ORIGINALARTICLE

Colonizing the Caribbean: biogeographyand evolution of livebearing fishes ofthe genus Limia (Poeciliidae)Pablo F. Weaver1,2*, Alexander Cruz2, Steven Johnson1, Julia Dupin2 andKathleen F. Weaver1

1Department of Biology, University of La

Verne, 1950 3rd St., La Verne, CA 91750,

USA, 2Department of Ecology and

Evolutionary Biology, University of Colorado,

Boulder, CO 80309-0334, USA

*Correspondence: Pablo Weaver, BiologyDepartment, 1950 3rd St., La Verne, CA 91750,USA.E-mails: pweaver@laverne.edu;weaverp@colorado.edu

ABSTRACT

Aim We investigate the origin and colonization of the West Indian endemicfreshwater fish group Limia. We evaluate the leading hypotheses for the origins

of West Indian life, including trans-oceanic dispersal, late Cretaceous vicari-

ance, and the GAARlandia land bridge at the Eocene/Oligocene boundary.

Location Greater Antilles, with extensive sampling in the Dominican Republic.

Methods We obtained DNA from wild sampling and the aquarium trade. We

sequenced three mitochondrial (12S, ND2 and Cytb) and two nuclear genes

(Rh, MYH6) for a combined molecular phylogenetic analysis to evaluate spe-cies relationships and the timing of divergence events between islands and the

mainland. We used Bayesian and likelihood approaches to build phylogenies, a

BEAST analysis to establish the timing of colonization, and R package BioGeo-BEARS to perform a historical biogeographical reconstruction.

Results Relaxed molecular clock results show that the ancestor to the WestIndian clade, which includes the Limia and Hispaniolan Poecilia, diverged from

a South American ancestor at the Eocene/Oligocene boundary. The basal

Jamaican species, L. melanogaster, split from the rest of Limia at the Oligocene/Miocene boundary. Cuban and Cayman taxa are sister to a diverse species

group from Hispaniola. Historical biogeographical reconstruction supported

the GAARlandia DEC+j model as the best fitting model for colonization.

Main conclusions Our results support a colonization model for Limia that is

concordant with the timing of GAARlandia and climate change during theEocene/Oligocene boundary. Limia colonization was most likely a result of

facilitated dispersal during a period of lower sea levels and shorter passage

along the Aves Ridge. These results are also consistent with other recent molec-ular clock studies of dispersal limited cichlids, toads and frogs, indicating a

growing body of support for the significance of Eocene/Oligocene climate

change for the historical biogeography of West Indian life.

Keywordscolonization, GAARlandia, Hispaniola, historical biogeographical reconstruc-

tion, Limia, relaxed molecular clocks, vicariance, West Indian biogeography

INTRODUCTION

After nearly two centuries of debate, biogeographical recon-

struction in the West Indies continues to be a contentious

issue, with wide disagreement over the relative importance of

dispersal–vicariance. The islands of the West Indies exhibit

exceptional biodiversity and are ranked in the top five of the

world’s most important biodiversity hotspots, with endemism

reaching over 90% in some groups (Hedges, 1996; Myers et al.,

2000). However, the island ecosystems are relatively depauper-

ate and some major mainland groups, including marsupials,

carnivores, lagomorphs, salamanders and most families of

frogs, turtles, and snakes are missing (Hedges, 1996, 2001).

These patterns are typical of isolated oceanic islands, in which

a few successful dispersers radiate in situ. Molecular clock

analyses across taxa, including some reptiles, amphibians, and

ª 2016 John Wiley & Sons Ltd http://wileyonlinelibrary.com/journal/jbi 1doi:10.1111/jbi.12798

Journal of Biogeography (J. Biogeogr.) (2016)

mammals, show a pattern of random and recent divergences

from mainland sources (mostly from South America), concor-

dant with a dispersal model of colonization (see Hedges, 1996,

2001 for figure showcasing random colonization patterns).

The primary mechanism of dispersal for these groups is

thought to be a combination of large flooding events in South

America, floating mats of debris, and the prevailing north-wes-

terly oceanic currents (King, 1962; Vonhof et al., 1998;

Hedges, 2001; Glor et al., 2005). Dispersal continues in the

present day, as evidenced by a colony of iguanas (Iguana

iguana) arriving to the island of Anguilla from nearby Guada-

lupe in the wake of Hurricane Luis in 1995 (Lawrence, 1998).

