final report genetic characterization of populations of the

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FINAL REPORT GENETIC CHARACTERIZATION OF POPULATIONS OF THE

NONINDIGENOUS BURMESE PYTHON IN EVERGLADES NATIONAL PARK

Prepared by: Timothy M. Collins and Barbie Freeman

Department of Biological Sciences Florida International University

University Park Miami Florida, FL 33199

And

Skip Snow U.S. National Park Service Everglades National Park

400l SR 9336 Homestead, FL 33034

Prepared for the South Florida Water Management District

2008

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Summary The Burmese Python, Python molurus bivittatus has become established in

Everglades National Park (ENP). Because pythons have been sampled from geographically disparate locations in ENP, it is possible that these populations are the result of independent introductions. The full extent of the invasion of Python molurus bivittatus in ENP is not known. Burmese pythons were first reported as established in ENP by Meshaka et al. (2000). Since then, the number of pythons captured or found dead in and around ENP has increased dramatically. Young snakes as well as female snakes with eggs have been captured, further providing evidence of reproduction. Knowledge of population structure is fundamental for developing a control and management plan for an invasive species, but the size, geographical extent, or degree of connectedness among populations in and around ENP is not known. Molecular data including mitochondrial DNA sequences and microsatellite loci were used to (1) examine the genetic diversity of populations of Python molurus bivittatus in ENP, (2) determine whether the distribution of genetic diversity suggests that the populations sampled to date are heterogeneous, to be managed separately, or are likely part of a single large population, and (3) determine if there is genetic evidence for parthenogenetic reproduction in Python molurus bivittatus in ENP. Multiple analyses including assignment methods and F-statistics revealed that the ENP Burmese pythons are genetically distinct from pythons sampled from Vietnam, but display little genetic differentiation within the Park, with the exception of a few outliers. The lack of genetic differentiation of most Burmese Pythons in ENP may indicate either a panmictic freely interbreeding population in the Park, or alternatively, limited genetic variation in the captive-bred populations that are the likely source of these snakes. Sampling of mothers and offspring provided no evidence of parthenogenetic reproduction in ENP pythons. Management strategies should focus on controlling an established population that is with a few exceptions, not genetically differentiated.

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INTRODUCTION

Burmese pythons have been found in Everglades National Park (ENP) since the early 1980’s (Meshaka et al., 2000), but have shown a dramatic increase in numbers since 1995 (Snow, 2006). Because pythons have been sampled from geographically disparate locations in Everglades National Park (Snow, 2006), it is believed that these introductions are the result of several instances of pet release. Python molurus is a constrictor with a general diet that in its native range of Southeast Asia can reach up to 6 meters in length and over 90 kilograms in weight, with a lifespan of up to 25 years (Ernst and Zug, 1996). Effects of this invasion have not yet been studied extensively, but preliminary evidence suggests that pythons may compete with native species for habitat and prey, may have a negative impact on the recovery of some rare and endangered wildlife species.

Genetics and phylogenetics provide valuable information needed for the study and management of invasive species, especially with the development of high-resolution genetic markers such as microsatellites, and the increasing ease of high-throughput DNA sequencing. Population genetics can provide information on population structure dispersal, breeding, and migration patterns. Phylogenetics can provide information on the taxonomy of the introduced species and the relatedness among the introduced populations, and can link the introduced population(s) to their source populations if native-range samples are available (Baker and Moeed, 1987; Collins et al., 2002; Hufbauer et al., 2004; Stepien and Tumeo, 2006).

Molecular evidence for parthenogenesis has been observed in a study of captive Python molurus bivittatus in the Artis Zoo in Amsterdam (Groot et al., 2003) but has not been demonstrated in wild populations. In that study, comparisons of microsatellite and AFLP markers showed that a female who had been separated from males had offspring that were all genetically identical. Limited genetic variation may suggest that this python species has the ability to develop parthenogenetically in their introduced habitat. Parthenogenesis should result in an excess of homozygotic and female individuals. Parthenogenesis has not been verified in Burmese Pythons in Florida. Female snakes captured with eggs provide an opportunity to explore this phenomenon, as offspring should be genetically identical females if parthenogenetically produced. This mode of reproduction may have important implications for the establishment and spread of populations of this species.

