The Effect of Pleistocene Glacial

UNLV Theses, Dissertations, Professional Papers, and Capstones

12-15-2019

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

Andre Nguyen

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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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This Thesis has been accepted for inclusion in UNLV Theses, Dissertations, Professional Papers, and Capstones by an authorized administrator of Digital Scholarship@UNLV. For more information, please contact [email protected]. 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

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

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

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

iii

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.

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.

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

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

vii

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

viii

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

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

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

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; Gü nther, 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

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

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.

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.

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

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;

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

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

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.

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

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.

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 t 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 t 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

(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

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.

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

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.

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

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.

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

(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).

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

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.

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

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).

Systematic status of Leptodactylus dominicensis

Phenotypical, behavioral, and genetic data clearly indicate that Leptodactylus dominicensis (Cochran, 1823) is closely related to L. albilabris (Gü nther, 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.

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).

Table 1. Species, population number, field number, country, state, island, municipality, latitude (h = degrees, min = minutes), and longitude of the localities of the specimens of Leptodactylus poecilochilus, L. dominicensis, and L. albilabris used in this study. For L.

poecilochilus, the museum catalog number is in parentheses; for L. dominicensis, the GenBank accession number is in parentheses.

BB = Brittany Barker Field Series, BVI = British Virgin Islands, CU = Culebra, JAR = Javier A. Rodríguez Field Series, JVD = Jost

Van Dyke, PR = Puerto Rico, SBH = S. Blair Hedges Field Series, STC = Saint Croix, STJ = Saint John, STT = Saint Thomas, TO =

Tortola, UK = United Kingdom, US = United States of America, USNM = National Museum of Natural History, Smithsonian

Institution, Washington, D.C., USVI = United States Virgin Islands, VI = Vieques. Samples of L. albilabris are listed in order by

population number, from west to east (Figure 1).

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Outgroup

Leptodactylus — — Panamá — — Panamá — — — — poecilochilus Province (USNM 565029)

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Ingroup

Leptodactylus — SBH 192453 Dominican Hispaniola El Seibo — — — — dominicensis Republic Province (EF091393)

Leptodactylus 1 JAR 2905 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2906 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2907 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2908 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2909 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2910 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2911 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2912 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2913 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris Leptodactylus 1 JAR 2914 US PR PR Cabo Rojo 18° 01.030' N 67° 08.955' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 2 JAR 2740 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 2 JAR 2741 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 2 JAR 2742 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 2 JAR 2743 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 2 JAR 2744 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 2 JAR 2745 US PR PR Moca 18° 23.477' N 67° 06.078' W

albilabris Leptodactylus 2 JAR 2747 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 2 JAR 2748 US PR PR Moca 18° 23.477' N 67° 06.078' W albilabris Leptodactylus 3 JAR 2625 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2626 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2627 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2628 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2629 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2630 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 3 JAR 2631 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2632 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2633 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 3 JAR 2634 US PR PR Isabela 18° 30.636' N 67° 05.728' W albilabris Leptodactylus 4 JAR 3070 US PR PR Mayagüez 18° 11.303' N 67° 03.626' W albilabris Leptodactylus 4 JAR 3072 US PR PR Mayagüez 18° 11.303' N 67° 03.626' W

albilabris Leptodactylus 4 JAR 3074 US PR PR Mayagüez 18° 11.303' N 67° 03.626' W albilabris Leptodactylus 4 JAR 3075 US PR PR Mayagüez 18° 11.303' N 67° 03.626' W albilabris Leptodactylus 4 JAR 3076 US PR PR Mayagüez 18° 11.303' N 67° 03.626' W albilabris Leptodactylus 5 JAR 2893 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2894 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2895 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2896 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2897 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 5 JAR 2898 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2899 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2900 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2901 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 5 JAR 2902 US PR PR Guánica 17° 58.751' N 66° 57.089' W albilabris Leptodactylus 6 JAR 3077 US PR PR Yauco 18° 09.100' N 66° 53.099' W

albilabris Leptodactylus 6 JAR 3078 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3079 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3080 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3081 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3083 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3084 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3085 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 6 JAR 3086 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 6 JAR 3087 US PR PR Yauco 18° 09.100' N 66° 53.099' W albilabris Leptodactylus 7 JAR 2854 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2855 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2856 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2857 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2858 US PR PR Hatillo 18° 29.175' N 66° 49.912' W

albilabris Leptodactylus 7 JAR 2859 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2860 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2861 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2862 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 7 JAR 2863 US PR PR Hatillo 18° 29.175' N 66° 49.912' W albilabris Leptodactylus 8 JAR 2878 US PR PR Arecibo 18° 22.730' N 66° 44.963' W albilabris Leptodactylus 8 JAR 2879 US PR PR Arecibo 18° 22.730' N 66° 44.963' W albilabris Leptodactylus 8 JAR 2880 US PR PR Arecibo 18° 22.730' N 66° 44.963' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 8 JAR 2881 US PR PR Arecibo 18° 22.730' N 66° 44.963' W albilabris Leptodactylus 8 JAR 2882 US PR PR Arecibo 18° 22.730' N 66° 44.963' W albilabris Leptodactylus 9 JAR 3095 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 9 JAR 3096 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 9 JAR 3097 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 9 JAR 3098 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W