Alternatively, the vicariance argument highlights the pres-

ence of organisms with poor dispersal abilities and relies on

the correlation between evolutionary patterns and geological

reconstructions. Advocates of West Indian vicariance bio-

geography envision entire ecosystems, trapped on drifting

island fragments, splitting from a proto-Central America in

the late Cretaceous (65 Ma) through plate tectonics (Rosen,

1975, 1985; Nelson & Platnick, 1981; Guyer & Savage, 1986;

Page & Lydeard, 1994). The presence of freshwater fish on

the islands of the West Indies has sparked much interest over

the years because of their diversity and endemism, as well as

their presumed limited dispersal abilities (Rosen & Bailey,

1963; Rosen, 1975; Rivas, 1986; Poeser, 2003), and they were

instrumental in the development of vicariance biogeography

as a discipline (Rosen, 1975). Previous taxonomic studies of

West Indian freshwater fish show that species on Hispaniola

and Cuba are polyphyletic; northern Hispaniolan species are

more closely related to eastern Cuban and Cayman island

species than they are to southern Hispaniolan species

(Rauchenberger, 1988; Burgess & Franz, 1989; Rodriguez,

1997; Hamilton, 2001). These patterns seemed concordant

with proposed geological models of island formation and a

late Cretaceous connection with the mainland (Pitman et al.,

1993; Pindell, 1994). However, the late Cretaceous vicariance

model has received much criticism, mainly because of a lack

of molecular clock support and the unlikely survival of life

in this region during and shortly after the late Cretaceous

bolide impact near the Yucatan peninsula (Hildebrand &

Boynton, 1990).

An alternative geological reconstruction that shows a more

recent land connection between the mainland and the West

Indies, is the GAARlandia model (Greater Antilles Aves

Ridge) (Iturralde-Vinent & MacPhee, 1999; Iturralde-Vinent,

2006). In this reconstruction, any late Cretaceous land for-

mations in the Caribbean would have been inundated by

subsequent sea level change, and permanent land was only

present after the Middle Eocene (< 40 Ma). During the

Eocene/Oligocene transition (30–35 Ma), a rapidly cooling

planet caused global declines in sea level upwards of 60 m

(Haq et al., 1987; Miller et al., 2008). Coupled with general

tectonic uplift in the region, this may have allowed for

potential overland colonization of the West Indies from

South America across the Aves Ridge (Iturralde-Vinent &

MacPhee, 1999; Iturralde-Vinent, 2006).

One of the groups for which colonization routes is still

unknown is Limia (family Poeciliidae), a genus of livebearing

freshwater fishes endemic to the islands of the West Indies

(Rauchenberger, 1988; Burgess & Franz, 1989; Rodriguez,

1997; Hamilton, 2001). With 17 described endemic species

on Hispaniola and one endemic species on each of the

islands of Cuba, Jamaica and Grand Cayman, Limia is the

dominant freshwater fish group in the Greater Antilles (Bur-

gess & Franz, 1989). However, the historical biogeography of

Limia has not been critically assessed using molecular tech-

niques and the timing and mechanism of colonization for

Limia remains unresolved. Our hope is that by reconstruct-

ing the colonization of Limia, we will gain a vital piece in

the ever-evolving debate on the origins of West Indian life

and gain insight into other organisms with limited long-dis-

tance dispersal abilities.

Freshwater fish in the Limia genus provide an ideal oppor-

tunity to evaluate colonization hypotheses because of their

diversity and presence on multiple islands. Using Limia spe-

cies from Hispaniola (Dominican Republic and Haiti),

Jamaica, Cuba, as well as poeciliid species from the main-

land, evaluation of multiple divergence events is possible:

West Indies from the mainland (all Limia), Jamaica from

Cuba/Hispaniola (L. melanogaster split) and Cuba from His-

paniola (L. vittata split). We test the above colonization sce-

narios using a combined gene molecular phylogenetic

approach with a robust sampling of poeciliids from the

islands and the mainland, together with a relaxed molecular

clock analysis and historical biogeographical reconstruction.

The goal for this study was to test biogeographical hypothe-

ses of the West Indies through an updated multigene phy-

logeny of Limia. We examine three alternative scenarios, with

regard to Limia colonization. First, long-distance transoceanic

dispersal models, which predict colonization across the Carib-

bean sea by salt tolerant taxa from diverse mainland sources,

with subsequent evolution of the fresh-water clades (Myers,

1966; Briggs, 1984, 1987; Hedges, 1996, 2001, 2006). Second,

late Cretaceous vicariance models, which predict colonization

during the late Cretaceous (65–85 Ma) and persistence of

biota on incipient island fragments (Rosen, 1975, 1985; Nelson

& Platnick, 1981; Guyer & Savage, 1986; Rauchenberger, 1988;

Page & Lydeard, 1994). Finally, the GAARlandia model, which

postulates inundation of the incipient island fragments after

the late Cretaceous and until the Middle Eocene (< 40 Ma),

with range expansion of South American groups along a short-

lived, exposed Aves Ridge, which was created by sea level

decline and Caribbean plate uplift during the Eocene/Oligo-

cene boundary (33–35 Ma) (Iturralde-Vinent & MacPhee,

1999; Iturralde-Vinent, 2006).