The full extent of the range of Python molurus bivittatus in ENP is unknown. Young snakes as well as female snakes with eggs and broods have been captured, providing evidence of reproduction. Pythons have been captured and sighted in different geographical areas of Everglades National Park. It is unclear whether this distribution results from separate introductions, or a single population that has become established in ENP. Molecular evidence can determine if the population consists of one or several different genetic units, and whether the distribution is patchy or uniform. Knowledge of population structure is fundamental for developing a control and management plan for an invasive species (Cowled et al., 2006; Abdelkrim et al., 2007; Cowled et al., 2007; Scalici and Gherardi, 2007), but the size, geographical extent, or degree of connectedness among populations in ENP is not known.

The Objectives of this project were to: • Determine the genetic diversity and population structure of Python molurus bivittatus in ENP, specifically, whether the distribution of genetic diversity suggest that the

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populations sampled to date are distinct, to be managed separately, or are they likely part of a single large population. • Determine whether there is genetic evidence for parthenogenetic reproduction in Python molurus bivittatus in ENP.

II. METHODS

Sample collection

Python molurus bivittatus tissue samples from 156 individuals were collected from

various regions of Everglades National Park (Figure 1). The sample included two mothers with their broods (one with six and one with fourteen embryos). UTM and/or a description of geographic location were recorded for all but six individuals. When possible information on sex, size, and reproductive condition such as testis size in males and number and size of oviductal eggs in females was recorded. These data provided information on sex ratio, estimated age and reproductive status to indicate breeding patterns. We also sampled thirteen snake skins from Python molurus bivittatus provided by a local reptile dealer and reported to be from a wild population in Vietnam, as well as a skin shed by a Burmese python for sale at a local pet store (see Appendix and Figure 1 for collection details). DNA was isolated from muscle tissue from the head and “neck”, tail, mid-body ribs; the entire embryo; or snake skin using standard phenol chloroform extraction methods (Saghai-Maroof et al., 1984). Mitochondrial DNA

In a subset of sixteen Burmese pythons from different regions of ENP a 360 base pair

region of the mitochondrial cytochrome b gene was amplified with the polymerase chain reaction (PCR). In a subset of eleven ENP pythons from different regions, a skin from a Burmese python for sale at a local reptile store, and two of the Vietnam skins, a 1300 bp control region sequence was determined using species-specific primers. Microsatellite genotyping

Jordan et al. (2002) developed 27 microsatellite loci conserved across 13 different

Australian python species, which were tested on the ENP Python molurus bivittatus. Ten of these loci were suitable for population analysis, displaying clean amplification and polymorphism, and were amplified by PCR. Microsatellite alleles were compared visually with gel electrophoresis, and each locus was sequenced to confirm correct amplification of the expected repeat sequence. Microsatellite PCR products were run for fragment analysis on an ABI Prism 3100 or 3130 Genetic Analyzer using POP4 polymer, and fragment sizes were analyzed with GeneScan3.1 and 3.7 or GeneMapper4 software (Applied Biosystems). Population analyses

Microsatellite data for both the entire data set (170 individual Burmese pythons) and the

data set without the Vietnam pythons (157 individuals) were analyzed using STRUCTURE (Pritchard et al., 2000) to determine the number of populations and assignment of individuals. Each run included 1 million iterations with a burnin of 10,000. Pritchard et al. (2000) used

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1,000,000 iterations in simulation studies to test their program. While various studies have used different burnin values for STRUCTURE, Evanno et al (2005) found that burnins longer than 10,000 did not change results significantly, and we observed that results did not vary greatly over separate, independent runs. Values for K (number of populations) were set equal to 1-15, with 10 runs for each K, using the admixture model with allele frequencies correlated. It has been observed that the admixture model and correlated allele frequencies is best suited for populations that may have similar frequencies due to migration or common ancestry, and therefore have low differentiation and subtle population structure (Falush et al., 2003; Latch et al., 2006; Rowe and Beebee, 2007). A second order rate change with respect to K (ΔK) as defined in Evanno et al., 2005 was calculated.

