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ORIGINALARTICLE

Reconstructing the lionfish invasion:insights into Greater Caribbeanbiogeography

Ricardo Betancur-R.1,2*, Andrew Hines3, Arturo Acero P.2, Guillermo Ortı1,

Ami E. Wilbur3 and D. Wilson Freshwater3

1The George Washington University, 2023 G

Street NW suite 340, Washington, DC 20052,

USA, 2Universidad Nacional de Colombia sede

Caribe (CECIMAR), Cerro Punta Betın, Santa

Marta, Colombia, 3Center for Marine Science,

University of North Carolina Wilmington,

5600 Marvin Moss Lane, Wilmington, NC

28409, USA

*Correspondence: Ricardo Betancur-R.,

Department of Biological Sciences, The George

Washington University, 2023 G Street NW suite

340, Washington, DC 20052, USA.

E-mail: [email protected]

ABSTRACT

Aim Lionfish (Pterois volitans and P. miles) are popular ornamental fishes native

to the Indo-Pacific that were introduced into Florida waters and are rapidly

spreading and establishing throughout the Western Atlantic (WA). Although

unfortunate, this invasion provides an excellent system in which to test

hypotheses on conservation biology and marine biogeography. The goals of

this study are: (1) to document the geographical extent of P. volitans and P. miles;

(2) to determine whether the progression of the lionfish invasion is the result of

expansion following the initial introduction event or the consequence of multiple

introductions at various WA locations; and (3) to analyse the chronology of the

invasion in conjunction with the genetic data in order to provide real-time

assessments of hypotheses of marine biogeography.

Location The Greater Caribbean, including the US east coast, Bermuda, the

Bahamas and the Caribbean Sea.

Methods Mitochondrial control region sequences were obtained from lionfish

individuals collected from Bermuda and three Caribbean locations and analysed

in conjunction with previously published data from five native and two non-

native locations (US east coast and the Bahamas; a total of six WA locations).

Genetic variation within and among groups was quantified, and population

structure inferred via spatial analyses of molecular variance, pairwise FST, exact

tests, Mantel tests and haplotype networks.

Results Mitochondrial DNA screening of WA lionfish shows that while P. miles

is restricted to the northernmost locations (Bermuda and the US east coast),

P. volitans is ubiquitous and much more abundant. Invasive populations of

P. miles and P. volitans have significantly lower levels of genetic diversity relative

to their native counterparts, confirming that their introduction resulted in a

strong founder effect. Despite the relative genetic homogeneity across the six WA

locations, population structure analyses of P. volitans indicate significant

differentiation between the northern (US east coast, the Bahamas and

Bermuda) and the Caribbean populations.

Main conclusions Our findings suggest that the ubiquity of WA lionfish is the

result of dispersal from a single source of introduction in Florida and not of

multiple independent introductions across the range. In addition, the progression

of the lionfish invasion (as documented from sightings), integrated with the

genetic evidence, provides support for five of six major scenarios of connectivity

and phylogeographical breaks previously inferred for Caribbean organisms.

Keywords

Biological invasions, Greater Caribbean biogeography, lionfish, Pterois miles,

Pterois volitans, Western Atlantic.

Journal of Biogeography (J. Biogeogr.) (2011) 38, 1281–1293

ª 2011 Blackwell Publishing Ltd http://wileyonlinelibrary.com/journal/jbi 1281doi:10.1111/j.1365-2699.2011.02496.x

INTRODUCTION

Biological invasions have dramatic effects on biodiversity and

alter the balance of natural habitats (Mack et al., 2000). Yet

they also provide, from an evolutionary standpoint, excellent

natural experiments for studying the genetic factors and

ecological forces that shape colonization and expansion of

invasive species into new habitats. Lionfish (Pterois volitans

Linnaeus and Pterois miles Bennett) are popular ornamental

fishes native to the Indo-Pacific that were introduced into

Florida waters as a result of aquarium releases (Courtenay,

1995; Whitfield et al., 2002; Semmens et al., 2004). Lionfish

are the first non-native marine fishes to become established in

the Western Atlantic (WA), and their populations are

expanding rapidly through the warm-temperate north-west

Atlantic and the Caribbean Sea (Whitfield et al., 2007;

Schofield, 2009). The US Geological Survey Nonindigenous

Aquatic Species database (USGS-NAS; http://nas.er.usgs.gov/)

has recently documented the chronology and extent of the

lionfish invasion based on confirmed occurrences (Schofield,

2009; Fig. 1). A fisherman reported the first specimen off

Dania, Florida, in 1985 (Morris & Akins, 2009). Six specimens

may have escaped Florida aquaria during Hurricane Andrew in

1992 (Courtenay, 1995); however, W. Courtenay has expressed

reservations over the accuracy of this report (see Discussion).

By 2001, lionfish populations were established along the US

Atlantic coast from Florida to North Carolina, and juvenile

specimens had also been reported as far north as Rhode Island.

By this time, lionfish had also established populations in

Bermuda (Whitfield et al., 2002). They were detected and

subsequently became established in the Bahamas between 2004

and 2006, and have been spreading rapidly into the Caribbean

Sea since 2007 (Ruiz-Carus et al., 2006; Whitfield et al., 2007;

Schofield, 2009). The southernmost records to date are from

the southern Caribbean Sea (Costa Rica to Venezuela), with

hundreds of sightings documented just a few months after the

first lionfish were reported in May 2009 off the coast of South

America (Gonzalez et al., 2009; Lasso-Alcala & Posada, 2010).

Lionfish are voracious predators that feed on post-settlement

reef fishes, causing severe but poorly understood impacts on

native coral reef diversity (Albins & Hixon, 2008). Although

eradication of invasive lionfish is virtually impossible, control

strategies need to be developed and implemented (Morris

et al., 2011).

This unfortunate biological invasion provides an opportu-

nity for addressing questions within the fields of conservation

biology and marine biogeography. For instance, despite recent

efforts to examine patterns of genetic diversity in invaders, very

little is known about genetic changes that may occur in general

over the course of an invasion (Dlugosch & Parker, 2008).

Mitochondrial DNA (mtDNA) analyses of lionfish samples

collected along the US east coast and the Bahamas during

earlier stages of the invasion have shown a strong founder

effect that resulted in a significant decrease in the genetic

diversity of the introduced population relative to two native

populations (Philippines and Western Indonesia; Hamner

et al., 2007; Freshwater et al., 2009b). Previous studies also

revealed that two lionfish species have been introduced into

the WA, but that P. volitans is over an order of magnitude

more abundant than P. miles (Hamner et al., 2007; Freshwater

et al., 2009a). Furthermore, Western Indonesia is the likely

source population for the invasive P. volitans in the WA, and

the Bahamian P. volitans population may have originated by

larval dispersal from the United States east coast population

(Freshwater et al., 2009b). These previous studies, however,

were restricted to two locations sampled at earlier stages of the

invasion, and no genetic data are available that document the

current extent of the lionfish invasion in the WA.