albilabris Leptodactylus 9 JAR 3099 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 9 JAR 3100 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 9 JAR 3101 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 9 JAR 3102 US PR PR Peñuelas 18° 04.586' N 66° 43.557' W albilabris Leptodactylus 10 JAR 3037 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris Leptodactylus 10 JAR 3039 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris Leptodactylus 10 JAR 3040 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris Leptodactylus 10 JAR 3043 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 10 JAR 3044 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris Leptodactylus 10 JAR 3045 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris Leptodactylus 10 JAR 3046 US PR PR Juana Díaz 18° 09.087' N 66° 32.023' W albilabris Leptodactylus 11 JAR 2993 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 2994 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 2995 US PR PR Ciales 18° 14.805' N 66° 31.215' W

albilabris Leptodactylus 11 JAR 2996 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 2997 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 2998 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 2999 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 3000 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 3001 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 3002 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 11 JAR 3003 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 11 JAR 3004 US PR PR Ciales 18° 14.805' N 66° 31.215' W albilabris Leptodactylus 12 JAR 2709 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2710 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2711 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2712 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2713 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W

albilabris Leptodactylus 12 JAR 2714 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2715 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2716 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2717 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 12 JAR 2718 US PR PR Juana Díaz 18° 00.395' N 66° 29.538' W albilabris Leptodactylus 13 JAR 2699 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2700 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2701 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 13 JAR 2702 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2703 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2704 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2705 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2707 US PR PR Manatí 18° 23.200' N 66° 29.119' W albilabris Leptodactylus 13 JAR 2708 US PR PR Manatí 18° 23.200' N 66° 29.119' W

albilabris Leptodactylus 14 JAR 2915 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2916 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2917 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2918 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2919 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2920 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2921 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2922 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 14 JAR 2923 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 14 JAR 2924 US PR PR Dorado 18° 27.731' N 66° 16.399' W albilabris Leptodactylus 15 JAR 2686 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2687 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2688 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2689 US PR PR Aibonito 18° 06.931' N 66° 13.999' W

albilabris Leptodactylus 15 JAR 2690 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2691 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2692 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2693 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 15 JAR 2694 US PR PR Aibonito 18° 06.931' N 66° 13.999' W albilabris Leptodactylus 16 JAR 2685 US PR PR Aibonito 18° 06.574' N 66° 13.572' W albilabris Leptodactylus 17 JAR 2556 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2557 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 17 JAR 2558 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2559 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2560 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2561 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2562 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2563 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W

albilabris Leptodactylus 17 JAR 2564 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 17 JAR 2565 US PR PR Toa Alta 18° 20.222' N 66° 12.848' W albilabris Leptodactylus 18 JAR 880 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 18 JAR 881 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 18 JAR 882 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 18 JAR 883 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 18 JAR 884 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 18 JAR 885 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 18 JAR 886 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 18 JAR 887 US PR PR Guayama 17° 57.200' N 66° 10.872' W albilabris Leptodactylus 19 JAR 2750 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2751 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2752 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris

Leptodactylus 19 JAR 2753 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2754 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2755 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2756 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2757 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2758 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 19 JAR 2759 US PR PR Guayama 17° 57.863' N 66° 06.706' W albilabris Leptodactylus 20 JAR 2726 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2727 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 20 JAR 2728 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2729 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2730 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2731 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2732 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2733 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W

albilabris Leptodactylus 20 JAR 2734 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 20 JAR 2735 US PR PR Aguas Buenas 18° 14.403' N 66° 06.239' W albilabris Leptodactylus 21 JAR 2987 US PR PR Cayey 18° 07.339' N 66° 04.698' W albilabris Leptodactylus 21 JAR 2988 US PR PR Cayey 18° 07.339' N 66° 04.698' W albilabris Leptodactylus 21 JAR 2989 US PR PR Cayey 18° 07.339' N 66° 04.698' W albilabris Leptodactylus 21 JAR 2990 US PR PR Cayey 18° 07.339' N 66° 04.698' W albilabris Leptodactylus 21 JAR 2991 US PR PR Cayey 18° 07.339' N 66° 04.698' W albilabris Leptodactylus 21 JAR 2992 US PR PR Cayey 18° 07.339' N 66° 04.698' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 22 JAR 2982 US PR PR Cayey 18° 06.578' N 66° 04.289' W albilabris Leptodactylus 22 JAR 2983 US PR PR Cayey 18° 06.578' N 66° 04.289' W albilabris Leptodactylus 22 JAR 2984 US PR PR Cayey 18° 06.578' N 66° 04.289' W albilabris Leptodactylus 22 JAR 2985 US PR PR Cayey 18° 06.578' N 66° 04.289' W albilabris Leptodactylus 22 JAR 2986 US PR PR Cayey 18° 06.578' N 66° 04.289' W albilabris Leptodactylus 23 JAR 3005 US PR PR Gurabo 18° 18.145' N 65° 59.238' W