The entire data set was examined using the program TESS (Francois et al., 2006; Chen et

al., 2007) adding UTM coordinates for each individual where possible, using the admixture model, 100,000 iterations with a burnin of 10,000 for each run for maximal number of clusters (K) equal to 1-15, 10 runs for each K. The entire data set was also run in TESS not using the admixture model also for values of K equal to 1-15, 10 runs for each K, 100,000 iterations with a burnin of 15,000. These initial runs without the admixture model resulted in estimated population numbers of 2, 3, and 4, so 100 more runs with the same parameters and with the maximum value for K set to 4 were performed. The TESS manual recommends a burnin of 10,000 and 50,000 iterations, starting at a maximal number of 2 clusters. After examining the point of stabilization in the log likelihood history of preliminary runs, a burnin value of 15,000 seemed more appropriate for the non-admixture runs. Again variation was minimal across different, independent runs for the same K. The data set without the Vietnam pythons was also run in TESS using the same parameters for non-admixture runs, K equal to 1-15, 10 runs for each K.

Using the value of K and the assignments determined by the first two programs the data was run in DOH (Paetkau et al., 1995) treating any outlier individuals or those with probabilities of assignment less than 0.9 as having unknown population origin to confirm their correct assignment. The data was run in GENALEX6 (Peakall and Smouse, 2006) and GENEPOP 3.4 (Raymond and Rousset, 1995) also using the number of populations and assignments from previous analyses to test for Hardy Weinberg equilibrium and determine observed and expected heterozygosity, allele patterns, number of migrants per generation (Nm) and FST. Embryos were omitted from these analyses because their inclusion violates the random sampling assumption of HW. MICRO-CHECKER (Van Oosterhout et al., 2004) was performed to check for genotyping errors in the ENP and Vietnam subpopulations determined through the previous analyses, using a 99% confidence interval. These errors commonly cause incorrect scoring and include null alleles (an allele fails to amplify during PCR), stutter (a product created from slip strand amplification in PCR), and large allele dropout (large alleles fail to or do not amplify efficiently compared to small alleles) (Bonin et al., 2004; Hoffman and Amos, 2005; Selkoe and Toonen, 2006).

III. RESULTS

Mitochondrial DNA Data from the subset of Burmese Python individuals captured in various areas of ENP showed no variation within the cytochrome b sequences among the ENP individuals, but some variation was

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present between the ENP pythons and Python molurus sequences downloaded from GenBank. SS127 and SS224 were the only individuals collected from ENP that had any differences within the approximately 1300 base pair control region sequence. SS127 and SS224 displayed 6 and 2 differences respectively from the other ENP pythons. There were only three phylogenetically (parsimony) informative sites in the control region sequence. These sites linked (1) the two samples from Vietnam, (2) Sample SS127 and the store skin, and (3) the Vietnam samples, SS127, the store skin, and SS224. The limited variation in these two regions among the ENP pythons suggests that mitochondrial DNA is not a useful molecular marker to assess genetic structure within the Park, but might prove useful in the determination of source populations, although there are too few variable characters to provide robust statistical support. Number of populations