The lionfish invasion also provides an excellent study system

for assessing marine connectivity and dispersal patterns. A

long-standing question in marine biology is whether Carib-

bean organisms are genetically homogeneous or geographically

segregated. While a few marine barriers have been proposed

within the Greater Caribbean Sea (Carlin et al., 2003; Baums

et al., 2005; Cowen et al., 2006; Taylor & Hellberg, 2003,

2006), empirical studies addressing this question directly are

scarce. Biophysical models have been developed to identify

connectivity patterns and potential barriers and to assess

spatial scales for larval dispersal among reef fish populations

(Schultz & Cowen, 1994; Paris & Cowen, 2004; Cowen et al.,

2006). Likewise, few genetic assessments of diverse marine taxa

provide evidence for both connectivity and phylogeographical

breaks in the Greater Caribbean (e.g. Avise, 1992; Carlin et al.,

2003; Taylor & Hellberg, 2003; Baums et al., 2005; Taylor &

Hellberg, 2006; see Fig. 2). Some of the scenarios proposed by

these studies include: (A) connectivity between the US east

coast and Bermuda via the Gulf Stream (Schultz & Cowen,

1994; Hare et al., 2002); (B) a barrier to gene flow between the

US east coast and the Bahamas owing to the fast northerly flow

of the Gulf Stream (Carlin et al., 2003; Freshwater et al.,

2009b); (C) a break between the US east coast and the Gulf of

Mexico as a result of ecological differences reflecting palaeo-

climatic factors (Avise, 1992; Palumbi, 1994; Soltis et al., 2006;

Mobley et al., 2010); (D) biogeographical discontinuity

between the Caribbean and the Bahamas, the Turks, and

Caicos islands, with minor exchange with northern Cuba and

Hispaniola (Cowen et al., 2006); (E) a north-western Carib-

bean barrier, isolating the Mesoamerican reef area, southern

Cuba, and Cayman Islands from the rest of the Caribbean

(Cowen et al., 2006; Salas et al., 2010); and (F) the eastern

Caribbean break bounded at the Mona passage between Puerto

Rico and Hispaniola in the north and around La Guajira

(Colombia) in the south, a region influenced by the outflow of

the Magdalena river as well as by strong seasonal upwelling and

offshore currents (Taylor & Hellberg, 2003; Baums et al., 2005;

Cowen et al., 2006; Taylor & Hellberg, 2006; Betancur-R. et al.,

2010; see Fig. 2).

Among marine fishes, lionfish have significant dispersal

capabilities as a result of their reproductive strategy. They

release buoyant gelatinous egg masses that facilitate planktonic

dispersal, with an average settlement age of 26 days (Imamura

& Yabe, 1996; Hare & Walsh, 2007; Arenholz & Morris, 2010).

R. Betancur-R. et al.

1282 Journal of Biogeography 38, 1281–1293ª 2011 Blackwell Publishing Ltd

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

N

CaribbeanSea

Gulf of Mexico

Figure 1 Chronological occurrences of lionfish (Pterois volitans and P. miles) in the Western Atlantic as of December 2010 (see also

Schofield, 2009). Reproduced with permission from P. Schofield (USGS-NAS); update for 2010 provided by A. Benson (USGS-NAS).

Reconstructing the lionfish invasion

Journal of Biogeography 38, 1281–1293 1283ª 2011 Blackwell Publishing Ltd

In addition, invasive lionfish spawn year-round, at a frequency

of every 4 days, with an annual fecundity of over 2 million

eggs (Morris & Whitfield, 2009). These characteristics, coupled

with recurrent dispersal via oceanic currents, may explain their

establishment and rapid range expansion throughout the

Caribbean in less than a decade. Alternatively, multiple

independent introductions across locations in the WA could

account for their broad distribution. Integrating the chronol-

ogy of lionfish invasion based on sightings with population

genetic data can provide real-time assessment of dispersal and

connectivity across the Greater Caribbean as well as validations

for model predictions.

Here, we expand upon previous genetic studies of invasive

lionfish along the US east coast and the Bahamas by adding

data from Bermuda and three Caribbean locations, to cover a

wider range of the WA invasion. We examine mtDNA control

region sequences for three main purposes. First, we aim to

establish the geographical extent of the invasion for both

P. volitans and P. miles using DNA taxonomy and phylo-

geography. While both species are well differentiated at the

mitochondrial level (Freshwater et al., 2009a), morphological

differentiation is subtle, with some degree of overlap in

diagnostic features (Schultz, 1986), making it difficult to

discriminate current lionfish reports taxonomically. Second,

we aim to determine whether the ubiquity of lionfish in the

WA is (1) the result of range expansion from the original

location of introduction (i.e. the US east coast), a scenario

consistent with the chronology of the occurrences (Fig. 1); or

(2) the result of multiple independent introductions at various

locations throughout the WA, potentially reflecting an increase

in genetic diversity. Finally, in the light of scenario 1, we aim to

analyse patterns of population structure in conjunction with

the geographical records of the lionfish invasion (Fig. 1) to test

hypotheses of connectivity and breaks for reef organisms in the

Greater Caribbean (Fig. 2).

MATERIALS AND METHODS

Sampling and laboratory protocols

Tissue samples and, whenever possible, voucher specimens

were collected from four locations in the north-western

Atlantic and the Caribbean, to cover a broad geographic range

of the lionfish invasion (Fig. 3a). Samples from Bermuda (BM)

and Grand Cayman (KY, north-western Caribbean) were

obtained through local collaborators during 2007 and 2008.

Samples from the San Andres Archipelago (SA, San Andres

and Old Providence Islands, western Caribbean off Nicaragua,

Colombian territory) and Santa Marta (SM, southern Carib-

bean, continental Colombia) were collected between June and

December 2009, soon after lionfish were first reported from

these locations in December 2008 and May 2009, respectively

Caribbean Sea

A

CB

D

EF

Gulf of Mexico The Bahamas

Bermuda

US east coast

Colombia-Panama gyre

Eastern Caribbean

Northwestern Caribbean

Figure 2 Major scenarios of connectivity

(white double arrow) and phylogeographical

breaks (white dashed lines) for Greater

Caribbean reef organisms. Grey lines depict

connectivity networks based on biophysical

models by Cowen et al. (2006; above 0.05

level of surviving proportion). Circles denote

sampling locations for invasive lionfish

(Pterois volitans and P. miles; see details in

Fig. 3). A, US east coast – Bermuda connec-

tion; B, Florida Peninsula – the Bahamas

break; C, US east coast – Gulf of Mexico

break; D, the Bahamas and the Turks and

Caicos – Caribbean break; E, Western

Caribbean break; F, Eastern Caribbean break

(see details in Table 4).

R. Betancur-R. et al.

1284 Journal of Biogeography 38, 1281–1293ª 2011 Blackwell Publishing Ltd

(Gonzalez et al., 2009; Schofield, 2009). Colombian samples

were obtained by scuba diving by R.B.R. and A.A.P. or

purchased locally from fishermen or diving centres. Voucher

specimens were deposited at the Museo de Historia Natural

Marina de Colombia (INVEMAR-PEC), Santa Marta, Colom-

bia. All tissue samples were preserved in 95% ethanol and

taken to the laboratory for DNA extraction and sequencing.

The newly collected sequences augment and complement

existing mtDNA datasets for P. miles (166 bp; GenBank

accession numbers AJ628896–AJ628938) and P. volitans

(680 bp; GenBank accession numbers FJ516407–FJ516454)

previously obtained from five native (Indian Ocean, Red Sea,

Gulf of Aqaba, Western Indonesia and Philippines) and two

WA [North Carolina (NC) and the Bahamas (BS)] locations by

Kochzius & Blohm (2005) and Freshwater et al. (2009b).

Molecular data were generated at The George Washington

University (GWU; SM and SA samples) and the University of

North Carolina Wilmington (UNCW; KY and BM samples).

The targeted gene region included c. 750 bp of the mtDNA

control region (d-loop). Nucleic acid extractions, primers used

for polymerase chain reaction (PCR) amplification (LionA-H:

5¢-CCA TCT TAA CAT CTT CAG TG-3¢; LionB-L: 5¢-CAT

ATC AAT ATG ATC TCA GTAC-3¢; L-PROF: 5¢-AAC TCT

CAC CCC TAG CTC CCA AAG-3¢) and PCR conditions were

as described in Freshwater et al. (2009b). The PCR amplicons

obtained at GWU were submitted for purification and

sequencing at High Throughput Sequencing Solutions, Uni-

versity of Washington, Seattle, Washington; UNCW samples

were sequenced as described in Freshwater et al. (2009b).

