Habitat Affiliation Determines Population Structure and Genetic Diversity in Costa Rican and Reptiles Analisa Shields-Estrada May 2015

Habitat Affiliation Determines Population Structure and Genetic Diversity in Costa Rican Amphibians and Reptiles

An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY

by ANALISA SHIELDS-ESTRADA MAY 2015

Table of Contents

Acknowledgements………………………………….………………………….………..i Abstract…………………………………………………………………………………..ii Introduction………………………………………………………………………………1 Methods…………………………………………………………………………………..6 Results……………………………………………………………………………………9 Discussion………………………………………………………………………………..11 References………………………………….. ……………………………………………17 Tables………………………………………………………………………………..…...20 Figures……………………………………………………………………………………21

Acknowledgements

This research was supported and helped carried out in large part by Dr. Luke O. Frishkoff. I would like to thank my research advisor, Dr. Elizabeth A. Hadly and my second reader, Dr. Gretchen C. Daily, without whom none of this work would have been possible. I would also like to thank the rest of the Daily & Hadly labs, in particular Chase Mendenhall, Hannah Frank, and Katie Solari. Furthermore, I must thank my field assistants in Las Cruces, , Randy and Jeisson Figueroa, and the entire staff at the Las Cruces Field Station. They made my fieldwork in Costa Rica physically possible. Lastly, I thank the Stanford University UAR for funding much of my work.

i ABSTRACT The genetic diversity held within populations can indicate much about the past and present state of a species. Population genetic structure can be an indicator of current or past dispersal barriers that are disrupting gene flow. Documenting and understanding barriers to gene flow allows us to determine what ecological factors play a role in halting genetic dispersal. Habitat affiliation can directly affect population structure and interact with ecological factors to determine whether a species experiences certain barriers as limiting to genetic dispersal. In addition, understanding gene flow and genetic diversity can help us predict how such factors might vary between species with different habitat affiliations, and allow us to answer questions about species’ life histories. For a herptile community in tropical Costa Rica, I asked three main questions. First, I asked for which and reptile species does elevation act as dispersal barrier and restrict gene flow over evolutionary time? Second, I asked what ecological traits do these species share which discriminate them from species that were able to experience undisrupted gene flow across elevational barriers? Lastly, I asked how habitat affiliation of species affects their population structure. To answer these questions I sampled three species of amphibians, ebraccatus, D. microcephalus, Centrolene prosoblepon, and one species of lizard, Norops polylepis. Exhibiting an array of habitat affiliations in southern Costa Rica, from ponds to streams to terrestrial habitats, and an array of macrohabitat requirements, from warmer, low elevations to cool, higher elevations, they allowed for good models to understand the intersection of gene flow, dispersal barriers, and habitat affiliation. Dendropsophus ebraccatus and D. microcephalus, two pond- affiliated species that prefer warmer, lower elevation habitats, were found to have very low levels of genetic diversity. These results supported evidence for evolutionarily low population size in both species. Another possible explanation is a population bottleneck and then subsequent recent migration in both species. Norops polylepis, a terrestrial species was found to have very high genetic diversity that was organized spatially. This allowed me to surmise that N. polylepis might be experiencing a barrier to dispersal, and that this barrier was likely either fixed or organized spatially. However, large population size could also explain these results. Lastly, Centrolene prosoblepon, a stream-affiliated species with a higher elevation tolerance was found to have high levels of genetic

ii diversity, that was not organized spatially. These results indicated that C. prosoblepon experienced a barrier to dispersal and that this barrier was either widespread or mobile due to the lack of spatial organization. Environmental barriers to dispersal are extremely hard to test, however genetic diversity held within each species, and why these levels of diversity are so different from one another can give us possible insight into population structure and dispersal barriers.