The average number of alleles for each microsatellite locus was eleven with a range from

seven to sixteen. Of 170 individuals thirteen had some missing data, the majority missing only one or two loci and one individual missing five loci. Of these individuals, probability of assignment or Q, also described as the estimated membership coefficient of an individual to or proportion of individual’s genome originating from a cluster (Pritchard et al., 2000), was less than 0.9 in only two snakes. The estimate for number of populations (K) from the program STRUCTURE as determined by highest log probability of K was five with a log probability of -4363.07, however the values reached a plateau at two after which standard deviation increased (Figure 2a). These data are consistent with observations that the log probability of K tends to plateau and climb slightly while variance among runs increases (Pritchard et al., 2000; Evanno et al., 2005; Latch et al., 2006). A better indicator of the true value of K in this situation is Evanno et al’s (2005) second order rate change (ΔK). ΔK was calculated to be two clusters (Figure 2b). The majority of individuals were assigned to their clusters with Q values of 0.9 or more (Figure 3). One cluster included the majority of the Everglades National Park pythons (ENP cluster) while the other cluster included all of the Vietnam pythons, the store snake, and three of the ENP pythons: SS127, SS224, and SS384 (Vietnam cluster). Note that this latter group fits the phylogenetic placement based on mtDNA for the individuals assayed. However SS384’s assignment is not definite as the average Q values were 0.4299 to the ENP cluster and 0.5701 to the Vietnam cluster. When the data was run without the Vietnam pythons STRUCTURE grouped the remaining pythons (ENP samples and store sample) into four clusters with an average log probability of K of –3884. This was the highest log probability of K, but also had the greatest standard deviation (Figure 4a). Delta K also indicates four as the number of clusters (Figure 4b). However, individuals do not have decidedly large proportions of their genomes belonging to their cluster (average Q value was 0.5888) and the clusters divide up SS006 and her set of embryos and distribute them into all four clusters (Figure 5). The four clusters show no geographic pattern.

Using the geographical coordinates as an a priori parameter, and when using the admixture model the program TESS always estimated number of clusters to be equal to the maximal number of clusters input for each run. When run without the admixture model, the estimated number of clusters was two, three, or four (excluding runs where maximum cluster number was 1) with the majority of the runs displaying two clusters. When the entire data set was rerun with the maximal number of clusters equal to four, the estimated number of clusters was two 76 out of 100 runs with an average log-likelihood of -4384, and three 24 out of 100 runs

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with an average log-likelihood of –4383. The majority of one cluster represented an Everglades National Park (ENP) cluster and the other a Vietnam cluster including the store snake and ENP pythons SS127 and SS224. The intermittent third cluster represented five outliers. Two of these outliers, SS127 from the East Everglades Ranger Station and SS224 from the L-67 Ext. Canal Levee (see Figure 1b), have probabilities of 0.178 and 0.004 respectively of their genotype belonging to the Vietnam cluster. Each has zero probability of belonging to the ENP cluster and a greater probability of belonging to neither of the two clusters. Two other outliers, SS369 from Coptic Hammock, ENP and SS384 from Main Park Rd., Rock Reef Pass, have probabilities of 0.190 and 0.155 respectively of their genotype belonging to the ENP cluster and 0 probability of belonging to the Vietnam cluster. Each also has a greater probability of belonging to neither of the two clusters. The final outlier is the Burmese python from a local reptile store that has a probability of only 0.010 of their genotype belonging to the Vietnam cluster and zero probability of belonging to the ENP cluster with a greater probability of belonging to neither of the two clusters. In the 10 runs where the maximal number of clusters was set to two, SS127, SS224, and the store python were grouped with the Vietnam cluster and SS369 and SS384 were grouped with the ENP cluster (Figure 6). TESS grouped the data set without the Vietnam pythons into 1 cluster in 13% of the runs with an average log-likelihood of –3936 and into 2 clusters in 87% of the runs with an average log-likelihood of –3276. SS006 and her embryos were separated between these two clusters, and the clusters show no geographical pattern (Figure 8). Population assignment