Contigs were made from forward and reverse sequences using

CodonCode Aligner 3.5.4 (CodonCode Corporation; GWU

samples) or Sequencher 4.9 (Gene Codes Corporation;

UNCW samples) and aligned by visual inspection.

Genetic variation and population structure

Genetic diversity measures (haplotype diversity, Nei, 1987),

nucleotide diversity (p, the average number of nucleotide

differences per site between two sequences, Nei, 1987) and

sequence diversity (j, the average number of nucleotide

differences between two sequences, Tajima, 1989) were

estimated in DnaSP 5 (Librado & Rozas, 2009). The assump-

tion of marker neutrality was tested using Tajima’s D (Tajima,

1989) as implemented in Arlequin 3.0 (Excoffier et al., 2005).

Population differentiation was examined using several

approaches available in Arlequin 3.0 (Excoffier et al., 2005).

12-45-10

11-25 42>200

H01 H05H02

H03

H08

H09

H04

H06

H07

PhilippinesW. Indonesia

BS

KY

SA

SM

NC

(b)(a)

Northern group

Caribbean group

BM

NCBM

Pterois miles

Pterois volitans

Figure 3 (a) Sampling locations for invasive lionfish (Pterois volitans and P. miles) in the Western Atlantic. Dashed ellipses delineate

approximate population groupings for P. volitans, as defined by structure analyses (Tables 2 and 3). Dark grey arrows depict oceanographic

currents. (b) Minimum spanning network (95% set of plausible cladograms) for P. volitans based on mtDNA d-loop sequences (680 bp).

Small connecting circles indicate internal nodes in the network that were not present in the sample (i.e. inferred intermediate haplotypes).

Western Atlantic haplotypes are labelled numerically following ‘H’ (see also Table 1). Pie charts represent relative frequencies of haplo-

types by location (colour-coded locations follow part a). The dashed line indicates the approximate separation of two major clusters [based on

the number of intermediate haplotypes and phylogenetic analyses (branches not to scale); see also fig. 3 in Freshwater et al., 2009b]. BM,

Bermuda; NC, North Carolina; BS, the Bahamas; KY, Grand Cayman; SA, San Andres Islands; SM, Santa Marta.

Reconstructing the lionfish invasion

Journal of Biogeography 38, 1281–1293 1285ª 2011 Blackwell Publishing Ltd

Tests of differentiation between populations were conducted

using pairwise FST statistics (based on haplotype frequencies

and molecular divergence) and exact tests (based on haplotype

frequency distributions only). An analysis of molecular vari-

ance (AMOVA, Excoffier et al., 1992) was performed using the

Tamura and Nei model of DNA sequence evolution with

among-site variation (TN+G; Gamma = 0.59), as selected by

the Akaike information criterion (AIC) in Modeltest 3.7

(Posada & Crandall, 1998). Given that a priori groupings in

AMOVA may be artificial, this study also conducted a spatial

analysis of molecular variance (SAMOVA) using samova 1.0,

which delineates groups of populations that are geographically

homogeneous and maximally differentiated (Dupanloup et al.,

2002). Approximate geographical coordinates for locations

were defined in Google Earth 5. One thousand annealing

processes were used as starting conditions for SAMOVA. To

assess the correct number of groups (K) for SAMOVA, the FCT

statistic from AMOVA(calculated a posteriori) was compared

for values of K ranging from 2 to 5 (Dupanloup et al., 2002).

To test for isolation by distance, the relationship between

genetic and geographical distances was analysed via Mantel

tests in Arlequin. Pairwise genetic distances based on FST

values were used as input for this analysis. These genetic

distances were tested against two geographical distance

matrixes estimated with Google Earth: shortest overwater

distances and distances following the path of Caribbean

currents. Statistical significance for pairwise FST statistics,

the exact test, AMOVA, the Mantel test and Tajima’s D was

assessed via 10,000 permutations in Arlequin (significance

level a = 0.05). Relationships among haplotypes were esti-

mated with a minimum spanning network under the parsi-

mony criterion in Arlequin. This analysis was conducted

under default settings, providing the 95% parsimoniously

plausible branch connections between haplotypes.

RESULTS

Native versus invasive populations of Pterois miles

and P. volitans

Mitochondrial control region sequences from 755 invasive

lionfish specimens were analysed in this study, producing a

final alignment length of 680 bp. Sequences corresponding to

P. miles were observed in only 21 individuals from two

locations: 17 of the 281 NC specimens (6.05%) and four of the

49 BM specimens (8.16%) carried P. miles d-loop sequences

(Fig. 3a; GenBank accession number FJ516408). A single

haplotype was observed among all these sequences, resulting

in genetic diversity indices for the invasive populations in the

WA equal to zero (see details in Table 1). In contrast, a study

by Kochzius & Blohm (2005) reported 38 haplotypes for

P. miles sampled from three localities in its native range.

The combined dataset for P. volitans, including the d-loop

sequences generated by Freshwater et al. (2009b), comprised

70 samples from native localities and 734 invasive individuals

from six WA locations (Table 1; Fig. 3a). Notably, no new

haplotypes were found among the 343 newly sequenced

individuals, preserving unchanged the previously reported

total of 36 native and nine invasive haplotypes. Likewise, no

haplotypes were shared between the native Indo-Pacific and

the invasive WA populations. When native and invasive

populations were analysed separately or combined into major

regions (total 12 units of comparison), negative values of

Tajima’s D were obtained in five calculations; the remaining

Table 1 Genetic diversity indices and tests of neutrality among native and non-native populations of Pterois volitans and P. miles.

Source n h HD p j Tajima’s D

P. volitans

North Carolina (NC)1,2 264 8 (H1–7, H9) 0.704 0.0038 2.54 0.717

Bermuda (BM)3 45 5 (H1–3, H6–7) 0.627 0.0030 2.02 )0.050

Bahamas (BS)2 127 8 (H1–8) 0.648 0.0033 2.21 )0.004

NC–BM–BS (northern) 436 9 (H1–9) 0.681 0.0035 2.40 0.739

Grand Cayman (KY)3 79 4 (H1–4) 0.432 0.0021 1.41 )0.588

San Andres Islands (SA)3 50 3 (H1–2, H4) 0.541 0.0029 1.92 0.613

Santa Marta (SM)3 169 3 (H1–2, H4) 0.524 0.0031 2.12 1.579

Caribbean (KY, SA, SM) 298 4 (H1–4) 0.504 0.0028 1.91 0.720

Invasive 734 9 (H1–9) 0.633 0.0033 2.27 0.769

Western Indonesia (ID)1,2 42 26 (H10–30, H32, H38, H40, H42–43) 0.962 0.0129 8.74 )0.930

Philippines (PH)1,2 28 12 (H18, H31, H33–39, H41, H44–45) 0.886 0.0150 10.17 )0.128

Native 70 36 (H10–H45) 0.965 0.0190 12.95 0.022

P. miles

Invasive 21 1 0.000 0.000 0.000 –

Native* 94 38 0.853 0.0199 3.30 –

n, number of sampled individuals; h, number of haplotypes (haplotypes observed given in parentheses; see also Fig. 3b); HD, haplotype diversity; p,

nucleotide diversity; j, sequence diversity; 1Hamner et al. (2007); 2Freshwater et al. (2009); 3this study.

*See Kochzius & Blohm (2005) for details.