iii INTRODUCTION Gene flow along with genetic drift, migration, and natural selection, is one of the four mechanisms influencing genetic diversity. Genetic drift and natural selection are two evolutionary forces acting to weed out variation and genetically homogenize populations of species, effectively lowering genetic diversity (Lacy, 2005). In contrast, gene flow (migration) and mutation act to contribute variation to local populations, and thus increase genetic diversity. The interplay of these four forces, and the ecological factors affecting them, such as barriers to dispersal, are key in determining genetic diversity and structure of a species (Charlesworth, 2010). Therefore, data on genetic diversity and structure can give us insight into the workings of drift, selection, migration, mutation on the evolutionary history of species and their fates. Both high and low levels of gene flow will impact the genetic diversity of species. High gene flow allows for long-range genetic dispersal (Allendorf, 1983), and results in decreased genetic differentiation within species possessing large geographic ranges (Hered, 1998). Low gene flow allows for greater genetic differentiation within a species, and across space, creating low levels of local genetic diversity, while potentially increasing the total amount of diversity held within the species across its entire range. Low levels of gene flow can also facilitate speciation, and in turn an increase in species density (the number of species per a given geographical area) (Ghalambor, 2006; Janzer, 1967; Simpson, 1964). Cessation of gene flow is required for allopatric speciation, or the generation of spatially segregated “descendant species” due to long-term geographical isolation (Doebeli, 2003). Disruption of natural rates of gene flow can occur due to a number of factors. Environmental factors, such as temperature, slope, humidity, distance to a water source, tree cover, and soil moisture, have the ability to increase or decrease gene flow. These environmental factors manifest themselves in distinct areas of a landscape, such as mountain ridges, rivers, and low elevation valleys that act as barriers of dispersal for some species. Phylogeography is the study of the “spatial arrangement of genetic lineages” (Avise, 2009). If no breaks in gene flow exist, and high gene flow prevails over great distances, we can conclude that genes have been exchanged between populations without disruption from environmental barriers or isolation of populations caused of

1 distance. High gene flow can result in low genetic diversity across populations since genes are being shared between populations (Chung, 1994). If, on the other hand, there are many interruptions in gene flow, a likely cause is that environmental or behavioral barriers have halted dispersal between populations. A decrease in gene flow between populations, also deemed population structure, may result in higher genetic diversity between populations over long periods of time since genes are not being shared amongst all populations and therefore the accumulations and deletions of mutations are able to occur separately in different populations without necessarily being swamped out by high levels of gene flow (Kraaijeveld-Smit, 2005). Another possible cause of population structure could be a consequence of the differential extinction of populations between existing populations. This would cause population structure to be the result of past extinctions instead of current limitations of gene flow (Maruyama, 1980). This could especially be the case in a human disturbed, fragmented landscape where population extinctions, and diseases, such as chytrid are likely (Thomas, 2000). Thus certain landscapes and life histories will serve to increase or decrease barriers within species over evolutionary time. These barriers have the potential to restrict dispersal, therefore limiting gene flow and in turn potentially increasing genetic variation. This in turn can lead to an increase in genetic differences, and reduced population size, fitness (Madsen, 1999) and viability of the overall population (Barret, 2007). The disruption of gene flow across environments has been specifically observed in high elevation tropical regions (Ghalambor, 2006). High elevation areas such as mountains, or ridges often act as barriers to dispersal between populations, due to environmental conditions (e.g., humidity, temperature, rainfall) that stay relatively constant across the tropics, and give tropical species a tendency to be only well adapted for narrow, but consistent environmental conditions (Janzen, 1967). Cheviron and Brumfield (2009), investigated the effects of elevation on gene flow. They found that elevation plays an important role in the genetic structure of populations (Cheviron, 2009). When distinct differences are found among the genes of populations from different sides of this ridgeline it suggests that elevation is acting as a dispersal barrier and that populations on either side have been evolving independently from one another in the

2 absence of gene flow to connect them. Another example of this can be seen with elevation and temperature. Although temperatures cool as elevation increases, in the tropics temperature is relatively constant at any given elevation (Ghalambor, 2006). Evidence suggests that tropical species have adapted to this lack of environmental variation, and therefore can tolerate only small temperature ranges, which limits the elevational extent that they can inhabit. As a result of these narrow temperature tolerances, some studies have shown that it is difficult for species to disperse across different elevations (Ghalambor, 2006). Therefore, environmental barriers have the potential to result in substantially decreased gene flow among populations of a species. However, there are other contingent factors that could contribute to variance in gene flow in the absence of environmental barriers. High gene flow could be characteristic of a species with life histories based on high rates of mobility, high intrinsic rates of population growth, or affinity for common habitat types. Low gene flow could be characteristic of a species with low individual mobility, low rates of population growth, affinity with rare habitat types, or possessing highly territorial behaviors (Charlesworth, 2010). Selection can also be a factor influencing gene flow and how it affects genetic diversity. High levels of positive selection, for example, have the ability to wipe out genetic diversity as the beneficial alleles fixes (Whitlock, 2007). My study focuses on amphibians and reptiles in the province of Puntarenas, southern Costa Rica (Figure 1). I focused my work on two pond-affiliated , Dendropsophus ebraccatus and Dendropsophus microcephalus, one stream affiliated , Centrolene prosoblepon, and one terrestrial lizard, Norops polylepis. Research conducted on two sister species of amphibians C. crassidigitus and C. fitzingeri by Frishkoff (Frishkoff et al., unpublished data) found that the Coto Brus Ridge in southern Costa Rica has functioned as a barrier to dispersal for C. fitzingeri but not for C. crassidigitus. Intriguingly these data suggest that this ridge acted as a barrier to gene flow for some species, even though species that experience this genetic barrier exist up to and on top of this ridge. For my thesis research, I am investigating whether multiple reptile and amphibian species sense elevation as a barrier to gene flow by using population genetics.