While each of these programs assigns individuals to clusters (subpopulations) the

assignment test DOH was used to confirm the assignment of outliers and individuals that had a Q value of less then 0.9 (according to STRUCTURE) of belonging to their cluster, and ensure all other individuals were assigned correctly. Individuals that had Q values higher than 0.9 were assigned to populations 1 (ENP) or 2 (Vietnam) as determined from STRUCTURE and TESS analyses, while the others were not assigned a population. DOH confirmed all the individual assignments from STRUCTURE and TESS, excepting SS384’s uncertain assignment from STRUCTURE, grouping SS127 and SS224 with the Vietnam cluster and SS369 and SS384 with the ENP cluster. F-statistics and Hardy Weinberg equilibrium

Results from GENEALEX6 revealed an average FST between the ENP and Vietnam populations of 0.088, representing moderate genetic differentiation. In population 1 (ENP) average expected heterozygosity (He) = 0.657 and average observed heterozygosity (Ho) = 0.634 and for population 2 (Vietnam) He = 0.805 and Ho = 0.735. GENEPOP generated an average FST

of 0.1824 between the two populations, still considered moderate genetic differentiation. Tests for Hardy Weinberg equilibrium are only reported from GENEPOP as it offers a more exact test with standard error provided. These analyses show that two out of ten loci (MS16 and MS22) in the ENP population and four out of ten loci (MS06, MS16, MS22, MS24) in the Vietnam population were not in Hardy Weinberg equilibrium at the P<0.05 level of significance. In the ENP population, five loci show heterozygote deficiency while one locus shows an excess of heterozygotes, and in the Vietnam population four loci show heterozygote deficiency while one locus shows an excess of heterozygotes. GENEALEX average FST among all four populations

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from STRUCTURE results for the data set without Vietnam pythons was 0.060. Average pairwise FST was 0.04. GENEPOP average FST among all four populations was 0.0646 and average pairwise FST was 0.0595. Between the two populations from TESS results for the data set without Vietnam pythons, GENEALEX average FST was 0.027, and GENEPOP average FST was 0.0453. It has been observed that STRUCTURE and Evanno et al’s (2005) second order rate change (ΔK) can correctly identify the true number of populations when FST is greater than 0.03, but below this does not correctly delineate subpopulations (Latch et al., 2006). Geneotyping errors

Results from MICRO-CHECKER showed no evidence for large allele dropout or scoring

error due to stutter in any of the loci for either subpopulation. According to MICRO-CHECKER null alleles may be present in locus MS22 in the ENP population based on an excess of homozygotes (expected homozygotes = 51.452, observed homozygotes = 72). In the Vietnam population null alleles may be present in locus MS09 (expected homozygotes = 2.156, observed homozygotes = 9) and MS24 (expected homozygotes = 3.25, observed homozygotes = 9). However, homozygous excess may be due to a departure from HWE as opposed to the presence of null alleles. A departure from HWE is likely in both populations because the ENP population may be from an inbred captive bred source and the Vietnam population consists of a very small sample size.

IV. DISCUSSION

Our results indicate that the Python molurus bivittatus populations in Everglades National Park are not genetically structured. However, there are competing hypotheses for this lack of structure. It is possible that the python population in the Park is freely interbreeding or panmictic. There are no significant geographical barriers in ENP to this species as the Burmese pythons are adept swimmers and climbers, and radio tracking of individual snakes has revealed movement across large distances in the park (Mazotti et al., 2007). Alternatively, lack of genetic differentiation may indicate a population originating from a genetically depauperate source population in the pet trade. However, even though native Burmese python populations are considered threatened and are CITES II listed, as are all snakes from the family Boidae, it is legal to import native caught pythons, for example over 12,000 were imported between 1989-2000 (Reed, 2005), and were sold by breeders or reptile suppliers.

P. m. bivittatus captive-bred populations originally came from a native population, but have had time to genetically differentiate, although it is difficult to know how long ago the captive bred populations were first introduced from the native and how many subsequent introductions have occurred. The differentiation of the captive bred from the native populations is likely the result of artificial selection (docility, breeding schedule, color morphs) and extensive inbreeding (http://www.anapsid.org/conserv.html). These factors could contribute to the ENP population’s deviation from Hardy Weinberg equilibrium. In the future an extensive comparison of the genetic structuring and composition of the captive bred populations and the introduced ENP population could further elucidate the genetic structure and evolution of the ENP pythons. The single representative of a Burmese python from a reptile store in this study is genetically distinct from the ENP pythons, but like the other four outliers (SS127, SS224, SS369, SS384) the majority of its genotype does not belong to either the ENP or Vietnam cluster. While it is not

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possible to determine from our data whether each individual snake is a recent introduction or a snake from subsequent generations born in the park, SS127, SS224, SS369, and SS384 may be examples of genetically distinct recent introductions. Of course, if the genotypes of recently introduced snakes match the common types found in the Park, these introductions will be difficult to recognize.

Limited genetic variation in the ENP Burmese pythons in mitochondrial DNA, especially the highly variable control region, and in some microsatellite loci may suggest that the captive bred population of Burmese pythons from which the ENP pythons originated has very limited genetic variation. Other possible reasons for limited variation include extensive dispersal and interbreeding throughout the park, or reduced rates of evolution in the control region for this species. Limited variation may also be an indication that this python species has reproduced parthenogenetically, however there was not an excess of females (55% male, 45% female) or homozygotic individuals (observed heterozygosity = 0.634). Two sets of embryos sampled from known mothers possessed novel alleles relative to the mother clearly indicating they were not parthenogenetically produced clones. These results indicate that the female Burmese pythons sampled in ENP were not reproducing parthenogenetically as seen in the Amsterdam zoo python (Groot et al., 2003). It may be possible however that this reproductive strategy is only pursued in extreme circumstances, for example, in the absence of males. This may be an important consideration if attempting to implement strategies for management that include removal or sterilization of males. In conclusion, our results indicate that there is limited genetic differentiation among populations of Python molurus bivittatus in Everglades National Park, suggesting that the Burmese python populations are not genetically differentiated, with the exception of the outliers noted above, which may represent independent introductions. This lack of genetic differentiation could be the result of a freely interbreeding panmictic population, or alternatively, isolated populations separately introduced from a genetically uniform captive-bred source population. Further studies of genetic variation among native range Burmese pythons as well as those in the reptile trade could further clarify provenance and genetic heritage of the ENP pythons. Additional investigations should also include samples from pythons now occurring throughout South Florida on the periphery of the “core” distribution as described in this report.

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a Figure 1a: GIS satellite photo of collection sites (with field Ids) in ENP

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b Figure 1b – approximate collection sites on ENP map from the National Park Service downloadable at http://www.nps.gov/ever/planyourvisit/maps.htm (blue diamonds represent outliers SS127 and SS224).

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ro

bK

Figure 2: Results from STRUCTURE with the entire data set (ENP and Vietnam); a – plot of the mean log probability of K (population number) for number of populations 1-15 (±SD) over 10 runs for each K. b – plot of Evanno et al’s (2005) delta K for number of populations 2-15.

17

a

b

c

Figure 3: Results from STRUCTURE, K=2; a – cluster assignment, probability of belonging to a cluster on vertical axis, individuals on horizontal axis seen in a single line or; b – multiple lines. Colors represent different clusters (green=ENP cluster 1, red=Vietnam cluster 2). Individual numbers refer to order of individuals in STRUCTURE data, see appendix I for corresponding field Ids; c – triangle plot showing clustering of individuals.

18

a

K vs lnprob of K

-4250

-4200

-4150

-4100

-4050

-4000

-3950

-3900

-3850

-3800

0 2 4 6 8 10 12 14 16

K

ln p

rob

of

K

b

K vs deltaK

-50

0

50

100

150

200

250

0 2 4 6 8 10 12 14 16

K

delt

aK

Figure 4: Results from STRUCTURE for the data set without the Vietnam pythons; a – plot of the mean log probability of K (population number) for number of populations 1-15 (±SD) over 10 runs for each K. b – plot of Evanno et al’s (2005) delta K for number of populations 2-15.

