledo Atoll): 4:
eastern Pacific Ocean (Isla San Beneditco).
a 500 bp fragment (Domain I ¼ 283 bp, Domain II ¼
217 bp) in all available samples. PCR amplifications were
performed in 25–50 lL reaction volumes containing
5 mM Tris–HCl pH 8.8, 5 mM Tris–HCl pH 8.0, 50 mM
KCl, 0.01% gelatin, 125 lg BSA, 2.5 mM MgCl2, 0.2 mM
dNTPs, 0.4 lM of each primer, 0.5–1 U Taq DNA Polymerase (Roche, Diagnostics Corporation, Indianapolis, IN,
USA), and c. 100 ng of extracted DNA using the following
temperature profile: 30 s at 95 C, 30 s at 55 C–60 C
and 45 s–1 min at 72 C for 30 cycles preceded by a
2 min denaturing step at 95 C and followed by a 3 min
extension step at 72 C. PCR products were sequenced
using standard cycle sequencing protocols (Mobix,
McMaster University) and visualized using an ABI
373A automated sequencer (Perkin-Elmer Corporation,
Applied Biosystems Division, Foster City, CA, USA).
Data analyses
Control region sequences were aligned manually using
Se-Al (Rambaut, 1996) and all variable sites were
confirmed by visual inspection of the chromatograms.
Twelve ambiguous sites in 16 individuals were resolved
as follows: if the haplotype sequence containing the
ambiguous site (e.g. C/T, haplotype 1) was otherwise
identical to a haplotype sequence without the ambiguous
site (e.g. C, haplotype 2), then the nucleotide was
assigned the nonambiguous base (e.g. haplotype 1 ¼
haplotype 2; Moum & Árnason, 2001); if the haplotype
sequence containing the ambiguous site (e.g. C/T, haplotype 1) was otherwise identical to two haplotypes
without the ambiguous site (e.g. C, haplotype 2; T,
haplotype 3), then the nucleotide was assigned the
nonambiguous base present in the most common and/or
most basal haplotype (e.g. if haplotype 3 was more
common and basal to haplotype 2 then haplotype 1 ¼
haplotype 3). Excluding these 16 individuals from all
subsequent analysis does not alter our conclusions (data
not shown). Relationships among control region haplotypes were inferred by constructing statistical parsimony
networks in TCS (Version 1.13, Clement et al., 2000).
For population-level analyses, we defined six putative
populations: Caribbean (excluding secondary contact
birds), Caribbean (secondary contact birds only), Atlantic
(excluding secondary contact birds), Atlantic (secondary
contact birds only), western Indian, and eastern Pacific.
Net genetic distance among population pairs was estimated as d ¼ pxy – 0.5 (px + py), where pxy is mean
percentage sequence divergence between populations x
and y, and px and py are mean percentage sequence
divergence within populations x and y, respectively (Nei,
1987) using ARLEQUIN (Version 2.001; Schneider et al.,
2001). Statistical significance of genetic distances was
assessed by randomization using 1000 permutations.
Nielsen & Wakeley (2001) recently developed a
program (MDIV) based on coalescent theory (Kingman,
1982a,b) that employs Bayesian inference to simultaneously estimate theta (Q, measured as 2Nel where Ne is
the effective mitochondrial population size and l is the
mutation rate per gene per year), divergence time
(T, measured as t/Ne where t is the time since population
divergence), and time to most recent common ancestor
(TMRCA, measured as tMRCA/Ne where tMRCA is the
time since gene divergence) for population pairs under a
finite sites model. Whereas T estimates when two
populations diverged from one another (i.e. population
divergence), TMRCA estimates when genes last shared a
common ancestor (i.e. gene divergence; Edwards &
Beerli, 2000) and can be used as a proxy for ancestral
population age (Griswold & Baker, 2002). We used MDIV
to estimate Q, T, and TMRCA among population pairs on
either side of the Isthmus of Panama and the southern tip
of Africa. For each pairwise comparison, a minimum of
three chains (length of Markov chain ¼ 5 000 000
cycles; burn-in time ¼ 500 000 cycles) with different
random seeds were run using the following parameters:
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Physical barriers to gene flow in masked boobies
I
1003
III
Sd_100
Fig. 2 Statistical parsimony networks (I–III)
among masked booby haplotypes estimated
by TCS. Circle sizes are proportional to
frequencies of haplotypes given in Table S1.