3 Based on the ideas of population structure, gene flow, and genetic diversity outlined above, we can think about habitat affiliation as a key factor affecting population structure, gene flow, and genetic diversity (Slatkin, 1987). In my study system, ponds are usually created by cattle farmers as watering holes for their cattle. Since Dendropsophus ebraccatus and Dendropsophus microcephalus are both pond affiliated species we might expect high gene flow within ponds, but very low gene flow between ponds, since mobility is limited. This could result in low genetic diversity within ponds and high diversity between ponds (Charlesworth, 2010). On the other hand, if a species is affiliated with a terrestrial habitat, such as Norops polylepis, we would expect it to have high mobility and thus evidence of high gene flow (Charlesworth, 2010). I assume Centrolene prosoblepon, a stream affiliated species would have medium levels of gene flow and genetic diversity between those of terrestrial and pond affiliated species. My objective was three-fold. First, I aimed to identify species that experience environmental barriers. I asked three questions: 1) For which amphibian and reptile species does the high elevation Coto Brus ridge and other elevational barriers in the surrounding area act as a dispersal barrier and restrict gene flow over evolutionary time? I wanted to understand what species traits (elevation limits, temperature tolerances, etc.) permit dispersal despite potential environmental barriers, and thereby differentiate them from species that do experience the particular environmental barrier. To answer this I asked the question: 2) What ecological traits do these species share, which differentiates them from species that were able to experience undisrupted gene flow across this ridge, or other potential environmental barriers? I hypothesized that amphibian and reptile species that prefer or can withstand open canopied, warmer, and lower elevation environments, will experience restricted gene flow over evolutionary time due to the ridge acting as a dispersal barrier. Norops polylepis, Dendropsophus ebraccatus, and Dendropsophus microcephalus, all prefer lower elevation sites, and Dendropsophus ebraccatus, and Dendropsophus microcephalus prefer warmer sites, therefore I hypothesized that these three species, when compared to Centrolene prosoblepon, which can tolerate higher elevation environments, would experience elevation as a barrier to gene flow (Savage, 2002). In particular, the ecological factors of Dendropsophus ebraccatus, and Dendropsophus

4 microcephalus are opposite of those provided by the high elevation ridge, therefore I hypothesized that they will experience a set of conditions they are not adapted to and gene flow will not be able to occur across the ridge. My third objective was to understand the gene flow and diversity dynamics between terrestrial, stream, and pond species of amphibians and lizards. I asked the question, 3) How does habitat affiliation affect population structure? I also aimed to understand the life history behind the four species identified that could lead to unanticipated results regarding gene flow and genetic diversity. I hypothesized that due to the mobility of terrestrial species, terrestrial species do indeed experience environmental barriers to gene flow to a greater extent than species affiliated with other habitats. I predicted terrestrial species, in this case N. polylepis, do indeed experience environmental barriers to gene flow, resulting in far lower gene flow and far higher genetic diversity than would be expected of a terrestrial species when compared to a less mobile, pond or stream affiliated species. I further hypothesized that not only would Dendropsophus ebraccatus, and Dendropsophus microcephalus, experience restricted gene flow across elevational barriers due to preferring lower elevation and warmer environments, but that due to population structure and habitat affiliation, they would experience high levels of gene flow and low genetic diversity within populations, and the opposite between populations. Finally, I hypothesized that Centrolene prosoblepon would experience relatively high levels of gene flow, however lower than those of N. polylepis and low genetic diversity due to its population structure and habitat affiliation. Looking at these questions among a group of varied species, allows us to understand why different species experience gene flow, genetic diversity, and dispersal barriers in such different ways. Focusing on a wide array of habitat affinities in which to understand gene flow, diversity, life history and dispersal barriers, is one way in which to do that.