19

a

b

c

Figure 5: Results from STRUCTURE for data set without Vietnam pythons, K=4; a – cluster assignment, probability of belonging to a cluster on vertical axis, individuals on horizontal axis seen in a single line or; b – multiple lines. Colors represent different clusters. Individual numbers refer to order of individuals in STRUCTURE data, see appendix I for corresponding field Ids; c – triangle plot showing clustering of individuals.

20

a

b

Figure 6: Results from TESS with entire data set (ENP and Vietnam), number of clusters=2; a – cluster assignment, individuals along horizontal axis (see Appendix I for corresponding field Ids) probability the individual is in a cluster on vertical axis, colors represent different clusters (green=ENP, red=Vietnam); b – clusters showing geographical distance, group of individuals (black dots) at the top in green represent pythons caught in ENP and the store sample (in red), individuals in the middle in red are Vietnam pythons, and individuals at the bottom in green are ENP pythons missing coordinates or geographical description (Appendix 1).

21

a

b

Figure 7: Results from TESS for the data set without the Vietnam pythons, number of clusters=2; a – cluster assignment, individuals along horizontal axis (see Appendix I for corresponding field Ids) probability the individual is in a cluster on vertical axis, colors represent different clusters; b – clusters showing geographical distance, group of individuals (black dots) at the top represent pythons caught in ENP and the store sample (in red), and individuals at the bottom (in blue) are ENP pythons missing coordinates or geographical description (Appendix 1).

22

Appendix I: Specimen No. (STRUCTURE and BAPS), Field ID, Collection site and coordinates where available (coordinates with* are estimated from location data), total length, snout to vent length, tissue type, and notes Spec No. Field ID

Collection Date Collection Location UTM - E UTM - N

TL (cm)

SVL (cm) Sex

Tissue Sampled Notes

1 SS006 274.5 242 F Tail 2 SS006ae embryo embryo of SS006 3 SS006be embryo embryo of SS006 4 SS006ce embryo embryo of SS006 5 SS006de embryo embryo of SS006 6 SS006ee embryo embryo of SS006 7 SS006fe embryo embryo of SS006 8 SS006ge embryo embryo of SS006 9 SS006he embryo embryo of SS006 10 SS016 11 SS026e 25-Jan-06 521570 2813641 embryo embryo of SS354 12 SS027e 25-Jan-06 521570 2813641 embryo embryo of SS354 13 SS030e 25-Jan-06 521570 2813641 embryo embryo of SS354 14 SS031e 25-Jan-06 521570 2813641 embryo embryo of SS354 15 SS032e 25-Jan-06 521570 2813641 embryo embryo of SS354 16 SS033e 25-Jan-06 521570 2813641 embryo embryo of SS354 17 SS035e 25-Jan-06 521570 2813641 embryo embryo of SS354 18 SS036e 25-Jan-06 521570 2813641 embryo embryo of SS354 19 SS037e 25-Jan-06 521570 2813641 embryo embryo of SS354 20 SS038e 25-Jan-06 521570 2813641 embryo embryo of SS354 21 SS039e 25-Jan-06 521570 2813641 embryo embryo of SS354 22 SS040e 25-Jan-06 521570 2813641 embryo embryo of SS354 23 SS041e 25-Jan-06 521570 2813641 embryo embryo of SS354 24 SS043e 25-Jan-06 521570 2813641 embryo embryo of SS354