Filled circles represent missing haplotypes.
Gray line indicates the only alternative
connection. Arrow denotes haplotype
Sd_100 (see text for details).
II
Caribbean
finite sites model (HKY, Hasegawa et al., 1985), Mmax ¼
5 or 10 (where M ¼ migration rate), and Tmax ¼ 5, 10 or
20. For each pairwise comparison, values for Mmax and
Tmax were selected as those that generated a posterior
distribution of the correct shape (e.g. a bell-shaped
curve), but minimized the relative number of data points
on the right-hand tail. T and Q were estimated as the
mode of their respective posterior distributions (MDIV
provides a point estimate only for TMRCA; Nielsen &
Wakeley, 2001). We calculated a Bayesian confidence
interval, or credibility interval (CI), for Q (Nielsen &
Wakeley, 2001), but we did not calculate a credibility
interval for T: credibility intervals for T are highly
sensitive to set values of Tmax because the right-hand
tail of the probability distribution decreases to zero very
slowly. Rather, we compared pairwise estimates of T by
plotting their respective posterior distributions and
visually inspecting the degree of overlap. Pairwise
estimates of T and TMRCA were converted to time since
divergence in years before present (y bp) using t ¼ TQ/
2l and tMRCA ¼ TMRCAQ/2l assuming a typical avian
divergence rate of 15% per million years (my) for
Domain I and Domain II combined (for 500 bp, l ¼
0.0000375; Wenink et al., 1996).
To test the assumption that our estimates of time since
initial divergence reflect historical isolation and not past
selective pressures (i.e. mitochondrial DNA sequence
variation in masked boobies is neutral), Tajima’s D
(Tajima, 1989) and Fu & Li’s D* and F* (Fu & Li, 1993)
were calculated for all populations (excluding secondary
contact birds) and statistical significance of test statistics
were assessed using DnaSP (Version 4.0; Rozas et al.,
2003). Whereas significant positive values may indicate
Atlantic
W. Indian
E. Pacific
balancing selection or population subdivision, significant
negative values may indicate recent selective sweeps or
population bottlenecks (Tajima, 1989; Fu & Li, 1993).
Excluding one test statistic for one population (Atlantic
masked boobies, D* ¼ 1.58, P < 0.05; positive significant
value likely due to one relatively frequent divergent
haplotype, see Fig. 2), all test statistics for all populations
were nonsignificant. Thus, we found little evidence to
contradict the assumption of neutrality.
Results
Among the 120 masked booby samples, we found
52 control region haplotypes defined by 83 variable sites
including 75 transitions, 7 transversions, and 3 indels
(Table S1). All haplotypes were unique to a single
sampling location (Table S1). Three statistical parsimony
networks were estimated by TCS (95% parsimony probability connection limit equals nine steps; Fig. 2).
Network I contained the majority of haplotypes from
the Caribbean and Atlantic, network II contained one
haplotype from the Caribbean (Sd_104), and network III
contained all haplotypes from the western Indian and
eastern Pacific as well as one haplotype from the
Caribbean (Sd_100). The minimum number of steps
between networks I and II, I and III, and II and III was
11, 35 and 36, respectively. Within network I, we found
two relatively distinct phylogeographic clades: one clade
contained the majority of haplotypes from the Caribbean
and the other contained the majority of haplotypes from
the Atlantic. Within network III, we also found two
relatively distinct clades: one clade contained all of the
haplotypes from the eastern Pacific and one haplotype
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
1004
T. E. STEEVES ET AL.
from the western Indian (Sd_64), and the other clade
contained the remainder of the haplotypes from the
western Indian and one haplotype from the Caribbean
(Sd_100). Sd_100 was found in all Caribbean samples
with secondary contact cytochrome b haplotype m-B and
was more closely related to haplotypes from the western
Indian Ocean than to haplotypes from the eastern Pacific
(i.e. Sd_100 was connected by a minimum of three steps
to interior haplotypes that were closely connected to
haplotypes from the western Indian, whereas it was
connected by a minimum of nine steps to interior
haplotypes that were closely connected to haplotypes
from the eastern Pacific).