R. Betancur-R. et al.

1286 Journal of Biogeography 38, 1281–1293ª 2011 Blackwell Publishing Ltd

seven had positive values (see details in Table 1). However,

permutation tests indicated no significant departure from

marker neutrality for any of the Tajima’s D estimates.

All haplotype diversity and nucleotide diversity values were

smaller in the six invasive populations (haplotype diversity,

HD = 0.50–0.70) than in the Western Indonesia and Philip-

pines populations of P. volitans (HD = 0.89–0.96; see details

in Table 1). A three-level AMOVA contrasting Indonesia,

Philippines and WA regions suggested that genetic variation

is partitioned mainly among regions (65.6%) rather than

among populations within regions (1.2%) or within popu-

lations (33.2%; Table 2). Fixation index values were signif-

icant in all AMOVA comparisons (P < 0.01; Table 2).

Likewise, pairwise FST statistics and exact tests indicated

significant differentiation among populations of these major

regions (Table 3).

Two main clusters were evident in the minimum spanning

network (Fig. 3a), coinciding with the geographical partition-

ing of genetic variation in WA and native populations of

P. volitans. One cluster includes haplotypes found in invasive

and Western Indonesian locations, and most connections

between haplotypes require the inclusion of only one to up to

five non-sampled intermediate haplotypes. The other cluster

includes haplotypes found mostly in Philippine specimens as

well as some Western Indonesian specimens, and many of the

inter-haplotype connections require a large number (up to

nine) of hypothetical intermediates. Likewise, a previous

phylogenetic assessment found a similar clustering scheme,

with haplotypes in the WA nested within the Western

Indonesian population and distantly related to the Philippine

haplotypes (see fig. 3 in Freshwater et al., 2009b).

Western Atlantic populations of Pterois volitans

D-loop sequences of P. volitans were found in the six WA

locations examined (Fig. 3a). Among the invasive populations,

genetic diversity values were highest in NC and BS (eight

haplotypes each), followed by BM (five haplotypes), SA (three

Table 2 Analysis of molecular variance (AMOVA) for native and non-native populations of Pterois volitans.

Source of variation d.f.

Sum of

squares

Variance

component

Percentage of

variation P-value

Native versus WA populations

Among regions: ID versus PH versus WA 2 378.75 2.84 65.59 0.000*

Among populations within regions: ID, PH, WA

(BM versus NC versus BS versus KY versus SA versus SM)

5 35.45 0.05 1.16 0.000*

Within populations: ID, PH, BM, NC, BS, KY, SA, SM 797 1147.57 1.44 33.25 0.000*

TOTAL 803 1561.77 4.34

WA populations only (based on SAMOVA partitions)

Among regions: northern versus Caribbean groups 1 28.41 0.07 6.21 0.000*

Among populations within regions: northern

(BM versus NC versus BS), Caribbean (KY versus SA versus SM)

4 7.04 0.01 0.53 0.100

Within populations: BM, NC, BS, KY, SA, SM 728 812.67 1.12 93.26 0.000*

TOTAL 733 848.11 1.20

d.f., degrees of freedom; BM, Bermuda; BS, the Bahamas; ID, Western Indonesia; KY, Grand Cayman; NC, North Carolina; PH, the Philippines; SA,

San Andres Islands; SM, Santa Marta; WA, Western Atlantic. *Significant P-values (<0.05).

Table 3 Pairwise differentiation tests for native and non-native populations of Pterois volitans. Pairwise FST comparisons (below the

diagonal) using Tamura & Nei distance and gamma correction (0.59), and P-values for exact tests of pairwise haplotype frequency

comparisons (above the diagonal).

NC BM BS KY SA SM ID PH

North Carolina (NC) – 0.468 ± 0.022 0.228 ± 0.037 0.000 ± 0.000* 0.068 ± 0.013 0.000 ± 0.000* 0.000 ± 0.000* 0.000 ± 0.000*

Bermuda (BM) 0.003 – 0.089 ± 0.010 0.004 ± 0.002* 0.038 ± 0.005* 0.000 ± 0.000* 0.000 ± 0.000* 0.000 ± 0.000*

Bahamas (BS) 0.001 0.015 _ 0.000 ± 0.000* 0.043 ± 0.011* 0.000 ± 0.000* 0.000 ± 0.000* 0.000 ± 0.000*

Grand Cayman (KY) 0.086* 0.059* 0.128* _ 0.160 ± 0.012 0.011 ± 0.007* 0.000 ± 0.000* 0.000 ± 0.000*

San Andres Islands (SA) 0.021* 0.003 0.041* 0.015 _ 0.283 ± 0.012 0.000 ± 0.000* 0.000 ± 0.000*

Santa Marta (SM) 0.057* 0.044* 0.090* 0.017 0.005 _ 0.000 ± 0.000* 0.000 ± 0.000*

Western Indonesia (ID) 0.263* 0.202* 0.247* 0.321* 0.233* 0.325* _ 0.000 ± 0.000*

Philippines (PH) 0.778* 0.691* 0.757* 0.771* 0.706* 0.788* 0.454* _

BM, Bermuda; BS, the Bahamas; ID, Western Indonesia; KY, Grand Cayman; NC, North Carolina; PH, the Philippines; SA, San Andres Islands; SM,

Santa Marta.

*Significant P-values (<0.05).

Reconstructing the lionfish invasion

Journal of Biogeography 38, 1281–1293 1287ª 2011 Blackwell Publishing Ltd

haplotypes), SM (three haplotypes) and KY (four haplotypes;

see details in Table 1). Two dominant haplotypes (H01 and

H02; see Fig. 3a and Table 1) were found in all invasive

populations (75.8–97.5%).

As suggested by simulation experiments on SAMOVA, the

largest value of FCT is probably associated with the correct

number of population groups (Dupanloup et al., 2002). The

largest value of FCT (0.62) was obtained at K = 2, with

progressively decreasing values as the parameter K increases

(K = 3, FCT = 0.59; K = 4, FCT = 0.55; K = 5, FCT = 0.52).

Thus, under K = 2, SAMOVA defined one group including the

northern locations (BM, BS, NC) and another including the

Caribbean sensu stricto locations (KY, SA and SM; Fig. 3a).

Genetic diversity values were considerably smaller in the

Caribbean (four haplotypes) than in the northern (nine

haplotypes) groupings (Table 1).

A hierarchical AMOVA (following SAMOVA partitions)

found the greatest genetic variation at the within-population

(93.3%) and among-regions (6.2%) levels, with significant

fixation indices for the two comparisons (Table 2). Roughly,

FST estimates and exact tests supported SAMOVA partitions,

indicating significant genetic structure between populations of

the northern and Caribbean regions, with no intraregional

differentiation (0.5%). Among the 30 possible pairwise com-

parisons for FST statistics and exact tests (15 each), there were

three exceptions to the abovementioned trend: no apparent

segregation between the Caribbean SA and the northern BM

and NC (based on FST and exact tests, respectively), and

significant differentiation between the Caribbean SM and KY

(based on exact tests) (Table 3). Given that these exceptions

are inconsistent between the two tests and that SA is

geographically disjunct in the sampling context and distant

from both BM and NC (i.e. KY is intermediate; see Fig. 3a), we

believe that failure to detect structure more probably repre-

sents lack of statistical power and has no actual biological

significance (i.e. continuous gene flow).