METHODS

Sampling

For an analysis of Limia diversity within the West Indies, we

sampled poeciliids from across the region, including wild

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P. F. Weaver et al.

collections and species we could obtain through the aquar-

ium trade. Wild collections were focused on the island of

Hispaniola to sample from the greatest diversity of poeciliids

and to ensure that each of the major clades of Limia was

included. A total of 30 Dominican populations were sampled

using seine and dip nets during trips in 2000, 2003, 2004,

2010, 2012 and 2013 (Fig. 1, see Appendix S1 in Supporting

Information). All appropriate permits for collection and

transport were obtained through local wildlife agencies. In

addition, we included the Haitian species (L. nigrofasciata),

the Jamaican species (L. melanogaster), the Cuban species

(L. vittata) and the Grand Cayman species (L. caymanensis),

which were obtained through aquarium stock at the Univer-

sity of Colorado (see Appendix S1). All specimens were pre-

served in 80% ethanol prior to DNA extraction, in

accordance with IACUC approval at the University of La

Verne. DNA sequences from additional outgroup taxa from

the mainland genera of Pamphorichthys, Micropoecilia, Poecil-

ia and Xiphophorus were obtained through Genbank (see

Appendix S1).

Molecular methods

Genomic DNA was extracted from ethanol-preserved caudal

peduncle muscle tissue following the manufacturer’s protocol

for the DNeasy Kit (Qiagen Inc., Valencia, CA, USA). To

provide resolution at both the shallower and deeper

divergence nodes, we amplified both mitochondrial and

nuclear loci (Hrbek et al., 2007). Five gene fragments, three

mitochondrial (12S, ND2 and Cytb) and two nuclear (Rh,

MYH6), were amplified and sequenced for the 68 ingroup

individuals. Primer sequences for polymerase chain reaction

(PCR) are listed in Appendix S1.

Amplifications were performed in an Eppendorf Mastercy-

cler (Eppendorf, Westbury, NY, USA) under the thermal

conditions listed in Appendix S1. PCR products were puri-

fied using ExoSAP enzymes (USB Corp., Cleveland, OH,

USA). Purified PCR products were cycle sequenced using Big

Dye chemistry v.3.1 (Applied Biosystems Inc., Foster City,

CA, USA) and visualized on an ABI 377XL (Life Technolo-

gies, Grand Island, NY, USA). DNA sequences were edited

with Geneious Pro 5.6.6 and alignments were performed

with Clustal W (Thompson et al., 1997).

Phylogenetic analyses

Phylogenetic analyses were performed using both Bayesian

analyses (BA) and maximum likelihood (ML) methods. Sub-

stantial discussion has revolved around the contrasting

approaches of multigene concatenated data sets versus con-

sensus gene trees (see Gadagkar et al., 2005; Degnan &

Rosenberg, 2009). To evaluate the utility of combining the

(a)

(b)

Figure 1 Locality map for populations sampled for this study. Limia is endemic to the West Indies, with one species occurring on eachof the islands of Cuba, Grand Cayman and Jamaica; the majority of the species (possibly 17) are found on Hispaniola. We sampledheavily from the south-west and central Dominican Republic where biodiversity is high. Additional species were obtained from Cuba,Jamaica, and Grand Cayman thru aquarium stock.

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3

Biogeography of Limia (Poeciliidae)

nuclear and mitochondrial data sets, we ran preliminary

analyses on the mitochondrial and nuclear data sets indepen-

dently using Mr. Bayes 3.1.2 (Ronquist & Huelsenbeck,

2003). Doubts about the utility of incongruence length dif-

ference tests (Barker & Lutzoni, 2002; Darlu & Lecointre,

2002; Alonso et al., 2012) lead us to visually inspect the

mitochondrial and nuclear trees for congruence. The mito-

chondrial and nuclear trees were overall similar in their

topologies, with the mitochondrial tree showing better reso-

lution near the tips and the nuclear tree showing better reso-

lution at deeper nodes (see Appendix S2).

We used PartitionFinder (Lanfear et al., 2012) to evalu-

ate the appropriate nucleotide substitution models and parti-

tion scheme on our combined data set. PartitionFinder

allowed us to evaluate various partition schemes, including

the fragment of 12S rDNA and each codon position indepen-

dently in all of the protein coding genes (ND2, Cytb, Rh and

MYH6). The optimal partition scheme for the data, as out-

lined by PartitionFinder, was a 7-way partition strategy

with the following models: subset 1 = K80+I for 12S and

MYH6 pos 2, subset 2 = GTR+I+G for Cytb pos 1, ND2

pos 1, subset 3 = HKY+G for Cytb pos 2 and ND2 pos 2,

subset 4 = HKY + I for Cytb pos 3, ND2 pos 3 and Rh

pos 1, subset 5 = JC+I for Rh pos 2, subset 6 = F81 for Rh

pos 3, subset 7 = HKY+I for MYH6 pos 1 and MYH6 pos 3.