Because network III did not contain Atlantic control
region haplotypes, we inferred that secondary contact
cytochrome b haplotype m-B was not present in the
Atlantic (results confirmed in subsequent analysis of
cytochrome b sequence variation for a subset of Atlantic
samples; data not shown). Similarly, we inferred that
neither cytochrome b haplotype m-C or m-D was present
in either the Pacific or the Indian Ocean, based on
subsequent analysis of control region sequence variation
among nine additional Indo-Pacific populations (n ¼
228; T.E. Steeves, D.J. Anderson & V.L. Friesen, unpublished). Thus, we estimated sequence divergence within
and between five populations only: Caribbean (excluding
secondary contact birds), Caribbean (secondary contact
birds only), Atlantic, western Indian, and eastern Pacific.
Estimates of px were relatively high (px ¼ 0.91–1.38;
Table 1), excluding the secondary contact birds (px ¼
0.00; Table 1). All pairwise estimates of d were significantly greater than zero: estimates between the Caribbean–Atlantic region and the Indian–Pacific region were
relatively high and comparable to estimates between
secondary contact birds and birds in the Caribbean and
the Atlantic (d ¼ 6.80–7.37; Table 1); in contrast, estimates between the Caribbean and the Atlantic, and
between the Indian and the Pacific were relatively low,
and comparable to estimates between secondary contact
Table 1 Estimates of mean percentage sequence divergence within
populations (px, along diagonal in bold), mean percentage sequence
divergence among populations (pxy, above diagonal), and corrected
mean percentage divergence among populations (d, below diagonal)
for masked booby populations on either side of the Isthmus of
Panama and the southern tip of Africa.
Caribbean*
Caribbean Atlantic
Western Indian
Eastern Pacific
Caribbean*
Caribbean Atlantic
Western
Indian
Eastern
Pacific
1.26
7.10
0.80
6.80
7.27
7.73
0.00
7.10
1.00
2.03
1.89
7.55
0.91
6.86
7.37
8.12
1.69
8.00
1.38
1.00
8.38
2.50
8.30
2.17
0.95
All pairwise estimates of d were significantly greater than zero (all
P < 0.01).
*Excluding secondary contact birds.
Secondary contact birds only.
birds and birds in the western Indian and the eastern
Pacific (d ¼ 0.80–2.03; Table 1).
Because we aimed to compare estimates of time since
initial divergence and time since secondary divergence,
we restricted our population pairwise comparisons to
Caribbean (excluding secondary contact birds) vs. western Indian and eastern Pacific, and Caribbean (secondary
contact birds only) vs. western Indian and eastern Pacific,
respectively. For all pairwise comparisons, although very
large values of T cannot be excluded, probability distributions for T and Q showed a small degree of overlap
(Figs 3 and 4). For the Caribbean (excluding secondary
contact birds) comparisons, whereas the point estimate of
T for the Caribbean vs. the eastern Pacific was c. twice as
high as for the Caribbean vs. the western Indian (5.64
and 2.88, respectively; Fig. 3a), the point estimate of Q
was c. 50% lower (6.37 (CI ¼ 4.36–10.72) and 11.58
(CI ¼ 7.99–18.37), respectively; Fig. 4a). Thus, estimates
of time since initial divergence of masked boobies on
either side of the Isthmus of Panama and the southern tip
of Africa were similar; estimates of tMRCA showed a
concordant pattern (Table 2). For the Caribbean (secondary contact birds only) comparisons, the point estimate
of Q for the Caribbean vs. the eastern Pacific was also
c. half that for the Caribbean vs. the western Indian (4.80
(CI ¼ 2.87–8.85) and 9.13 (CI ¼ 6.04–16.34), respectively; Fig. 4b), but the point estimate of T was c. six times
higher (2.08 and 0.35, respectively; Fig. 3b). Thus,
the estimate of time since secondary contact for the
Caribbean vs. western Indian comparison was c. three
times more recent than for the Caribbean vs. the eastern
Pacific comparison; in contrast, estimates of tMRCA were
similar (Table 2).