5 METHODS Study location Amphibian and reptile species were sampled in southern Costa Rica, centered within the Coto Brus valley, and reaching north to the slopes of the Talamanca mountain range, and south to the Bay of Golfito (Figure 1). Coto Brus, is a mid elevation (800m-1400m) originally premontane tropical forest, but has experienced substantial deforestation in the last half century, leading to a patched landscape of forest, cattle pasture, and coffee plantations (Daily, 2003). North towards La Amistad, the elevation slowly increases, with the highest elevation site at approximately 1800m with a tropical humid forest South towards the Bay of Golfito the elevation steadily decreases with the lowest elevation site at 300m with which was compromised of lowland tropical forest.

Study species I have focused my analysis on three amphibian and one lizard species. This includes two very closely related pond species of frog, Dendropsophus ebraccatus and Dendropsophus microcephalus, one stream species of frog, Centrolene prosoblepon, and one scansorial (climbing) lizard species, Norops polylepis. These species display a wide array of habitat affinities in which to understand genetic flow and structure. Dendropsophus ebraccatus, is a small frog (23-35mm Snout-Vent Length (SVL)) belonging to the family of tree frogs, (Savage, 2002). It occurs in a wide variety of habitats from primary lowland humid tropical forest to heavily deforested areas and occupies an elevation range of sea level to 1320m (Savage, 2002). This species lays its eggs above natural or man-made ponds or pools of freshwater and is frequently encountered perched on branches, leaves, sticks, and blades of grass, typically only a half a meter, or at maximum a couple meters above surface level (Savage, 2002). Dendropsophus microcephalus is a similar species, but is found across a smaller elevation range (7m-780m), with a smaller body size (18-31mm SVL). This species occurs in lowland forests and pre-montane forests around temporary ponds, frequently in human-disturbed landscapes (Savage, 2002). Like D. ebraccatus it also lays its eggs above still freshwater and is therefore found on flora overhanging ponds or pools

6 (Savage, 2002). Dendropsophus ebraccatus and D. microcephalus are frequently found to occupy and coexist in the same ponds and pools. Centrolene prosoblepon, on the other hand, is associated with the moving water of streams and rivers in lowland tropical forests and premontane forests (20m-1900m). It belongs to the glass frog family, Centrolenidae, is has a body size range of 21-31mm SVL (Savage, 2002), and is completely nocturnal. Similar to the two Dendropsophus species, C. prosoblepon lays its eggs over freshwater (Savage, 2002). However, it prefers fast-moving water and individuals tend to be found in low vegetation such as trees and branches hanging above fast-moving streams (Savage, 2002). Norops (=Anolis) polylepis is a terrestrial lizard found primarily in pre-montane primary, or intact secondary forests (1m-1330m), and also in some disturbed secondary forests (Savage, 2002). It perches on small plants, shrubs and saplings in premontane primary forests relatively close to the ground, approximately 0.75m-3m off the surface (Savage, 2002). They prefer forest shade and limited exposure to direct sunlight. However, this lizard is quite resilient to variation in temperature, allowing for a large temperature range of 21°C -32°C (Savage, 2002). This species is completely diurnal and approximately 41mm-57mm in length and belongs to the Iguandiae, one of the most specious families of lizards (Savage, 2002).

Sampling procedure From June 2014 to August 2014, I visited a total of 31 sites across southern Costa Rica. Two to three sites were visited each evening, and genetic samples were collected from between 12-30 individuals of each of the four species, when encountered, at each site. All individuals were captured between the hours of 18:00 and 1:00 by hand for genetic sampling. I used flame-sterilized scissors to take a tissue sample (toe or tail clipping), which I immediately placed in 95% ethanol filled Eppendorf tubes. Samples were stored at 4°C until extraction. DNA was extracted from 126 samples of D. ebraccatus, 45 samples of D. microcephalus, 40 samples of C. prosoblepon, and 40 samples of N. polylepis. Then I amplified mitochondrial cytochrome b using polymerase chain reaction (PCR). Cytochrome b was chosen as a good marker due to the existence of primers that function across a wide range of species, and for its

7 abundance in the mitochondrion, and therefore tendency for variation due to a high mutation rate. It is also quite standard for assessing within-species variation in herptiles (Johns, 1998). The length of the regions amplified varied across the four species: A 720- base pair (bp) region was amplified in D. ebraccatus, a 733-bp region in D. microcephalus, a 725-bp region in C. prosoblepon, and a 723-bp region in N. polylepis. Primers for the amphibians were designed using MVZ15-L and CB3Xen-H degenerate primers already designed for cytochrome b (Goebel, 1999). These primers were tested with each amphibian and then the starting and ending position of the primers were adjusted based on sequencing results. For N. polylepis, primers already designed for cytochrome b in N. carolinensis were similarly tested and adjusted (Alfoldi, 2011). PCRs were run in 10 μLvolumes with 5.8μL of H20, 0.2μL of MgCl2 at 25mM, 2.0μL of Taq Ready Mix (5X; Invitrogen) and 0.5μL of both the forward and reverse primers (10μM). A thermocycler set at 95°C for 30 seconds, followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 47°C, 1 minute at 69°C, and 5 final minutes at 69°C, amplified the corresponding region in each of the four species. After amplification, PCR products were sent to Elim Biopharm (Hayward, CA) for sequencing using the identical respective primers at lower concentrations. For D. ebraccatus and D. microcephalus sequencing was run from only the forward direction, due to the difficulty making a reverse primer. For C. prosoblepon and N. polylepis, sequencing was run from both the forward and reverse directions.

Data analysis Within each species sequences were aligned using the program Geneious V6.1.6 (1). From these alignments haplotype networks were created for each species separately using the package “pegas” in R. Unique haplotypes are defined by a specific sequence of base pairs. Using the basic haplotype networks of each species, I proceeded to create haplotype accumulation curves that allowed me to determine whether I was close to sampling the majority of haplotypes among my sites. When this curve begins to level off it shows that as we add more sequences (from additional individuals of that species) no more haplotypes are found. I used this asymptote as an indication that I had sampled the majority of possible haplotypes.

8 Next, I ran a nucleotide diversity test, nuc.div {pegas} to determine genetic diversity within each species as well as among each population within each species. This function computes nucleotide diversity by summing the number of differences between pairs of sequences and dividing that by the number of comparisons. These tests allowed me to look at genetic diversity within species to see whether there was evidence of population structuring. To look at the genetic diversity within and between, I ran AMOVA tests and constructed haplotype networks. Different haplotypes are based on segregating sites among these base pairs. In haplotype networks, the distance between haplotypes demonstrates how genetically distant the haplotypes are. The more mutational steps between haplotypes, the more genetically removed one haplotype is from another. Haplotype networks provide information that when analyzed can aid in understanding the gene flow and genetic diversity within these each of the four species. In order to look at potential environmental barriers, haplotype networks, with populations grouped by site, elevation, and latitude, were also constructed for each of the four species. The AMOVA tests were run on all four species based on site, elevation, and latitude. I used these tests to determine if there were genetic differences between amphibians and reptiles found among different sites (Figure 1), different elevations (Figure 1), or different latitudes (Figure 1). Finally, to look at the relationship between genetic distance and geographic distance as an indicator for gene flow, I ran isolation by distance tests on all four species. Furthermore, I ran global Fst tests on all four species to determine whether the majority of variation held within each species was organized spatially or not.

RESULTS I genotyped 30 Norops polylepis and found 20 different haplotypes, 40 Centrolene prosoblepon possessed 9 different haplotypes, 126 D. ebraccatus had 7 haplotypes, and 45 D. microcephalus had 4 haplotypes (TABLE?). Haplotype sampling of both pond species (D. ebraccatus and D. microcephalus) shows that I have captured the majority of the existing haplotypic variation for this portion of cyt b (Figure 2).

9 Centrolene prosoblepon also demonstrates evidence of almost complete haplotype sampling, however room still exists for more haplotypes to be identified. Norops polylepis, has many more haplotypes to be sampled, as indicated by the steeper slope, and failure to asymptote (Figure 2).

Nucleotide Diversity Nucleotide diversity was measured across all populations for each species and showed substantial variation between species. Centrolene prosoblepon had the highest nucleotide diversity levels (0.009, Figure 3) followed closely by Centrolene prosoblepon (0.008, Figure 3). Dendrosophous ebraccatus and Dendrosophous microcephalus, had much lower nucleotide diversity levels (0.0004, 0.002, Figure 3). No significant differences in nucleotide diversity values were found between different populations of the same species for any of the four species (all p > 0.05).

Relationships between haplotypes The haplotype networks showed relatively high levels of sequence diversity among N. polylepis but low diversity was found among D. ebraccatus and D. microcephalus, with C. prosoblepon species falling in the middle. (Figure 4). The diversity shown in the haplotype networks is partially reflected by the nucleotide diversity levels found in the last section. The haplotype network of D. ebraccaatus showed one haplotype encompassing 90% of the individuals sequenced, demonstrating extremely low haplotypic diversity (Figure 4). Dendropsophus microcephalus exhibited a similar lack of diversity with the great majority of the sequenced individuals falling into one of two closely related haplotypes. Norops polylepis, in contrast, had both closely related and distantly related haplotypes equally represented by the sequenced individuals (Figure 4). Centrolene prosoblepon demonstrated a similar pattern to N. polylepis but with fewer haplotypes.

Genetic Variation, Distance, & Population Structure No patterns were observed between haplotype and site, elevation, or latitude when looking at haplotype networks, with populations grouped by site, elevation, and latitude,

10 as exemplified in Figure 5 & Table 1. All AMOVA tests resulted in non-significant p- values (p > 0.05), supporting a null hypothesis that the potential environmental barriers examined in this study are not playing a significant role in restricting gene flow among, Dendrosophous ebraccatus, Dendrosophous microcephalus, and Centrolene prosoblepon (Table 1). However, the AMOVA test resulted in a borderline significant p-value when testing for genetic differences based on populations grouped by sample site for Dendrosophous microcephalus (p = 0.06), indicating that there could be a significant difference between genetic diversity among sampling sites for Dendrosophous microcephalus. From Figure 2 we know that Norops polylepis has more haplotypes to be sampled, therefore potentially more samples are needed to determine if environmental barriers are also not significant in restricting gene flow among Norops polylepis. Global Fst tests resulted in high measures for both Norops polylepis (0.695) and Dendrosophous microcephalus (0.590), indicating unique genetic variants are held within distinct populations. Contrastingly, the Fst test resulted in low measures for Dendrosophous ebraccatus (0.053) and Centrolene prosoblepon (0.181) indicating that the majority of variation is held within all populations, and is not structured in this landscape. Sampling levels in most populations were too low to robustly calculate pairwise Fst values between populations. As a result analyses of isolation by distance were conducted at the individual level, by comparing the genetic distance between individuals with their geographic distance. Isolation by distance tests resulted in significant positive correlation between geographic distance and genetic distance in Dendrosophous ebraccatus (p = 0.001) and Dendrosophous microcephalus (p = 0.001) (Figure 6). There was no significant correlation between geographic distance and genetic distance in Centrolene prosoblepon or Norops polylepis (Figure 6).

DISCUSSION Gene Flow & Environmental Barriers I found that my original hypothesis regarding elevational barriers was not supported (p>0.05, Table 1). My results instead supported the counter hypothesis that amphibian and reptile species that prefer or can withstand open canopied, warmer, and

11 lower elevation environments do not experience restricted gene flow over evolutionary time due to the Coto Brus ridge, or other elevational barriers acting as dispersal barriers. (p>0.05, Table 1). Furthermore, when within each species, populations were grouped based on latitude, no significant patterns were observed for any of the four species (p>0.05, Figure 5 & Table 1). The same was true when populations were grouped based on sampling site (p>0.05, Table 1). Since none of these results were significantly significant this neither supported or denied the hypothesis that among these four species in this landscape, their macrohabitat requirements are key indicators in determining whether a species experiences restricted gene flow due to environmental barriers. However, these results could lead to a number of conclusions. First, the number and identity of the species I selected may not have not encompassed the broadest differences in macrohabitats of the herpetofauna of the region. In addition, the geographical area (limited sites in southwestern Costa Rica) did not include the most extreme potential environmental barriers of each species’ range. Furthermore, much of southeastern Costa Rica has a uniquely fragmented landscape, ranging from monoculture coffee plantations, to integrated agriculture amongst forest patches, to secondary and primary forests (Daily, 2003). When anthropogenic change disrupts the natural habitat of an area, not only are the flora and fauna affected, but humidity, temperature, and other factors crucial to a species habitat can change as well (Lewis 1998 & McGuiffe 1995). Ground surface temperature levels have been found to increase after deforestation and remain at heightened levels in a number of habitats ranging from southern British Columbia to the Amazon (Lewis 1998 & McGuiffe 1995). In particular, most of my lower elevation sites were primary and old secondary forest, whereas many of my higher elevation sites were true secondary forest and cattle pastures (Esquinas Rainforest Lodge, 2014 & Organization for Tropical Studies, 2015). This could have affected the temperatures at each site and made higher than expected temperatures due to anthropogenic change in relatively high elevation sites, a confounding factor. This is an example of anthropogenic chance at a local scale (Friskoff, unpublished data). We can also think of anthropogenic change at the global level in the form of climate change and consider how that may be affecting our results. Many studies have

12 found that climate change is causing a global poleward shift for many species as the globe warms (Colwell, 2008). However, in the tropics, where there are shallow latitudinal temperature gradients, these shifts tend to be elevational instead of latitudinal (Colwell, 2008). As the globe warms, species in tropical regions are seeking higher elevations, and thus cooler habitats (Colwell, 2008). Both of these factors can cause our previous assumptions that as elevation increases, climates cool, and therefore species that prefer warmer climates will experience barriers to gene flow at higher elevations, to be wrong.

Gene Flow & Habitat Affinity My second hypothesis, that as a terrestrial species, N. polylepis will experience environmental barriers to gene flow to a greater extent than species affiliated with other habitats, was partially supported by my results. My third, closely related hypothesis that Dendropsophus ebraccatus, and Dendropsophus microcephalus, due to population structure and habitat affiliation, would experience high levels of gene flow and low genetic diversity within populations, and the opposite between populations, was, however, not supported by my results (Figure 4 & Table 1). Finally, my final hypothesis that Centrolene prosoblepon would experience relatively high levels of gene flow (though lower than those of the terrestrial N. polylepis) and low genetic diversity due to its population structure and habitat affiliation, was not supported by my results. These three closely related results were due to a multitude of factors. First, the very high levels of genetic diversity in N. polylepis and C. prosblepon, would not naturally be anticipated given medium past population sizes and the absence of dispersal barriers. Both looking at the haplotype networks and the nucleotide diversity, N. polylepis and C. prosblepon demonstrate very high levels of genetic diversity. (Figures 3 & 4). One possible explanation for these results is that some barrier is acting to restrict gene flow and cause high genetic diversity across populations. However, if this is the case, this barrier was not tested for, or not tested for properly in the first part of my experiment, as no significant results were found between populations grouped by elevation, latitude, or sampling site in N. polylepis. Other potential barriers could include habitat fragmentation and bodies of water (Gantenbein, 2003). Furthermore, these results may suggest that N. polylepis and C. prosblepon have had evolutionarily large total

13 population sizes, which have allowed for high overall genetic diversity. With larger population sizes you have more chances for polymorphisms to arise, meaning there will generally be greater genetic diversity in larger population sizes (Nei, 1993). Therefore, the high genetic diversity results seen in N. polylepis and C. prosblepon could be simply explained by large population sizes. The puzzling results of low genetic diversity in Dendropsophus ebraccatus, and Dendropsophus microcephalus, (Figures 3 & 4), could possibly be explained by a recent bottleneck, although we have no statistical evidence for this. These species naturally inhabit pond like pools of standing water in forests. As forests in Costa Rica have experienced anthropogenic change and decreased in magnitude, density, and abundance, the natural habitat of these two species could have been reduced, resulting in a bottleneck. Very low genetic diversity would be characteristic of a species that recently experienced a population bottleneck (Nei, 1975), since population bottlenecks are known to cause drastic reductions in genetic diversity among the species experiencing the bottleneck (Nei, 1975 & Houlden, 2009). However, as deforested lands were turned into cattle pastures, which have increased drastically in the last century in Costa Rica, man made ponds that in many regards mimic the ones naturally found in forests, have become widespread (Holl, 2001). Therefore, after the population bottleneck of these two species, I surmise there was a population boom and very recent migration to these man made sites, causing genetic diversity to still reflect the diversity right after the bottle neck, before population expansion occurred. Post bottleneck migration typically results in an increase in genetic diversity (Nei, 1975). Since some genetic diversity, yet very little, exists within Dendropsophus ebraccatus, and Dendropsophus microcephalus, very recent migration following a population bottleneck is very possible. This would allow for very low genetic diversity and little genetic differentiation between or within populations of Dendropsophus ebraccatus, and Dendropsophus microcephalus. However, similarly to large population size being a likely explanation for high genetic diversity, low population size could also explain the low genetic diversity found among Dendropsophus ebraccatus and Dendropsophus microcephalus. If these two species always existed at relatively low population sizes, this alone could be evidence for low genetic diversity between populations of both species (Ellstrand, 1993).