albilabris Leptodactylus 23 JAR 3006 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3007 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3008 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3009 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3010 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3011 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3012 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 23 JAR 3013 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 23 JAR 3014 US PR PR Gurabo 18° 18.145' N 65° 59.238' W albilabris Leptodactylus 24 JAR 2669 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2670 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2671 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2672 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2673 US PR PR Carolina 18° 26.549' N 65° 57.415' W

albilabris Leptodactylus 24 JAR 2674 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2675 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2676 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2677 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 24 JAR 2678 US PR PR Carolina 18° 26.549' N 65° 57.415' W albilabris Leptodactylus 25 JAR 238 US PR PR Maunabo 18° 01.623' N 65° 54.888' W albilabris Leptodactylus 25 JAR 239 US PR PR Maunabo 18° 01.623' N 65° 54.888' W albilabris Leptodactylus 25 JAR 240 US PR PR Maunabo 18° 01.623' N 65° 54.888' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 25 JAR 241 US PR PR Maunabo 18° 01.623' N 65° 54.888' W albilabris Leptodactylus 26 JAR 2577 US PR PR Maunabo 18° 01.586' N 65° 54.822' W albilabris Leptodactylus 26 JAR 2578 US PR PR Maunabo 18° 01.586' N 65° 54.822' W albilabris Leptodactylus 26 JAR 2579 US PR PR Maunabo 18° 01.586' N 65° 54.822' W albilabris Leptodactylus 26 JAR 2580 US PR PR Maunabo 18° 01.586' N 65° 54.822' W albilabris Leptodactylus 26 JAR 2581 US PR PR Maunabo 18° 01.586' N 65° 54.822' W

albilabris Leptodactylus 26 JAR 2582 US PR PR Maunabo 18° 01.586' N 65° 54.822' W albilabris Leptodactylus 27 JAR 2960 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2961 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2962 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2963 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2964 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2965 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2966 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 27 JAR 2967 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2968 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 27 JAR 2969 US PR PR Juncos 18° 14.006' N 65° 52.088' W albilabris Leptodactylus 28 JAR 2929 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2930 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2931 US PR PR Río Grande 18° 23.427' N 65° 46.837' W

albilabris Leptodactylus 28 JAR 2932 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2933 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2934 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2935 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2936 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2937 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 28 JAR 2938 US PR PR Río Grande 18° 23.427' N 65° 46.837' W albilabris Leptodactylus 29 JAR 2636 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 29 JAR 2637 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2638 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2639 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2640 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2641 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2642 US PR PR Fajardo 18° 16.246' N 65° 42.882' W

albilabris Leptodactylus 29 JAR 2643 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2644 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 29 JAR 2645 US PR PR Fajardo 18° 16.246' N 65° 42.882' W albilabris Leptodactylus 30 JAR 3049 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3050 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3051 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3052 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3053 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 30 JAR 3054 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3055 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3056 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3057 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 30 JAR 3058 US PR VI VI 18° 06.088' N 65° 32.347' W albilabris Leptodactylus 31 JAR 3059 US PR VI VI 18° 07.261' N 65° 26.204' W

albilabris Leptodactylus 32 JAR 2943 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2944 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2945 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2946 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2947 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2948 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2949 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2950 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 32 JAR 2951 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 32 JAR 2952 US PR CU CU 18° 18.631' N 65° 17.969' W albilabris Leptodactylus 33 JAR 3131 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3132 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3133 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3134 US USVI STT — 18° 20.401' N 64° 58.124' W

albilabris Leptodactylus 33 JAR 3135 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3136 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3137 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3138 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3139 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3140 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 33 JAR 3141 US USVI STT — 18° 20.401' N 64° 58.124' W albilabris Leptodactylus 34 BB 7788 US USVI STT — 18° 19.375' N 64° 54.649' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 35 JAR 3181 US USVI STC — 17° 43.898' N 64° 51.645' W albilabris Leptodactylus 35 JAR 3182 US USVI STC — 17° 43.898' N 64° 51.645' W albilabris Leptodactylus 35 JAR 3183 US USVI STC — 17° 43.898' N 64° 51.645' W albilabris Leptodactylus 35 JAR 3184 US USVI STC — 17° 43.898' N 64° 51.645' W albilabris Leptodactylus 35 JAR 3185 US USVI STC — 17° 43.898' N 64° 51.645' W albilabris Leptodactylus 36 JAR 3170 US USVI STC — 17° 45.544' N 64° 45.912' W

albilabris Leptodactylus 36 JAR 3171 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3172 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3173 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3174 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3175 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3176 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3177 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3178 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 36 JAR 3179 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 36 JAR 3180 US USVI STC — 17° 45.544' N 64° 45.912' W albilabris Leptodactylus 37 BB 7847 US USVI STC — 17° 44.293' N 64° 42.910' W albilabris Leptodactylus 37 BB 7848 US USVI STC — 17° 44.293' N 64° 42.910' W albilabris Leptodactylus 38 JAR 3247 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3248 US USVI STJ — 18° 19.668' N 64° 47.481' W

albilabris Leptodactylus 38 JAR 3249 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3250 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3251 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3252 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3253 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3254 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3255 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris Leptodactylus 38 JAR 3256 US USVI STJ — 18° 19.668' N 64° 47.481' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 39 JAR 3239 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 39 JAR 3240 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 39 JAR 3241 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 39 JAR 3242 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 39 JAR 3243 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 39 JAR 3244 UK BVI JVD — 18° 26.786' N 64° 45.078' W

albilabris Leptodactylus 39 JAR 3245 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 39 JAR 3246 UK BVI JVD — 18° 26.786' N 64° 45.078' W albilabris Leptodactylus 40 BB 7521 UK BVI JVD — 18° 26.861' N 64° 44.404' W albilabris Leptodactylus 40 BB 7522 UK BVI JVD — 18° 26.861' N 64° 44.404' W albilabris Leptodactylus 40 BB 7523 UK BVI JVD — 18° 26.861' N 64° 44.404' W albilabris Leptodactylus 41 BB 7491 UK BVI TO — 18° 25.351' N 64° 38.671' W albilabris Leptodactylus 42 JAR 3233 UK BVI TO — 18° 25.919' N 64° 38.268' W albilabris Leptodactylus 42 JAR 3234 UK BVI TO — 18° 25.919' N 64° 38.268' W albilabris

Table 1. Continued.

Population Field Latitude Latitude Longitude Longitude Species Country State Island Municipality number number (h) (min) (h) (min)

Leptodactylus 42 JAR 3235 UK BVI TO — 18° 25.919' N 64° 38.268' W albilabris Leptodactylus 42 JAR 3236 UK BVI TO — 18° 25.919' N 64° 38.268' W albilabris Leptodactylus 42 JAR 3237 UK BVI TO — 18° 25.919' N 64° 38.268' W albilabris Leptodactylus 42 JAR 3238 UK BVI TO — 18° 25.919' N 64° 38.268' W albilabris

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).

Population Phylogroup Population(s) Haplotypes number(s) 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, 4, 5, 6, 7, 9, 10, 85, 91, 103,

Juana Díaz, Ciales, Juana Díaz, Manatí, 11, 12, 13, 16, 104, 114, 115,

Aibonito 2, Toa Alta, Guayama 1, Guayama 2, 17, 18, 19, 20, 118, 127, 128,

Aguas Buenas, Cayey 1, Cayey 2, Gurabo, 21, 22, 23, 26, 129, 131, 132,

Maunabo 2, Juncos, Culebra 27, 32 133, 136, 137,

138, 139, 140

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

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

Table 2. Continued.

Population Phylogroup Population(s) Haplotypes number(s) PG 11 Aibonito 1, Guayama 1, Guayama 2, 15, 18, 19, 20, 21, 1, 2, 3, 4, 72,

Aguas Buenas, Cayey 1, Cayey 2, Gurabo, 22, 23, 24, 25, 26, 84, 88, 90, 95,

Carolina, Maunabo 1, Maunabo 2, Juncos, 27, 29, 30, 31 97, 98, 99, 101,

Fajardo, Vieques 1, Vieques 2 102, 105, 134

PG 12 Isabela, Arecibo 3, 8 40, 42

PG 13 Dorado 14 12, 34

PG 14 Juana Díaz, Ciales, Manatí, Toa Alta, 10, 11, 13, 17, 21 20, 38, 54

Cayey 1

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

Table 3. Island, population number, municipality, and unique cytochrome b haplotype(s) detected in 42 populations of Leptodactylus albilabris.

Population Island Municipality Haplotype number 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

Table 3. Continued.

Population Island Municipality Haplotype number 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

Table 3. Continued.

Population Island Municipality Haplotype number

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

Table 4. Island, population number, municipality, sample size, number of unique haplotypes, haplotype diversity (Hd), and nucleotide

diversity (πd) estimated using a cytochrome b fragment (828 base pairs) from 322 specimens of Leptodactylus albilabris from 42 localities across the Puerto Rican Bank. I estimated Hd and πd using ARELEQUIN 3.5.2.2 (Excoffier and Lischer 2010) for localities represented by ≥ 5 individuals.