Discussion
Secondary contact in masked boobies
We found that all Caribbean masked boobies with the
secondary contact cytochrome b haplotype m-B had the
same control region haplotype (Sd_100) and, in accord
with Steeves et al. (2003), Sd_100 grouped with haplotypes from the western Indian–eastern Pacific region
(rather than with haplotypes from the Caribbean–Atlantic
region; Fig. 2). We also found that time since secondary
divergence was more recent than time since initial
divergence of extant masked booby populations on
either side of the Isthmus of Panama and the southern
tip of Africa (Table 2). More importantly, the secondary
contact haplotype (Sd_100) was more closely related to
western Indian haplotypes than to eastern Pacific haplotypes (Fig. 2), and the secondary contact birds diverged
more recently from birds in the western Indian than from
birds in the eastern Pacific (Table 2). Our combined
results indicate that the presence of Sd_100 in the
Caribbean is due to a secondary dispersal event from
the western Indian Ocean around the southern tip of
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
(a) 0.006
(a)
0.020
Posterior probability
Physical barriers to gene flow in masked boobies
0.015
1005
Posterior probability
0.005
0.004
0.003
0.002
0.010
0.005
0.001
0.000
0.000
0
5
10
15
0
20
10
20
40
Θ
(b) 0.004
(b)
0.003
Posterior probability
T
Posterior probability
30
0.002
0.012
0.008
0.004
0.001
0.000
0.000
0
2
4
6
8
10
0
5
Africa into the Caribbean Sea, likely during a period
when the cold Benguela Current off southwestern Africa
was relatively weak and the warm Agulhas Current off
southeastern Africa was relatively strong.
Assuming that pairwise estimates of tMRCA are a good
proxy for ancestral population age and that population
divergence occurs after gene flow ceases, we estimate
15
20
25
Θ
T
Fig. 3 Posterior probabilities for divergence time (T) among masked
boobies on either side of the Isthmus of Panama and the southern tip
of Africa. (a) Excluding secondary contact birds: pairwise comparisons between Caribbean vs. western Indian (open triangles) and
Caribbean vs. eastern Pacific (open circles); (b) Secondary contact
birds only: pairwise comparisons between Caribbean vs. western
Indian (open triangles) and Caribbean vs. eastern Pacific (open
circles).
10
Fig. 4 Posterior probabilities for theta (Q) among masked boobies on
either side of the Isthmus of Panama and the southern tip of Africa.
(a) Excluding secondary contact birds: pairwise comparisons
between Caribbean vs. western Indian (open triangles) and Caribbean vs. eastern Pacific (open circles); (b) Secondary contact birds
only: pairwise comparisons between Caribbean vs. western Indian
(open triangles) and Caribbean vs. eastern Pacific (open circles).
that the secondary contact event occurred within the last
c. 43 000 – 183 000 years. Although these dates are
approximate and should be confirmed using additional
unlinked loci (e.g. multiple single nucleotide polymorphisms; Brumfield et al., 2003), we suggest that secondary dispersal likely occurred during the transition from
the glacial conditions of marine isotope stage 6 and the
interglacial conditions of marine isotope substage 5e (i.e.
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
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T. E. STEEVES ET AL.
Table 2 Estimates of time since divergence (t) and time to most
recent common ancestor (tMRCA) among masked booby populations on either side of the Isthmus of Panama and the southern tip of
Africa.
Caribbean*
Caribbean Region
T (ybp)
tMRCA (ybp)
t (ybp)
tMRCA (ybp)
Western Indian
Eastern Pacific
444 672
479 024
595 984
641 413
42 606
133 120
182 600
197 120
*Excluding secondary contact birds.
Secondary contact birds only.
Termination II: c. 130 000 years ago). Multiple paleoindicators (i.e. foraminifera assemblages and foraminifera
oxygen isotopes) in sediment cores collected from the
Benguela Current (Pierre et al., 2001; Chen et al., 2002)
and from the Agulhas Current Retroflection (Flores et al.,
1999) indicate that during several major Pleistocene
deglaciation events, including Termination II, warm
water pulsed around the southern tip of Africa.