The Mantel test revealed no significant isolation by distance,

either for shortest overwater distance or for shortest distance

following Caribbean current paths (r2 = 0.057, P = 0.14; and

r2 = 0.07, P = 0.16, respectively; see Fig. 4). This is the

expected outcome in the presence of a single detected barrier

(see Discussion) restricting gene flow between northern and

Caribbean regions. The lack of correlation is more evident

when comparing genetic and geographical distances at inter-

regional versus intraregional levels. For instance, whereas

KY–BM (northern and Caribbean regions, respectively) and

KY–SM (Caribbean) location pairs have similar overwater

distances (c. 1450 km of current paths), their corresponding

pairwise FST values are roughly one order of magnitude

different (0.128 and 0.017, respectively).

DISCUSSION

The geographical extent of the Pterois miles

and P. volitans invasion

As shown in other studies on fish and animal groups with

cryptic morphology (e.g. Schlei et al., 2008; Vandersea et al.,

2008), mtDNA ‘barcoding’ provides a useful tool to discrim-

inate the current geographical range of invasive P. miles and

P. volitans. Although modal differences in dorsal- and anal-fin

counts between the two species are evident, the overlapping

meristic counts preclude unambiguous identifications (see

Tables 1 and 2 in Schultz, 1986). Notably, intermediate

morphotypes seem to be more common in the WA (Whitfield

et al., 2007), and thus reports from sightings are uncertain

regarding whether the two species are present at all locations

(Schofield, 2009; Fig. 1). Furthermore, while previous genetic

studies showed the presence of the two species in the WA

(Hamner et al., 2007; Freshwater et al., 2009b), their limited

geographical sampling (BS and NC locations only) prevents us

from distributional extrapolations at the current stage of the

invasion.

The mtDNA screening of 755 invasive lionfish from six

invasive locations indicates that P. miles has a restricted

distribution and is confined to the northernmost areas

0.00

0.02

0.04

0.06

0.08

0.10

0.14

0.12

0 500 1000 2000 2500 3000

ΦST

1500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000

(a) (b)

Overwater distance (km)Overwater distance (km)

ΦST

0.00

0.02

0.04

0.06

0.08

0.10

0.14

0.12

Figure 4 Relationship between geographical and genetic distances (based on pairwise FST values) for Western Atlantic populations of

Pterois volitans: (a) shortest marine distance (r2 = 0.057; n = 15; Mantel test P = 0.14); (b) shortest overwater distance following Caribbean

current paths (r2 = 0.07; n = 15; Mantel test P = 0.16).

R. Betancur-R. et al.

1288 Journal of Biogeography 38, 1281–1293ª 2011 Blackwell Publishing Ltd

sampled in this study (NC and BM), whereas P. volitans is

ubiquitous throughout the north-western Atlantic and the

Caribbean (Fig. 3a). The absence of P. miles in the Bahamas

and the Caribbean may indicate that the southern distribution

limit of this species along the US east coast is too far north or

that their population numbers are too low for them to have

overcome the barriers to dispersal across the Florida Strait.

Pterois volitans is also more abundant than P. miles

(P. volitans:P. miles = 35:1), even at co-occurring locations

(P. volitans:P. miles = 17:1 for NC and 12:1 for BM), and is

genetically more diverse (nine haplotypes in P. volitans versus

one in P. miles; see Table 1).

It is noteworthy that, given the ambiguous morphology

and the matrilineal properties of mtDNA, our study was

unable to detect possible instances of introgression among

non-related populations of the same species or between the

two lionfish species. Intraspecific hybridization among dis-

tantly related populations seems unlikely in the light of two

circumstances. (1) Phylogenetic analyses on the P. volitans

mtDNA sequences suggest that the source of WA populations

is Western Indonesia and all invasive haplotypes appear to be

closely related (Hamner et al., 2007; Freshwater et al., 2009b;

see Fig. 3b). Although none of the WA haplotypes were

found to occur in the native populations, implying that the

actual source population has not been sampled, a previous

study utilizing cytochrome b sequences obtained from the

same individuals as examined here revealed haplotype overlap

between Indonesia and the WA (Hamner et al., 2007). (2)

Only one mitochondrial haplotype characterizes the WA

P. miles populations. On the other hand, interspecific

hybridization between invasive P. miles and P. volitans

cannot be ruled out and may indeed provide an interpreta-

tion to the invasion success of lionfish (i.e. hybrid vigour).

Although this study examined no nuclear markers to test

genomic introgression, as noted above, the diagnostic mor-

phological characteristics for P. miles and P. volitans listed by

Schultz (1986) are equivocal for many WA specimens

(Whitfield et al., 2007; Freshwater et al., 2009a). To test

interspecific hybridization further, assignment tests on

nuclear markers are essential.

Genetic diversity, introduction events, and the myth

of Hurricane Andrew

As previously shown by analyses of mtDNA cytochrome b and

control region sequences obtained from lionfish at two non-

native (NC and BS) and two native (Western Indonesia and

Philippines; Hamner et al., 2007; Freshwater et al., 2009b)

locations, examination of a broader geographical range of the

current invasion in this study confirms a reduction of genetic

diversity in the WA lionfish associated with a strong founder

effect. While the population genetic study by Kochzius &

Blohm (2005) on P. miles from three native locations detected

38 haplotypes, only one haplotype was observed in the WA

populations. Likewise, measures of genetic variation among

P. volitans populations in the native range were substantially

greater than those for the WA populations (nine invasive

versus 36 native haplotypes; Table 1).

The observed patterns of mtDNA diversity suggest that the

continued expansion of lionfish (particularly P. volitans) in the

north-western Atlantic and the Caribbean is likely to be the

result of dispersal from the initial invading population.

Considering the origin of WA P. volitans, multiple indepen-

dent introductions of Indonesian lionfish across the WA at

various times (according to the chronology of reports; Fig. 1)

could alternatively explain their ubiquity. However, this is an

unlikely explanation given the high genetic variability observed

in the Western Indonesian population (24 observed haplotypes

plus multiple intermediate non-sampled haplotypes) and the

fact that two haplotypes dominate at the six WA locations

sampled (H01 and H02; see Fig. 3b and Table 1). Further-

more, as suggested by AMOVAs, most of the genetic variation

is partitioned among native and invasive regions (65.6%) or

within populations (33.2%), rather than among populations

within regions (1.2%). Thus, both the genetic homogeneity

across WA populations (but see below) and the observed

founder effect suggest that all P. volitans populations share a

common geographical origin of introduction (i.e. Florida).

With only one haplotype in NC and BM locations versus at

least 38 in the native range (Kochzius & Blohm, 2005; see

Table 1), similar conclusions can be drawn for P. miles.

There is a popular myth regarding the unconfirmed report

of six specimens that apparently escaped from a large

aquarium at Florida’s Biscayne Bay owing to Hurricane

Andrew in 1992 and suggesting that these escaped specimens

are the source of the catastrophic lionfish invasion (Courtenay,

1995). In a popular on-line article in ScienceInsider (http://

news.sciencemag.org/scienceinsider/2010/04/mystery-of-the-

lionfish-dont-bla.html) W. Courtenay has, however, recently

questioned the accuracy of such testimony. In addition, Morris

& Akins (2009) and Schofield (2009) argued that the first

documented lionfish report by a Lobster fisherman off Dania,

Florida, pre-dates Hurricane Andrew by seven years (specimen

preserved by the USGS-NAS, ID no. 261964). Further evidence

refuting the ‘hurricane myth’ comes from the mitochondrial

data. The minimum number of founding individuals required

to explain the observed genetic diversity in WA lionfish (nine

d-loop haplotypes in P. volitans plus one in P. miles) is

somewhere between eight and twelve (not six). The upper limit

of this estimate assumes that the origin of all mtDNA

haplotypes pre-dates that of the invasion, requiring ten

founding individuals of P. volitans (nine females and one

male) plus two of P. miles (one male and one female). On the

other hand, the lower limit implies that haplotypes H07, H08,

H05 and H09, the least abundant haplotypes and often placed

at the tips in the parsimony network (except for H08, which is

internal), originated in the WA population of P. volitans by a

single mutation from the otherwise more abundant and

internally placed H02, H04 and H01, respectively (Fig. 3b).