Number of Population Sample Haplotype Nucleotide Island Municipality unique number size diversity (Hd) diversity (π) haplotypes

Puerto Rico 1 Cabo Rojo 10 8 0.96 +/- 0.06 0.0039 +/- 0.0025

Puerto Rico 2 Moca 8 7 0.96 +/- 0.08 0.0051 +/- 0.0032

Puerto Rico 3 Isabela 10 6 0.89 +/- 0.08 0.0059 +/- 0.0035

Puerto Rico 4 Mayagüez 5 4 0.90 +/- 0.16 0.0053 +/- 0.0037

Puerto Rico 5 Guánica 10 7 0.91 +/- 0.08 0.0047 +/- 0.0029

Puerto Rico 6 Yauco 10 7 0.91 +/- 0.09 0.0048 +/- 0.0029

Puerto Rico 7 Hatillo 10 7 0.91 +/- 0.10 0.0063 +/- 0.0038

Puerto Rico 8 Arecibo 5 4 0.90 +/- 0.16 0.0051 +/- 0.0035

Puerto Rico 9 Peñuelas 8 2 0.25 +/- 0.18 0.0006 +/- 0.0006

Table 4. Continued.

Number of Population Sample Haplotype Nucleotide Island Municipality unique number size diversity (Hd) diversity (π) haplotypes

Puerto Rico 10 Juana Díaz 1 7 4 0.86 +/- 0.10 0.0078 +/- 0.0048

Puerto Rico 11 Ciales 12 9 0.95 +/- 0.05 0.0088 +/- 0.0050

Puerto Rico 12 Juana Díaz 2 10 7 0.87 +/- 0.11 0.0045 +/- 0.0028

Puerto Rico 13 Manatí 9 7 0.92 +/- 0.09 0.0074 +/- 0.0044

Puerto Rico 14 Dorado 10 6 0.89 +/- 0.08 0.0085 +/- 0.0049

Puerto Rico 15 Aibonito 1 9 5 0.86 +/- 0.09 0.0058 +/- 0.0035

Puerto Rico 16 Aibonito 2 1 — — —

Puerto Rico 17 Toa Alta 10 8 0.96 +/- 0.06 0.0073 +/- 0.0043

Puerto Rico 18 Guayama 1 8 4 0.82 +/- 0.10 0.0058 +/- 0.0036

Puerto Rico 19 Guayama 2 10 6 0.89 +/- 0.08 0.0053 +/- 0.0032

Puerto Rico 20 Aguas Buenas 10 10 1.00 +/- 0.04 0.0121 +/- 0.0068

Puerto Rico 21 Cayey 1 6 6 1.00 +/- 0.10 0.0110 +/- 0.0068

Puerto Rico 22 Cayey 2 5 5 1.00 +/- 0.13 0.0104 +/- 0.0068

Table 4. Continued.

Number of Population Sample Haplotype Nucleotide Island Municipality unique number size diversity (Hd) diversity (π) haplotypes

Puerto Rico 23 Gurabo 10 6 0.87 +/- 0.09 0.0092 +/- 0.0053

Puerto Rico 24 Carolina 10 7 0.93 +/- 0.06 0.0111 +/- 0.0063

Puerto Rico 25 Maunabo 1 4 4 — —

Puerto Rico 26 Maunabo 2 6 5 0.93 +/- 0.12 0.0066 +/- 0.0043

Puerto Rico 27 Juncos 10 10 1.00 +/- 0.04 0.0072 +/- 0.0042

Puerto Rico 28 Río Grande 10 8 0.96 +/- 0.06 0.0082 +/- 0.0048

Puerto Rico 29 Fajardo 10 8 0.93 +/- 0.08 0.0104 +/- 0.0060

Vieques 1 30 — 10 6 0.89 +/- 0.08 0.0074 +/- 0.0044

Vieques 2 31 — 1 — — —

Culebra 32 — 10 5 0.84 +/- 0.08 0.0097 +/- 0.0056

Saint Thomas 1 33 — 11 2 0.51 +/- 0.10 0.0000 +/- 0.0000

Saint Thomas 2 34 — 1 — — —

Table 4. Continued.

Number of Population Sample Haplotype Nucleotide Island Municipality unique number size diversity (Hd) diversity (π) haplotypes

Saint Croix 1 35 — 5 1 0.00 +/- 0.00 0.0000 +/- 0.0000

Saint Croix 2 36 — 11 2 0.18 +/- 0.14 0.0002 +/- 0.0003

Saint Croix 3 37 — 2 — — —

Saint John 38 — 10 3 0.69 +/- 0.10 0.0011 +/- 0.0009

Jost Van Dyke 1 39 — 8 2 0.25 +/- 0.18 0.0003 +/- 0.0004

Jost Van Dyke 2 40 — 2 — — —

Tortola 1 41 — 1 — — —

Tortola 2 42 — 6 3 0.60 +/- 0.22 0.0004 +/- 0.0005

Table 5. Pairwise Fst values among 35 populations of Leptodactylus albilabris represented by ≥ 5 individuals. Values were estimated from 322 cytochrome b sequences. Populations are listed from west to east.

Population Cabo Rojo Moca Isabela Mayagüez Guánica Yauco Hatillo Arecibo

Cabo Rojo 0.00 — — — — — — —

Moca 0.0402 0.0 — — — — — —

Isabela 0.0778 0.0747 0.0 — — — — —

Mayagüez 0.0687 0.0159 0.1063 0.0 — — — —

Guánica 0.0071 0.0632 0.1000 0.0937 0.0 — — —

Yauco 0.0667 0.0391 0.1000 0.0150 0.0703 0.0 — —

Hatillo 0.0667 0.0513 0.1000 0.0560 0.0307 0.0409 0.0 —

Arecibo 0.0687 0.0159 0.1063 0.0217 0.0937 0.0560 0.0752 0.0

Peñuelas 0.3748 0.3929 0.4092 0.4815 0.2748 0.3420 0.1924 0.4815

Juana Díaz 1 0.0907 0.0880 0.1260 0.1231 0.0607 0.0882 0.0315 0.1231

Ciales 0.0450 0.0204 0.0775 0.0028 0.0667 0.0346 0.0509 0.0369

Juana Díaz 2 0.0797 0.0746 0.1222 0.0821 0.0232 0.0544 -0.0217 0.1010

Table 5. Continued.

Population Cabo Rojo Moca Isabela Mayagüez Guánica Yauco Hatillo Arecibo

Manatí 0.0636 0.0467 0.0975 0.0485 0.0862 0.0324 0.0654 0.0701

Dorado 0.0778 0.0747 0.1111 0.1063 0.1000 0.1000 0.1000 0.1063

Aibonito 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

Guayama 1 0.1090 0.1071 0.1435 0.1436 0.0619 0.0983 0.0225 0.1436

Guayama 2 0.0685 0.0747 0.1111 0.1063 0.0722 0.0909 0.0722 0.1063

Aguas Buenas 0.0222 0.0174 0.0556 0.0440 0.0444 0.0444 0.0444 0.0440

Cayey 1 0.0239 0.0187 0.0602 0.0480 0.0481 0.0481 0.0481 0.0480

Cayey 2 0.0248 0.0194 0.0625 0.0500 0.0498 0.0498 0.0498 0.0500

Gurabo 0.0889 0.0746 0.1222 0.0821 0.0544 0.0643 0.0124 0.1010

Carolina 0.0556 0.0517 0.0889 0.0812 0.0778 0.0778 0.0778 0.0812

Maunabo 2 0.0546 0.0504 0.0909 0.0826 0.0469 0.0631 0.0302 0.0826

Table 5. Continued.

Population Cabo Rojo Moca Isabela Mayagüez Guánica Yauco Hatillo Arecibo

Juncos 0.0124 0.0174 0.0363 0.0440 0.0149 0.0348 0.0149 0.0440

Río Grande 0.0249 0.0402 0.0778 0.0687 0.0572 0.0667 0.0667 0.0687

Fajardo 0.0460 0.0517 0.0889 0.0812 0.0685 0.0778 0.0778 0.0812

Vieques 0.0778 0.0747 0.1111 0.1063 0.1000 0.1000 0.1000 0.1063

Culebra 0.1000 0.0978 0.1333 0.1318 0.1222 0.1222 0.1222 0.1318

Saint Thomas 0.2724 0.2805 0.3053 0.3390 0.2944 0.2944 0.2944 0.3390

Saint Croix 1 0.4158 0.4381 0.4521 0.5500 0.4399 0.4399 0.4399 0.5500

Saint Croix 2 0.4424 0.4667 0.4753 0.5642 0.4643 0.4643 0.4643 0.5642

Saint John 0.1778 0.1795 0.2111 0.2235 0.2000 0.2000 0.2000 0.2235

Jost Van Dyke 0.3748 0.3929 0.4092 0.4815 0.3977 0.3977 0.3977 0.4815

Tortola 2 0.2018 0.2051 0.2379 0.2584 0.2258 0.2258 0.2258 0.2584

Table 5. Continued.

Population Peñuelas Juana Díaz 1 Ciales Juana Díaz 2 Manatí Dorado Aibonito Toa Alta

Cabo Rojo — — — — — — — —

Moca — — — — — — — —

Isabela — — — — — — — —

Mayagüez — — — — — — — —

Guánica — — — — — — — —

Yauco — — — — — — — —

Hatillo — — — — — — — —

Arecibo — — — — — — — —

Peñuelas 0.0 — — — — — — —

Juana Díaz 1 0.2761 0.0 — — — — — —

Ciales 0.3597 0.0209 0.0 — — — — —

Juana Díaz 2 0.1227 0.0266 0.0729 0.0 — — — —

Table 5. Continued.

Population Peñuelas Juana Díaz 1 Ciales Juana Díaz 2 Manatí Dorado Aibonito Toa Alta

Dorado 0.4092 0.1260 0.0775 0.1222 0.0663 0.0 — —

Aibonito 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 -0.0024 0.0596 0.0

Guayama 1 0.2027 0.0605 0.1078 0.0063 0.1299 0.1435 0.1343 0.0373

Guayama 2 0.3544 0.1003 0.0775 0.0760 0.0975 0.1111 0.0841 0.0590

Aguas Buenas 0.3520 0.0674 0.0232 0.0667 0.0411 0.0556 0.0684 0.0124

Cayey 1 0.4106 0.0736 0.0249 0.0725 0.0445 0.0602 0.0568 0.0073

Cayey 2 0.4370 0.0767 0.0258 0.0752 0.0461 0.0625 0.0561 0.0248

Gurabo 0.2227 0.0570 0.0729 0.0038 0.0987 0.1222 0.1063 0.0307

Carolina 0.3863 0.1024 0.0558 0.1000 0.0749 0.0889 0.1022 0.0556

Maunabo 2 0.3420 0.0612 0.0550 0.0386 0.0756 0.0909 0.0710 0.0221

Table 5. Continued.

Population Peñuelas Juana Díaz 1 Ciales Juana Díaz 2 Manatí Dorado Aibonito Toa Alta

Juncos 0.2923 0.0403 0.0232 0.0175 0.0411 0.0556 0.0364 0.0023

Fajardo 0.3863 0.1024 0.0558 0.0909 0.0749 0.0889 0.0495 0.0556

Vieques 0.4092 0.1260 0.0775 0.1222 0.0975 0.1111 0.1248 0.0778

Culebra 0.4322 0.1496 0.0991 0.1444 0.1201 0.1333 0.1474 0.1000

Saint Thomas 0.6048 0.3380 0.2641 0.3163 0.2969 0.3053 0.3238 0.2724

Saint Croix 1 0.8446 0.5143 0.3983 0.4643 0.4488 0.4521 0.4791 0.4158

Saint Croix 2 0.7891 0.5343 0.4223 0.4862 0.4742 0.4753 0.5011 0.4424

Saint John 0.5136 0.2339 0.1741 0.2222 0.1996 0.2111 0.2269 0.1778

Jost Van Dyke 0.7500 0.4603 0.3597 0.4207 0.4047 0.4092 0.4331 0.3748

Tortola 2 0.5956 0.2659 0.1970 0.2500 0.2261 0.2379 0.2559 0.2018

Table 5. Continued.

Population Guayama 1 Guayama 2 Aguas Buenas Cayey 1 Cayey 2 Gurabo Carolina Maunabo 2

Cabo Rojo — — — — — — — —

Moca — — — — — — — —

Isabela — — — — — — — —

Mayagüez — — — — — — — —

Guánica — — — — — — — —

Yauco — — — — — — — —

Hatillo — — — — — — — —

Arecibo — — — — — — — —

Peñuelas — — — — — — — —

Juana Díaz 1 — — — — — — — —

Ciales — — — — — — — —

Juana Díaz 2 — — — — — — — —

Table 5. Continued.

Population Guayama 1 Guayama 2 Aguas Buenas Cayey 1 Cayey 2 Gurabo Carolina Maunabo 2

Manatí — — — — — — — —

Dorado — — — — — — — —

Aibonito — — — — — — — —

Toa Alta — — — — — — — —

Guayama 1 0.0 — — — — — — —

Guayama 2 0.0616 0.0 — — — — — —

Aguas Buenas 0.0862 0.0556 0.0 — — — — —

Cayey 1 0.0943 0.0602 -0.0170 0.0 — — — —

Cayey 2 0.0499 0.0228 0.0 0.0 0.0 — — —

Gurabo 0.0206 0.0760 0.0667 0.0725 0.0560 0.0 — —

Carolina 0.1205 0.0889 0.0333 0.0360 0.0174 0.1000 0.0 —

Maunabo 2 -0.0244 0.0078 0.0307 0.0333 -0.0345 0.0209 0.0667 0.0

Table 5. Continued.

Population Guayama 1 Guayama 2 Aguas Buenas Cayey 1 Cayey 2 Gurabo Carolina Maunabo 2

Juncos 0.0258 0.0162 0.0 0.0 -0.0417 0.0278 0.0236 -0.0199

Fajardo 0.0496 0.0203 0.0333 0.0360 -0.0248 0.0722 0.0572 -0.0370

Vieques 0.1435 0.1111 0.0556 0.0602 0.0228 0.1222 0.0703 0.0909

Culebra 0.1666 0.1333 0.0778 0.0848 0.0881 0.1444 0.1111 0.1154

Saint Thomas 0.3480 0.3053 0.2505 0.2828 0.2969 0.3163 0.2834 0.3127

Saint Croix 1 0.5153 0.4521 0.3919 0.4643 0.5000 0.4643 0.4278 0.4988

Saint Croix 2 0.5339 0.4753 0.4204 0.4931 0.5237 0.4862 0.4534 0.5224

Saint John 0.2482 0.2111 0.1556 0.1724 0.1252 0.2222 0.1638 0.2029

Jost Van Dyke 0.4643 0.4092 0.3520 0.4106 0.3062 0.4207 0.3295 0.4421

Tortola 2 0.2805 0.2379 0.1781 0.2000 0.0870 0.2500 0.1597 0.2333

Table 5. Continued.

Population Juncos Rio Grande Fajardo Vieques Culebra Saint Thomas Saint Croix 1

Cabo Rojo — — — — — — —

Moca — — — — — — —

Isabela — — — — — — —

Mayagüez — — — — — — —

Guánica — — — — — — —

Yauco — — — — — — —

Hatillo — — — — — — —

Arecibo — — — — — — —

Peñuelas — — — — — — —

Juana Díaz 1 — — — — — — —

Ciales — — — — — — —

Juana Díaz 2 — — — — — — —

Table 5. Continued.

Population Juncos Rio Grande Fajardo Vieques Culebra Saint Thomas Saint Croix 1

Manatí — — — — — — —

Dorado — — — — — — —

Aibonito — — — — — — —

Toa Alta — — — — — — —

Guayama 1 — — — — — — —

Guayama 2 — — — — — — —

Aguas Buenas — — — — — — —

Cayey 1 — — — — — — —

Cayey 2 — — — — — — —

Gurabo — — — — — — —

Carolina — — — — — — —

Maunabo 2 — — — — — — —

Table 5. Continued.

Population Juncos Rio Grande Fajardo Vieques Culebra Saint Thomas Saint Croix 1

Juncos 0.0 — — — — — —

Río Grande 0.0023 0.0 — — — — —

Fajardo -0.0069 0.0556 0.0 — — — —

Vieques 0.0363 0.0394 0.0889 0.0 — — —

Culebra 0.0778 0.1000 0.1111 0.1333 0.0 — —

Saint Thomas 0.2505 0.2724 0.2834 0.3053 0.3272 0.0 —

Saint Croix 1 0.3919 0.4158 0.4278 0.4521 0.4766 0.1791 0.0

Saint Croix 2 0.4204 0.4424 0.4534 0.4753 0.4971 0.1804 -0.0891

Saint John 0.1294 0.1253 0.1889 0.1608 0.2333 0.4034 0.5646

Jost Van Dyke 0.2923 0.2476 0.3863 0.2885 0.4322 0.6048 0.8446

Tortola 2 0.1220 0.0842 0.2138 0.1247 0.2622 0.4543 0.6739

Table 5. Continued.

Population Saint Croix 2 Saint John Jost Van Dyke Tortola 2

Cabo Rojo — — — —

Moca — — — —

Isabela — — — —

Mayagüez — — — —

Guánica — — — —

Yauco — — — —

Hatillo — — — —

Arecibo — — — —

Peñuelas — — — —

Juana Díaz 1 — — — —

Ciales — — — —

Juana Díaz 2 — — — —

Table 5. Continued.

Population Saint Croix 2 Saint John Jost Van Dyke Tortola 2

Manatí — — — —

Dorado — — — —

Aibonito — — — —

Toa Alta — — — —

Guayama 1 — — — —

Guayama 2 — — — —

Aguas Buenas — — — —

Cayey 1 — — — —

Cayey 2 — — — —

Gurabo — — — —

Carolina — — — —

Maunabo 2 — — — —

Table 5. Continued.

Population Saint Croix 2 Saint John Jost Van Dyke Tortola 2

Juncos — — — —

Río Grande — — — —

Fajardo — — — —

Vieques — — — —

Culebra — — — —

Saint Thomas — — — —

Saint Croix 1 — — — —

Saint Croix 2 0.0 — — —

Saint John 0.5733 0.0 — —

Jost Van Dyke 0.7891 0.3454 0.0 —

Tortola 2 0.6609 0.1882 -0.0011 0.0

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.

Population Number of Number Island Municipality Heterozygosity number samples 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

Table 6. Continued.

Population Number of Number Island Municipality Heterozygosity number samples 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

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.

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.

Figure 2. Continued.

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.

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.

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.

Figure 5. Continued.

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.

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., CzekanskiǦMoir, 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 IndoǦPacific 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.

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.

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.

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.

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.

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.

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 glacialǦinterglacial 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.

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.

100

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.

101

Papadopoulou, A., and L. L. Knowles. 2015. Genomic tests of the speciesǦpump 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.

102

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.

103

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.

104

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.

105

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

106

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.

107

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.

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Curriculum Vitae Andre Nguyen

Personal Information

Email: [email protected]

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. x 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

x Awards: Dean’s List – Fall 2014 & Spring 2015 (San Diego State University)

x 2018 Aquatic Biology Endowment ($5000)

x 2018 Graduate Professional Student Association Research Sponsorship ($689)

x 2018 Nevada INBRE Service Award ($5000)

x 2018 Nevada INBRE Service Award ($2300)

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