Initial divergence of masked boobies
We estimate that the initial divergence of extant masked
booby populations on either side of the Isthmus of
Panama and the southern tip of Africa occurred within
the last c. 479 000 years (Table 2). Steeves et al. (2003)
estimated that masked booby populations in the Caribbean Sea and the Pacific Ocean diverged within the last
c. 460 000 – 640 000 years, assuming a sulid-specific
divergence rate of 2.8%my)1 for cytochrome b or a
more typical avian rate of 2%my)1, respectively.
Although the latter estimates were derived using traditional population genetic methods (Wilson et al., 1985)
and reflect both gene and population divergence (i.e.
total divergence; Edwards & Beerli, 2000; Arbogast et al.,
2002), we attribute the similarity of our estimates and
those of Steeves et al. (2003) to the relatively young age
of ancestral masked booby populations (Table 2). Edwards & Beerli (2000) and Arbogast et al. (2002) cautioned that the ratio of gene divergence to population
divergence between recently diverged populations can be
extremely high, depending on the size of the ancestral
population. We add that this ratio will also depend on the
age of the ancestral population: if the ancestral population is relatively young, then population divergence may
contribute to a larger fraction of the total divergence
between two recently diverged populations.
Although masked boobies in the Caribbean Sea (excluding secondary contact birds) appear to have diverged
more recently from birds in the western Indian Ocean
than from birds in the eastern Pacific Ocean (Table 2),
overlapping credibility intervals for both T and Q (Figs 3a
and 4a) preclude the conclusion that the Isthmus of
Panama has never been breached by masked boobies. In
addition, because the paleoceanographic record for fora-
minifera is largely restricted to the last c. 500 000 years
(Flores et al., 1999; Pierre et al., 2001; Chen et al., 2002),
further speculation regarding the initial dispersal route of
masked boobies, either in or out of the Caribbean Sea,
c. 445 000 – 641 000 years ago (Table 2) is difficult.
Regardless, the Isthmus of Panama has clearly not been
breached by several species of highly mobile pantropical
seabirds in hundreds of thousands of years (Avise et al.,
2000; Steeves et al., 2003, this study). In contrast,
fluctuating oceanographic conditions around the southern tip of Africa likely enabled secondary dispersal by
masked boobies from the Indian Ocean into the Caribbean Sea within the last c. 183 000 years.
Acknowledgments
We gratefully acknowledge the following individuals and
organizations for their invaluable contributions to this
study: G. Rocamora for collecting tissue samples; US Fish
and Wildlife Service, US Coast Guard, and Puerto Rico
Department of Natural and Environmental Resources for
logistical support in Puerto Rico; Royal Society for the
Protection of Birds for logistical support on Ascension;
Island Conservation and Ecology Group, Grupo de
Ecologı́a y Conservación de Islas, and Spight Family
Foundation for logistical support in Mexico; T. Loxton,
N. Ratcliffe, and R. White for field assistance on Ascension;
M. Amin Ordoñez, A. Hebshi, and S. Hebshi for field
assistance in Mexico; T. Birt, S. Garner, O. Haddrath, and
R. Kristensen for laboratory assistance; J. Pillardy for
technical assistance regarding MDIV analyses; J. Brown
and R. Nielsen for helpful discussions regarding Bayesian
statistics; A. Mix and P. Werner for advice regarding
interpretation of paleoceanographic data, and T. Burg for
many helpful general discussions and comments on
earlier drafts of the manuscript. Funding was supplied by
American Museum of Natural History (Frank M. Chapman
Memorial Grant to TES), Natural Sciences and Engineering Research Council of Canada (Discovery Grants to VLF
and Post-Graduate Scholarship to TES), Premier’s
Research Excellence Award (to VLF), Queen’s University
(Graduate Dean’s Doctoral Field Travel Grants and
Pearl E. Williams and Llewellyn Hillis Grant to TES), and
Wake Forest University Sullivan Fund (to DJA).
Supplementary material
The following material is available from http://
www.blackwellpublishing.com/products/journals/suppmat/
JEB/JEB906/JEB906sm.htm
Table S1 Frequencies and variable sites among
52 masked booby control region haplotypes.
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Received 11 February 2004; revised 8 November 2004; accepted 20
January 2005
J. EVOL. BIOL. 18 (2005) 1000–1008 ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY