UNLV Theses, Dissertations, Professional Papers, and Capstones

12-15-2019

The Effect of Pleistocene Glacial - Interglacial Cycles on Patterns The Effect of Pleistocene Glacial - Interglacial Cycles on Patterns

of Genetic Diversity and Differentiation of Populations of of Genetic Diversity and Differentiation of Populations of

Leptodactylus Albilabris (White-Lipped Frog) in the Puerto Rican Leptodactylus Albilabris (White-Lipped Frog) in the Puerto Rican

Bank Bank

Andre Nguyen

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Repository Citation Repository Citation Nguyen, Andre, "The Effect of Pleistocene Glacial - Interglacial Cycles on Patterns of Genetic Diversity and Differentiation of Populations of Leptodactylus Albilabris (White-Lipped Frog) in the Puerto Rican Bank" (2019). UNLV Theses, Dissertations, Professional Papers, and Capstones. 3830. http://dx.doi.org/10.34917/18608737

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THE EFFECT OF PLEISTOCENE GLACIAL - INTERGLACIAL CYCLES ON PATTERNS

OF GENETIC DIVERSITY AND DIFFERENTIATION OF POPULATIONS OF

LEPTODACTYLUS ALBILABRIS (WHITE-LIPPED FROG)

IN THE PUERTO RICAN BANK

By

Andre Nguyen

Bachelor of Science – Biological Sciences San Diego State University

2015

A thesis submitted in partial fulfillment of the requirements for the

Master of Science – Biological Sciences

School of Life Sciences College of Sciences

The Graduate College

University of Nevada, Las Vegas December 2019

ii

Thesis Approval

The Graduate College The University of Nevada, Las Vegas

October 02, 2019

This thesis prepared by

Andre Nguyen

entitled

The Effect of Pleistocene Glacial - Interglacial Cycles on Patterns of Genetic Diversity and Differentiation of Populations of Leptodactylus Albilabris (White-Lipped Frog) in the Puerto Rican Bank

is approved in partial fulfillment of the requirements for the degree of

Master of Science – Biological Sciences School of Life Sciences

Javier Rodriguez, Ph.D. Kathryn Hausbeck Korgan, Ph.D. Examination Committee Chair Graduate College Dean Dennis Bazylinski, Ph.D. Examination Committee Member Mira Han, Ph.D. Examination Committee Member Tereza Jezkova, Ph.D. Examination Committee Member Matthew Lachniet, Ph.D. Graduate College Faculty Representative

iii

Abstract

The historical distributional shifts of various species have been attributed to the glacial-

interglacial cycles of the Pleistocene Epoch (2.6 mya – 10,000 ya). Sea level changes caused by

Pleistocene glacial-interglacial cycles impacted the total area of landmasses, particularly in

island systems. Cooler, glacial periods increased emergent (subaerial) island area via lower sea

levels. Adjacent islands may have become connected through emergent land bridges, resulting in

a larger subaerial landmass. Warmer interglacial periods led to higher sea levels, which in turn

reduced island area, and in some cases, submerged land bridges, fragmenting larger islands into

separate, smaller units. The Puerto Rican Bank (PRB) is an island system in the eastern

Caribbean Sea consisting of more than 180 islands, islets, and cays. The largest islands of the

PRB are Puerto Rico, Vieques, Culebra, Saint Thomas, Saint John, Jost Van Dyke, Tortola,

Virgin Gorda, and Anegada (Saint Thomas, Saint John, and Saint Croix form part of the United

States Virgin Islands, but Saint Croix lays in its own bank, and has not had a land connection to

the PRB.) Changing sea levels influenced the degree of land exposure and connectivity across

the PRB. Lower sea levels revealed the shelf, and connected all the present-day islands of the

PRB into a single landmass. Conversely, higher sea levels submerged the shelf, fragmenting the

PRB into numerous islands, as in its current configuration. The repeated episodes of island

connectivity and isolation of the PRB may have left a genetic imprint on native species. I relied

on mitochondrial DNA and nuclear genomic sequences to assess whether historic sea levels

associated with Pleistocene glacial-interglacial cycles have shaped patterns of present-day

genetic diversity in Leptodactylus albilabris (White-lipped Frog) in the PRB. I tested four

specific hypotheses. (i) The boundaries of the five physiographic regions of Puerto Rico present

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a physical or ecological barrier to migration among L. albilabris from the different regions.

(ii) There is a positive correlation between geographic and genetic distance, or “isolation by

distance” (IBD), among populations of L. albilabris across the PRB. (iii) Puerto Rico is the

ancestral area of L. albilabris. (iv) Because Saint Croix has not had a land connection to the

PRB, the origin of the Saint Croix populations of L. albilabris is either the result of natural

colonization or human-mediated introduction, or of in situ evolution of the species on Saint

Croix. My findings are partially supported, nuclear genomic analyses revealed distinct genetic

groups corresponding to the Cordillera Central and the rest of Puerto Rico. I did not detect an

association between geographic and genetic distance among populations of L. albilabris across

the PRB, which suggests that this frog does not exhibit IBD across its distribution. Genetic

diversity and heterozygosity estimates from mitochondrial and nuclear genomic DNA data sets

suggested that Puerto Rico may be the ancestral area of L. albilabris. Both mitochondrial and

nuclear genomic analyses suggested that the Saint Croix populations of L. albilabris are the

result of introduction. The Saint Croix populations of L. albilabris are the least genetically

diverse of all populations of the frog, and are genetically very similar to the Saint Thomas

populations, suggesting that Saint Thomas is the source of the Saint Croix L. albilabris. My

study characterized the genetic architecture of L. albilabris across its distribution in the PRB, and

contributed to our understanding of how historical changes in Pleistocene sea levels have shaped

patterns of present-day genetic diversity in island systems.

v

Acknowledgements

I thank my graduate committee members: Dr. Javier Rodríguez (School of Life Sciences),

for accepting me as a student and helping me cultivate my interest and knowledge in

biodiversity; Dr. Dennis Bazylinski (School of Life Sciences), for providing resources that taught

me new skills that I had not had opportunities to develop before coming to UNLV; Dr. Mira Han

(School of Life Sciences), for guiding me in the right direction with bioinformatics data analysis

and introducing me to a different aspect of the biological sciences, Dr. Matthew Lachniet

(Department of Geoscience), for providing expertise about aspects of historic global climate

relevant to this project; and Dr. Tereza Jezkova (Department of Biology, Miami University,

Ohio), for guidance with the computationally intensive analyses used in this project. I also thank

Dr. Viviana Morillo López (formerly at UNLV’s School of Life Sciences and presently at

Marine Biological Laboratory, University of Chicago), for taking the time to mentor me and

teach me laboratory techniques; and Dr. Richard Tillett (Ecology, Evolution and Conservation

Biology Graduate Program, University of Nevada, Reno), for providing me with hands-on

bioinformatics training that I have developed a great interest for. I also acknowledge the

committee members from the School of Life Sciences Aquatic Biology Endowment for granting

me funding to conduct fieldwork, and the Nevada INBRE service awards for funding my

bioinformatics training. These funding opportunities have provided me opportunities to become a

more experienced and well-rounded biologist. Many thanks to my fellow graduate students, and

friends in the School for being a part of my experience at UNLV. Special thanks to my

girlfriend, Shannon Aurigemma, for her never-ending support. Finally, I thank my family

Phuong, Dung, and Andrea Nguyen, for their love and support is the foundation that I stand on.

vi

Table of Contents

Abstract .......................................................................................................................................... iii Acknowledgements ......................................................................................................................... v Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix CHAPTER 1: Introduction ............................................................................................................. 1 CHAPTER 2: Materials and Methods ............................................................................................ 6

Taxon sampling ........................................................................................................................... 6 Mitochondrial DNA - Laboratory Methods ................................................................................ 6 Mitochondrial DNA - Analyses .................................................................................................. 7 Double-digest restriction site associated DNA-sequencing - Laboratory Methods ................... 8 Double-digest restriction site associated DNA-sequencing - Analyses .................................... 10

CHAPTER 3: Results ................................................................................................................... 12 Bayesian phylogenetic analysis of mitochondrial DNA ........................................................... 12 Haplotype network of mitochondrial DNA .............................................................................. 13 Haplotype diversity and nucleotide diversity of mitochondrial DNA ...................................... 14 Population structuring and association between geographic and genetic distance calculated

using mitochondrial DNA ......................................................................................................... 15 Double-digest restriction site associated DNA-sequencing SNP-filtering ............................... 16 Population genomic analyses – heterozygosity and phylogenomic structure ........................... 17

vii

CHAPTER 4: Discussion .............................................................................................................. 19 The effect of Puerto Rico’s physiography and Pleistocene climate on patterns of genetic

variation in Leptodactylus albilabris ........................................................................................ 19 Isolation by distance of Leptodactylus albilabris populations across the Puerto Rican Bank . 22 Ancestral area of Leptodactylus albilabris ............................................................................... 23 Origins of the Saint Croix populations of Leptodactylus albilabris ......................................... 25 Systematic status of Leptodactylus dominicensis ..................................................................... 27 Concluding remarks .................................................................................................................. 28

References ..................................................................................................................................... 92 Curriculum Vitae ........................................................................................................................ 109

viii

List of Tables

Table 1. Sample list....................................................................................................................... 29 Table 2. Unique Mitochondrial DNA haplotypes by phylogroup ................................................ 54 Table 3. Mitochondrial DNA haplotypes by population .............................................................. 56 Table 4. Mitochondrial DNA genetic diversity statistics............................................................. 59 Table 5. Pairwise fixation index values among populations of Leptodactylus albilabris ............ 63 Table 6. Single nucleotide polymorphism heterozygosity statistics ............................................. 78

ix

List of Figures

Figure 1. Map of the 42 sampling localities for Leptodactylus albilabris .................................... 80 Figure 2. Bayesian inferrence phylogenetic tree of unique cytochrome b haplotypes ................. 82 Figure 3. Haplotype network map of cytochrome b haplotypes ................................................... 85 Figure 4. Individual sample ancestry matrix and relative population ancestry proportions

generated from single nucleotide polymorphisms ........................................................................ 87 Figure 5. Configurations of the Puerto Rican Bank at 10 meter intervals (0 m – -60 m) ............. 89

1

CHAPTER 1: Introduction

Resolving the geographic distribution of genetic lineages, that is, the phylogeographic

patterns of a species, can provide insight into the historical events that have shaped modern

species distributions. Making inferences that incorporate natural, historical, and recent processes

requires integrating data from various scientific fields (Kidd & Ritchie, 2006; Hassine et al.,

2016) such as geology, population genetics, and ecology. The climate and sea level oscillations

that occurred throughout the Pleistocene Epoch (2.6 mya – 10,000 ya) are a well-documented

example of intermittent episodes that influenced the demographic history of biota (Hewitt, 1996;

Leal et al., 2016; Gehara et al., 2017; Silva et al., 2017; Parvizi et al., 2018; Potter et al., 2018;

Hyseni & Garrick, 2019). Climate may have impacted the vagility of organisms, whereas sea

level changes affected land area configurations globally. Land area configuration changes via

eustatic sea levels (i.e. uniformly global variation of sea level) facilitated population isolations

through habitat fragmentations and land area contractions. Through increased land exposure,

eustatic sea levels paradoxically also led to range expansion and secondary contact of previously

separated populations (Hewitt, 1996, 2000, 2004; Petit et al., 2003; Krysko et al., 2016).

The sea level changes of the Pleistocene Epoch resulted from alternating episodes of

global cooling and warming, or glacial-interglacial cycles. The extensive, cooler glacial periods

froze ocean water as massive ice sheets, lowering sea levels for up to ca. -134 m (Rohling et al.,

1998; Bintanja et al., 2005; Lambeck et al., 2014), compared to present-day sea levels. On the

contrary, the shorter, warmer interglacial periods of the Pleistocene Epoch led to smaller ice

volumes and higher sea levels (Weigelt et al., 2016). The demographic consequences of these sea

level changes can be inferred using phylogenetic and population genetic methods that detect

2

molecular signals in present-day taxa (e.g. Davison & Chiba, 2008; Barrow et al., 2017;

Moriyama et al., 2018; Deli et al., 2019).

Islands are considered natural platforms of evolution (Losos & Ricklefs, 2009).

Populations on islands are enclosed by abiotic boundaries, presenting opportunities for

evolutionary processes to occur undisturbed from non-native biotic forces (Slatkin, 1987; Wade

& McCauley, 1988; Whitlock & McCauley, 1990; Reynolds et al., 2017). Processes such as

immigration, extinction, population differentiation, and speciation can occur within individual

islands and archipelagos (Clouse et al., 2015; Fernández-Palacios et al., 2016; Hirano et al.,

2019), contributing to the complexities of evolutionary history of island species. During

interglacial periods of the Pleistocene, rising sea levels submerged land bridges that connected

islands systems. Rising sea levels thus caused islands to fragment into smaller, more isolated

units. Island fragmentation may have led to population isolation, which can influence the genetic

architecture of a species. On the other hand, the lower sea levels of glacial periods increased

island area by exposing submerged land bridges (Weigelt et al., 2016). Newly adjoined islands

presented opportunities for colonization or reconnection of previously isolated populations (Garg

et al., 2018). Sea level changes of the Pleistocene also impacted marine currents, and therefore

influenced the paths of organisms on flotsam (Fernández-Palacios et al., 2016), altering paths of

colonization and genetic exchange between island populations.

The Puerto Rican Bank (PRB) is a 350 km long island system located in the eastern

Caribbean Sea. This group of islands consists of Puerto Rico and the “Eastern Islands:” Vieques,

Culebra, the United States Virgin Islands (Saint Thomas and Saint John), the British Virgin

Islands (Jost Van Dyke, Tortola, Virgin Gorda, Anegada), and ca. 180 associated islands, islets

and cays (Barker et al., 2012). This archipelago is connected by a submerged shelf less than 120

3

m below current sea level (Heatwole & MacKenzie, 1967; Hedges, 1999; Renken et al.,

2002). Glacial periods of the Pleistocene revealed the shelf, and united all the modern islands of

the PRB into a contiguous landmass approximately twice the area (ca. 18,000 km2) of Puerto

Rico, the largest island of the archipelago (Thomas, 1999; Figure 1). Politically, Saint Croix is

part of the United States Virgin Islands, but this island lays in its own bank and has not had a

land connection to the PRB (Heatwole & MacKenzie, 1967).

Leptodactylus albilabris (White-lipped frog; Gunther, 1859) is a native, widespread

species in the PRB. The maximum reported snout-to-vent length of adults is 49 mm (Schwartz &

Henderson, 1991; Hedges & Heinicke, 2007). This frog typically occurs in habitats with standing

fresh water, where adult males can be found in dense aggregations advertising for mates (López

et al., 1988). Adult females deposit eggs in foam nests; rain may wash away the eggs from the

nests into nearby ponds in which free-living aquatic larva (i.e. tadpoles) hatch (Dent, 1956;

Schwartz & Henderson, 1991; Joglar, 2005; Hedges & Heinicke, 2007). Amphibians are

excellent models to investigate phylogeographic patterns in island systems. Their relatively low

rates of dispersal and great climatic and environmental sensitivity present opportunities to

explore the impact of glacial cycles on biogeography and genealogies (Zeisset & Beebee, 2008;

Garcia-Porta et al., 2012). I characterized the population genetic architecture of L. albilabris

throughout the PRB to investigate the potential effect of eustatic sea level changes during the

Pleistocene on the population genetic structure of this frog.

I predicted that the boundaries between the physiographic regions of Puerto Rico restrict

gene flow among populations of L. albilabris from different areas of the island. Puerto Rico has

five major physiographic regions: (i) the Cordillera Central, (ii) Sierra de Cayey, (iii) Sierra de

Luquillo, (iv) Cuchilla de Pandura, and (v) the Lowlands (Hedges, 1999). The boundaries of

4

these areas may present a physical and/or ecological barrier to dispersal of L. albilabris across

physiographic regions. If this hypothesis is correct, population genetic analyses would reveal

distinctive genetic groups (i.e. clades) corresponding to these physiographic regions.

The population genetic architecture of L. albilabris across the PRB may reflect “isolation

by distance” (IBD). This pattern is exhibited when geographic distance facilitates gradual genetic

differentiation across a species’ distribution, so that there is a positive relationship between

geographic distance and genetic divergence between populations (Wright, 1943; Kimura, &

Weiss, 1964; de Aguiar et al., 2009; Baptestini et al., 2013). If this scenario is accurate, Puerto

Rican populations of L. albilabris should display a greater degree of genetic divergence from the

more distant populations of the British Virgin Islands, compared to populations from the closer

United States Virgin Islands. I also expect the intermediately located United States Virgin

Islands populations of L. albilabris to exhibit genetic similarities with both Puerto Rican and

British Virgin Islands populations of this frog.

I hypothesized that Puerto Rico, the largest island of the PRB, is the ancestral area of L.

albilabris. If this hypothesis is correct, population genetic analyses should show genetic markers

from Puerto Rican populations located at the base of the L. albilabris species phylogeny, and

Puerto Rican populations having greater genetic diversity than the Eastern Islands populations.

Alternatively, analyses may detect greater genetic diversity and basal positioning of genetic

markers from the Eastern Islands populations of L. albilabris, implying that the frog originated

on the Eastern Islands and dispersed westward.

As stated earlier, Saint Croix (U.S. Virgin Islands) lays in its own bank and has not had a

land connection to the PRB (Heatwole & MacKenzie, 1967). Because L. albilabris occurs on

Saint Croix, I hypothesized that the frog colonized Saint Croix from the PRB, naturally or via

5

human-mediated introduction(s), either accidentally (Perry et al., 2006) or deliberately (cf.

Barker & Rodríguez-Robles, 2017). If this prediction is correct, genetic markers from Saint

Croix should be identical to, or derived from L. albilabris populations in the PRB. On the

contrary, greater levels of genetic variation in Saint Croix L. albilabris may indicate that the frog

evolved in situ on the island, from which it colonized the PRB. In this scenario, oceanic currents

could have carried frogs on flotsam to the PRB. If Saint Croix is the ancestral area of L.

albilabris, genetic markers from Saint Croix individuals would be positioned basally on the L.

albilabris species phylogeny.

In 1923, Cochran described Leptodactylus dominicensis based on a single specimen from

Hispaniola (Cochran, 1923), an island (ca. 76,200 km2) located west of Puerto Rico. In her

original description and in a later systematic monograph on the herpetology of Hispaniola

(Cochran, 1941), Cochran stated that L. dominicensis, known only from a small area in

northeastern Hispaniola, is closely related to L. albilabris. Heyer (1978) synonymized L.

dominicensis with L. albilabris, a taxonomic rearrangement supported by Hedges and Heinicke

(2007) and by de Sá et al. (2014), based on L. dominicensis’ marked morphological and genetic

similarity to L. albilabris. I relied on a denser sampling of L. albilabris across the PRB to assess

the systematic position of L. dominicensis.

6

CHAPTER 2: Materials and Methods

Taxon sampling

I collected 322 individuals of L. albilabris from 42 localities across eight islands: Puerto

Rico (n = 243), Vieques (n = 11), Culebra (n = 10), Saint Thomas (n = 12), Saint John (n = 10),

Saint Croix (n = 18), Jost Van Dyke (n = 11), and Tortola (n = 7) (Figure 1; Table 1). Sample

size varied from 5 – 17 frogs per locality.

Mitochondrial DNA - Laboratory Methods

I extracted genomic DNA from tissue samples preserved in 95% ethanol (liver, heart,

stomach, muscle) with the DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA). I used the

primers CBL1 (TCTGCYTGATGAAAYTTTGG) and CBH15

(ACTGGTTGDCCYCCRATYAK; Hedges & Heinecke, 2007) to amplify 900 base pairs (bp) of

the mitochondrial DNA marker cytochrome b (cyt b). I carried out PCR reactions in 50 μl

volumes consisting of 2 μl of template DNA, 0.5 μl of each primer (10 μM), 25 μl of GoTaq®

Green Master Mix (Promega Corp., Madison, WI), and 22 μl of ddH2O. I denatured DNA at

95°C for 2.5 min, then performed 32 cycles of amplification as follows: denaturation at 95°C for

1 min, annealing at 51°C for 1 min, and extension at 72°C for 1.5 min, followed by a final

extension at 72°C for 10 min. I cleaned the PCR products with the Zymo Genomic DNA Clean

and ConcentratorTM kit (Zymo Research Corporation, Irvine, CA, USA). Sequencing of cyt b

was performed at the Genomics Core Facility of the University of Nevada, Las Vegas, with the

same primers used for amplification.

7

I aligned the sequences using SEQUENCHER 5.3 (Gene Codes Corporation, Ann Arbor,

MI), and verified the alignment visually. Missing data were treated as a fifth character state at the

particular site. The final mitochondrial DNA (mtDNA) data set consisted of 828 bp of cyt b.

I used DnaSP 6.10.04 (Rozas et al., 2017) to collapse the 322 cyt b sequences to 140 unique

haplotypes. I incorporated cyt b sequence data from one specimen of L. dominicensis

(GenBank accession number EF 091393.1) into the ingroup.

Mitochondrial DNA - Analyses

Prior to conducting phylogenetic analyses, I determined best fitting models of nucleotide

substitution for each codon position with jModelTest 2.1.10 (Darriba et al., 2012; Guindon &

Gascuel, 2003). The Akaike Information Criteria identified K80 (for the first and third codon

positions) (Kimura, 1980), and JC (for the second codon position) (Jukes & Cantor, 1969) as the

most appropriate models. I inferred phylogenetic relationships among the unique cyt b

haplotypes using Bayesian Inference (BI) criteria, as implemented in the program MrBayes 3.2.6

(Ronquist et al., 2012). I produced posterior probability distributions by allowing four Monte

Carlo Markov Chains to proceed for 40 million generations each, with samples taken every 100

generations, a procedure that yielded 120,002 trees. After visual evaluation, I discarded the first

25% of trees as “burn-in” samples, and combined the remaining samples to estimate tree

topology, posterior probability values, and branch lengths. I confirmed convergence of all

parameters in Tracer 1.6 (http://tree.bio.ed.ac.uk/software/tracer/). I used Figtree 1.4.3 (Rambaut

& Drummond, 2010) to illustrate the tree topology.

I visualized the cyt b haplotype distribution by creating a median-joining network using

NETWORK 5.0.0.3. The median-joining method uses a maximum parsimony approach to search

8

for all the shortest phylogenetic trees for a given dataset (Bandelt et al., 1999). I weighted

transversions twice as high as transitions, and used the maximum parsimony option to remove

excessive links and median vectors (Polzin & Daneshmand, 2003). I included all 322 cyt b

sequences of L. albilabris, and 1 cyt b sequence for L. dominicensis in this analysis.

I calculated haplotype diversity (Hd) and nucleotide diversity (πd) for the cyt b data using

ARLEQUIN 3.5.2.2 (Excoffier & Lischer, 2010). I assigned prior population designation based

on the specific locality where the samples were collected on each of the eight islands. I

calculated genetic diversity statistics for all sampling sites represented by at least five specimens.

I inferred gene flow between populations by calculating the fixation index (Fst). Fst values range

from 0 to 1. A value of 0 indicates no population structuring, that is, complete interbreeding

between two populations (panmixia), whereas a value of 1 indicates that all genetic variation is

explained by the population structure, and that the two populations do not share any genetic

diversity. Values reported are means ± 1 SD.

I tested for isolation by distance using the program Alleles in Space (Miller, 2005).

Specifically, the Mantel test option evaluates the correlation between genetic and geographic

distance matrices, using 1,000 permutations and logarithmic transformations. I conducted

analyses for all populations of L. albilabris (PRB and Saint Croix), populations across the PRB,

populations only located in Puerto Rico, and populations only from the Eastern Islands.

Double-digest restriction site associated DNA-sequencing - Laboratory Methods

I selected 119 samples of L. albilabris for double-digest restriction site associated DNA-

sequencing (ddRAD-seq). These samples span the geographic range of the frog in the PRB

(Puerto Rico, n = 70; Vieques, n = 5; Culebra, n = 6; Saint Thomas, n = 7; Saint John, n = 10;

9

Saint Croix, n = 7; Jost Van Dyke, n = 8; and Tortola, n = 6). The ddRAD-seq libraries were

prepared and sequenced at Floragenex Inc. (Portland, OR), as described by Truong et al. (2012),

with the minor adjustments described below. I double-digested 100 – 500 ng of genomic DNA at

37°C using the rare-cutting restriction enzyme Pst1 (5’-CTGCA*G-3’) and the frequent-cutting

restriction enzyme Mse1 (5’-T*TAA-3’). Pst1 P5 adapters containing unique 11 bp sample

identifiers were ligated to individual samples. Next, PCR amplified samples were pooled (5 μl

each) to make the libraries. Fragments were size-selected for the range of 200 – 800 base pairs.

Size-selected samples were amplified in a total reaction volume of 20 μl containing 5 μl of 10-

fold diluted restriction-ligation mixture, 5 ng Illumina P5 primer (5′-

AATGATACGGCGACCACCG-3′), 30 ng Illumina P7 primer (5′-

CAAGCAGAAGACGGCATACGA-3′), 0.2 mM dNTPs, 0.4 U AmpliTaq® (Applied Biosystems),

and 1× AmpliTaq® buffer. The Illumina P7 primer contained an additional +GC to decrease loci

density. Indexed pools were mixed in equimolar ratios and were sequenced together using 101

bp single-end reads on an Illumina HiSeq 2000. The 119 samples were part of two separate

libraries; the first library consisted of 95 samples (Library 1), and the second library consisted of

24 samples (Library 2).

Genomic sequencing methods provide large datasets that may contain artifacts (e.g.

sequencing error, allelic drop out, PCR bias) and uninformative markers that may saturate

relevant population genomic information (Roesti et al., 2012; da Fonesca et al., 2016). Therefore,

I implemented the following bioinformatics protocol to process the data and identify informative

markers. I used STACKS v2.2 to process raw sequence reads (Rochette & Catchen, 2017). I

demultiplexed the raw reads using the PROCESS_RADTAGS command, which identifies sample-

specific barcodes. This command also removes reads with a Phred score < 10 (quality score less

10

than 90%); with default 15% sliding window, as well as ambiguous barcodes. I used the

following series of STACKS commands to assemble and map the demultiplexed sequence data,

and to call single nucleotide polymorphisms (SNPs): the USTACKS command aligned reads,

generated putative de novo loci, and identified SNPs at each locus (Hohenlohe et al., 2010;

Rochette & Catchen, 2017); the CSTACKS command generated a catalog of consensus loci among

all individuals; and the SSTACKS command matched the putative loci of each individual to the

catalog. The POPULATIONS command filtered out SNPs with a maximum observed heterozygosity

of 0.7 and minor allele frequency of 0.05 (Rochette & Catchen, 2017), while focusing on only

single-SNP loci detected in at least two populations and 65% of individuals per population. Next,

I applied the filtering protocol of Puritz et al. (2014). The latter protocol removed individual

samples with more than 40% missing data, as well as genotypes detected in less than half the

total populations (Puritz et al., 2014a, b). I generated a variant calling format (vcf) file containing

16,225 SNPs for analysis. I assessed potential batch affects by assigning the filtered samples to

groups based on the respective sequence library (i.e. Library 1 or Library 2). I removed SNPs

with ≤ 10% representation between the two libraries, and visually inspected the results by using

Principal Component Analysis (PCA) (Luu et al., 2017).

Double-digest restriction site associated DNA-sequencing - Analyses

I calculated observed heterozygosity for each population using the vcf of 16,225 SNPs

and the R package hierfstat (Goudet, 2005). I removed missing data and used the basic.stats

function to calculate observed heterozygosity for each population. I then estimated standard error

in the sciplot R package (Morales, 2017). I assessed population genetic structure within L.

albilabris using the R package LEA (Frichot & François, 2015). This analysis allows for the

11

assessment of population structure among samples, and indicates whether samples represent one

or multiple genetic clusters. Accurate analyses with LEA require missing data to be imputed, or

replaced, with the value “9”. Using a custom script, I further filtered SNPs to only include those

with at least 50% representation among all samples, and imputed the remaining missing data

with the value “9”. I also analyzed population structure with the function snmf. This function

uses a sparse non-negative matrix factorization algorithm to estimate the number of genetic

clusters within the data set, by providing least-squares estimates of ancestry proportions and

calculating an entropy criterion (Frichot et al., 2014). In snmf, the cross-entropy criterion

provides a quality of fit to the statistical model, and can identify the most likely number of

genetic clusters (Frichot et al., 2014). The number of possible genetic cluster(s) was 1 – 10, a

range of values that I predetermined.

12

CHAPTER 3: Results

Bayesian phylogenetic analysis of mitochondrial DNA

Bayesian analysis of mtDNA sequences of L. albilabris revealed a phylogeny with

shallow divergences, and a topology that effectively represents a basal polytomy (Figure 2). I

recovered 28 well-supported (≥ 90% posterior probability values) nodes, or clades. Excluding the

two most inclusive clades, I designated 18 of the remaining 26 well-supported nodes as distinct

phylogroups (PG) (Figure 2). The remainder eight well-supported nodes are nested within five

(i.e. Phylogroups 7, 8, 11, 14, 17) of the 18 phylogroups. The populations and haplotypes that

comprise each phylogroup are listed in Table 2.

Ten of the 18 phylogroups of L. albilabris are comprised of populations exclusively

associated with two of the five physiographic areas of Puerto Rico, the Cordillera Central or the

Lowlands. However, the only phylogroup in the Cordillera Central is Phylogroup 6, which is

made up of a single population from central Puerto Rico (Ciales, Population 11; Figure 2). Of the

18 populations in the Lowlands of Puerto Rico, 14 comprise the remaining nine phylogroups:

Phylogroups 3, 7, 9, 12, 13, 15, 16, 17, 18 (Table 2). There are no well-supported phylogroups

restricted to the Sierra de Cayey, Sierra de Luquillo, or Cuchilla de Pandura physiographic

regions.

I discovered two well-supported nodes in the Eastern Islands, one from Vieques

(Populations 30, 31), and another one from Culebra (Population 32, Phylogroup 1) (Figure 2).

The Vieques clade is nested within a larger phylogroup (Phylogroup 11) that includes

Populations 15, 18–27, and 29 from eastern Puerto Rico (Table 2). The Vieques and Culebra

clades of L. albilabris do not include all the haplotypes identified in each of these islands. One

13

additional haplotype occurs in Vieques (H 64, Population 30); this haplotype does not belong in

any well-supported clade, and is shared with populations from eastern Puerto Rico (Populations

21, 24, 25, 27, 28), Saint John (Population 38), Jost Van Dyke 1, 2 (Populations 39, 40,

respectively), and Tortola 1, 2 (Populations 41, 42, respectively). Culebra harbors three

additional haplotypes (H 45, H 104, H 136) that do not belong within Phylogroup 1. H 45 is

sister to the Culebra clade (Phylogroup 1) (Figure 2), although that node (86% posterior

probability value) did not meet the ≥ 90% threshold to be considered well-supported. H 104 and

H 136 nest within Phylogroup 8, a clade that includes 19 populations (Populations 4–7, 9–13,

16–23, 26–27) across Puerto Rico.

Haplotype network of mitochondrial DNA

The median-joining network for L. albilabris depicts 114 haplotypes private to Puerto

Rico (Figure 3). The most frequent haplotype (H 115) was detected in 30 individuals from 12

different populations across Puerto Rico. Each of the Eastern Islands harbors at least one private

haplotype (Table 3; Figure 3). Six haplotypes (H 3, H 4, H 64, H 97, H 98, H 99) are private to

Vieques 1, 2 (Populations 30 and 31, respectively), and five (H 3, H 4, H 97, H 98, H 99) of

them form a cluster separated by 1–4 mutational steps. I detected five private haplotypes on

Culebra (Population 32), where three haplotypes (H 43, H 45, H 60) form a cluster separated by

1–3 mutations. Saint Thomas 2 (Population 34, H 8) and Saint Croix 2 (Population 36, H 30)

each harbors a single private haplotype. Saint John (Population 38) has two private haplotypes

(H 5, H 109), whereas Jost Van Dyke 1 (Population 39, H 111) and Tortola 2 (Population 42, H

107) each has a single private haplotype.

14

Network analysis detected four haplotypes present on more than one island (Table 3;

Figure 3). Haplotype 6 occurs on Puerto Rico, Vieques, Saint John, Jost Van Dyke, and Tortola.

Haplotype 10 occurs on Saint Thomas and Saint Croix. Haplotypes 28, H 108, and H 121 are

recognized as equivalent in NETWORK because they differ by three unspecified base pairs, and

they occur on Saint Thomas, Tortola, and Puerto Rico (Río Grande, Population 28), respectively.

Leptodactylus dominicensis (Hispaniola) and two samples from northeastern Puerto Rico

(Dorado, Population 14) share H 51.

Haplotype diversity and nucleotide diversity of mitochondrial DNA

I calculated haplotype diversity (Hd) for all populations represented by 5 individuals

(Table 4). Hd is highly variable for L. albilabris populations across the PRB and Saint Croix

(average Hd = 0.81, range = 0.0 – 1.0; Table 4). The populations with the greatest haplotype

diversity (Hd = 1.0) occur in eastern Puerto Rico: Aguas Buenas (Population 20), Cayey 1

(Population 21), Cayey 2 (Population 22), and Juncos (Population 27). The Saint Croix

populations 1, 2 (Populations 35, 36, respectively) lack haplotype diversity (Hd = 0.0).

Within Puerto Rico, average Hd = 0.89 (range = 0.25 – 1.0). As previously stated, four

populations from Puerto Rico displayed the highest haplotype diversity, whereas Peñuelas

(Population 9) had the lowest Hd value. The Eastern Island populations of L. albilabris had lower

Hd estimates (average Hd = 0.63, range = 0.25 – 0.89) than those from Puerto Rico. Vieques 1

(Population 30) had the highest Hd, whereas Jost Van Dyke 1 (Population 39) had the lowest one.

I also calculated nucleotide diversity (πd) for all populations represented by 5

individuals (Table 4). πd among populations of L. albilabris across the PRB and Saint Croix

averaged 0.0059 (range = 0.0 – 0.0121). The greatest πd estimate corresponded to Aguas Buenas

15

(Population 20, Puerto Rico), whereas the smallest πd values occur in Saint Thomas 1

(Population 33, πd = 0.0), and Saint Croix 1, 2 (Population 35, πd = 0.0, and Population 36, πd =

0.0002).

Within Puerto Rico, average πd = 0.007 (range = 0.0006 – 0.0121). As I mentioned,

Aguas Buenas (Population 20) had the highest πd estimate, whereas Peñuelas (Population 9) had

the lowest one. The πd estimates among the Eastern Islands populations of L. albilabris averaged

0.0032 (range = 0.0 – 0.0097), a value noticeably smaller than that for the Puerto Rican

populations. In the Eastern Islands, Culebra (Population 32) had the greatest πd estimate, whereas

Saint Thomas 1 (Population 33) had the smallest one, as reported above.

Population structuring and association between geographic and genetic distance calculated

using mitochondrial DNA

Fixation index (Fst) estimates for populations of L. albilabris across the PRB and Saint

Croix averaged 0.15 (range = 0.0 – 0.84; Table 5). Two localities from Saint Croix, Population

35 (average Fst = 0.46, range = -0.09 – 0.84) and Population 36 (average Fst = 0.48, range = -0.09

– 0.79) had the greatest overall Fst estimates. Interestingly, these Saint Croix localities do not

exhibit genetic structuring between them (Fst = 0.0). The Puerto Rican locality of Juncos

(Population 27) exhibited the lowest average Fst value (0.03, range -0.04 – 0.42). The Mantel test

indicated no correlation between genetic and geographic distance among all (PRB and Saint

Croix) populations of L. albilabris (r = 0.01; P = 0.34), or among populations from the PRB (r =

0.02; P = 0.19).

Puerto Rican populations of L. albilabris exhibited relatively low population genetic

structuring (average Fst = 0.08, range = 0.0 – 0.48). Peñuelas (Population 9) displayed the highest

16

values (average Fst = 0.35, range = 0.12 – 0.48), and Juncos (Population 27) had the lowest

estimates, as reported above. The Mantel test did not detect a correlation between genetic and

geographic distance within Puerto Rico (r = 0.01; P = 0.28).

The Fst estimates for L. albilabris populations in the Eastern Islands (average = 0.35,

range = 0.0 – 0.84) indicate higher levels of population genetic structuring than among Puerto

Rican populations of this frog. Jost Van Dyke 1 (Population 39) exhibited the highest average Fst

value (0.47, range = 0.0 – 0.84). On the contrary, and interestingly, this Jost Van Dyke

population and Tortola 2 (Population 42) exhibited zero genetic structuring between them (Fst =

0.0). Except for the aforementioned Jost Van Dyke 1 and Tortola 2 populations, Vieques 1

(Population 30) displayed the smallest Fst values (average = 0.28, range = 0.12 – 0.48). The

Mantel test indicates a correlation between genetic and geographic distance among L. albilabris

populations in the Eastern Islands (r = 0.47; P = 0.001).

Double-digest restriction site associated DNA-sequencing SNP-filtering

I recovered 341,505,569 reads from the first library, and 313,493,233 reads from the

second library, for a total of 654,998,802 reads from the 119 samples. Read depth for all samples

averaged 26.52 reads per sample (range = 16.86 – 45.22 reads). Prior to filtering, my

bioinformatics protocol generated a catalog of 1,570,383 genotyped loci and 2,497,178 SNPs.

After my filtering procedure I retained 16,225 single-SNP loci from 114 samples in the final vcf

file. My bioinformatics protocol detected five samples that did not pass the filtering criteria, and

I removed those samples from the dataset.

17

The filtering protocol to assess batch effects resulted in 4,046 SNPs. I visually inspected

the results through PCA, and the patterns were largely unchanged. Therefore, I determined that

my filtering protocol was thorough, and I conducted further analyses using all 16,225 SNPs.

Population genomic analyses – heterozygosity and phylogenomic structure

Heterozygosity estimates among all populations of L. albilabris (PRB and Saint Croix)

were low (average = 0.17, range = 0.10 – 0.23; Table 6). Aguas Buenas (Population 20, Puerto

Rico) and Saint Croix 2 (Population 36) exhibited the greatest and smallest heterozygosity

estimates, respectively.

Within Puerto Rico, heterozygosity estimates showed little variation (average = 0.19,

range 0.17 – 0.23; Table 6). Aguas Buenas (Population 20) and Moca (Population 2) displayed

the highest and lowest estimates, respectively. Heterozygosity estimates of the Eastern Islands

populations of L. albilabris (average = 0.12, range = 0.10 – 0.17) were lower than those of

Puerto Rican populations. Vieques 1 (Population 30) and Jost Van Dyke 1 (Population 39)

displayed the greatest and smallest heterozygosity values, respectively.

After filtering for missing data and conducting imputation, I generated a dataset with

16,140 SNPs representing 114 individuals. Clustering analyses using snmf identified five L.

albilabris genetic clusters with little admixture (Figure 4). Two genetic clusters were detected on

Puerto Rico, although only the first cluster was exclusive to this island. The first genetic cluster

(Cluster 1) consisted of three of the six populations from the Cordillera Central, specifically

Yauco (Population 6), Juana Díaz (Population 10), and Ciales (Population 11). The second

genetic cluster (Cluster 2) was formed by all other populations from the remaining four

physiographic regions of Puerto Rico (i.e. Sierra de Cayey, Sierra de Luquillo, Cuchilla de

18

Pandura, Lowlands), and the neighboring islands of Vieques 1 (Population 30) and Culebra

(Population 32). The third genetic cluster (Cluster 3) comprised populations from Saint Thomas

1 (Population 33) and Saint Croix 2 (Population 36). The fourth genetic cluster (Cluster 4)

corresponded to Saint John (Population 38), the only island that comprised an independent,

distinct genetic cluster. The fifth genetic cluster (Cluster 5) was formed by populations from Jost

Van Dyke 1, 2 (Populations 39, 40) and Tortola (Population 42).

Genetic clustering analysis revealed incongruences of cluster membership at the

population level. I detected two instances in which populations had individuals that belong to

different genetic clusters. (i) In Saint John (Population 38), nine individuals belong to Cluster 4,

and one belongs in Cluster 5. (ii) In Jost Van Dyke 1 (Population 39), one individual belongs to

Cluster 4, and the remaining six belong in Cluster 5.

19

CHAPTER 4: Discussion

Phylogeographic and population genetic studies can be more informative when the spatial

and temporal history of the study system are taken into consideration. The sea level changes of

Pleistocene glacial-interglacial cycles are associated with distributional shifts of taxa on a global

basis (Hewitt, 2000; Vasconcellos et al., 2019). Lower sea levels during glacial periods revealed

land bridges in island systems, providing opportunities for organisms to migrate between

formerly separated islands (Garg et al., 2018; Weigelt et al., 2016). On the other hand, higher sea

levels during interglacial periods submerged land bridges and fragmented islands (Weigelt et al.,

2016), potentially isolating previously connected populations. Repeated episodes of island

connectivity and fragmentation constitute a dynamic scenario that may have influenced the

evolutionary history of organisms native to island systems (Potter et al., 2018; Parvizi et al.,

2018; Sánchez-Montes et al., 2019). The Puerto Rican Bank (PRB) is an archipelago in the

eastern Caribbean Sea that exhibited periodic connectivity and fragmentation during the

Pleistocene (Heatwole & MacKenzie, 1967; Hedges, 1999; Renken et al., 2002). I herein

conducted a genetic assessment of populations of Leptodactylus albilabris across the PRB and

Saint Croix, to determine the potential effect of changes in island configuration on the

demographic history of this frog.

The effect of Puerto Rico’s physiography and Pleistocene climate on patterns of genetic

variation in Leptodactylus albilabris

I hypothesized that the boundaries of the five physiographic regions of Puerto Rico

(Cordillera Central, Sierra de Cayey, Sierra de Luquillo, Cuchilla de Pandura, Lowlands)

facilitate geographic structuring among populations of L. albilabris from those regions. The

20

results from the mtDNA and ddRAD-seq analyses only provided partial, incongruent support for

this hypothesis. Phylogenetic analysis of cytochrome b haplotypes detected well-supported

phylogroups associated with the Cordillera Central and the Lowlands (Figure 2). However, the

only phylogroup in the Cordillera Central compromises a single population from central Puerto

Rico, implying that the vast majority of populations from the Cordillera Central do not form a

distinct genetic cluster. Conversely, populations from the Lowlands formed nine different

phylogroups, reflecting a higher degree of structuring of populations of L. albilabris from low

elevation areas around Puerto Rico. The observation that several mtDNA haplotypes occur in

more than one physiographic region, coupled with the low Fst values among Puerto Rican

populations, suggest relatively unrestricted, recent or ongoing gene flow among L. albilabris

from much of the island.

The findings from the ddRAD-seq clustering analyses coincided in part with those from

the mitochondrial sequences. Specifically, genetic clustering of the much larger genomic dataset

also identified two genetic groups in Puerto Rico. Cluster 1 comprises three populations from the

Cordillera Central (Yauco, Population 6; Juana Díaz, Population 10; Ciales, Population 11)

(Figure 4), reminiscent of the mtDNA-inferred Phylogroup 6. However, in contrast to the

mtDNA dataset, the second genetic group (Cluster 2) identified by the genomic data is made up

of populations from the four remainder regions of Puerto Rico (Sierra de Cayey, Sierra de

Luquillo, Cuchilla de Pandura, Lowlands), and the neighboring islands of Vieques and Culebra.

Individuals collected in Juana Díaz, southcentral Puerto Rico, exemplify the distinction between

the two clusters. The four samples collected in the northern, mountainous section of this

municipality (Population 10) belong to Cluster 1, whereas the individual collected in the coastal

Lowlands in southern Juana Díaz (Population 12) belongs in Cluster 2. Interestingly, L.

21

albilabris from higher elevations are larger than those from lower elevations (Heatwole et al.,

1968), and coastal individuals exhibit a higher salinity tolerance (Gómez-Mestre & Tejedo,

2003). These biological differences may facilitate genetic divergence between L. albilabris

populations from the Cordillera Central and those from other regions of Puerto Rico. In regards

to L. albilabris from Vieques and Culebra, receding coastlines of these two islands during glacial

periods likely allowed coastal populations in eastern Puerto Rico to expand into these adjacent

islands (Figure 5D).

Collectively, analyses of the mtDNA and ddRAD-seq datasets revealed that although

some degree of genetic diversification has occurred, most Puerto Rican populations of L.

albilabris are genetically homogeneous. This finding is not intuitive, given that the occurrence of

L. albilabris depends on at least temporal availability of freshwater, because these frogs have a

free-living aquatic tadpole (Dent, 1956; Heatwole et al., 1968). Moisture and precipitation are

not evenly distributed throughout Puerto Rico; regional habitat characteristics of the island range

from higher elevation wet forests in the Cordillera Central, Sierra de Cayey and Sierra de

Luquillo, to lower elevation moist areas in the Cuchilla de Pandura, and to low elevation

subtropical dry forests predominantly located in the Lowlands of southern Puerto Rico, in the

rain shadow of the Cordillera Central (Ewel & Whitmore, 1973; Helmer et al., 2002; Renken et

al., 2002). (Subtropical dry forests also occur on the northeast coast of Puerto Rico, and on

Vieques, Culebra, and the United States and British Virgin Islands [Murphy et al., 1995].) Due to

its reproductive mode, arid conditions are expected to limit the distribution and mobility of L.

albilabris, and thus represent barriers to gene flow that will facilitate genetic divergence among

populations. These circumstances would have been especially true during the late Pleistocene

(ca. 95 – 10 kya), because the Caribbean was then cooler, but drier than at the present time

22

(Royer et al., 2017; Warken et al., 2019). However, L. albilabris tadpoles have higher heat

tolerance, which suggests that they are adapted to breeding in ephemeral and/or shallow bodies

of water (Heatwole et al., 1968). In fact, in Jost Van Dyke (Population 39) I collected specimens

in a small ditch next to an unpaved road. The larval stage of L. albilabris is relatively short,

tadpoles can metamorphose within 21–35 days after hatching (Dent, 1956; Joglar 2005; Flores-

Nieves et al., 2014). Additionally, L. albilabris at various stages of metamorphosis can be found

in muddy areas or under debris when standing water is no longer present (Heatwole et al., 1968).

These physiological traits limit the frogs’ dependence on freshwater, and thus increase

population connectivity and gene flow throughout much of Puerto Rico.

Isolation by distance of Leptodactylus albilabris populations across the Puerto Rican Bank

I hypothesized that populations of L. albilabris exhibit “isolation by distance” (IBD)

across the PRB. That is, I expected populations from Puerto Rico to be most divergent from the

populations from the eastern most British Virgin Islands (i.e. Jost Van Dyke and Tortola).

Mantel tests of the mtDNA dataset conducted at different spatial scales did not detect any

correlations between genetic and geographic distance among populations of L. albilabris within

Puerto Rico, across the PRB, or among all populations across the PRB and Saint Croix. Failure

to detect a signal for IBD suggests that gene flow occurred across wide spatial scales during

periods of connectivity in the PRB, or that the White-lipped frog experienced recent population

(spatial) expansion, perhaps facilitated by increasing land area during glacial periods (Reynolds

et al., 2017). Anolis cristatellus (Puerto Rican crested anole) has a similar distribution to L.

albilabris, and Eastern Islands populations of this lizard do not exhibit IBD either (Reynolds et

al., 2017).

23

On the contrary, Mantel tests restricted to the Eastern Islands populations of L. albilabris

detected IBD among the localities. Additionally, these populations displayed relatively high Fst

values. These findings suggest limited gene flow among Eastern Islands L. albilabris, and

possible genetic drift, because genetic drift is a phenomenon typically associated with small,

isolated populations (Wright, 1931; Nei et al., 1975; Frankham, 1997). The divergence among

the Eastern Islands populations of L. albilabris may be attributed to isolation caused by island

fragmentation during interglacial periods, and/or to limited gene flow even during periods of

island connectivity. As I mentioned, arid climate and xeric habitats characterized the PRB during

the Pleistocene (Pregill & Olson, 1981; Royer et al., 2017; Warken et al., 2019). Therefore, the

frog’s ability to move among localities corresponding to the present-day Eastern Islands may

have been limited by scarcity of suitable habitat on the land bridges. Ecological barriers to gene

flow, rather than geographic distance, may then explain genetic divergence among Eastern

Islands L. albilabris.

Ancestral area of Leptodactylus albilabris

As I stated, L. albilabris has a widespread distribution across the PRB, particularly in

Puerto Rico, where the species occurs in natural, residential, and commercial areas. I proposed

that L. albilabris evolved in Puerto Rico, the largest island of the PRB. Biogeographic theory

posits that greater genetic diversity is indicative of a longer demographic history; that is, older

populations have had more time to evolve (Taberlet et al., 2002; Provan & Bennett, 2008) and

accumulate genetic differences. Genetic diversity estimates calculated from mtDNA and

ddRAD-seq support the prediction that Puerto Rico is the ancestral area of L. albilabris, because

the Puerto Rican populations of this frog exhibit greater genetic diversity than those from the

24

Eastern Islands and Saint Croix (Tables 4, 6). Further, more private mtDNA haplotypes were

detected in Puerto Rico than in the Eastern Islands (Figure 3). Eleutherodactylus antillensis

(Red-eyed Coquí) is a frog with a similar distribution to that of L. albilabris, and genetic

analyses also suggest that E. antillensis evolved in Puerto Rico, and colonized the Eastern

Islands from sources in eastern Puerto Rico via land bridges during island connectivity (Barker et

al., 2012).

Nevertheless, the lower genetic diversity of Eastern Islands L. albilabris may be a

consequence of island area. Island biogeographic theory asserts that larger islands harbor more

ecological diversity, whether it is due to total physical area or to habitat heterogeneity

(MacArthur & Wilson, 1967; Ricklefs & Lovett, 1999). It is thus possible that the greater levels

of genetic diversity in Puerto Rican L. albilabris may simply result from the greater land area of

Puerto Rico, and that the lower genetic diversity in Eastern Islands L. albilabris is a demographic

consequence of island fragmentation (Frankham, 1997; White & Searle, 2007; Furlan et al.,

2012; Wang et al., 2014) in the PRB. Specifically, range contraction and population isolation by

rising sea levels could have led to population bottleneck and genetic drift (Hoffman & Blouin,

2004; Wang et al., 2014), causing a reduction in the genetic diversity of Eastern Islands

populations (Table 4, 6). Phylogenetic analysis of unique mtDNA haplotypes recovered a

topology that effectively represents a basal polytomy (Figure 2), and therefore is uninformative

regarding the question of the geographic origin of L. albilabris. All these scenarios allow for the

possibility that the White-lipped frog may have originated in the Eastern Islands, and then

colonized and differentiated genetically in Puerto Rico. In conclusion, the ancestral area of L.

albilabris remains equivocal.

25

Origins of the Saint Croix populations of Leptodactylus albilabris

The origin of the Saint Croix populations of L. albilabris is unclear, given that this island

has not had a land connection to the PRB (Gill et al., 1989). Ocean currents around the PRB

generally flow in a northwestern direction (Heatwole & Mackenzie, 1967), suggesting that L.

albilabris could have evolved in situ on Saint Croix and later colonized the PRB. Typically,

ancestral populations exhibit relatively high levels of genetic diversity and population structuring

(Reilly et al., 2019), but Saint Croix L. albilabris do not display these patterns. I sampled three

localities across the island (Figure 1), and detected only two haplotypes among 18 individuals:

one private mtDNA haplotype in one frog, and another haplotype shared by the remaining 17

animals from Saint Croix. The latter haplotype is also shared by seven individuals from Saint

Thomas (Figure 3; Table 3). Clustering analyses of the genomic data confirmed the genetic

similarity between Saint Thomas (Population 33) and Saint Croix (Populations 36) L. albilabris,

and depicted the two populations as a single cluster with no admixture (Figure 4). The low

genetic diversity in populations of L. albilabris in Saint Croix suggests two possible scenarios:

the species experienced a selective sweep across the island (Tajima, 1989; Nguiffo et al., 2019),

or it arrived on Saint Croix relatively recently, possibly from a source population in Saint

Thomas. The selective sweep scenario in turn implies that the Saint Thomas and Saint Croix

populations evolved the same cytochrome b haplotype through convergence, perhaps due to a

similar selective pressure (Losos, 2011; Messer & Petrov, 2013). The latter evolutionary vista

may not represent the more parsimonious explanation to account for the presence of L. albilabris

on Saint Croix.

Perhaps a more likely scenario is that L. albilabris originated in the PRB, and

subsequently arrived on Saint Croix by either natural means or through human-mediated

26

introduction(s). Natural immigration requires L. albilabris to have rafted to Saint Croix. As

mentioned before, the surface currents in the eastern Caribbean Sea generally have a

northwestern direction (Heatwole & Mackenzie, 1967), making rafting southward from a source

in the PRB to Saint Croix an improbable event. Nevertheless, ocean dynamics can be impacted

during tropical storms and hurricanes (Ezer, 2019), and thus surface currents could temporarily

change course and cause flotsam to drift in atypical directions. Even when rafting occurs, the

survival of rafting organisms in the PRB is low (Heatwole & Levins, 1972; Ricklefs &

Bermingham, 2008). Salt water is physiologically stressful for anurans (Balinsky, 1981;

Duellman & Trueb, 1994), and the probability of oceanic dispersal of different frog species can

vary, because of differences in physiological tolerance to salinity and habitat requirements

(Duryea et al., 2015). Yet, oceanic dispersal by anurans has been documented (e.g. Hedges et al.,

1992; Vences et al., 2003; Heinicke et al., 2007; Bell et al., 2015), and there are published

accounts of oceanic dispersal by nonavian reptiles in the Caribbean Sea (e.g. Corallus

hortulanus, Garden tree boa, Henderson & Hedges, 1995; Green iguana, Iguana iguana, Censky

et al., 1998; Anolis sagrei, Brown anole, Calsbeek & Smith, 2003). Accidental or even deliberate

introduction of L. albilabris to Saint Croix is also possible. Maritime travel by humans has had a

noticeable impact on biodiversity globally (Reilly et al. 2019), including the transportation of

frog species among Caribbean islands (MacLean, 1982; Kaiser et al., 2002; Rödder, 2009;

Barker et al., 2012; Barker & Rodríguez-Robles, 2017). For example, E. antillensis and

Osteopilus septentrionalis (Cuban Tree frog) have been documented ‘hitchhiking’ on

landscaping materials that were being transported between islands of the PRB and Saint Croix

(Townsend et al., 2000; Powell et al., 2005; Powell, 2006; Perry et al., 2006; Platenberg, 2007;

Powell et al., 2011).

27

Systematic status of Leptodactylus dominicensis

Phenotypical, behavioral, and genetic data clearly indicate that Leptodactylus

dominicensis (Cochran, 1823) is closely related to L. albilabris (Gunther, 1859). In fact, several

authors have placed L. dominicensis in the synonymy of L. albilabris (Heyer, 1978; Hedges &

Heinicke, 2007; de Sá et al., 2008). I conducted the most inclusive genetic assessment of L.

albilabris throughout its range to date, and relied on this sampling to assess the taxonomic

position of L. dominicensis. Phylogenetic analysis conclusively demonstrated that the lone

available cytochrome b sequence of L. dominicensis nests within those of L. albilabris. Indeed,

two individuals of L. albilabris from Puerto Rico’s northern coast (Dorado, Population 14) share

the same mtDNA haplotype (H 51) with L. dominicensis (Figure 2). My findings thus support the

conclusion that L. dominicensis should not be considered a separate species, a taxonomic

arrangement that I follow hereafter.

Leptodactylus albilabris is known only from a small area in northeastern Hispaniola.

Individuals of this frog have been collected from El Seibo Province, Dominican Republic

(Cochran, 1941; Heyer, 1978; Hedges & Heinicke, 2007), which is adjacent to Samaná Bay. As

with L. albilabris in Saint Croix, the presence of L. albilabris on Hispaniola likely represents a

relatively recent colonization event, by natural or anthropogenic means. Oceanic currents in the

eastern Caribbean Sea could have transported L. albilabris via flotsam west into Samaná Bay.

Accidental or deliberate introduction of L. albilabris to Hispaniola may also have occurred, as

human-mediated introduction of Eleutherodactylus johnstonei, Lesser Antillean Frog (Barbour,

1930; Censky, 1989; Lever, 2003; Rödder, 2009), and Osteopilus septentrionalis, Cuban

Treefrog (Townsend et al., 2000; Powell et al., 2005; Powell, 2006; Perry et al., 2006), to various

islands in the Caribbean Sea has been documented.

28

Concluding remarks

Elucidating the genetic architecture of L. albilabris across the PRB illustrated the

importance of integrating geologic information into studies of the evolutionary history of a

species. I used molecular data to evaluate how eustatic sea level changes during the Pleistocene

Epoch may have shaped the genetic profile of this frog. I detected interesting genetic patterns in

L. albilabris, both within Puerto Rico and throughout the species’ range in the PRB. The

ddRAD-seq clustering analyses uncovered a divergence between L. albilabris populations from

Puerto Rico’s Cordillera Central and the rest of the island. Future studies should investigate

which biological and/or environmental factor(s) may facilitate this differentiation, and assess

whether traits other than body size and salinity tolerance vary between populations from higher

and lower elevation areas in Puerto Rico. Additionally, little is known about the dispersal ability

of L. albilabris. It will therefore be informative to conduct radiotracking and mark-recapture

studies to characterize the spatial ecology (e.g. movement patterns, home range size), and by

extension, the degree of connectivity among populations of the species. As a final point, my

study contributed to our increasing understanding of the biogeography of the Puerto Rican Bank

(e.g. Barker et al., 2012; Falk & Perkins, 2013; Papadopoulou & Knowles, 2015, 2017;

Rodríguez-Robles et al., 2015; Reynolds et al., 2017).

29

Tab

le 1

. Spe

cies

, pop

ulat

ion

num

ber,

field

num

ber,

coun

try, s

tate

, isl

and,

mun

icip

ality

, lat

itude

(h =

deg

rees

, min

= m

inut

es),

and

long

itude

of t

he lo

calit

ies o

f the

spec

imen

s of L

epto

dact

ylus

poe

cilo

chilu

s, L.

dom

inic

ensi

s, an

d L.

alb

ilabr

is u

sed

in th

is st

udy.

For

L.

poec

iloch

ilus,

the

mus

eum

cat

alog

num

ber i

s in

pare

nthe

ses;

for L

. dom

inic

ensi

s, th

e G

enB

ank

acce

ssio

n nu

mbe

r is i

n pa

rent

hese

s.

BB

= B

ritta

ny B

arke

r Fie

ld S

erie

s, B

VI =

Brit

ish

Virg

in Is

land

s, C

U =

Cul

ebra

, JA

R =

Javi

er A

. Rod

rígue

z Fi

eld

Serie

s, JV

D =

Jos

t

Van

Dyk

e, P

R =

Pue

rto R

ico,

SB

H =

S. B

lair

Hed

ges F

ield

Ser

ies,

STC

= S

aint

Cro

ix, S

TJ =

Sai

nt Jo

hn, S

TT =

Sai

nt T

hom

as, T

O =

Torto

la, U

K =

Uni

ted

Kin

gdom

, US

= U

nite

d St

ates

of A

mer

ica,

USN

M =

Nat

iona

l Mus

eum

of N

atur

al H

isto

ry, S

mith

soni

an

Inst

itutio

n, W

ashi

ngto

n, D

.C.,

USV

I = U

nite

d St

ates

Virg

in Is

land

s, V

I = V

iequ

es. S

ampl

es o

f L. a

lbila

bris

are

list

ed in

ord

er b

y

popu

latio

n nu

mbe

r, fr

om w

est t

o ea

st (F

igur

e 1)

.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

O

utgr

oup

Le

ptod

acty

lus

poec

iloch

ilus

(USN

M 5

6502

9)

—

—

Pana

má

—

—

Pana

má

Prov

ince

—

—

—

—

30

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

In

grou

p

Lept

odac

tylu

s do

min

icen

sis

(EF0

9139

3)

—

SBH

192

453

D

omin

ican

R

epub

lic

H

ispa

niol

a El

Sei

bo

Prov

ince

—

—

—

—

Le

ptod

acty

lus

albi

labr

is

1 JA

R 2

905

US

PR

PR

Cab

o R

ojo

18°

01.0

30' N

67

° 08

.955

' W

Lept

odac

tylu

s al

bila

bris

1

JAR

290

6 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

290

7 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

290

8 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

290

9 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

291

0 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

291

1 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

291

2 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

291

3 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

Lept

odac

tylu

s al

bila

bris

1

JAR

291

4 U

S PR

PR

C

abo

Roj

o 18

° 01

.030

' N

67°

08.9

55' W

31

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

2 JA

R 2

740

US

PR

PR

Moc

a 18

° 23

.477

' N

67°

06.0

78' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

1 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

2 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

3 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

4 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

5 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

7 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

2

JAR

274

8 U

S PR

PR

M

oca

18°

23.4

77' N

67

° 06

.078

' W

Lept

odac

tylu

s al

bila

bris

3

JAR

262

5 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

262

6 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

262

7 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

262

8 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

262

9 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

263

0 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

32

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

3 JA

R 2

631

US

PR

PR

Isab

ela

18°

30.6

36' N

67

° 05

.728

' W

Lept

odac

tylu

s al

bila

bris

3

JAR

263

2 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

263

3 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

3

JAR

263

4 U

S PR

PR

Is

abel

a 18

° 30

.636

' N

67°

05.7

28' W

Lept

odac

tylu

s al

bila

bris

4

JAR

307

0 U

S PR

PR

M

ayag

üez

18°

11.3

03' N

67

° 03

.626

' W

Lept

odac

tylu

s al

bila

bris

4

JAR

307

2 U

S PR

PR

M

ayag

üez

18°

11.3

03' N

67

° 03

.626

' W

Lept

odac

tylu

s al

bila

bris

4

JAR

307

4 U

S PR

PR

M

ayag

üez

18°

11.3

03' N

67

° 03

.626

' W

Lept

odac

tylu

s al

bila

bris

4

JAR

307

5 U

S PR

PR

M

ayag

üez

18°

11.3

03' N

67

° 03

.626

' W

Lept

odac

tylu

s al

bila

bris

4

JAR

307

6 U

S PR

PR

M

ayag

üez

18°

11.3

03' N

67

° 03

.626

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

289

3 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

289

4 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

289

5 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

289

6 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

289

7 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

33

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

5 JA

R 2

898

US

PR

PR

Guá

nica

17

° 58

.751

' N

66°

57.0

89' W

Lept

odac

tylu

s al

bila

bris

5

JAR

289

9 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

290

0 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

290

1 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

5

JAR

290

2 U

S PR

PR

G

uáni

ca

17°

58.7

51' N

66

° 57

.089

' W

Lept

odac

tylu

s al

bila

bris

6

JAR

307

7 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bri s

6

JAR

307

8 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

307

9 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

308

0 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

308

1 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

308

3 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

308

4 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

308

5 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

Lept

odac

tylu

s al

bila

bris

6

JAR

308

6 U

S PR

PR

Y

auco

18

° 09

.100

' N

66°

53.0

99' W

34

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

6 JA

R 3

087

US

PR

PR

Yau

co

18°

09.1

00' N

66

° 53

.099

' W

Lept

odac

tylu

s al

bila

bris

7

JAR

285

4 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

285

5 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

285

6 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

285

7 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

285

8 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

285

9 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

286

0 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

286

1 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

286

2 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

7

JAR

286

3 U

S PR

PR

H

atill

o 18

° 29

.175

' N

66°

49.9

12' W

Lept

odac

tylu

s al

bila

bris

8

JAR

287

8 U

S PR

PR

A

reci

bo

18°

22.7

30' N

66

° 44

.963

' W

Lept

odac

tylu

s al

bila

bris

8

JAR

287

9 U

S PR

PR

A

reci

bo

18°

22.7

30' N

66

° 44

.963

' W

Lept

odac

tylu

s al

bila

bris

8

JAR

288

0 U

S PR

PR

A

reci

bo

18°

22.7

30' N

66

° 44

.963

' W

35

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

8 JA

R 2

881

US

PR

PR

Are

cibo

18

° 22

.730

' N

66°

44.9

63' W

Lept

odac

tylu

s al

bila

bris

8

JAR

288

2 U

S PR

PR

A

reci

bo

18°

22.7

30' N

66

° 44

.963

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

309

5 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

309

6 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

309

7 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

309

8 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

309

9 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

310

0 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

310

1 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

9

JAR

310

2 U

S PR

PR

Pe

ñuel

as

18°

04.5

86' N

66

° 43

.557

' W

Lept

odac

tylu

s al

bila

bris

10

JA

R 3

037

US

PR

PR

Juan

a D

íaz

18°

09.0

87' N

66

° 32

.023

' W

Lept

odac

tylu

s al

bila

bris

10

JA

R 3

039

US

PR

PR

Juan

a D

íaz

18°

09.0

87' N

66

° 32

.023

' W

Lept

odac

tylu

s al

bila

bris

10

JA

R 3

040

US

PR

PR

Juan

a D

íaz

18°

09.0

87' N

66

° 32

.023

' W

Lept

odac

tylu

s al

bila

bris

10

JA

R 3

043

US

PR

PR

Juan

a D

íaz

18°

09.0

87' N

66

° 32

.023

' W

36

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

10

JAR

304

4 U

S PR

PR

Ju

ana

Día

z 18

° 09

.087

' N

66°

32.0

23' W

Lept

odac

tylu

s al

bila

bris

10

JA

R 3

045

US

PR

PR

Juan

a D

íaz

18°

09.0

87' N

66

° 32

.023

' W

Lept

odac

tylu

s al

bila

bris

10

JA

R 3

046

US

PR

PR

Juan

a D

íaz

18°

09.0

87' N

66

° 32

.023

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

993

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

994

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

995

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

996

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

997

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

998

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 2

999

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 3

000

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 3

001

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 3

002

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

Lept

odac

tylu

s al

bila

bris

11

JA

R 3

003

US

PR

PR

Cia

les

18°

14.8

05' N

66

° 31

.215

' W

37

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

11

JAR

300

4 U

S PR

PR

C

iale

s 18

° 14

.805

' N

66°

31.2

15' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

709

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

710

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

711

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

712

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

713

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

714

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

715

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

716

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

717

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

12

JA

R 2

718

US

PR

PR

Juan

a D

íaz

18°

00.3

95' N

66

° 29

.538

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

699

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

700

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

701

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

38

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

l abr

is

13

JAR

270

2 U

S PR

PR

M

anat

í 18

° 23

.200

' N

66°

29.1

19' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

703

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

704

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

705

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

707

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

13

JA

R 2

708

US

PR

PR

Man

atí

18°

23.2

00' N

66

° 29

.119

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

915

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

916

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

917

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

918

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

919

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

920

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

921

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

922

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

39

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

14

JAR

292

3 U

S PR

PR

D

orad

o 18

° 27

.731

' N

66°

16.3

99' W

Lept

odac

tylu

s al

bila

bris

14

JA

R 2

924

US

PR

PR

Dor

ado

18°

27.7

31' N

66

° 16

.399

' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

686

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

687

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

688

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

689

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

690

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

691

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

692

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

693

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

15

JA

R 2

694

US

PR

PR

Aib

onito

18

° 06

.931

' N

66°

13.9

99' W

Lept

odac

tylu

s al

bila

bris

16

JA

R 2

685

US

PR

PR

Aib

onito

18

° 06

.574

' N

66°

13.5

72' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

556

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

557

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

40

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

17

JAR

255

8 U

S PR

PR

To

a A

lta

18°

20.2

22' N

66

° 12

.848

' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

559

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

560

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

561

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

562

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

563

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

564

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

17

JA

R 2

565

US

PR

PR

Toa

Alta

18

° 20

.222

' N

66°

12.8

48' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

80

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

81

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

82

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

83

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

84

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

85

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

41

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

18

JAR

886

U

S PR

PR

G

uaya

ma

17°

57.2

00' N

66

° 10

.872

' W

Lept

odac

tylu

s al

bila

bris

18

JA

R 8

87

US

PR

PR

Gua

yam

a 17

° 57

.200

' N

66°

10.8

72' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

750

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

751

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

752

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

753

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

754

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

755

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

756

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

757

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

758

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

19

JA

R 2

759

US

PR

PR

Gua

yam

a 17

° 57

.863

' N

66°

06.7

06' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

726

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

727

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

42

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

20

JAR

272

8 U

S PR

PR

A

guas

Bue

nas

18°

14.4

03' N

66

° 06

.239

' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

729

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

730

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

731

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

732

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

733

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

734

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

20

JA

R 2

735

US

PR

PR

Agu

as B

uena

s 18

° 14

.403

' N

66°

06.2

39' W

Lept

odac

tylu

s al

bila

bris

21

JA

R 2

987

US

PR

PR

Cay

ey

18°

07.3

39' N

66

° 04

.698

' W

Lept

odac

tylu

s al

bila

bris

21

JA

R 2

988

US

PR

PR

Cay

ey

18°

07.3

39' N

66

° 04

.698

' W

Lept

odac

tylu

s al

bila

bris

21

JA

R 2

989

US

PR

PR

Cay

ey

18°

07.3

39' N

66

° 04

.698

' W

Lept

odac

tylu

s al

bila

bris

21

JA

R 2

990

US

PR

PR

Cay

ey

18°

07.3

39' N

66

° 04

.698

' W

Lept

odac

tylu

s al

bila

bris

21

JA

R 2

991

US

PR

PR

Cay

ey

18°

07.3

39' N

66

° 04

.698

' W

Lept

odac

tylu

s al

bila

bris

21

JA

R 2

992

US

PR

PR

Cay

ey

18°

07.3

39' N

66

° 04

.698

' W

43

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

22

JAR

298

2 U

S PR

PR

C

ayey

18

° 06

.578

' N

66°

04.2

89' W

Lept

odac

tylu

s al

bila

bris

22

JA

R 2

983

US

PR

PR

Cay

ey

18°

06.5

78' N

66

° 04

.289

' W

Lept

odac

tylu

s al

bila

bris

22

JA

R 2

984

US

PR

PR

Cay

ey

18°

06.5

78' N

66

° 04

.289

' W

Lept

odac

tylu

s al

bila

bris

22

JA

R 2

985

US

PR

PR

Cay

ey

18°

06.5

78' N

66

° 04

.289

' W

Lept

odac

tylu

s al

bila

bris

22

JA

R 2

986

US

PR

PR

Cay

ey

18°

06.5

78' N

66

° 04

.289

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

005

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

006

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

007

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

008

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

009

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

010

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

011

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

012

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

Lept

odac

tylu

s al

bila

bris

23

JA

R 3

013

US

PR

PR

Gur

abo

18°

18.1

45' N

65

° 59

.238

' W

44

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

23

JAR

301

4 U

S PR

PR

G

urab

o 18

° 18

.145

' N

65°

59.2

38' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

669

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

670

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

671

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

672

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

673

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

674

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

675

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

676

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

677

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

24

JA

R 2

678

US

PR

PR

Car

olin

a 18

° 26

.549

' N

65°

57.4

15' W

Lept

odac

tylu

s al

bila

bris

25

JA

R 2

38

US

PR

PR

Mau

nabo

18

° 01

.623

' N

65°

54.8

88' W

Lept

odac

tylu

s al

bila

bris

25

JA

R 2

39

US

PR

PR

Mau

nabo

18

° 01

.623

' N

65°

54.8

88' W

Lept

odac

tylu

s al

bila

bris

25

JA

R 2

40

US

PR

PR

Mau

nabo

18

° 01

.623

' N

65°

54.8

88' W

45

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

25

JAR

241

U

S PR

PR

M

auna

bo

18°

01.6

23' N

65

° 54

.888

' W

Lept

odac

tylu

s al

bila

bris

26

JA

R 2

577

US

PR

PR

Mau

nabo

18

° 01

.586

' N

65°

54.8

22' W

Lept

odac

tylu

s al

bila

bris

26

JA

R 2

578

US

PR

PR

Mau

nabo

18

° 01

.586

' N

65°

54.8

22' W

Lept

odac

tylu

s al

bila

bris

26

JA

R 2

579

US

PR

PR

Mau

nabo

18

° 01

.586

' N

65°

54.8

22' W

Lept

odac

tylu

s al

bila

bris

26

JA

R 2

580

US

PR

PR

Mau

nabo

18

° 01

.586

' N

65°

54.8

22' W

Lept

odac

tylu

s al

bila

bris

26

JA

R 2

581

US

PR

PR

Mau

nabo

18

° 01

.586

' N

65°

54.8

22' W

Lept

odac

tylu

s al

bila

bris

26

JA

R 2

582

US

PR

PR

Mau

nabo

18

° 01

.586

' N

65°

54.8

22' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

960

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

961

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

962

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

963

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

964

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

965

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

966

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

46

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

27

JAR

296

7 U

S PR

PR

Ju

ncos

18

° 14

.006

' N

65°

52.0

88' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

968

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

27

JA

R 2

969

US

PR

PR

Junc

os

18°

14.0

06' N

65

° 52

.088

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

929

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

930

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

931

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

932

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

933

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

934

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

935

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

936

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

937

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

28

JA

R 2

938

US

PR

PR

Río

Gra

nde

18°

23.4

27' N

65

° 46

.837

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

636

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

47

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

a lbi

labr

is

29

JAR

263

7 U

S PR

PR

Fa

jard

o 18

° 16

.246

' N

65°

42.8

82' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

638

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

639

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

640

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

641

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

642

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

643

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

644

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

29

JA

R 2

645

US

PR

PR

Faja

rdo

18°

16.2

46' N

65

° 42

.882

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

049

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

050

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

051

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

052

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

053

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

48

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

30

JAR

305

4 U

S PR

V

I V

I 18

° 06

.088

' N

65°

32.3

47' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

055

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

056

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

057

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

30

JA

R 3

058

US

PR

VI

VI

18°

06.0

88' N

65

° 32

.347

' W

Lept

odac

tylu

s al

bila

bris

31

JA

R 3

059

US

PR

VI

VI

18°

07.2

61' N

65

° 26

.204

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

943

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

944

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

945

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

946

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

947

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

948

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

949

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

950

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

49

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

32

JAR

295

1 U

S PR

C

U

CU

18

° 18

.631

' N

65°

17.9

69' W

Lept

odac

tylu

s al

bila

bris

32

JA

R 2

952

US

PR

CU

C

U

18°

18.6

31' N

65

° 17

.969

' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

131

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

132

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

133

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

134

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

135

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

136

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

137

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

138

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

139

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

140

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

33

JA

R 3

141

US

USV

I ST

T —

18

° 20

.401

' N

64°

58.1

24' W

Lept

odac

tylu

s al

bila

bris

34

B

B 7

788

US

USV

I ST

T —

18

° 19

.375

' N

64°

54.6

49' W

50

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

35

JAR

318

1 U

S U

SVI

STC

—

17

° 43

.898

' N

64°

51.6

45' W

Lept

odac

tylu

s al

bila

bris

35

JA

R 3

182

US

USV

I ST

C

—

17°

43.8

98' N

64

° 51

.645

' W

Lept

odac

tylu

s al

bila

bris

35

JA

R 3

183

US

USV

I ST

C

—

17°

43.8

98' N

64

° 51

.645

' W

Lept

odac

tylu

s al

bila

bris

35

JA

R 3

184

US

USV

I ST

C

—

17°

43.8

98' N

64

° 51

.645

' W

Lept

odac

tylu

s al

bila

bris

35

JA

R 3

185

US

USV

I ST

C

—

17°

43.8

98' N

64

° 51

.645

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

170

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

171

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

172

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

173

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

174

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

175

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

176

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

177

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

178

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

51

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

36

JAR

317

9 U

S U

SVI

STC

—

17

° 45

.544

' N

64°

45.9

12' W

Lept

odac

tylu

s al

bila

bris

36

JA

R 3

180

US

USV

I ST

C

—

17°

45.5

44' N

64

° 45

.912

' W

Lept

odac

tylu

s al

bila

bris

37

B

B 7

847

US

USV

I ST

C

—

17°

44.2

93' N

64

° 42

.910

' W

Lept

odac

tylu

s al

bila

bris

37

B

B 7

848

US

USV

I ST

C

—

17°

44.2

93' N

64

° 42

.910

' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

247

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

248

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

249

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

250

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

251

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

252

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

253

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

254

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

255

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

Lept

odac

tylu

s al

bila

bris

38

JA

R 3

256

US

USV

I ST

J —

18

° 19

.668

' N

64°

47.4

81' W

52

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

39

JAR

323

9 U

K

BV

I JV

D

—

18°

26.7

86' N

64

° 45

.078

' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

240

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

241

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

242

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

243

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

244

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

245

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

39

JA

R 3

246

UK

B

VI

JVD

—

18

° 26

.786

' N

64°

45.0

78' W

Lept

odac

tylu

s al

bila

bris

40

B

B 7

521

UK

B

VI

JVD

—

18

° 26

.861

' N

64°

44.4

04' W

Lept

odac

tylu

s al

bila

bris

40

B

B 7

522

UK

B

VI

JVD

—

18

° 26

.861

' N

64°

44.4

04' W

Lept

odac

tylu

s al

bila

bris

40

B

B 7

523

UK

B

VI

JVD

—

18

° 26

.861

' N

64°

44.4

04' W

Lept

odac

tylu

s al

bila

bris

41

B

B 7

491

UK

B

VI

TO

—

18°

25.3

51' N

64

° 38

.671

' W

Lept

odac

tylu

s al

bila

bris

42

JA

R 3

233

UK

B

VI

TO

—

18°

25.9

19' N

64

° 38

.268

' W

Lept

odac

tylu

s al

bila

bris

42

JA

R 3

234

UK

B

VI

TO

—

18°

25.9

19' N

64

° 38

.268

' W

53

Tab

le 1

. Con

tinue

d.

Spec

ies

Popu

latio

n nu

mbe

r Fi

eld

nu

mbe

r C

ount

ry

Stat

e Is

land

M

unic

ipal

ity

Lat

itude

(h

) L

atitu

de

(min

) L

ongi

tude

(h

) L

ongi

tude

(m

in)

Le

ptod

acty

lus

albi

labr

is

42

JAR

323

5 U

K

BV

I TO

—

18

° 25

.919

' N

64°

38.2

68' W

Lept

odac

tylu

s al

bila

bris

42

JA

R 3

236

UK

B

VI

TO

—

18°

25.9

19' N

64

° 38

.268

' W

Lept

odac

tylu

s al

bila

bris

42

JA

R 3

237

UK

B

VI

TO

—

18°

25.9

19' N

64

° 38

.268

' W

Lept

odac

tylu

s al

bila

bris

42

JA

R 3

238

UK

B

VI

TO

—

18°

25.9

19' N

64

° 38

.268

' W

54

Table 2. Phylogroup and associated population(s), population number(s), and haplotypes of

Leptodactylus albilabris. Phylogroups (PG) are clades supported by ≥ 90% posterior probability

values in the Bayesian Inference tree estimated for 140 unique cytochrome b haplotypes of L.

albilabris (Figure 2).

Phylogroup Population(s) Population number(s) Haplotypes

PG 1 Culebra 32 43, 60

PG 2 Carolina, Fajardo 24, 29 116, 126, 130

PG 3 Juana Díaz 2, Guayama 1 12, 18 68, 81

PG 4 Hatillo, Juana Díaz 1, Ciales 7, 10, 11 63, 82

PG 5 Aibonito 1, Aguas Buenas, Cayey 1 15, 20, 21 61, 100

PG 6 Ciales 11 21, 48

PG 7 Moca, Isabela, Yauco 2, 3, 6 17, 26, 73, 74

PG 8 Mayagüez, Guánica, Yauco, Hatillo, Peñuelas,

Juana Díaz, Ciales, Juana Díaz, Manatí,

Aibonito 2, Toa Alta, Guayama 1, Guayama 2,

Aguas Buenas, Cayey 1, Cayey 2, Gurabo,

Maunabo 2, Juncos, Culebra

4, 5, 6, 7, 9, 10,

11, 12, 13, 16,

17, 18, 19, 20,

21, 22, 23, 26,

27, 32

85, 91, 103,

104, 114, 115,

118, 127, 128,

129, 131, 132,

133, 136, 137,

138, 139, 140

PG 9 Cabo Rojo, Hatillo 1, 7 110, 119

PG 10 Manatí, Aguas Buenas 13, 20 83, 93

55

Table 2. Continued.

Phylogroup Population(s) Population number(s) Haplotypes

PG 11 Aibonito 1, Guayama 1, Guayama 2,

Aguas Buenas, Cayey 1, Cayey 2, Gurabo,

Carolina, Maunabo 1, Maunabo 2, Juncos,

Fajardo, Vieques 1, Vieques 2

15, 18, 19, 20, 21,

22, 23, 24, 25, 26,

27, 29, 30, 31

1, 2, 3, 4, 72,

84, 88, 90, 95,

97, 98, 99, 101,

102, 105, 134

PG 12 Isabela, Arecibo 3, 8 40, 42

PG 13 Dorado 14 12, 34

PG 14 Juana Díaz, Ciales, Manatí, Toa Alta,

Cayey 1

10, 11, 13, 17, 21 20, 38, 54

PG 15 Moca 2 19, 23

PG 16 Cabo Rojo, Guánica, Juncos, Río Grande 1, 5, 27, 28 9, 29, 31, 32, 76

PG 17 Guayama 2, Gurabo, Juncos, Río Grande 19, 23, 27, 28 14, 15, 36, 49,

69, 75

PG 18 Carolina, Río Grande 24, 28 55, 57

56

Table 3. Island, population number, municipality, and unique cytochrome b haplotype(s)

detected in 42 populations of Leptodactylus albilabris.

Island Population number Municipality Haplotype

Puerto Rico 1 Cabo Rojo 9, 11, 29, 31, 32, 37, 44, 110

Puerto Rico 2 Moca 19, 23, 24, 35, 73, 92, 96

Puerto Rico 3 Isabela 7, 17, 25, 41, 42, 74

Puerto Rico 4 Mayagüez 18, 70, 96, 127

Puerto Rico 5 Guánica 9, 11, 16, 29, 44, 46, 115

Puerto Rico 6 Yauco 26, 47, 71, 96, 115, 117, 120

Puerto Rico 7 Hatillo 82, 86, 87, 96, 115, 119, 125

Puerto Rico 8 Arecibo 35, 40, 94, 96

Puerto Rico 9 Peñuelas 115, 138

Puerto Rico 10 Juana Díaz 1 20, 38, 63, 115

Puerto Rico 11 Ciales 21, 38, 39, 48, 63, 96, 103, 106, 139

Puerto Rico 12 Juana Díaz 2 11, 68, 85, 96, 115, 128, 140

Puerto Rico 13 Manatí 20, 52, 71, 83, 86, 96, 133

Puerto Rico 14 Dorado 12, 27, 34, 51, 52, 123

Puerto Rico 15 Aibonito 1 1, 11, 96, 100, 124

Puerto Rico 16 Aibonito 2 129

Puerto Rico 17 Toa Alta 13, 20, 27, 52, 91, 115, 124, 133

Puerto Rico 18 Guayama 1 1, 81, 115, 131

57

Table 3. Continued.

Island Population number Municipality Haplotype

Puerto Rico 19 Guayama 2 1, 11, 15, 22, 75, 115

Puerto Rico 20 Aguas Buenas 53, 61, 66, 72, 84, 88, 89, 91, 93, 134

Puerto Rico 21 Cayey 1 2, 6, 33, 54, 91, 100, 137

Puerto Rico 22 Cayey 2 1, 79, 118, 135

Puerto Rico 23 Gurabo 1, 49, 78, 80, 96, 115

Puerto Rico 24 Carolina 6, 50, 55, 67, 95, 105, 126

Puerto Rico 25 Maunabo 1 6, 102, 112, 122

Puerto Rico 26 Maunabo 2 1, 101, 114, 115, 132

Puerto Rico 27 Juncos 1, 6, 11, 25, 65, 69, 76, 77, 113, 115

Puerto Rico 28 Río Grande 6, 9, 14, 36, 56, 57, 58, 121

Puerto Rico 29 Fajardo 1, 11, 59, 62, 90, 116, 126, 130

Vieques 1 30 – 3, 6, 64, 97, 98, 99

Vieques 2 31 – 4

Culebra 32 – 43, 45, 60, 104, 136

Saint Thomas 1 33 – 10, 28

Saint Thomas 2 34 – 8

Saint Croix 1 35 – 10

Saint Croix 2 36 – 10, 30

Saint Croix 3 37 – 10

58

Table 3. Continued.

Island Population number Municipality Haplotype

Saint John 38 — 5, 6, 109

Jost Van Dyke 39 — 6, 111

Jost Van Dyke 40 — 6

Tortola 41 — 6

Tortola 42 — 6, 107, 108

59

Tab

le 4

. Isl

and,

pop

ulat

ion

num

ber,

mun

icip

ality

, sam

ple

size

, num

ber o

f uni

que

hapl

otyp

es, h

aplo

type

div

ersi

ty (H

d), a

nd n

ucle

otid

e

dive

rsity

(πd)

est

imat

ed u

sing

a c

ytoc

hrom

e b

frag

men

t (82

8 ba

se p

airs

) fro

m 3

22 sp

ecim

ens o

f Lep

toda

ctyl

us a

lbila

bris

from

42

loca

litie

s acr

oss t

he P

uerto

Ric

an B

ank.

I es

timat

ed H

d and

πd u

sing

AR

ELEQ

UIN

3.5

.2.2

(Exc

offie

r and

Lis

cher

201

0) fo

r loc

aliti

es

repr

esen

ted

by ≥

5 in

divi

dual

s.

Isla

nd

Popu

latio

n nu

mbe

r M

unic

ipal

ity

Sam

ple

size

Num

ber

of

uniq

ue

hapl

otyp

es

Hap

loty

pe

dive

rsity

(Hd)

N

ucle

otid

e di

vers

ity (π

)

Puer

to R

ico

1 C

abo

Roj

o 10

8

0.96

+/-

0.06

0.

0039

+/-

0.00

25

Puer

to R

ico

2 M

oca

8 7

0.96

+/-

0.08

0.

0051

+/-

0.00

32

Puer

to R

ico

3 Is

abel

a 10

6

0.89

+/-

0.08

0.

0059

+/-

0.00

35

Puer

to R

ico

4 M

ayag

üez

5 4

0.90

+/-

0.16

0.

0053

+/-

0.00

37

Puer

to R

ico

5 G

uáni

ca

10

7 0.

91 +

/- 0.

08

0.00

47 +

/- 0.

0029

Puer

to R

ico

6 Y

auco

10

7

0.91

+/-

0.09

0.

0048

+/-

0.00

29

Puer

to R

ico

7 H

atill

o 10

7

0.91

+/-

0.10

0.

0063

+/-

0.00

38

Puer

to R

ico

8 A

reci

bo

5 4

0.90

+/-

0.16

0.

0051

+/-

0.00

35

Puer

to R

ico

9 Pe

ñuel

as

8 2

0.25

+/-

0.18

0.

0006

+/-

0.00

06

60

Tab

le 4

. Con

tinue

d.

Isla

nd

Popu

latio

n nu

mbe

r M

unic

ipal

ity

Sam

ple

size

Num

ber

of

uniq

ue

hapl

otyp

es

Hap

loty

pe

dive

rsity

(Hd)

N

ucle

otid

e di

vers

ity (π

)

Puer

to R

ico

10

Juan

a D

íaz

1

7 4

0.86

+/-

0.10

0.

0078

+/-

0.00

48

Puer

to R

ico

11

Cia

les

12

9 0.

95 +

/- 0

.05

0.00

88 +

/- 0.

0050

Puer

to R

ico

12

Juan

a D

íaz

2 10

7

0.87

+/-

0.11

0.

0045

+/-

0.00

28

Puer

to R

ico

13

Man

atí

9 7

0.92

+/-

0.09

0.

0074

+/-

0.00

44

Puer

to R

ico

14

Dor

ado

10

6 0.

89 +

/- 0.

08

0.00

85 +

/- 0.

0049

Puer

to R

ico

15

Aib

onito

1

9 5

0.86

+/-

0.09

0.

0058

+/-

0.00

35

Puer

to R

ico

16

Aib

onito

2

1 —

—

—

Puer

to R

ico

17

Toa

Alta

10

8

0.96

+/-

0.06

0.

0073

+/-

0.00

43

Puer

to R

ico

18

Gua

yam

a 1

8 4

0.82

+/-

0.10

0.

0058

+/-

0.00

36

Puer

to R

ico

19

Gua

yam

a 2

10

6 0.

89 +

/- 0.

08

0.00

53 +

/- 0.

0032

Puer

to R

ico

20

Agu

as B

uena

s 10

10

1.

00 +

/- 0.

04

0.01

21 +

/- 0.

0068

Puer

to R

ico

21

Cay

ey 1

6

6 1.

00 +

/- 0.

10

0.01

10 +

/- 0.

0068

Puer

to R

ico

22

Cay

ey 2

5

5 1.

00 +

/- 0.

13

0.01

04 +

/- 0.

0068

61

Tab

le 4

. Con

tinue

d.

Isla

nd

Popu

latio

n nu

mbe

r M

unic

ipal

ity

Sam

ple

size

Num

ber

of

uniq

ue

hapl

otyp

es

Hap

loty

pe

dive

rsity

(Hd)

N

ucle

otid

e di

vers

ity (π

)

Puer

to R

ico

23

Gur

abo

10

6 0.

87 +

/- 0.

09

0.00

92 +

/- 0.

0053

Puer

to R

ico

24

Car

olin

a 10

7

0.93

+/-

0.06

0.

0111

+/-

0.00

63

Puer

to R

ico

25

Mau

nabo

1

4 4

—

—

Puer

to R

ico

26

Mau

nabo

2

6 5

0.93

+/-

0.12

0.

0066

+/-

0.00

43

Puer

to R

ico

27

Junc

os

10

10

1.00

+/-

0.04

0.

0072

+/-

0.00

42

Puer

to R

ico

28

Río

Gra

nde

10

8 0.

96 +

/- 0.

06

0.00

82 +

/- 0.

0048

Puer

to R

ico

29

Faja

rdo

10

8 0.

93 +

/- 0.

08

0.01

04 +

/- 0.

0060

Vie

ques

1

30

—

10

6 0.

89 +

/- 0.

08

0.00

74 +

/- 0.

0044

Vie

ques

2

31

—

1 —

—

—

Cul

ebra

32

—

10

5

0.84

+/-

0.0

8 0.

0097

+/-

0.00

56

Sain

t Tho

mas

1

33

—

11

2 0.

51 +

/- 0

.10

0.00

00 +

/- 0.

0000

Sain

t Tho

mas

2

34

—

1 —

—

—

62

Tab

le 4

. Con

tinue

d.

Isla

nd

Popu

latio

n nu

mbe

r M

unic

ipal

ity

Sam

ple

size

Num

ber

of

uniq

ue

hapl

otyp

es

Hap

loty

pe

dive

rsity

(Hd)

N

ucle

otid

e di

vers

ity (π

)

Sain

t Cro

ix 1

35

—

5

1 0.

00 +

/- 0.

00

0.00

00 +

/- 0.

0000

Sain

t Cro

ix 2

36

—

11

2

0.18

+/-

0.1

4 0.

0002

+/-

0.00

03

Sain

t Cro

ix 3

37

—

2

—

—

—

Sain

t Joh

n 38

—

10

3

0.69

+/-

0.10

0.

0011

+/-

0.00

09

Jost

Van

Dyk

e 1

39

—

8 2

0.25

+/-

0.18

0.

0003

+/-

0.00

04

Jost

Van

Dyk

e 2

40

—

2 —

—

—

Torto

la 1

41

—

1

—

—

—

Torto

la 2

42

—

6

3 0.

60 +

/- 0.

22

0.00

04 +

/- 0.

0005

63

Tab

le 5

. Pai

rwis

e F s

t val

ues a

mon

g 35

pop

ulat

ions

of L

epto

dact

ylus

alb

ilabr

is re

pres

ente

d by

≥ 5

indi

vidu

als.

Val

ues w

ere

estim

ated

from

322

cyt

ochr

ome

b se

quen

ces.

Popu

latio

ns a

re li

sted

from

wes

t to

east

.

Popu

latio

n C

abo

Roj

o M

oca

Isab

ela

May

agüe

z G

uáni

ca

Yau

co

Hat

illo

Are

cibo

C

abo

Roj

o 0.

00

—

—

—

—

—

—

—

Moc

a 0.

0402

0.

0 —

—

—

—

—

—

Isab

ela

0.07

78

0.07

47

0.0

—

—

—

—

—

May

agüe

z 0.

0687

0.

0159

0.

1063

0.

0 —

—

—

—

Guá

nica

0.

0071

0.

0632

0.

1000

0.

0937

0.

0 —

—

—

Yau

co

0.06

67

0.03

91

0.10

00

0.01

50

0.07

03

0.0

—

—

Hat

illo

0.06

67

0.05

13

0.10

00

0.05

60

0.03

07

0.04

09

0.0

—

Are

cibo

0.

0687

0.

0159

0.

1063

0.

0217

0.

0937

0.

0560

0.

0752

0.

0

Peñu

elas

0.

3748

0.

3929

0.

4092

0.

4815

0.

2748

0.

3420

0.

1924

0.

4815

Juan

a D

íaz

1 0.

0907

0.

0880

0.

1260

0.

1231

0.

0607

0.

0882

0.

0315

0.

1231

Cia

les

0.04

50

0.02

04

0.07

75

0.00

28

0.06

67

0.03

46

0.05

09

0.03

69

Juan

a D

íaz

2 0.

0797

0.

0746

0.

1222

0.

0821

0.

0232

0.

0544

-0

.021

7 0.

1010

64

Tab

le 5

. Con

tinue

d.

Popu

latio

n C

abo

Roj

o M

oca

Isab

ela

May

agüe

z G

uáni

ca

Yau

co

Hat

illo

Are

cibo

M

anat

í 0.

0636

0.

0467

0.

0975

0.

0485

0.

0862

0.

0324

0.

0654

0.

0701

Dor

ado

0.07

78

0.07

47

0.11

11

0.10

63

0.10

00

0.10

00

0.10

00

0.10

63

Aib

onito

0.

0703

0.

0622

0.

1248

0.

0358

0.

0933

0.

0723

0.

0933

0.

0808

Toa

Alta

0.

0444

0.

0402

0.

0778

0.

0687

0.

0278

0.

0476

0.

0071

0.

0687

Gua

yam

a 1

0.10

90

0.10

71

0.14

35

0.14

36

0.06

19

0.09

83

0.02

25

0.14

36

Gua

yam

a 2

0.06

85

0.07

47

0.11

11

0.10

63

0.07

22

0.09

09

0.07

22

0.10

63

Agu

as B

uena

s 0.

0222

0.

0174

0.

0556

0.

0440

0.

0444

0.

0444

0.

0444

0.

0440

Cay

ey 1

0.

0239

0.

0187

0.

0602

0.

0480

0.

0481

0.

0481

0.

0481

0.

0480

Cay

ey 2

0.

0248

0.

0194

0.

0625

0.

0500

0.

0498

0.

0498

0.

0498

0.

0500

Gur

abo

0.08

89

0.07

46

0.12

22

0.08

21

0.05

44

0.06

43

0.01

24

0.10

10

Car

olin

a 0.

0556

0.

0517

0.

0889

0.

0812

0.

0778

0.

0778

0.

0778

0.

0812

Mau

nabo

2

0.05

46

0.05

04

0.09

09

0.08

26

0.04

69

0.06

31

0.03

02

0.08

26

65

Tab

le 5

. Con

tinue

d.

Popu

latio

n C

abo

Roj

o M

oca

Isab

ela

May

agüe

z G

uáni

ca

Yau

co

Hat

illo

Are

cibo

Ju

ncos

0.

0124

0.

0174

0.

0363

0.

0440

0.

0149

0.

0348

0.

0149

0.

0440

Río

Gra

nde

0.02

49

0.04

02

0.07

78

0.06

87

0.05

72

0.06

67

0.06

67

0.06

87

Faja

rdo

0.04

60

0.05

17

0.08

89

0.08

12

0.06

85

0.07

78

0.07

78

0.08

12

Vie

ques

0.

0778

0.

0747

0.

1111

0.

1063

0.

1000

0.

1000

0.

1000

0.

1063

Cul

ebra

0.

1000

0.

0978

0.

1333

0.

1318

0.

1222

0.

1222

0.

1222

0.

1318

Sain

t Tho

mas

0.

2724

0.

2805

0.

3053

0.

3390

0.

2944

0.

2944

0.

2944

0.

3390

Sain

t Cro

ix 1

0.

4158

0.

4381

0.

4521

0.

5500

0.

4399

0.

4399

0.

4399

0.

5500

Sain

t Cro

ix 2

0.

4424

0.

4667

0.

4753

0.

5642

0.

4643

0.

4643

0.

4643

0.

5642

Sain

t Joh

n 0.

1778

0.

1795

0.

2111

0.

2235

0.

2000

0.

2000

0.

2000

0.

2235

Jost

Van

Dyk

e 0.

3748

0.

3929

0.

4092

0.

4815

0.

3977

0.

3977

0.

3977

0.

4815

Torto

la 2

0.

2018

0.

2051

0.

2379

0.

2584

0.

2258

0.

2258

0.

2258

0.

2584

66

Tab

le 5

. Con

tinue

d.

Popu

latio

n Pe

ñuel

as

Juan

a D

íaz

1 C

iale

s Ju

ana

Día

z 2

Man

atí

Dor

ado

Aib

onito

To

a A

lta

C

abo

Roj

o —

—

—

—

—

—

—

—

Moc

a —

—

—

—

—

—

—

—

Isab

ela

—

—

—

—

—

—

—

—

May

agüe

z —

—

—

—

—

—

—

—

Guá

nica

—

—

—

—

—

—

—

—

Yau

co

—

—

—

—

—

—

—

—

Hat

illo

—

—

—

—

—

—

—

—

Are

cibo

—

—

—

—

—

—

—

—

Peñu

elas

0.

0 —

—

—

—

—

—

—

Juan

a D

íaz

1 0.

2761

0.

0 —

—

—

—

—

—

Cia

les

0.35

97

0.02

09

0.0

—

—

—

—

—

Juan

a D

íaz

2 0.

1227

0.

0266

0.

0729

0.

0 —

—

—

—

67

Tab

le 5

. Con

tinue

d.

Popu

latio

n Pe

ñuel

as

Juan

a D

íaz

1 C

iale

s Ju

ana

Día

z 2

Man

atí

Dor

ado

Aib

onito

To

a A

lta

D

orad

o 0.

4092

0.

1260

0.

0775

0.

1222

0.

0663

0.

0 —

—

Aib

onito

0.

4331

0.

1408

0.

0555

0.

0959

0.

0886

0.

1248

0.

0 —

Toa

Alta

0.

2476

0.

0213

0.

0450

0.

0097

0.

0201

-0

.002

4 0.

0596

0.

0

Gua

yam

a 1

0.20

27

0.06

05

0.10

78

0.00

63

0.12

99

0.14

35

0.13

43

0.03

73

Gua

yam

a 2

0.35

44

0.10

03

0.07

75

0.07

60

0.09

75

0.11

11

0.08

41

0.05

90

Agu

as B

uena

s 0.

3520

0.

0674

0.

0232

0.

0667

0.

0411

0.

0556

0.

0684

0.

0124

Cay

ey 1

0.

4106

0.

0736

0.

0249

0.

0725

0.

0445

0.

0602

0.

0568

0.

0073

Cay

ey 2

0.

4370

0.

0767

0.

0258

0.

0752

0.

0461

0.

0625

0.

0561

0.

0248

Gur

abo

0.22

27

0.05

70

0.07

29

0.00

38

0.09

87

0.12

22

0.10

63

0.03

07

Car

olin

a 0.

3863

0.

1024

0.

0558

0.

1000

0.

0749

0.

0889

0.

1022

0.

0556

Mau

nabo

2

0.34

20

0.06

12

0.05

50

0.03

86

0.07

56

0.09

09

0.07

10

0.02

21

68

Tab

le 5

. Con

tinue

d.

Popu

latio

n Pe

ñuel

as

Juan

a D

íaz

1 C

iale

s Ju

ana

Día

z 2

Man

atí

Dor

ado

Aib

onito

To

a A

lta

Ju

ncos

0.

2923

0.

0403

0.

0232

0.

0175

0.

0411

0.

0556

0.

0364

0.

0023

Faja

rdo

0.38

63

0.10

24

0.05

58

0.09

09

0.07

49

0.08

89

0.04

95

0.05

56

Vie

ques

0.

4092

0.

1260

0.

0775

0.

1222

0.

0975

0.

1111

0.

1248

0.

0778

Cul

ebra

0.

4322

0.

1496

0.

0991

0.

1444

0.

1201

0.

1333

0.

1474

0.

1000

Sain

t Tho

mas

0.

6048

0.

3380

0.

2641

0.

3163

0.

2969

0.

3053

0.

3238

0.

2724

Sain

t Cro

ix 1

0.

8446

0.

5143

0.

3983

0.

4643

0.

4488

0.

4521

0.

4791

0.

4158

Sain

t Cro

ix 2

0.

7891

0.

5343

0.

4223

0.

4862

0.

4742

0.

4753

0.

5011

0.

4424

Sain

t Joh

n 0.

5136

0.

2339

0.

1741

0.

2222

0.

1996

0.

2111

0.

2269

0.

1778

Jost

Van

Dyk

e 0.

7500

0.

4603

0.

3597

0.

4207

0.

4047

0.

4092

0.

4331

0.

3748

Torto

la 2

0.

5956

0.

2659

0.

1970

0.

2500

0.

2261

0.

2379

0.

2559

0.

2018

69

Tab

le 5

. Con

tinue

d.

Popu

latio

n G

uaya

ma

1 G

uaya

ma

2 A

guas

Bue

nas

Cay

ey 1

C

ayey

2

Gur

abo

Car

olin

a M

auna

bo 2

C

abo

Roj

o —

—

—

—

—

—

—

—

Moc

a —

—

—

—

—

—

—

—

Isab

ela

—

—

—

—

—

—

—

—

May

agüe

z —

—

—

—

—

—

—

—

Guá

nica

—

—

—

—

—

—

—

—

Yau

co

—

—

—

—

—

—

—

—

Hat

illo

—

—

—

—

—

—

—

—

Are

cibo

—

—

—

—

—

—

—

—

Peñu

elas

—

—

—

—

—

—

—

—

Juan

a D

íaz

1 —

—

—

—

—

—

—

—

Cia

les

—

—

—

—

—

—

—

—

Juan

a D

íaz

2 —

—

—

—

—

—

—

—

70

Tab

le 5

. Con

tinue

d.

Popu

latio

n G

uaya

ma

1 G

uaya

ma

2 A

guas

Bue

nas

Cay

ey 1

C

ayey

2

Gur

abo

Car

olin

a M

auna

bo 2

M

anat

í —

—

—

—

—

—

—

—

Dor

ado

—

—

—

—

—

—

—

—

Aib

onito

—

—

—

—

—

—

—

—

Toa

Alta

—

—

—

—

—

—

—

—

Gua

yam

a 1

0.0

—

—

—

—

—

—

—

Gua

yam

a 2

0.06

16

0.0

—

—

—

—

—

—

Agu

as B

uena

s 0.

0862

0.

0556

0.

0 —

—

—

—

—

Cay

ey 1

0.

0943

0.

0602

-0

.017

0 0.

0 —

—

—

—

Cay

ey 2

0.

0499

0.

0228

0.

0 0.

0 0.

0 —

—

—

Gur

abo

0.02

06

0.07

60

0.06

67

0.07

25

0.05

60

0.0

—

—

Car

olin

a 0.

1205

0.

0889

0.

0333

0.

0360

0.

0174

0.

1000

0.

0 —

Mau

nabo

2

-0.0

244

0.00

78

0.03

07

0.03

33

-0.0

345

0.02

09

0.06

67

0.0

71

Tab

le 5

. Con

tinue

d.

Popu

latio

n G

uaya

ma

1 G

uaya

ma

2 A

guas

Bue

nas

Cay

ey 1

C

ayey

2

Gur

abo

Car

olin

a M

auna

bo 2

Ju

ncos

0.

0258

0.

0162

0.

0 0.

0 -0

.041

7 0.

0278

0.

0236

-0

.019

9

Faja

rdo

0.04

96

0.02

03

0.03

33

0.03

60

-0.0

248

0.07

22

0.05

72

-0.0

370

Vie

ques

0.

1435

0.

1111

0.

0556

0.

0602

0.

0228

0.

1222

0.

0703

0.

0909

Cul

ebra

0.

1666

0.

1333

0.

0778

0.

0848

0.

0881

0.

1444

0.

1111

0.

1154

Sain

t Tho

mas

0.

3480

0.

3053

0.

2505

0.

2828

0.

2969

0.

3163

0.

2834

0.

3127

Sain

t Cro

ix 1

0.

5153

0.

4521

0.

3919

0.

4643

0.

5000

0.

4643

0.

4278

0.

4988

Sain

t Cro

ix 2

0.

5339

0.

4753

0.

4204

0.

4931

0.

5237

0.

4862

0.

4534

0.

5224

Sain

t Joh

n 0.

2482

0.

2111

0.

1556

0.

1724

0.

1252

0.

2222

0.

1638

0.

2029

Jost

Van

Dyk

e 0.

4643

0.

4092

0.

3520

0.

4106

0.

3062

0.

4207

0.

3295

0.

4421

Torto

la 2

0.

2805

0.

2379

0.

1781

0.

2000

0.

0870

0.

2500

0.

1597

0.

2333

72

Tab

le 5

. Con

tinue

d.

Popu

latio

n Ju

ncos

R

io G

rand

e Fa

jard

o V

iequ

es

Cul

ebra

Sa

int T

hom

as

Sain

t Cro

ix 1

Cab

o R

ojo

—

—

—

—

—

—

—

Moc

a —

—

—

—

—

—

—

Isab

ela

—

—

—

—

—

—

—

May

agüe

z —

—

—

—

—

—

—

Guá

nica

—

—

—

—

—

—

—

Yau

co

—

—

—

—

—

—

—

Hat

illo

—

—

—

—

—

—

—

Are

cibo

—

—

—

—

—

—

—

Peñu

elas

—

—

—

—

—

—

—

Juan

a D

íaz

1 —

—

—

—

—

—

—

Cia

les

—

—

—

—

—

—

—

Juan

a D

íaz

2 —

—

—

—

—

—

—

73

Tab

le 5

. Con

tinue

d.

Popu

latio

n Ju

ncos

R

io G

rand

e Fa

jard

o V

iequ

es

Cul

ebra

Sa

int T

hom

as

Sain

t Cro

ix 1

Man

atí

—

—

—

—

—

—

—

Dor

ado

—

—

—

—

—

—

—

Aib

onito

—

—

—

—

—

—

—

Toa

Alta

—

—

—

—

—

—

—

Gua

yam

a 1

—

—

—

—

—

—

—

Gua

yam

a 2

—

—

—

—

—

—

—

Agu

as B

uena

s —

—

—

—

—

—

—

Cay

ey 1

—

—

—

—

—

—

—

Cay

ey 2

—

—

—

—

—

—

—

Gur

abo

—

—

—

—

—

—

—

Car

olin

a —

—

—

—

—

—

—

Mau

nabo

2

—

—

—

—

—

—

—

74

Tab

le 5

. Con

tinue

d.

Popu

latio

n Ju

ncos

R

io G

rand

e Fa

jard

o V

iequ

es

Cul

ebra

Sa

int T

hom

as

Sain

t Cro

ix 1

Junc

os

0.0

—

—

—

—

—

—

Río

Gra

nde

0.00

23

0.0

—

—

—

—

—

Faja

rdo

-0.0

069

0.05

56

0.0

—

—

—

—

Vie

ques

0.

0363

0.

0394

0.

0889

0.

0 —

—

—

Cul

ebra

0.

0778

0.

1000

0.

1111

0.

1333

0.

0 —

—

Sain

t Tho

mas

0.

2505

0.

2724

0.

2834

0.

3053

0.

3272

0.

0 —

Sain

t Cro

ix 1

0.

3919

0.

4158

0.

4278

0.

4521

0.

4766

0.

1791

0.

0

Sain

t Cro

ix 2

0.

4204

0.

4424

0.

4534

0.

4753

0.

4971

0.

1804

-0

.089

1

Sain

t Joh

n 0.

1294

0.

1253

0.

1889

0.

1608

0.

2333

0.

4034

0.

5646

Jost

Van

Dyk

e 0.

2923

0.

2476

0.

3863

0.

2885

0.

4322

0.

6048

0.

8446

Torto

la 2

0.

1220

0.

0842

0.

2138

0.

1247

0.

2622

0.

4543

0.

6739

75

Tab

le 5

. Con

tinue

d.

Popu

latio

n Sa

int C

roix

2

Sain

t Joh

n Jo

st V

an D

yke

Torto

la 2

C

abo

Roj

o —

—

—

—

Moc

a —

—

—

—

Isab

ela

—

—

—

—

May

agüe

z —

—

—

—

Guá

nica

—

—

—

—

Yau

co

—

—

—

—

Hat

illo

—

—

—

—

Are

cibo

—

—

—

—

Peñu

elas

—

—

—

—

Juan

a D

íaz

1 —

—

—

—

Cia

les

—

—

—

—

Juan

a D

íaz

2 —

—

—

—

76

Tab

le 5

. Con

tinue

d.

Popu

latio

n Sa

int C

roix

2

Sain

t Joh

n Jo

st V

an D

yke

Torto

la 2

M

anat

í —

—

—

—

Dor

ado

—

—

—

—

Aib

onito

—

—

—

—

Toa

Alta

—

—

—

—

Gua

yam

a 1

—

—

—

—

Gua

yam

a 2

—

—

—

—

Agu

as B

uena

s —

—

—

—

Cay

ey 1

—

—

—

—

Cay

ey 2

—

—

—

—

Gur

abo

—

—

—

—

Car

olin

a —

—

—

—

Mau

nabo

2

—

—

—

—

77

Tab

le 5

. Con

tinue

d.

Popu

latio

n Sa

int C

roix

2

Sain

t Joh

n Jo

st V

an D

yke

Torto

la 2

Ju

ncos

—

—

—

—

Río

Gra

nde

—

—

—

—

Faja

rdo

—

—

—

—

Vie

ques

—

—

—

—

Cul

ebra

—

—

—

—

Sain

t Tho

mas

—

—

—

—

Sain

t Cro

ix 1

—

—

—

—

Sain

t Cro

ix 2

0.

0 —

—

—

Sain

t Joh

n 0.

5733

0.

0 —

—

Jost

Van

Dyk

e 0.

7891

0.

3454

0.

0 —

Torto

la 2

0.

6609

0.

1882

-0

.001

1 0.

0

78

Table 6. Island, population number, municipality, number of samples, heterozygosity, and

number of loci calculated from 16,225 SNPs yielded from double digest restriction-site

associated DNA sequencing of 114 individuals of Leptodactylus albilabris.

Island Population number Municipality Number of

samples Heterozygosity Number of loci

Puerto Rico 1 Cabo Rojo 4 0.1778 +/- 0.0021 11311

Puerto Rico 2 Moca 3 0.1737 +/- 0.0021 14700

Puerto Rico 3 Isabela 4 0.174 +/- 0.002 14118

Puerto Rico 4 Mayagüez 2 0.206 +/- 0.0027 12951

Puerto Rico 6 Yauco 5 0.1793 +/- 0.0018 13206

Puerto Rico 10 Juana Díaz 5 0.1818 +/- 0.0018 13042

Puerto Rico 11 Ciales 4 0.2016 +/- 0.0021 12564

Puerto Rico 13 Manatí 4 0.2023 +/- 0.002 14590

Puerto Rico 14 Dorado 5 0.1894 +/- 0.0019 14419

Puerto Rico 15 Aibonito 3 0.2196 +/- 0.0023 14643

Puerto Rico 19 Guayama 5 0.1914 +/- 0.0018 14413

Puerto Rico 20 Aguas Buenas 4 0.2325 +/- 0.0023 12343

Puerto Rico 24 Carolina 5 0.1786 +/- 0.0018 13483

Puerto Rico 28 Río Grande 5 0.1919 +/- 0.0018 14689

Puerto Rico 29 Fajardo 5 0.2030 +/- 0.0018 14241

79

Table 6. Continued.

Island Population number Municipality Number of

samples Heterozygosity Number of loci

Vieques 1 30 — 5 0.1722 +/- 0.0018 14069

Culebra 32 — 5 0.1214 +/- 0.0017 14003

Saint Thomas 1 33 — 7 0.1085 +/- 0.0016 14129

Saint Croix 2 36 — 7 0.0979 +/- 0.0015 14112

Saint John 38 — 10 0.1039 +/- 0.0015 13637

Jost Van Dyke 1 39 — 11 0.0993 +/- 0.0015 14140

Tortola 2 42 — 6 0.1326 +/- 0.0017 14752

80

Figure 1. Map of the major islands of the Puerto Rican Bank, in the eastern Caribbean Sea.

Orange circles indicate the approximate geographic locations of the sampling localities of

Leptodactylus albilabris. The approximate land configuration at maximum sea level lowering

(ca. –134 m) during the Last Glacial Maximum (ca. 29,000 – 21,000 ya; Lambeck et al., 2014) is

shown in grey. Note the changes in area, isolation, and connectivity of islands. AN = Anegada,

CU = Culebra, JVD = Jost Van Dyke, PR = Puerto Rico, STC = Saint Croix, STJ = Saint John,

STT = Saint Thomas, TO = Tortola, VG = Virgin Gorda, VI = Vieques.

81

82

Figure 2. Bayesian Inference tree (generated by MrBayes 3.2.6; Ronquist et al., 2012) estimated

for 140 unique cytochrome b haplotypes of Leptodactylus albilabris, using L. poecilochilus as

the outgroup. Numbers at nodes designated by black circles are ≥ 90% posterior probability

values. Colored (green, yellow, blue, red, grey) branches represent the physiographic region(s) of

Puerto Rico where the haplotype occurs; white branches indicate that the haplotype is present in

one or more Eastern Island (CU = Culebra, VI = Vieques, TO = Tortola, STT = Saint Thomas,

STC = Saint Croix, STJ = Saint John, JVD = Jost Van Dyke). Taxon labels consist of the

population number(s) where a particular haplotype was detected, followed by the specific

haplotype number (preceded by the letter H) in parentheses. Asterisks indicate the two most

inclusive, well-supported phylogroups. The light grey box indicates the clade consisting of

Vieques 1, 2 (Populations 30, 31, respectively). PG = phylogroup.

83

84

Figure 2. Continued.

85

Figure 3. Maximum parsimony network (estimated with NETWORK 5.0.0.3; Bandelt et al.,

1999) representing the relationships among 140 unique cytochrome b haplotypes recovered from

322 samples of Leptodactylus albilabris. Colors indicate the island or region where the

haplotypes occur (i.e. Puerto Rico, Vieques, Culebra, Saint Thomas, Saint John, Saint Croix, Jost

Van Dyke, Tortola). Numbered circles represent unique haplotypes; hatch marks indicate single

mutations; black squares indicate missing (i.e. unsampled or extinct) haplotypes. Circle size is

proportional to haplotype frequency. The three haplotypes with an inner circle (i.e. 1, 88; 17, 73;

28, 108, 121) represent merged haplotypes, for NETWORK recognizes these haplotypes as

equivalent. Specifically, haplotypes 1 and 88 and haplotypes 17 and 73 differ by a single,

unspecified base pair (N), whereas haplotypes 28, 108, and 121 differ by three unspecified base

pairs.

86

87

Figure 4. Genetic clustering of 16,140 single nucleotide polymorphic loci estimated by the R

package LEA (Frichot and François, 2015). The five genetically distinct groups are color coded

(light grey, yellow, blue, red, dark grey). Pie charts indicate the approximate geographic

locations of the sampling localities, and illustrate the proportion of each genetic group in a given

population. Sparse non-negative matrix factorization (snmf) analysis shows the assignment of

each individual sample in a population to a particular genetic group. Populations are listed below

the bar plots, from west to east. PR = Puerto Rico, VI = Vieques, CU = Culebra, STT = Saint

Thomas, STJ = Saint John, STC = Saint Croix, JVD = Jost Van Dyke, TO = Tortola.

88

89

Figure 5. Approximate configurations of the Puerto Rican Bank (PRB) at six different sea levels

(-10 m – -60 m) during the Last Glacial Maximum (ca. 29,000 – 21,000 ya; Lambeck et al.,

2014). Bathymetric data were obtained from the General Bathymetric Chart of the Oceans

https://www.gebco.net/.

A) 0 m. Present-day sea levels, illustrating the separation of the largest islands of the PRB.

B) -10 m. Saint John, Jost Van Dyke, Tortola, Virgin Gorda, and Anegada are connected by a

land bridge.

C) -20 m. Puerto Rico and Vieques are now connected by a land bridge; Saint Thomas is now

partially connected to Saint John by a land bridge.

D) -30 m. Puerto Rico, Vieques, and Culebra, are now connected by a land bridge; Saint Thomas

is now completely connected to Saint John, Jost Van Dyke, Tortola, Virgin Gorda, and Anegada

by a more extensive land bridge.

E) -40 m. All the islands of the PRB are now connected by a land bridge.

F) -50 m. The PRB has slightly greater subaerial exposure.

G) -60 m. The configuration and exposure of the PRB are very similar to those during maximum

sea level lowering (-134 m; Figure 1), due to the steep slopes of the PRB (Heatwole &

MacKenzie, 1967). Note that Saint Croix is never connected to the PRB.

AN = Anegada, CU = Culebra, JVD = Jost Van Dyke, PR = Puerto Rico, STC = Saint Croix,

STJ = Saint John, STT = Saint Thomas, TO = Tortola, VG = Virgin Gorda, VI = Vieques.

90

91

Figure 5. Continued.

92

References

Balinsky, J. B. 1981. Adaptation of nitrogen metabolism to hyperosmotic environment in

Amphibia. Journal of Experimental Zoology 215:335–350.

Bandelt, H. J., Forster, P., and A. Röhl. 1999. Median-joining networks for inferring intraspecific

phylogenies. Molecular Biology and Evolution 16:37–48.

Baptestini, E. M., de Aguiar, M. A. M., and Y. Bar–Yam. 2013. Conditions for neutral speciation

via isolation by distance. Journal of Theoretical Biology 335:51–56.

Barbour, T. 1930. Some faunistic changes in the Lesser Antilles. Proceedings of the New

England Zoologists’ Club 11:73-85.

Barker, B. S., and J. A. Rodríguez-Robles. 2017. Origins and genetic diversity of introduced

populations of the Puerto Rican Red-eyed Coquí, Eleutherodactylus antillensis, in Saint

Croix (U.S. Virgin Islands) and Panamá. Copeia 105:220–228.

Barker, B. S., Rodríguez-Robles, J. A., Aran, V. S., Montoya, A., Waide, R. B., and J. A. Cook.

2012. Sea level, topography, and island diversity: phylogeography of the Puerto Rican

Red-eyed Coquí, Eleutherodactylus antillensis. Molecular Ecology 21:6033–6052.

Barrow, L. N., Soto-Centeno, J. A., Warwick, A. R., Lemmon, A. R., and E. M. Lemmon. 2017.

Evaluating hypotheses of expansion from refugia through comparative phylogeography

of south-eastern Coastal Plain amphibians. Journal of Biogeography 44:2692–2705.

Bell, R. C., Drewes, R. C., Channing, A., Gvoždík, V., Kielgast, J., Lötters, S., Stuart, B. L., and

K. R. Zamudio. 2015. Overseas dispersal of Hyperolius reed frogs from Central Africa to

the oceanic islands of São Tomé and Príncipe. Journal of Biogeography 42:65–75.

Bintanja, R., van de Wal, R. S. W., and J. Oerlemans. 2005. Modelled atmospheric temperatures

and global sea levels over the past million years. Nature 437:125–128.

93

Calsbeek, R., and T. B. Smith. 2003. Ocean currents mediate evolution in island lizards. Nature

426:552–555.

Censky, E. J. 1989. Eleutherodactylus johnstonei (Salientia: Leptodactylidae) from Anguilla,

West Indies. Caribbean Journal of Science 25:229-230.

Censky, E. J., Hodge, K., and J. Dudley. 1998. Over-water dispersal of lizards due to hurricanes.

Nature 395:556.

Clouse, R. M., Janda, M., Blanchard, B., Sharma, P., Hoffman, B. D., Andersen,

A.N., Czekanski Moir, J. E., Krushelnycky, P., Rabeling, C., Wilson, E. O., Economo, E.

P., Sarnat, E. M., General, D. M., Alpert, G. D., and W. C. Wheeler. 2015. Molecular

phylogeny of Indo Pacific carpenter ants (Hymenoptera: Formicidae, Camponotus)

reveals waves of dispersal and colonization from diverse source areas. Cladistics 31:424–

437.

Cochran, D. M. 1923. A new frog of the genus Leptodactylus. Journal of the Washington

Academy of Sciences 13:184–185.

Cochran, D. M. 1941. The herpetology of Hispaniola. Smithsonian Institution, Bulletin of the

United States National Museum 177:1–398.

da Fonesca, R. R., Albrechtsen, A., Themudo, G. E., Ramos-Madrigal, J., Sibbesen, J. A.,

Maretty, L., Zepeda-Mendoza, M. L., Campos, P. F., Heller, R., and R. J. Pereira. 2016.

Next-generation biology: Sequencing and data analysis approaches for non-model

organisms. Marine Genomics 30:3–13.

Darriba, D., Taboada, G. L., Doallo, R., and D. Posada. 2012. jModelTest 2: more models, new

heuristics and parallel computing. Nature Methods 9:772.

94

Davison, A., and S. Chiba. 2008. Contrasting response to Pleistocene climate change by ground-

living and arboreal Mandarina snails from the oceanic Hahajima archipelago.

Philosophical Transactions of the Royal Society B 363:3391–3400.

Deli, T., Kiel, C., and C. D. Schubart. 2019. Phylogeographic and evolutionary history analyses

of the warty crab Eriphia verrucosa (Decapoda, Brachyura, Eriphiidae) unveil genetic

imprints of a late Pleistocene vicariant event across the Gibraltar Strait, erased by

postglacial expansion and admixture among refugial lineages. BMC Evolutionary

Biology 19: https://doi.org/10.1186/s12862-019-1423-2.

de Aguiar, M. A. M., Baranger, M., Baptestini, E. M., Kaufman, L., and Y. Bar–Yam. 2009.

Global patterns of speciation and diversity. Nature 460:384–387.

de Sá, R. O., Grant, T., Camargo, A., Heyer, W. R., Ponssa, M. L., and E. Stanley. 2014.

Systematics of the neotropical genus Leptodactylus Fitzinger, 1826 (Anura:

Leptodactyilidae): phylogeny, the relevance of non-molecular evidence, and species

accounts. South American Journal of Herpetology 9:1–128.

Dent, J. N. 1956. Observations on the life history and development of Leptodactylus albilabris.

Copeia 4:207–210.

Duellman, W. E., and L. Trueb. 1994. Biology of Amphibians. The John Hopkins University

Press, Baltimore, Maryland.

Duryea, M. C., Zamudio, K. R., and C. A. Brasileiro. 2015. Vicariance and marine migration in

continental island populations of a frog endemic to the Atlantic Coastal forest. Heredity

115:225–234.

95

Ewel, J. J., and J. L. Whitmore. 1973. The Ecological Life Zones of Puerto Rico and the U.S.

Virgin Islands. USDA Forest Service, Institute of Tropical Forestry, Research Paper:1–

72.

Excoffier, L., and H. E. L. Lischer. 2010. Arlequin Suite ver 3.5, a new series of programs to

perform population genetics analyses under Linux and Windows. Molecular Ecology

Resources 10:564–567.

Ezer, T. 2019. Numerical modeling of the impact of hurricanes on ocean dynamics: sensitivity of

the Gulf Stream response to storm’s track. Ocean Dynamics 69:1053–1066.

Falk, B. G., and S. L. Perkins. 2013. Host specificity shapes population structure of pinworm

parasites in Caribbean reptiles. Molecular Ecology 22:4576

Fernández-Palacios, J. M., Rijsdijk, K. F., Norder, S. J., Otto, R., de Nascimento, L., Fernández-

Lugo, S., Tjørve, E., and R. J. Whittaker. 2016. Towards a glacial-sensitive model of

island biogeography. Global Ecology and Biogeography 25:817–830.

Flores-Nieves, C. Z., Logue, D. M., and C. J. Santos-Flores. 2014. Native White-lipped frog

larvae (Leptodactylus albilabris) outcompete introduced Cane toad larvae (Rhinella

marina) under laboratory conditions. Herpetological Conservation and Biology 9:378–

386.

Frankham, R. 1997. Do island populations have less genetic variation than mainland

populations? Heredity 78:311–327.

Frichot, E., and O. François. 2015. LEA: An R package for landscape and ecological association

studies. Methods in Ecology and Evolution 6:925–929.

Frichot, E., Mathieu, F., Trouillon, T., Bouchard, G., and O. François. 2014. Fast and efficient

estimation of individual ancestry coefficients. Genetics 196:973–983.

96

Furlan, E., Stoklosa, J., Griffiths, J., Gust, N., Ellis, R., Huggins, R. M., and A. R. Weeks. Small

population size and extremely low levels of genetic diversity in island populations of the

platypus, Ornithorhynchus anatinus. Ecology and Evolution 2:844–857.

Garcia-Porta, J., Litvinchuk, S. N., Crochet, P. A., Romano, A., Geniez, P. H., Lo-Valvo, M.,

Lymberakis, P., and S. Carranza. 2012. Molecular phylogenetics and historical

biogeography of the west-palearctic common toads (Bufo bufo species complex).

Molecular Phylogenetics and Evolution 63:113–130.

Garg, K. M., Chattopadhyay, B., Wilton, P. R., Prawiradilaga, D. M., and F. E. Rheindt. 2018.

Pleistocene land bridges act as semipermeable agents of avian gene flow in Wallacea.

Molecular Phylogenetics and Evolution 125:196–203.

Gehara, M., Garda, A. A., Werneck, F. P., Oliviera, E. F., da Fonseca, E. M., Camurugi, F.,

Magalhães, F. M., Lanna, F. M., Sites, J. W., Marques, R., Silveira-Filho, R., São Pedro,

V. A., Colli, G. R., Costa, G. C., and F. T. Burbrink. 2017. Estimating synchronous

demographic changes across populations using hABC and its application for a

herpetological community from northeastern Brazil. Molecular Ecology 26:4756–4771.

Gill, I. P., Hubbard, D. K., McLaughlin, P., and C. Moore. 1989. Sedimentological and tectonic

evolution of Tertiary St. Croix, pp. 49–71. In: Hubbard, D. K. (ed.), 12th Caribbean

Geological Conference, West Indies Laboratory. Teague Bay, Saint Croix.

Gómez-Mestre, I., and M. Tejedo. 2003. Local adaptation of an anuran amphibian to osmotically

stressful environments. Evolution 57:1889–1899.

Goudet, J. 2005. HIERFSTAT, a package for R to compute and test hierarchical F-statistics.

Molecular Ecology Notes 5:184–186.

97

Guindon, S., and O. Gascuel. 2003. A simple, fast and accurate method to estimate large

phylogenies by maximum-likelihood. Systematic Biology 52:696–704.

Hassine, J. B., Gutiérrez-Rodríguez, J., Escoriza, D., and I. Martínez-Solano. 2016. Inferring the

roles of vicariance, climate and topography in population differentiation in Salamandra

algira (Caudata, Salamandridae). Journal of Zoological Systematics and Evolutionary

Research 54:116–126.

Heatwole, H., De Austin, S. B., and R. Herrero. 1968. Heat tolerances of tadpoles of two species

of tropical anurans. Comparative Biochemistry and Physiology 27:807–815.

Heatwole, H., and R. Levins. 1972. Biogeography of the Puerto Rican Bank: flotsam transport of

terrestrial animals. Ecology 53:112–117.

Heatwole, H., and F. Mackenzie. 1967. Herpetogeography of Puerto Rico. IV. Paleogeography,

faunal similarity and endemism. Evolution 21:429–438.

Hedges, S. B. 1999. Distribution Patterns of Amphibians in the West Indies, pp. 211–254. In:

Duellman, W. E. (ed.), Regional Patterns of Amphibian Distribution: a Global

Perspective. Johns Hopkins University Press, Baltimore, Maryland.

Hedges, S. B., Hass, C. A., and L. R. Maxson. 1992. Caribbean biogeography: molecular

evidence for dispersal in West Indian terrestrial vertebrates. Proceedings of the National

Academy of Sciences, USA 89:1909–1913.

Hedges, S. B. and M. P. Heinicke. 2007. Molecular phylogeny and biogeography of West Indian

frogs of the genus Leptodactylus (Anura, Leptodactylidae). Molecular Phylogenetics and

Evolution 44:308–314.

98

Heinicke, M. P., Duellman, W. E., and S. B. Hedges. 2007. Major Caribbean and Central

American frog faunas originated by ancient oceanic dispersal. Proceedings of the

National Academy of Sciences, USA 104:10092–10097.

Helmer, E. H., Ramos, O., López T. D. M., Quiñones, M., and W. Diaz. 2002. Mapping the

forest type and land cover of Puerto Rico, a component of the Caribbean biodiversity

hotspot. Caribbean Journal of Science 38:165–183.

Henderson, R. W., and S. B. Hedges. 1995. Origin of West Indian populations of the

geographically widespread Boa Corallus enhydris inferred from mitochondrial DNA

sequences. Molecular Phylogenetics and Evolution 4:88–92.

Henderson, R. W., and R. Powell. 2009. Natural History of West Indian Reptiles and

Amphibians. University Press of Florida, Florida

Hewitt, G. M. 1996. Some genetic consequences of ice ages, and their role in divergence and

speciation. Biological Journal of the Linnean Society 58:247–276.

Hewitt, G. M. 2000. The genetic legacy of the Quaternary ice ages. Nature 405:907–913.

Hewitt, G. M. 2004. Genetic consequences of climatic oscillations in the Quaternary.

Philosophical Transactions of the Royal Society of London B-Biological Sciences

359:183–195.

Heyer, W. R. 1978. Systematics of the fuscus group of the frog genus Leptodactylus (Amphibia,

Leptodactylidae). Science Bulletin, Natural History Museum of Los Angeles County

29:1–85.

Hirano, T., Kameda, Y., Saito, T., and S. Chiba. 2019. Divergence before and after the isolation

of islands: Phylogeography of the Bradybaena land snails on the Ryukyu Islands of

Japan. Journal of Biogeography 46:1197–1213.

99

Hoffman, E. A., and M. S. Blouin. 2004. Evolutionary history of the Northern Leopard frog:

reconstruction of phylogeny, phylogeography, and historical changes in population

demography from mitochondrial DNA. Evolution 58:145–159.

Hohenlohe, P. A., Bassham, S., Etter, P. D., Stiffler, N., Johnson, E. A., and W. A. Cresko.

2010. Population genomics of parallel adaptation in threespine stickleback using

sequenced RAD tags. PLoS Genetics 6:e1000862

https://doi.org/10.1371/journal.pgen.1000862.

Hyseni, C., and R. C. Garrick. 2019. The role of glacial interglacial climate change in shaping

the genetic structure of eastern subterranean termites in the southern Appalachian

Mountains, USA. Ecology and Evolution 9:4621–4636.

Joglar, R. 2005. Anfibios, pp. 39–96. In: Joglar, R. (ed.), Biodiversidad de Puerto Rico:

Vertebrados Terrestres y Ecosistemas. Instituto de Cultura Puertorriqueña. San Juan,

Puerto Rico.

Jukes, T. H., and C. R. Cantor. 1969. Evolution of Protein Molecules, pp. 21–132. In: Munro, H.

N. (ed.), Mammalian Protein Metabolism. Academic Press, New York.

Kaiser, H., Barrio-Amorós, C. L., Trujillo, J. D., and J. D. Lynch. 2002. Expansion of

Eleutherodactylus johnstonei in northern South America: Rapid dispersal through human

interactions. Herpetological Review 33:290-294.

Kidd, D. M., and M. G. Ritchie. 2006. Phylogeographic information systems: putting the

geography into phylogeography. Journal of Biogeography 33:1851–1865.

Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions

through comparative studies of nucleotide sequences. Journal of Molecular Evolution

16:111–120.

100

Kimura, M., and G. H. Weiss. 1964. The stepping stone model of population structure and the

decrease of genetic correlation with distance. Genetics 49:561–576.

Krysko, K. L., Nuñez, L. P., Lippi, C. A., Smith, D. J., and M. C. Granatosky. 2016. Pliocene–

Pleistocene lineage diversifications in the Eastern Indigo Snake (Drymarchon couperi) in

the southeastern United States. Molecular Phylogenetics and Evolution 98:111–122.

Lambeck, K., Rouby, H., Purcell, A., Sun, Y., and M. Sambridge. 2014. Sea level and global ice

volumes from the Last Glacial Maximum to the Holocene. Proceedings of the National

Academy of Sciences, USA 111:15296–15303.

Leal, B. S. S., da Silva, C. P., and F. Pinheiro. 2016. Phylogeographic studies depict the role of

space and time scales of plant speciation in a highly diverse neotropical region. Critical

Reviews in Plant Sciences 35:215–230.

Lever, C. 2003. Naturalized reptiles and amphibians of the world. Oxford University Press.

Oxford, United Kingdom.

López, P. T., Narins, P. M., Lewis, E. R., and S. W. Moore. 1988. Acoustically induced call

modification in the White-lipped Frog, Leptodactylus albilabris. Animal Behavior

36:1295–1308.

Losos, J. B. 2011. Convergence, adaptation, and constraint. Evolution 65:1827–1840.

Losos, J. B. and R. E. Ricklefs. 2009. Adaptation and diversification on islands.

Nature 457:830–836.

Luu, K., Bazin, E., and M. G. B. Blum. 2016. pcadapt: an R package to perform genome scans

for selection based on principle component analysis. Molecular Ecology Resources

17:67–77.

101

MacArthur, R. H., and E. O. Wilson. 1967. The Theory of Island Biogeography. Princeton

University Press. Princeton, New Jersey.

MacLean, W. P. 1982. Reptiles and amphibians of the Virgin Islands. Macmillan Education Ltd.,

London.

Messer, P. W., and D. A. Petrov. 2013. Population genomics of rapid evolution adaptation by

soft selective sweeps. Trends in Ecology & Evolution 28:659–669.

Miller, M. P. 2005. Alleles in Space (AIS): Computer software for the joint analysis of

interindividual spatial and genetic information. Journal of Heredity 96:722–724.

Morales, M. 2017. Sciplot: scientific graphing functions for factorial designs. The R

Development Core Team and with general advice from the R-help listserv community.

Moriyama, J., Takeuchi, H., Ogura-Katayama, A., and T. Hikida. 2018. Phylogeography of the

Japanese ratsnake, Elaphe climacophora (Serpentes: Colubridae): impacts of Pleistocene

climatic oscillations and sea-level fluctuations on geographical range. Biological Journal

of the Linnean Society 124:174–187.

Murphy, P. G., Lugo, A. E., Murphy, A. J., and D. C. Nepstad. 1995. The dry forests of Puerto

Rico’s south coast, pp. 178–209. In: Lugo, A. E., and C. Lowe (ed.), Tropical Forests:

Management and Ecology. Springer–Verlag, New York.

Nguiffo, D. N., Mpoame, M., and C. S. Wondji. 2019. Genetic diversity and population structure

of goliath frogs (Conraua goliath) from Cameroon. Mitochondrial DNA Part A 30:657–

663.

Nei, M., Maruyama, T., and R. Chakraborty. 1975. The bottleneck effect and genetic variability

in populations. Evolution 29:1–10.

102

Papadopoulou, A., and L. L. Knowles. 2015. Genomic tests of the species pump hypothesis:

Recent island connectivity cycles drive population divergence but not speciation in

Caribbean crickets across the Virgin Islands. Evolution 69:1501–1517.

Papadopoulou, A., and L. L. Knowles. 2017. Linking micro- and macroevolutionary perspectives

to evaluate the role of Quaternary sea-level oscillations in island diversification.

Evolution 71:2901–2917.

Parvizi, E., Naderloo, R., Keikhosravi, A., Solhjouy-Fard, S., and C. D. Schubart. 2018. Multiple

Pleistocene refugia and repeated phylogeographic breaks in the southern Caspian Sea

region: Insights from the freshwater crab Potamon ibericum. Journal of Biogeography

45:1234–1245.

Perry, G., Powell, R., and H. Watson. 2006. Keeping invasive species off Guana Island, British

Virgin Islands. Iguana: Conservation, Natural History, and Husbandry of Reptiles

13:273–277.

Petit, R. J., Aguinagalde, I., de Beaulieu, J., Bittkau, C., Brewer, S., Cheddadi, R., Ennos, R.,

Fineschi, S., Grivet, D., Lascoux, M., Mohanty, A., Müller-Starck, G., Demesure-Musch,

B., Palmé, A., Martín, J. P., Rendell, S., and G. G. Vendramin. 2003. Glacial refugia:

hotspots but not melting pots of genetic diversity. Science 300:1563–1565.

Platenberg, R. J. 2007. Impacts of Introduced Species on an Island Ecosystem: Non-native

Reptiles and Amphibians in the U.S. Virgin Islands, pp. 168–174. In: Witmer, G. W.,

Pitt, W. C., and K. A Fagerstone (eds.), Managing Vertebrate Invasive Species:

Proceedings of an International Symposium. USDA/APHIS/WS National Wildlife

Research Center. Fort Collins, Colorado.

103

Polzin. T., and S. V. Daneshmand. 2003. On Steiner trees and minimum spanning trees in

hypergraphs. Operations Research Letters 31:12–20.

Potter, S., Xue, A. T., Bragg, J. G., Rosauer, D. F., Roycroft, E. J., and C. Moritz. 2018.

Pleistocene climatic changes drive diversification across a tropical savanna. Molecular

Ecology 27:520–532.

Powell, R. 2006. Conservation of the herpetofauna on the Dutch Windward Islands: St.

Eustatius, Saba, and St. Maarten. Applied Herpetology 3:293–306.

Powell, R., Henderson, R. W., Farmer, M. C., Breuil, M., Echternacht, A. C., van Buurt, G.,

Romagosa, C. M., and G. Perry. 2011. Introduced amphibians and reptiles in the greater

Caribbean: patterns and conservation implications, pp. 63–144. In: Hailey A., Wilson,

B.S., and J. A. Horrocks. (eds.), Conservation of Caribbean Island Herpetofaunas

Volume 1: Conservation Biology and the Wider Caribbean. Brill, Boston.

Powell, R., Henderson, R.W., and J. S. Parmerlee, Jr. 2005. Reptiles and Amphibians of the

Dutch Caribbean: St. Eustatius, Saba, and St. Maarten. St. Eustatius National Parks

Foundation, Gallows Bay, St. Eustatius, Netherlands Antilles.

Pregill, G. K., and S. L. Olson. 1981. Zoogeography of West Indian vertebrates in relation to

Pleistocene climatic cycles. Annual Review of Ecology and Systematics 12:75–98.

Provan, J., and K. D. Bennett. 2008. Phylogeographic insights into cryptic glacial refugia. Trends

in Ecology & Evolution 23:564–571.

Puritz, J. B., Hollenbeck, C. M., and J. R. Gold. 2014a. dDocent: a RADseq, variant-calling

pipeline designed for population genomics of non-model organisms. PeerJ 2:e431

https://doi.org/10.7717/peerj.431.

104

Puritz, J. B., Matz, M. V., Toonen, R. J., Weber, J. N., Bolnick, D. I., and C. E. Bird. 2014b.

Demystifying the RAD fad. Molecular Ecology 23:5937–5942.

Rambaut, A. and A. J., Drummond. 2010. FigTree v1.3.1. Institute of Evolutionary Biology,

University of Edinburgh, Edinburgh. http://tree.bio.ed.ac.uk/software/figtree/.

Reilly, S. B., Stubbs, A. L., Karin, B. R., Arida, E., Iskandar, D. T., and J. A. McGuire. 2019.

Recent colonization and expansion through the Lesser Sundas by seven amphibian and

reptile species. Zoologica Scripta 48:614–626.

Renken, R. A., Ward, W. C., Gill, I. P., Gómez-Gómez, F., and J. Rodríguez-Martínez. 2002.

Geology and hydrology of the Caribbean islands aquifer system of the Commonwealth of

Puerto Rico and the U.S. Virgin Islands. Regional Aquifer-System Analysis—Caribbean

Islands. U.S. Geological Survey Professional Paper 1419, U.S. Department of the

Interior, Reston, Virginia.

Reynolds, R. G., Strickland, T. R., Kolbe, J. J., Falk, B. G., Perry, G., Revell, L. J., and J. B.

Losos. 2017. Archipelagic genetics in a widespread Caribbean anole. Journal of

Biogeography 44:2631–2647.

Ricklefs, R., and E. Bermingham. 2008. The West Indies as a laboratory of biogeography and

evolution. Philosophical Transactions of the Royal Society B-Biological Sciences

363:2393–2413.

Ricklefs, R. E. and I. J. Lovette. 1999. The roles of island area per se and habitat diversity in the

species-area relationships of four Lesser Antillean faunal groups. Journal of Animal

Ecology 68:1142–1160.

Rochette, N. C., and J. M. Catchen. 2017. Deriving genotypes from RAD-seq short-read data

using Stacks. Nature Protocols 12:2640–2659.

105

Rödder, D. 2009. Human footprint, facilitated jump dispersal, and the potential distribution of

the invasive Eleutherodactylus johnstonei Barbour 1914 (Anura Eleutherodactylidae).

Tropical Zoology 22:205–217.

Rodríguez-Robles, J. A., Jezkova, T., Fujita, M. K., Tolson, P. J., and M. A. García. 2015.

Genetic divergence and diversity in the Mona and Virgin Islands Boas, Chilabothrus

monensis (Epicrates monensis) (Serpentes: Boidae), West Indian snakes of special

conservation concern. Molecular Phylogenetics and Evolution 88:144–153.

Roesti, M., Salzburger, W., and D. Berner. 2012. Uninformative polymorphisms bias genome

scans for signatures of selection. BMC Evolutionary Biology 12:94.

Rohling, E. J., Fenton, M., Jorissen, F. J., Bertrand, P., Ganssen, G., and J. P. Caulet. 1998.

Magnitudes of sea-level lowstands of the past 500,000 years. Nature 394:162–165.

Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D. L., Darling, A., Höhna, S., Larget, B.,

Liu, L., Suchard, M. A., and J. P. Huelsenbeck. 2012. MrBayes 3.2: Efficient Bayesian

phylogenetic inference and model choice across a large model space. Systematic Biology

61:539–542.

Royer, A., Malaizé, B., Lécuyer, C., Queffelec, A., Charlier, K., Caley, T., and A. Lenoble.

2017. A high-resolution temporal record of environmental changes in the Eastern

Caribbean (Guadeloupe) from 40 to 10 ka BP. Quaternary Science Reviews 155:198–

212.

Rozas, J., Ferrer-Mata, A., Sánchez-Del Barrio, J. C., Guirao-Rico, S., Librado, P., Ramos-

Onsins, S. E., and A. Sánchez-Gracia. 2017. DnaSP 6: DNA Sequence polymorphism

analysis of large datasets. Molecular Biology and Evolution 34:3299–3302.

106

Sánchez-Montes, G., Recuero, E., Barbosa, A. M., and Í. Martínez-Solano. 2019.

Complementing the Pleistocene biogeography of European amphibians: Testimony from

a southern Atlantic species. Journal of Biogeography 46:568–583.

Schwartz, A., and R. W. Henderson. 1991. Amphibians of the West Indies: Descriptions,

Distribution, and Natural History. University of Florida Press, Gainesville.

Silva, G. A. R., Antonelli, A., Lendel, A., Moraes, E. D. M., and M. H. Manfrin. 2017. The

impact of early Quaternary climate change on the diversification and population

dynamics of a South American cactus species. Journal of Biogeography 45:76–88.

Slatkin, M. 1987. Gene flow and the geographic structure of natural populations. Science

236:787–792.

Taberlet, P., Fumagalli, L., Wust-Saucy, A., and J. Cosson. 2002. Comparative phylogeography

and postglacial colonization routes in Europe. Molecular Ecology 7:453–464.

Tajima, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA

polymorphism. Genetics 123:585–595.

Thomas, R. 1999. The Puerto Rico Area, pp. 169–179. In: Crother, B. I. (ed.), Caribbean

Amphibians and Reptiles. Academic Press, San Diego.

Townsend, J. H., Eaton, J. M., Powell, R., Parmerlee, Jr., J. S., and R. W. Henderson. 2000.

Cuban Treefrogs (Osteopilus septentrionalis) in Anguilla, Lesser Antilles. Caribbean

Journal of Science 36:326–328.

Truong, H. T., Ramos, A. M., Yalcin, F., de Ruiter, M., van der Poel, H. J. A., Huvenaars, K. H.

J., Hogers, R. C. J., van Enckevort, L. J. G., Janssen, A., van Orsouw, N. J., and M. J. T.

van Eijk. 2012. Sequence-based genotyping for marker discovery and co-dominant

107

scoring in germplasm and populations. PLoS One 7:e37565

https://doi.org/10.1371/journal.pone.0037565.

Vasconcellos, M. M., Colli, G. R., Weber, J. N., Ortiz, E. M., Rodrigues, M. T., and D. C.

Cannatella. 2019. Isolation by instability: Historical climate change shapes population

structure and genomic divergence of treefrogs in the Neotropical Cerrado savanna.

Molecular Ecology 28:1748–1764.

Vences, M., Vieites, D. R., Glaw, R., Brinkmann, H., Kosuch, J., Veith, M., and A. Meyer. 2003.

Multiple overseas dispersal in amphibians. Proceedings of the Royal Society of London

B-Biological Sciences 270:2435–2442.

Wade, M. J., and D. E. McCauley. 1988. Extinction and recolonization: their effects on the

genetic differentiation of local populations. Evolution 42:995–1005.

Wang, S., Zhu, W., Gao, X., Li, X., Yan, S., Liu, X., Yang, J., Gao, Z., and Y. Li. 2014.

Population size and time since island isolation determine genetic diversity loss in insular

frog populations. Molecular Ecology 23:637–648.

Warken, S. F., Scholz, D., Spötl, C., Jochum, K. P., Pajón, J. M., Bahr, A., and A. Mangini.

2019. Caribbean hydroclimate and vegetation history across the last glacial period.

Quaternary Science Reviews 218:75–90.

Weigelt, P., Steinbauer, M. J., Cabral J. S., and H. Kreft. 2016. Late Quaternary climate change

shapes island biodiversity. Nature 532:99–102.

White, T. A., and J. B. Searle. 2007. Genetic diversity and population size: island populations of

the common shrew, Sorex araneus. Molecular Ecology 16:2005–2016.

108

Whitlock, M. C., and D. E. McCauley. 1990. Some population genetic consequences of colony

formation and extinction: Genetic correlations within founding groups. Evolution

44:1717–1724.

Wright, S. 1931. Evolution in Mendelian populations. Genetics 16:97–159.

Wright, S. 1943. Isolation by distance. Genetics 28:114–138.

Zeisset, I., and T. J. C. Beebee. 2008. Amphibian phylogeography: a model for understanding

historical aspects of species distributions. Heredity 101:109–119.

109

Curriculum Vitae

Andre Nguyen

Personal Information Email: andrenguyn@gmail.com

Education SAN DIEGO STATE UNIVERSITY, San Diego, CA. Bachelor of Science in Biology, Emphasis in Zoology, Spring 2015 UNIVERSITY OF NEVADA LAS VEGAS, Las Vegas, NV. Master of Science in Biology (Ecology and Evolution): Fall 2017 - present (Research Advisor: Dr. Javier A. Rodríguez) Publications Nguyen A., Richart, C.H. & Burns K.J. (2015) Black-chested Mountain-Tanager

(Cnemathraupis eximia), Neotropical Birds Online (Schulenberg TS, Ed). Ithaca: Cornell Lab of Ornithology, Ithaca, New York, USA.

Professional Experience 11/2019 Assistant Instructor, Data Carpentry Genomics Workshop 8/2019 – present Discussion Section Instructor, Biology 415 - Evolution University of Nevada, Las Vegas 3/2019 – 5/2019 Field technician BEC Environmental INC., Las Vegas, NV. 9/2017 – 6/2019 Laboratory Course Instructor, Biology 197 - Principles of Modern Biology II University of Nevada, Las Vegas

9/2016 – 3/2017 Naturalist Arrowhead Ranch Outdoor Science School, Lake Arrowhead, CA.

Responsible for teaching 5th, 6th, and 7th grade science curriculum provided by the California State Board of Education

3/2016 – 5/2016 Avian Field Technician TW Biological Services, Oceanside/Camp Pendleton, CA.

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10/2015 – 11/2015 Research Associate Great Basin Institute, Death Valley, CA. 5/2015 – 08/2015 Intern (Research Assistant) Death Valley National Park, CA. Awards

Awards: Dean’s List – Fall 2014 & Spring 2015 (San Diego State University)

2018 Aquatic Biology Endowment ($5000)

2018 Graduate Professional Student Association Research Sponsorship ($689)

2018 Nevada INBRE Service Award ($5000)

2018 Nevada INBRE Service Award ($2300)