Whatever the actual number of founders, the most likely

vector of the WA lionfish invasion is probably multiple

aquarium releases of fish and possibly eggs in waters off Florida

Reconstructing the lionfish invasion

Journal of Biogeography 38, 1281–1293 1289ª 2011 Blackwell Publishing Ltd

(Whitfield et al., 2002; Semmens et al., 2004; Morris & Akins,

2009).

Population structure in invasive Pterois volitans

and real-time assessments of Greater Caribbean

connectivity

Despite the relative genetic homogeneity across the six

sampling locations and the marked founder effect observed

in WA P. volitans (see above), SAMOVA and pairwise FST

analyses as well as exact tests on haplotype frequencies revealed

a break between the northern (BM, NC and BS) and the

Caribbean (KY, SA and SM) locations (Tables 2 and 3;

Fig. 3a). While the four d-loop haplotypes observed in the

Caribbean are also present in the north-western Atlantic, there

is greater genetic diversity in the latter region (nine haplotypes;

see Table 1 and Fig. 3), suggesting a secondary founder effect

into the Caribbean. This may be explained by a decrease in

genetic diversity associated with dispersal out of the epicentre

of introduction in Florida, and it is also supported by the

progression of sightings. Whereas lionfish became frequently

observed along the US east coast and Bermuda by 2000 and in

the Bahamas by 2004, the first Caribbean reports date from

2007 (Schofield, 2009; Fig. 1), evidencing a temporal lag in

their arrival into the Caribbean (see below).

It would be interesting to determine if over time the

frequency of haplotypes in the Caribbean populations remains

different from that seen near the source of the founder event.

Notably, all Caribbean samples analysed here were obtained

soon after lionfish were first reported from the various

locations. Thus, it may be that this Caribbean sampling

reflects an initial dispersal wave, or that differential post-

settlement survival owing to stochastic events resulted in the

observed haplotype differences. Under this scenario, subse-

quent waves of dispersal from the north may lead to the

genetic homogenization of the WA populations. On the other

hand, no significant correlation between geographical and

genetic distance was evident, as only two population groups

were detected by structure analyses. Therefore, there is far

more genetic variation between adjacent locations on either

side of the inferred barrier (e.g. KY and BS) than among

distant locations within each population group (e.g. KY and

SM; see Fig. 4). As predicted by the stepping-stone model,

multiple semi-permeable barriers may limit gene flow to

adjacent demes, ultimately resulting in populations concom-

itantly isolated by distance (e.g. Ramachandran et al., 2005).

Table 4 Utilization of the lionfish (Pterois volitans and P. miles) progression (Fig. 1; Schofield, 2009) and genetic structure analyses (see text

and Table 3) as tests for proposed scenarios of Greater Caribbean connectivity and phylogeographical breaks for reef organisms (see details

in Fig. 2).

Code Scenario Reference Support from the chronology of progression Support from genetic structure

A US east coast – Bermuda

connection

1–2 Supported. Simultaneous arrival along the

US coast and Bermuda by 2000 via the Gulf

Stream. Also supported by P. miles invasion

(only present at these locations)

Supported. No genetic

differentiation*

B US east coast – the

Bahamas break

3–4 Supported. Temporal lag in the arrival in the

Bahamas in 2004, four years after widely

observed along the US east coast. Also

supported by the absence of P. miles from

the Bahamas

Not supported. No differentiation*

(although unique haplotypes

observed at both NC and BM

locations)

C US east coast – Gulf

of Mexico break

5–8 Supported. Temporal lag in the arrival in the

Gulf in 2006, six years after widely observed

along the US east coast. Also, currently

dispersing slowly into the Gulf

No data from locations in the Gulf of

Mexico

D The Bahamas, the

Turks, and

Caicos – Caribbean

break

10 Supported. Temporal lag in the arrival in the

Caribbean in 2007, three years after

reported from the Bahamas.

Supported. Significant differentiation

E North-western

Caribbean break

10–11 Not supported. Simultaneous arrival at

locations east and west of the

break in 2008

Not supported. No genetic

differentiation* between KY and

SA/SM locations (although KY

includes one haplotype absent from

SA and SM samples)

F Eastern Caribbean

break (Mona; Guajira)

10, 12–15 Supported. Slowly dispersing eastwards from

the break (Lesser Antilles)

No data from locations east of

the break

1Schultz & Cowen (1994); 2Hare et al. (2002); 3Freshwater et al. (2009b); 4Carlin et al. (2003); 5Avise (1992); 6Palumbi (1994); 7Soltis et al. (2006);8Mobley et al. (2010); 10Cowen et al. (2006); 11Salas et al. (2010); 12Taylor & Hellberg (2003); 13Taylor & Hellberg (2006); 14Baums et al. (2005);15Betancur-R. et al. (2010). *Lack of genetic differentiation may be a result of low variability of mitochondrial control region sequences.

BM, Bermuda; KY, Grand Cayman; NC, North Carolina, SA, San Andres Archipelago; SM, Santa Marta.

R. Betancur-R. et al.

1290 Journal of Biogeography 38, 1281–1293ª 2011 Blackwell Publishing Ltd

However, previous studies on reef organisms in the Caribbean

have shown no significant isolation by distance (Shulman &

Bermingham, 1995; Geertjes et al., 2004), even those utilizing

highly variable microsatellite markers (Hepburn et al., 2009;

Salas et al., 2010; but see Purcell et al., 2006). These results

suggest that the dispersal of P. volitans may not be following a

stepping-stone model. Alternatively, the low variation of the

mtDNA control region sequences examined here may be

preventing fine-scale inferences as well as the detection of

additional breaks. The future inclusion of timeline samples as

well as the utilization of more sensitive markers would provide

a framework to test these predictions.

When coupled with genetic tools, the chronological

progression of the P. volitans invasion (Schofield, 2009;

Fig. 1) provides a natural experiment with which to test

hypotheses on Greater Caribbean connectivity and phylogeo-

graphical breaks for reef organisms in real time (e.g. Galindo

et al., 2006). Table 4 summarizes the six major scenarios

inferred from previous studies (e.g. Avise, 1992; Carlin et al.,

2003; Taylor & Hellberg, 2003, 2006; Baums et al., 2005),

indicating those for which the lionfish progression and the

genetic analyses provide validation (see also Introduction and

Fig. 2).

Assuming that a temporal lag in the arrival at particular

locations provides evidence for breaks, and that simultaneous

reports at adjacent locations provide evidence for connectivity,

the chronology of the P. volitans progression (Fig. 1) generally

validates the predictions (Table 4; Fig. 2). In addition, the

presence of P. miles along the US east coast (NC) and Bermuda

but not in the Bahamas or the Caribbean provides support for

connectivity between the US east coast and Bermuda (scenario

A) and for a biogeographical break between the US east coast

and the Bahamas (scenario B). While our genetic assessment

confirms some of these conclusions, a lack of genetic sampling

at key locations (i.e. the Gulf of Mexico and Lesser Antilles)

precludes inferences on population structure for scenarios C

(break between the US east coast and the Gulf of Mexico) and

F (eastern Caribbean break). Furthermore, the lack of popu-

lation structure between locations in the Bahamas (BS) and

along the US east coast (NC; scenario B) may not necessarily

reflect continuous gene flow but rather failure of mtDNA

control region sequences to detect structure (although unique

haplotypes were observed at both locations; Freshwater et al.,

2009b). Given the simultaneous lionfish reports in 2008 at

Belize, the Cayman Islands, Jamaica and the San Andres

Archipelago (Schofield, 2009), as well as the lack of genetic

differentiation between KY and SA/SM locations, the north-

western Caribbean discontinuity (scenario D) inferred from

biophysical models (Cowen et al., 2006; Salas et al., 2010) is

the only hypothesis supported neither by the chronology of the

progression nor by the genetic assessments (although haplo-

type H03 observed at KY is absent from SA and SM samples;

Fig. 3). Moreover, the Colombia–Panama gyre subregion

(roughly bounded by breaks in scenarios E and F), previously

hypothesized as the most isolated Caribbean subregion

(Cowen et al., 2006; Salas et al., 2010), is probably not as

isolated as the eastern Caribbean (Fig. 2), as shown by the

slower lionfish progression into the Lesser Antilles (Fig. 1) and

Venezuela (Lasso-Alcala & Posada, 2010), as well as by patterns

of genetic structure in Acropora (Baums et al., 2005; Galindo

et al., 2006). Further studies addressing broader geographical

sampling of the lionfish invasion in conjunction with fine-scale

microsatellite markers from multiple loci would provide

stronger empirical evidence with which to assess model

predictions.

ACKNOWLEDGEMENTS

Grand Cayman and Bermuda samples were generously provided

by Bradley Johnson of the Cayman Islands Department of

Environment and Chris Flook of the Bermuda Aquarium and

Zoo, respectively. We thank Pamela Schofield and Amy Benson

(USGS-NAS) for providing the updated map for the lionfish

progression. Funding from Colombian agencies was granted by

Fundacion para la Promocion de la Investigacion y la Tec-

nologıa, Banco de la Republica (grant no. 2648), and

COLCIENCIAS (grant no. 1361-521-28271). This is contribu-

tion no. 344 of Centro de Estudios en Ciencias del Mar,

CECIMAR, Universidad Nacional de Colombia sede Caribe.

REFERENCES

Albins, M.A. & Hixon, M.A. (2008) Invasive Indo-Pacific

lionfish Pterois volitans reduce recruitment of Atlantic coral-

reef fishes. Marine Ecology Progress Series, 367, 233–238.

Arenholz, D.W. & Morris, J.A. (2010) Larval duration of the

lionfish, Pterois volitans along the Bahamian Archipelago.

Environmental Biology of Fishes, 88, 305–309.

Avise, J.C. (1992) Molecular population structure and bioge-

ographic history of a regional fauna – a case history with

lessons for conservative biology. Oikos, 63, 62–76.

Baums, I.B., Miller, M.W. & Hellberg, M.E. (2005) Regionally

isolated populations of an imperiled Caribbean coral,

Acropora palmata. Molecular Ecology, 14, 1377–1390.

Betancur-R., R., Acero P, A., Duque-Caro, H. & Santos, S.R.

(2010) Phylogenetic and morphologic analyses of a coastal

fish reveals a marine biogeographic break of terrestrial origin

in the southern Caribbean. PLoS ONE, 5, e11566.

Carlin, J.L., Robertson, D.R. & Bowen, B.W. (2003) Ancient

divergences and recent connections in two tropical

Atlantic reef fishes Epinephelus adscensionis and Rypticus

saponaceous (Percoidei: Serranidae). Marine Biology, 143,

1057–1069.

Courtenay, W.R. (1995) Marine fish introductions in south-

eastern Florida. American Fisheries Society Introduced Fish

Section Newsletter, 1995, 2–3.

Cowen, R.K., Paris, C.B. & Srinivasan, A. (2006) Scaling of

connectivity in marine populations. Science, 311, 522–527.

Dlugosch, K.M. & Parker, I.M. (2008) Founding events in

species invasions: genetic variation, adaptive evolution, and

the role of multiple introductions. Molecular Ecology, 17,

431–449.

Reconstructing the lionfish invasion

Journal of Biogeography 38, 1281–1293 1291ª 2011 Blackwell Publishing Ltd

Dupanloup, I., Schneider, S. & Excoffier, L. (2002) A simulated

annealing approach to define the genetic structure of pop-

ulations. Molecular Ecology, 11, 2571–2581.

Excoffier, L., Smouse, P.E. & Quattro, J.M. (1992) Analysis of

molecular variance inferred from metric distances among

DNA haplotypes: application to human mitochondrial DNA

restriction data. Genetics, 131, 479–491.

Excoffier, L., Laval, G. & Schneider, S. (2005) Arlequin (ver-

sion 3.0): an integrated software package for population

genetics data analysis. Evolutionary Bioinformatics Online,

2005, 47–50.

Freshwater, D.W., Hamner, R.M., Parham, S. & Wilbur, A.E.

(2009a) Molecular evidence that the lionfishes Pterois miles

and Pterois volitans are distinct species. Journal of the North

Carolina Academy of Science, 125, 39–46.

Freshwater, D.W., Hines, A., Parham, S., Wilbur, A.E.,

Sabaoun, M., Woodhead, J., Akins, J.L., Purdy, B.,

Whitfield, P.E. & Paris, C.B. (2009b) Mitochondrial con-

trol region sequence analyses indicate dispersal from the

US East Coast as the source of the invasive Indo-Pacific

lionfish Pterois volitans in the Bahamas. Marine Biology,

156, 1213–1221.

Galindo, H.M., Olson, D.B. & Palumbi, S.R. (2006) Seascape

genetics: a coupled oceanographic-genetic model predicts

population structure of Caribbean corals. Current Biology,

16, 1622–1626.

Geertjes, G.J., Postema, J., Kamping, A., van Delden, W.,

Videler, J.J. & van de Zande, L. (2004) Allozymes and

RAPDs detect little genetic population substructuring in the

Caribbean stoplight parrotfish Sparisoma viride. Marine

Ecology Progress Series, 279, 225–235.

Gonzalez, J., Grijalba-Bendeck, M., Acero P, A. & Betancur-R.,

R. (2009) The invasive Indo-Pacific lionfish, Pterois volitans/

miles complex, in the southern Caribbean Sea. Aquatic

Invasions, 4, 507–510.

Hamner, R.M., Freshwater, D.W. & Whitfield, P.E. (2007)

Mitochondrial cytochrome b analysis reveals two invasive

lionfish species with strong founder effects in the western

Atlantic. Journal of Fish Biology, 71, 214–222.

Hare, J.A. & Walsh, H.J. (2007) Planktonic linkages among

marine protected areas on the south Florida and southeast

United States continental shelves. Canadian Journal of

Fisheries and Aquatic Sciences, 64, 1234–1247.

Hare, J.A., Churchill, J.H., Cowen, R.K., Berger, T.J., Cornil-

lon, P.C., Dragos, P., Glenn, S.M., Govoni, J.J. & Lee, T.N.

(2002) Routes and rates of larval fish transport from the

southeast to the northeast United States continental shelf.

Limnology and Oceanography, 47, 1774–1789.

Hepburn, R.I., Sale, P.F., Dixon, B. & Heath, D.D. (2009)

Genetic structure of juvenile cohorts of bicolor damselfish

(Stegastes partitus) along the Mesoamerican barrier reef:

chaos through time. Coral Reefs, 28, 277–288.

Imamura, H. & Yabe, M. (1996) Larval record of a red firefish,

Pterois volitans, from northwestern Australia (Pisces: Scor-

paeniformes). Bulletin of the Faculty of Fisheries Hokkaido

University, 47, 41–46.

Kochzius, M. & Blohm, D. (2005) Genetic population struc-

ture of the lionfish Pterois miles (Scorpaenidae, Pteroinae) in

the Gulf of Aqaba and northern Red Sea. Gene, 347, 295–

301.

Lasso-Alcala, O. & Posada, J.M. (2010) Presence of the invasive

red lionfish, Pterois volitans (Linnaeus, 1758), on the coast of

Venezuela, southeastern Caribbean Sea. Aquatic Invasions, 5,

S53–S59.

Librado, P. & Rozas, J. (2009) DnaSP v5: a software for

comprehensive analysis of DNA polymorphism data. Bio-

informatics, 25, 1451–1452.

Mack, R.N., Simberloff, D., Lonsdale, W.M., Evans, H., Clout,

M. & Bazzaz, F.A. (2000) Biotic invasions: causes, epide-

miology, global consequences, and control. Ecological

Applications, 10, 689–710.

Mobley, K.B., Small, C.M., Jue, N.K. & Jones, A.G. (2010)

Population structure of the dusky pipefish (Syngnathus

floridae) from the Atlantic and Gulf of Mexico, as revealed

by mitochondrial DNA and microsatellite analyses. Journal

of Biogeography, 37, 1363–1377.

Morris, J.A. & Akins, J.L. (2009) Feeding ecology of invasive

lionfish (Pterois volitans) in the Bahamian archipelago.

Environmental Biology of Fishes, 86, 389–398.

Morris, J.A. & Whitfield, A.K. (2009) Biology, ecology, control

and management of the invasive Indo-Pacific lionfish: an

updated integrated assessment. NOAA Technical Memoran-

dum NOS NCCOS, No. 99. NOAA, Beaufort, NC.

Morris, J.A., Shertzer, K.W. & Rice, J.A. (2011) A stage-based

matrix population model of invasive lionfish with implica-

tions for control. Biological Invasions, 13, 7–12.

Nei, M. (1987) Molecular evolutionary genetics. Columbia

University Press, New York.

Palumbi, S.R. (1994) Genetic divergence, reproductive isola-

tion, and marine speciation. Annual Review of Ecology and

Systematics, 25, 547–572.

Paris, C.B. & Cowen, R.K. (2004) Direct evidence of a bio-

physical retention mechanism for coral reef fish larvae.

Limnology and Oceanography, 49, 1964–1979.

Posada, D. & Crandall, K.A. (1998) MODELTEST: testing

the model of DNA substitution. Bioinformatics, 14, 817–

818.

Purcell, J.F.H., Cowen, R.K., Hughes, C.R. & Williams, D.A.

(2006) Weak genetic structure indicates strong dispersal

limits: a tale of two coral reef fish. Proceedings of the Royal

Society B: Biological Sciences, 273, 1483–1490.

Ramachandran, S., Deshpande, O., Roseman, C.C., Rosenberg,

N.A., Feldman, M.W. & Cavalli-Sforza, L.L. (2005) Support

from the relationship of genetic and geographic distance in

human populations for a serial founder effect originating in

Africa. Proceedings of the National Academy of Sciences USA,

102, 15942–15947.

Ruiz-Carus, R., Matheson, R.E., Roberts, D.E. & Whitfield,

P.E. (2006) The western Pacific red lionfish, Pterois volitans

(Scorpaenidae), in Florida: evidence for reproduction and

parasitism in the first exotic marine fish established in state

waters. Biological Conservation, 128, 384–390.

R. Betancur-R. et al.

1292 Journal of Biogeography 38, 1281–1293ª 2011 Blackwell Publishing Ltd

Salas, E., Molina-Urena, H., Walter, R.P. & Heath, D.D. (2010)

Local and regional genetic connectivity in a Caribbean coral

reef fish. Marine Biology, 157, 437–445.

Schlei, O.L., Crete-Lafreniere, A., Whiteley, A.R., Brown, R.J.,

Olsen, J.B., Bernatchez, L. & Wenburg, J.K. (2008) DNA

barcoding of eight North American coregonine species.

Molecular Ecology Resources, 8, 1212–1218.

Schofield, P.J. (2009) Geographic extent and chronology of the

invasion of non-native lionfish (Pterois volitans [Linnaeus

1758] and P. miles [Bennett 1828]) in the Western North

Atlantic and Caribbean Sea. Aquatic Invasions, 4, 443–449.

Schultz, E.T. (1986) Pterois volitans and Pterois miles: two valid

species. Copeia, 3, 686–690.

Schultz, E.T. & Cowen, R.K. (1994) Recruitment of coral-reef

fishes to Bermuda – local retention or long-distance trans-

port. Marine Ecology Progress Series, 109, 15–28.

Semmens, B.X., Buhle, E.R., Salomon, A.K. & Pattengill-

Semmens, C.V. (2004) A hotspot of non-native marine

fishes: evidence for the aquarium trade as an invasion

pathway. Marine Ecology Progress Series, 266, 239–244.

Shulman, M.J. & Bermingham, E. (1995) Early life histories,

ocean currents and the population genetics of Caribbean

reef fishes. Evolution, 49, 897–910.

Soltis, D.E., Morris, A.B., McLachlan, J.S., Manos, P.S. & Soltis,

P.S. (2006) Comparative phylogeography of unglaciated

eastern North America. Molecular Ecology, 15, 4261–4293.

Tajima, F. (1989) Statistical method for testing the neutral

mutation hypothesis by DNA polymorphism. Genetics, 123,

585–595.

Taylor, M.S. & Hellberg, M.E. (2003) Genetic evidence for

local retention of pelagic larvae in a Caribbean reef fish.

Science, 299, 107–109.

Taylor, M.S. & Hellberg, M.E. (2006) Comparative phylo-

geography in a genus of coral reef fishes: biogeographic and

genetic concordance in the Caribbean. Molecular Ecology,

15, 695–707.

Vandersea, M.W., Litaker, R.W., Marancik, K.E., Hare, J.A.,

Walsh, H.J., Lem, S., West, M.A., Wyanski, D.M., Labani,

E.H. & Tester, P.A. (2008) Identification of larval sea basses

(Centropristis spp.) using ribosomal DNA-specific molecular

assays. Fishery Bulletin, 106, 183–193.

Whitfield, P.E., Gardner, T., Vives, S.P., Gilligan, M.R.,

Courtenay, W.R., Ray, G.C. & Hare, J.A. (2002) Biological

invasion of the Indo-Pacific lionfish Pterois volitans along

the Atlantic coast of North America. Marine Ecology Progress

Series, 235, 289–297.

Whitfield, P.E., Hare, J.A., David, A.W., Harter, S.L., Munoz,

R.C. & Addison, C.M. (2007) Abundance estimates of the

Indo-Pacific lionfish Pterois volitans/miles complex in the

Western North Atlantic. Biological Invasions, 9, 53–64.

BIOSKETCH

Ricardo Betancur-R. is a postdoctoral researcher at The

George Washington University. This study is part of his

ongoing research on the Western Atlantic lionfish invasion.

His research interests are broadly concerned with the system-

atics and evolutionary history of fishes, with particular

emphasis on Caribbean fish biogeography (see also http://

www.fishphylogeny.org).

Authors contributions: R.B.R., D.W.F. and A.A.P. conceived

the study; R.B.R. and A.H. collected the data; R.B.R. and

A.E.W. analysed the data; D.W.F. and G.O. provided signif-

icant input for the analyses and Discussion; R.B.R. drafted the

manuscript; all authors contributed to the writing and

approved the final version of the manuscript.

Editor: Craig McClain

Reconstructing the lionfish invasion

Journal of Biogeography 38, 1281–1293 1293ª 2011 Blackwell Publishing Ltd