Bayesian analyses were then performed on the combined

data set, using the seven partition scheme provided by Par-

titionFinder. The Markov chain Monte Carlo (MCMC)

simulation was performed utilizing two independent runs,

using eight chains and 10 million generations, sampling

every 1000 generations. Convergence and stability of the runs

was detected using Tracer 1.5. (Rambaut & Drummond,

2007). The first 25% of each run was established as burn-in

and discarded. The remaining trees were used to compute a

50% majority rule consensus tree. Maximum likelihood trees

were built using PhyML (Guindon & Gascuel, 2003) as

implemented in Geneious Pro 5.6.6 and paup 4.0 (Swofford,

2003). Support for nodes for both methods was established

with 1000 bootstrap replicates. For the PhyML analysis, we

employed a general GTR+I+G substitution model, optimizing

topology, length and rate, and used the BEST topology

search strategy. In the Paup analysis, we used the seven par-

tition scheme as outlined by PartitionFinder. Tree search-

ing was performed with a branch and bound strategy, using

the FURTHEST addition method and bootstrapping was per-

formed using a random seed and branch and bound search

type. Trees were rooted with the Central American livebear-

ing species Xiphophorus hellerii after Hrbek et al. (2007).

Divergence time estimation

We chose single representatives from each of the main lineages

(34 in total) to maximize coverage of available gene sequences

and calculated divergence time estimates using a relaxed-clock

method implemented in beast 1.7.4 (Drummond & Rambaut,

2007). The data set was partitioned with the same nucleotide

substitution model as the seven partition scheme we used for

the MrBayes analysis, except that base frequencies were esti-

mated for all partitions. We used the uncorrelated lognormal

distribution model on the partitioned data set to account for

lineage specific rate heterogeneity. We used the Yule speciation

process as the tree prior. Because no fossil records were avail-

able for our taxa, we calibrated the tree based on the opening

of the Windward Passage, which split Cuba from Hispaniola

(L. vittata from the Hispaniolan Limia clade) 14–17 Ma (Itur-

ralde-Vinent, 2006). Additional analyses were run using an

alternative uniform prior distribution of 20–25 Ma for the

Windward Passage (Pitman et al., 1993) to evaluate the influ-

ence of this parameter (see Appendix S2). The estimation of

14–17 Ma is supported by the geological reconstruction of

Iturralde-Vinent (2006) as well as independent molecular

clock estimates for toads (Alonso et al., 2012) and cichlids

(Chakrabarty, 2006; P!erez et al., 2007; "R!ı"can et al., 2012). This

calibration point has also been used successfully in examina-

tion of the Cuban Girardinus group (Doadrio et al., 2009), as

well as the Central American Poecilia sphenops (Alda et al.,

2013). The analyses were performed with two independent

runs for 30 million generations, with trees selected every

10,000 estimations. We checked for convergence diagnostics

with Tracer 1.5 (Rambaut & Drummond, 2007) and com-

bined runs using Log-Combiner 1.7 (part of the beast pack-

age). The data were summarized using TreeAnnotator 1.7.4.

(part of the beast package) and visualized using FigTree

1.4.2. (Rambaut, 2009).

Biogeographical analyses

To investigate the historical biogeography of Limia, ancestral

ranges were estimated using the R package BioGeoBEARS

(Matzke, 2013); this allowed direct tests of the fit of dispersal

and GAARlandia models. For all of the hypotheses listed, we

applied the Dispersal-Extinction-Cladogenesis (DEC; Ree &

Smith, 2008) model and its modified version, DEC+j, whichallows for founder event speciation. Additionally, analyses

included a time-stratified approach that represents different

dispersal rates across time slices (see Appendix S3). Finally,

the relative likelihood of all resulting models were compared

using Akaike’s information criterion (AIC; Burnham &

Anderson, 2002). The best model was then used to estimate

the biogeographical history of Limia.

On the basis of the dated chronogram for Limia, we

pruned the tree to remove outgroups, but kept the sister

group comprised of the Hispaniolan Poecilia species, as well

as the closest mainland relative Pamphorichthys. We defined

five geographical areas occupied by Limia and its closest rela-

tives, including South America (SA), Hispaniola (HI),

Jamaica (JA), Cuba (CU) and Cayman Islands (CA)

(Table 1). We evaluated the likelihood of a dispersal model

versus a GAARlandia model of colonization, using time slices

that correspond with the proposed colonization pathway

applied to each model (see Appendix S3). We did not test

the late Cretaceous vicariance hypothesis, as the timing was

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4

P. F. Weaver et al.

not supported by the divergence time analysis. For the dis-

persal model, our probability matrices reflect no land con-

nection between the Greater Antilles and South America and

the emergence of Jamaica during the mid-Miocene (after

Robinson, 1994). For the GAARlandia model, time slices

reflect the reconstructions of Iturralde-Vinent & MacPhee

(1999) and Iturralde-Vinent (2006) and include facilitated

dispersal via a land connection between South America and

the landmass that included the proto-island fragments of

Cuba, Hispaniola and the Jamaican Blue Mountain terranes

from 35 to 30 Ma, followed by flooding and separation of

the Greater Antilles from the mainland from 30 Ma to the

present. Both models assume the opening of the Windward

Passage separating Eastern Cuba from Hispaniola at 15 Ma

(Iturralde-Vinent, 2006), as well as the formation of the Cay-

man Islands during late Miocene uplift, with a short-lived

connection to Cuba from 5 to 3 Ma (Jones, 1994).

RESULTS

Phylogenetic analysis

Our combined and concatenated mitochondrial (12S, ND2,

Cytb) and nuclear (Rh, MYH6) data sets yielded 2661 charac-

ters (1133 mitochondrial, 1528 nuclear). The resulting gene

sequences are available in GenBank, accession #s KX023907-

KX024245. The 7-way partitioned data set, which included

both nuclear and mitochondrial data, provided good resolu-

tion at both the shallower and deeper nodes of our ingroup

taxa with Bayesian posterior probabilities (PP) of 0.90–1.0 andmaximum likelihood bootstrap (MLB) support of 80–100%(Fig. 2, Appendix S2). Overall tree topologies of the Bayesian,

as well as ML reconstructions from both PhyML and PAUP

were similar at all nodes. Support for nodes was very similar

across ML methods. A discussion of Limia evolution and spe-

cies relationships within the genus are included in

Appendix S2.

Divergence time estimation

The West Indies clade of Limia and Poecilia is shown to split

from the South American Pamphorichthys group during the

Eocene/Oligocene transition approximately 32.9 Ma (95%

highest posterior densities (HPD) = 21.6–48.8) (Fig. 3, node

A). The Jamaican species L. melanogaster split from other

Limia species near the Oligocene/Miocene transition 22.8 Ma

(95% HPD = 17.2–32.0) (Fig. 3, node B). The tree was cali-

brated by the splitting of Cuban L. vittata and Grand Cay-

man L. caymanensis from the Hispaniolan Limia clade

(Fig. 3, node C), which corresponds with the opening of the

Windward Passage and separation of the proto-island land-

masses of eastern Cuba and northern Hispaniola 14–17 Ma.

The Grand Cayman species, L. caymanensis split from its sis-

ter taxon, the Cuban L. vittata at 3.64 Ma (95%

HPD = 1.28–7.85) (Fig. 3, node D). The large clade of His-

paniolan lineages, which include several putative L. perugiae

populations (see Appendix S2 for further discussion), as well

as a sympatric population of L. sulphurophila from La Zurza,

and several unidentified groups, is shown to be a recent

diversification event at 1.76 Ma (95% HPD = 0.69–3.10)(Fig. 3, node E). The correlation of our molecular clock

analyses with the GAARlandia model is shown in Fig. 4. The

timing of the West Indies clade divergence (A, Fig. 4), the

isolation of Blue Mountains terrane of Jamaica from south-

ern Hispaniola (B, Fig. 4) and the opening of the Windward

Passage separating eastern Cuba from Hispaniola (C, Fig. 4)

correlate with estimates from the Beast analysis.

Biogeographical analyses

The AIC model comparison (Table 2) supported the

GAARlandia DEC+j model as the best fitting model. This

model was 4.8 AIC units lower than the second best model,

no-GAARlandia DEC. This result shows that a model that

includes a land connection between South America and the

proto-island fragments during 35 to 30 Ma is better fitted for

the clade comprised of Limia, Poecilia and Pamphorichthys

than a model where the colonization of the Caribbean islands

happened only through dispersal. Moreover, the model with

the founder event parameter (j) shows a significant lower like-

lihood score than its simpler version, which suggests that range

expansions alone are not sufficient to explain movements to

new areas. For example, the results show that one jump disper-

sal (or, founder event) is estimated to explain the current

range of L. caymanensis (Fig. 5).

DISCUSSION

Biogeography of Limia

West Indian fauna likely colonized the islands during many

events and through multiple mechanisms. Here, we show

evidence that an ancestor to the West Indies clade of live-

bearing freshwater fishes (including all Limia and the

Table 1 Current species distributions used to reconstruct thebiogeographical history of Limia in the West Indies. The speciesincluded in this analysis were: Pamphorichthys hollandi, from theSouth America Biogeographic region, Limia dominicensis, Limiaperugiae, Limia nigrofasciata, Limia zonata, Limia versicolor,Poecilia elegans, Poecilia dominicensis and Poecilia hispaniolanafrom Hispaniola, Limia melanogaster from Jamaica, Limia vittatafrom Cuba and Limia caymanensis from Grand Cayman.

Biogeographicalregion Species occurrences

South America (SA) Pamphorichthys hollandiHispaniola (HI) L. dominicensis, L. perugiae, L. nigrofasciata,

L. zonata, L. versicolor, Poecilia elegans,Poecilia dominicensis, Poecilia hispaniolana

Jamaica (JA) L. melanogasterCuba (CU) L. vittata

Grand Cayman (CA) L. caymanensis

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Biogeography of Limia (Poeciliidae)

Figure 2 Phylogenetic reconstruction based on a Bayesian analysis of a combined data set for 88 individuals and five genes (threemitochondrial: 12S, ND2, Cytb and two nuclear: MYH6, Rh) with seven data partitions. Numbers above nodes represent BayesianPosterior Probabilities and numbers below nodes represent maximum likelihood bootstrap estimates from ML analysis. A version of thisphylogeny with labelled terminal nodes is available in Appendix S2.

Journal of Biogeographyª 2016 John Wiley & Sons Ltd

6

P. F. Weaver et al.

Hispaniolan Poecilia) diverged from mainland taxa at the

Eocene/Oligocene boundary (32.9 Ma) (Fig. 3). The timing

of the split between the West Indian clade and the mainland

precludes a late Cretaceous vicariant origin or recent transo-

ceanic dispersal. In addition, our historical biogeographical

reconstruction showed the most likely model for colonization

is a GAARlandia model, including the colonization of Jamai-

can and Cuban Limia species.

The GAARlandia model predicts a short-lived (~3 Myr)

subaerial land connection between South America and the

incipient landmasses of the West Indies across the Aves

Ridge during the Eocene/Oligocene transition (30–35 Ma)

(Iturralde-Vinent & MacPhee, 1999; Iturralde-Vinent, 2006).

This time frame marks an important transitional period in

the evolution of life on Earth, characterized by global climate

cooling and related changes in sea level. Transitioning from

the warm climate of the Eocene, the Earth experienced rapid

cooling of over 5 °C from the Eocene optimum to the start

of the Oligocene, accompanied by the formation of polar ice

caps and substantial sea level drops on the order of 60 m

(Haq et al., 1987; Miller et al., 2008). Supplemented by tec-

tonic uplift of the Caribbean plate, these conditions may

have resulted in either shallow banks or a land bridge con-

nection between proto-Antillean islands and the South

American mainland (Iturralde-Vinent & MacPhee, 1999;

Iturralde-Vinent, 2006). During this time frame, much of the

Greater Antilles existed as a single landmass, with connec-

tions between eastern Cuba, north and south Hispaniola, the

Blue Mountains of Jamaica, Puerto Rico, and the Aves Ridge

south to mainland South America. The paleogeographical

model of Iturralde-Vinent & MacPhee (1999) and Iturralde-

Vinent (2006) also predicts that after the lowest sea levels of

the Oligocene, the Aves Ridge land span was subsequently

inundated by rising sea levels before the Miocene (23.5 Ma),

severing the mainland connection and isolating several island

fragments, including the Blue Mountains of Jamaica (Fig. 4).

Further movement of Greater Antillean island fragments

acted to separate eastern Cuba from northern Hispaniola,

Figure 3 Chronogram of Limia and related groups derived from a relaxed molecular clock analysis of a combined data set (threemitochondrial: 12S, ND2, Cytb, and two nuclear: MYH6, Rh). Bars indicate the 95% highest posterior densities (HPD). Branch lengthsrepresent time since divergence and major nodes are indicated by lettered circles. Nodes A, B, and C represent significant splittingevents during the colonization of the West Indian livebearers and correspond with the geological timeline as illustrated in Fig. 4.

Journal of Biogeographyª 2016 John Wiley & Sons Ltd

7

Biogeography of Limia (Poeciliidae)

opening the Windward Passage in the mid-Miocene

(14–17 Ma).

The results of our analyses follow rather closely several

events predicted by the GAARlandia model. Our best model

of colonization supports the split of the West Indian/South

American clade during the Eocene/Oligocene (32.9 Ma). We

also demonstrate support for the separation of Jamaica near

the early Miocene (22.8 Ma). Divergence of the Jamaican

L. melanogaster corresponds well with the proposed timing

of rising sea levels and the separation of the Blue Mountain

block of Jamaica from southern Hispaniola (Iturralde-Vinent

& MacPhee, 1999; Fig 4). This result is interesting in itself,

as many palaeogeographical models debate the independence

of the eastern Blue Mountain block from a large western

block (part of the Nicaraguan rise) (see Lewis et al., 1990;

Robinson, 1994). In addition, orthodox views of Jamaican

geology find little evidence for persistence of any aerial

Jamaican terranes prior to the mid-Miocene (Robinson,

1965, 1994). Other studies evaluating the relationships

between Jamaican and Hispaniolan taxa may help to solidify

this pattern and better explain the unique geological history

of the Jamaican terranes.

Implications and future directions

While our divergence timing results are concordant with the

predictions of the GAARlandia model, we do not believe

there is sufficient evidence to rule out alternative methods of

colonization, such as a combination of dispersal and vicari-

ant events, or short distance, stepping stone dispersal. As dis-

cussed by Ali (2012), there is little geological evidence to

support the notion of a contiguous land bridge from South

America into the West Indies during the Eocene/Oligocene

transition. Furthermore, rather than crossing a land bridge,

any of the taxa used for support of the GAARlandia model

could have also used short distance dispersal to colonize the

West Indies. In the case of West Indian freshwater fish fauna,

the ability of poeciliids and cichlids to tolerate, and even

thrive, in saltwater has been widely demonstrated both on

the islands and in mainland taxa (Myers, 1938; Burgess &

Franz, 1989). Other taxa that may have used the GAARlandia

land span for colonization, including megalonychid sloths

and other mammals, were also physically capable of crossing

over a saltwater barrier, especially with the potentially shorter

distances between landmasses that would have occurred dur-

ing the Eocene/Oligocene transition. Coupled with the

absence of less tolerant groups on the islands, including pri-

mary division freshwater fish and caecilians (Hedges, 1996,

2001), it is difficult to rule out ancient dispersal, as argued

by Heinicke et al. (2007) and their divergence time estimate

for West Indian Eleutherodactylus lineage at 29–47 Ma. The

alternative, in which fish were confined to the freshwater

connections of a GAARlandia land span seems less likely and

would not have restricted the colonization of primary divi-

sion freshwater fishes.

Whether GAARlandia was a true land bridge or not, the

predictions of the GAARlandia model as a corridor for dis-

persal should not be discounted. The main historical critique

of the GAARlandia model was the absence of a clustering of

divergence time estimates around 30 to 35 Ma. Until very

recently, DNA sequencing and molecular clock techniques

did not allow for fine scale resolution of divergence time

(a)

(b)

(c)

Figure 4 Inferred colonization by the West Indies (WI) clade(represented by Limia and the Hispaniolan Poecilia). Mostrecent common ancestor (MRCA) estimates correspond with keynodes from the molecular clock analysis in Fig. 3. Divergenceevents are in concordance with a three-part model of Caribbeanpalaeogeography presented by Iturralde-Vinent & MacPhee(1999) and Iturralde-Vinent (2006). (a) The WI clade divergedfrom the South American Pamphorichthys group and reachedthe islands via the GAARlandia land span. (b) Rising sea levelsseparated southern Hispaniola from the Blue Mountains terraneof Jamaica, isolating the Jamaican species L. melanogaster. (c)Opening of the Windward Passage separated eastern Cuba fromthe Hispaniolan species.

Journal of Biogeographyª 2016 John Wiley & Sons Ltd

8

P. F. Weaver et al.

estimates. A poor fossil record and lack of reliable calibration

points lead to further inconsistency. As such, those estimates

that were available were often crude approximations relying

on percentage sequence divergence and seemed to show a

scattering of divergence events through time (Hedges, 2001).

However, many of these interpretations have changed with

recent phylogenetic work utilizing a combined mitochondrial

and nuclear DNA approach, in combination with broader

sampling, more accurate calibration and relaxed molecular

clock methods (see Hedges, 1996 and the updated study,

Heinicke et al., 2007).

Many recent re-evaluations of West Indian taxa give esti-

mates for colonization that are now consistent with Eocene/

Oligocene colonization, including cichlids (Hulsey et al.,

2011; "R!ı"can et al., 2012), bufonids of the Peltophryne genus

(Alonso et al., 2012), some frogs, for example, Syrrhophus

(Crawford & Smith, 2005) and Osteopilus (Moen & Wiens,

2009), Polistinae wasps (Silva & Noll, 2015), and dispersal

limited spiders (Crews & Gillespie, 2010) and butterflies

(Wahlberg, 2006). Additional earlier work using fossil and

phylogenetic evidence also support GAARlandia colonization,

including extinct primates (Horovitz & MacPhee, 1999;

Davalos, 2004) and megalonychid sloths (Macphee & Itur-

ralde-Vinent, 2000; White & MacPhee, 2001; Davalos, 2004),

several bat groups (Davalos, 2004), as well as hystricognath

rodents (Woods, 2001; MacPhee et al., 2003; Davalos, 2004;

but see Fabre et al. (2014) advocating mid-Miocene disper-

sal). Even without concrete evidence for a subaerial land

span, it seems clear that the time frame of the Eocene/Oligo-

cene transition and its associated climate and

Table 2 Biogeographical reconstruction inferred in BIOGEOBEARS and the relative probabilities of each model calculated from theAIC weights. Models include dispersal-extinction-cladogenesis (DEC) for both GAARlandia reconstruction and the overwater dispersalmodels, as well as complementary models that allow for founder event speciation (+J).

Model LnL No. of parameters d e j AIC D AIC AIC_weight Relative LL

GAARlandia DEC !15.1 2 0.0097 1E-12 0 34.297 6.574 0.032 0.037GAARlandia DEC+J !10.9 3 1E-12 1E-12 0.5534 27.723 0 0.862 1

No-GAARlandia DEC !15.3 2 1E-12 1E-12 0 34.605 6.881 0.028 0.032No-GAARlandia DEC+J !13.3 3 2.01E-10 2.1E-11 0.0751 32.514 4.791 0.079 0.091

LnL, log-likelihood; d, rate of dispersal; e, rate of extinction; j, relative probability of founder event speciation at cladogenesis; AIC, Akaike’sinformation criterion; D AIC, AIC-min(AIC); AIC weight, Normalized relative model likelihood; Relative LL, Model relative likelihood.

Figure 5 Maximum likelihood, ancestralrange estimations of historical biogeographyof Limia under the best model, GAARlandiaDEC+j. Geographical codes: SA: SouthAmerica, HI: Hispaniola. JA: Jamaica,CU: Cuba, CA: Grand Cayman.

Journal of Biogeographyª 2016 John Wiley & Sons Ltd

9

Biogeography of Limia (Poeciliidae)

palaeogeography have played a major part in West Indian

colonization and in shaping the remarkable present-day bio-

diversity.

A note on the taxonomy of Limia

As expected, we find support for the monophyly of Limia as a

group. Interestingly, however, we also show the closest sister

group of Limia to be the clade represented by the three His-

paniolan Poecilia species. Earlier studies have demonstrated

the sister group relationship between Limia and the Pam-

phorichthys group (Hamilton, 2001; Hrbek et al., 2007; Mered-

ith et al., 2010, 2011) but did not include the Hispaniolan

Poecilia species (P. elegans, P. dominicensis and P. hispan-

iolana) in their analyses. One consequence of this new phylo-

genetic arrangement is to further add to the confusion in the

taxonomic designations of Poecilia. Several studies have advo-

cated generic rankings for the taxa Poecilia, Limia, Mollienesia,

Pamphorichthys, Acanthophacelus and Micropoecilia (Rodri-

guez, 1997; Hamilton, 2001; Poeser, 2003), while others use

them as subgenera within the genus Poecilia (Meredith et al.,

2010, 2011). In either case, the designation of the Hispaniolan

Poecilia (P. elegans, P. dominicensis and P. hispaniolana) and

South American Poecilia (P. vivipara) as a group does not

accurately reflect evolutionary history and needs revision.

ACKNOWLEDGEMENTS

We give special thanks to colleagues at both the University

of La Verne and the University of Colorado for their input

and guidance. In particular, we thank Jerome Garcia,

Vanessa Morales, Roshan Gamage, Andrew Martin, David

Stock, Robert Guralnick and Dena Smith for the helpful sug-

gestions and support. We also thank our Dominican col-

leagues, Carlos Rodriguez, Arlen Marmolejo, Miguel

Landestoy and Marcos Rodriguez for assistance in the field

and in the lab, as well as the Ministerio de Medio Ambiente

y Recursos Naturales for permits. Additional thanks to the

referees of this manuscript for their constructive insight and

suggestions. Funding for this project was made possible by

the CU EBIO departmental graduate student grant, the CU

Museum Research grant and the ULV Faculty Research

grant.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Appendix S1 Additional details regarding localities and

protocols.

Appendix S2 Phylogenetic reconstructions and calibration

schemes.

Appendix S3 Matrices used in the historical biogeographi-

cal reconstructions.

BIOSKETCH

Pablo Weaver is a researcher and instructor at the Univer-

sity of La Verne in southern California and recently received

his doctoral degree from the Ecology and Evolutionary Biol-

ogy Department at the University of Colorado, Boulder. His

interests include ecology and evolution of livebearing fishes,

especially West Indian groups.

The main interests of all authors include evolution, phyloge-

netics and biogeography, with a focus on island systems.

Author contributions: P.W. and A.C. conceived of the study.

P.W., A.C., and S.J. collected specimens from the field, and

along with K.W., P.W. and S.J. conducted the gene sequenc-

ing and phylogenetic analyses. P.W. and S.J. led the writing

and provided the artwork. J.D. conducted the biogeographi-

cal analyses. A.C. and K.W. provided laboratory space and

financial assistance. All authors contributed to the writing of

the manuscript.

Editor: Liliana Katinas

Journal of Biogeographyª 2016 John Wiley & Sons Ltd

12

P. F. Weaver et al.

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