14 Genetic Variation & Population Structure Based on the results of the isolation by distance tests and the Fst measures, a few more rigorous conclusions can be drawn regarding genetic variation and population structure. These tests further support the hypothesis that N. polylepis has highly genetically different populations, leading us to hypothesize that in the area sampled N. polylepis could have low gene flow and could be interacting with a barrier to gene flow. Furthermore, we can conclude that the genetic differences are organized spatially, which allows us to conclude that these barriers seem to be fixed and organized. In regards to C. prosoblepon, the results indicate that their genetic diversity is not organized spatially, again supporting the conclusion that barriers to dispersal are at play, and that unlike in N. polylepis, there may be multiple types of prevalent barriers, or these barriers may be a mobile or widespread, such as competition with another species or habitat fragmentation, making them lack spatial organization. For Dendropsophus ebraccatus and Dendropsophus microcephalus, geographic distance and genetic difference correlate positively with one another meaning that as geographic distance increases so does genetic difference. This implies lower gene flow since we would expect genetic distance to stay constant with geographic distance if gene flow was very high. However, how low of gene flow can be predicted from this test is not an available measurement, so no conclusions can be drawn from this. In conclusion, we can say little about potential enivornmental barriers affecting gene flow for Dendropsophus ebraccatus, Dendropsophus microcephalus, Centrolene prosoblepon, and N. polylepis or which macrohabitat requirements are affiliated with species that tend to experience potential barriers to gene flow. However, we can conclude much about the genetic diversity held within populations of these four species and why that might be occurring. We can attempt to tie this diversity back to population structure and environmental barriers. Dendropsophus ebraccatus and Dendropsophus microcephalus, show evidence for evolutionarily low population sizes, and showed little to no evidence for restricted gene flow due to dispersal barriers. Low genetic diversity among these two species could also be due to a population bottle neck and recent migration, although I do not have statistical results to support this hypothesis. Norops polylepis and C. prosoblepon showed evidence for large past population sizes and/or high

15 genetic diversity due to experiencing a barrier to genetic dispersal. In conclusion, environmental barriers to dispersal are extremely hard to test, however genetic diversity held within each species, and why these levels of diversity are so different from one another can give us possible insight into population structure and dispersal barriers.

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APPENDIX A Table 1. AMOVA results across all three population types and all four species (where “Sampling Site” refers to unested populations, “Latitude “refers to populations grouped by latitude, and “Elevation” refers to populations grouped by elevation).

p-value D. ebraccatus D. microcephalus C. prosoblepon N. polylepis Sampling 0.14 0.01 0.40 0.27 Site Latitude 0.55 0.63 0.42 0.69 Elevation 0.85 0.28 0.41 0.99

Figure 1. Map of the study area and the sampling sites shown in red.

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Figure 2. Haplotype Accumulation Curve for D. ebraccatus, D. microcephalus, C. prosoblepon, and N. Polylepis

21 Nucleotide Diversity Among Four Speices 0.008 0.006 0.004 Nucleotide Diversity Nucleotide 0.002

C. prosblepon D. ebraccatus D. microcephalus N. polylepis

Species

Figure 3. Nucleotide Diversity values for D. ebraccatus, D. microcephalus, C. prosoblepon, and N. Polylepis

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Figure 4. D. ebraccatus, D. microcephalus, C. prosoblepon, and N. Polylepis haplotype networks. Each circle represents a different haplotype network. The size of each circle represent the relative number of individuals with that particular haplotype. The distance between the circles represent the genetic distance between haplotypes where each notch represents a single polymorphism.

23 North VII Mid VI South

II I V

III IV

Figure 5. Dendrosophous ebraccatus haplotype network based on latitude (the northern most sites were grouped into “North”, the southern most sites into “South,” etc.)

24 D. ebraccatus D. microcephalus 0.030 0.030 r = 0.28 r = 0.57 p = 0.001 p = 0.001 0.020 0.020 0.010 0.010 Genetic Distance Genetic Distance Genetic (Proportion Difference) (Proportion Difference) (Proportion 0.000 0.000 0.00 0.05 0.10 0.15 0.20 0.25 0.00 0.05 0.10 0.15 0.20 0.25 Geographic Distance (Degrees) Geographic Distance (Degrees)

C. prosoblepon N. polylepis 0.030 0.030 r = 0.01 p = 0.26 0.020 0.020 0.010 0.010 r = 0.03 Genetic Distance Genetic Distance Genetic p = 0.17 (Proportion Difference) (Proportion Difference) (Proportion 0.000 0.000 0.00 0.05 0.10 0.15 0.20 0.25 0.00 0.05 0.10 0.15 0.20 0.25 Geographic Distance (Degrees) Geographic Distance (Degrees)

Figure 6. Isolation by Distance results for D. ebraccatus, D. microcephalus, C. prosoblepon and N. polylepis.

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