25 SS082 19-Sep-03 Main Park Road, near Sisal Pond, ENP 522743 2811855

2'-3' est. Tail To FLMNH

23

26 SS084 18-Sep-03 Main Park Road, Nine Mile Pond, ENP 520295 2792782 66.5 58 Tail

27 SS092 19-May-03 Dan Beard Center, ENP 531906 2807886 3' est. Tail To FLMNH

28 SS093 1-May-03 Main Park Road, Flamingo Wells, ENP 520278 2796992 118 105 Tail To FLMNH

29 SS108 14-Dec-03 Main Park Road, Mahogany Ham., ENP 518676 2802509 107.5 Tail To FLMNH

30 SS111 27-Dec-03 Old Tamiami Trail (Dead Body Rd.) 524211 2849227 317 276 M "Neck" Testes turgid

31 SS112 23-Dec-03 Tamiami Trail 525850 2849018 299 258 F Head D.O.R. between Tamiami Ranger Station and Shark Valley

32 SS114 8-Jan-04 Royal Palm Rd. 538465 2808915 231 203 M Head, "neck"

Very black in coloration, irregular scale pattern

33 SS120 28-Jan-04 L-67 Ext. Canal Levee 532790 2846707 290.5 258.5 F "Neck" No oviductal eggs

34 SS121 28-Jan-04 L-67 Ext. Canal Levee 532979 2841343 337.5 297.5 M Head, "neck" Testes turgid

35 SS122 28-Jan-04 L-67 Ext. Canal Levee 532971 2841491 238 206.5 M Head, "neck" Testes turgid

36 SS123 28-Jan-04 L-67 Ext. Canal Levee 532957 2841934 299 260 M Head, "neck" Testes turgid

37 SS124 28-Jan-04 L-67 Ext. Canal Levee 532957 2841934 281 247 M Head, "neck" Testes turgid

38 SS125 28-Jan-04 L-67 Ext. Canal Levee 532899 2840896 336 293 M Head, "neck" Tested enlarged, somewhat turgid

39 SS126 1-Mar-04 L-67 Ext. Canal Levee 532707 2849031 317.5 279 M Head, "neck" Tested enlarged, somewhat turgid

40 SS127 5-Mar-04 Near East Everglades Ranger Station 542617 2833134 315 277 F

Tail (ventral) 41 large oviductal unshelled eggs

41 SS128 16-Mar-04 L-67 Ext. Canal Levee 532694 2849062 377 335 F Tail (ventral) 40 large oviductal unshelled eggs

42 SS129 9-Apr-04 Royal Palm Rd. 538830 2808071 272 235 M Tail (ventral) Testes not turgid

43 SS130 3-Apr-04 Shark Valley Tram Road, ENP 523410 2847955 4'-6' est. Tail To FLMNH

44 SS131 18-Apr-04 Hells Bay, Main Park Rd. 517065 2790203 325 287 F Tail 35 large oviductal unshelled eggs

24

(ventral)

45 SS132 26-Apr-04 Nine Mile Pond, Main Park Rd. 520297 2793310 385 343 F Tail (ventral) 46 large oviductal unshelled eggs

46 SS139 4-Jul-04 Near Dwarf Cypress, Main Park Rd. 524774 2813005 243 213 M Tail

Testes long but not enlarged or turgid

47 SS140 20-May-04 Paurotis Pond, Main Park Rd. 520295 2794759 143 126 F Tail Oviducts thin, no follicle development evident

48 SS141 28-Jun-04 Nine Mile Pond, Main Park Rd. 519722 2791991 222 191 M Tail Testes not turgid

49 SS142 15-Jul-04 Near jct. Royal Palm Rd., Main Park Rd. 537498 2810718 133 118 M Tail Testes not developed

50 SS143 19-Jul-04 Jct. Royal Palm Rd., Main Park Rd. 538969 2809875 69.5 60.5 F

Mid-body ribs

D.O.R. No follicle development evident

51 SS144 22-Jul-04 Main Park Road, West Lake, ENP 517234 2790312 3' est. Tail To FLMNH 52 SS145 22-Jul-04 Main Park Road, West Lake, ENP 515845 2789381 3' est. Tail To FLMNH

53 SS146 24-Jul-04 Main Park Road, Royal Palm Rd, ENP 538876 2809934 3' est. Tail To FLMNH

54 SS147 3-Aug-04 West Lake, Main Park Rd. 514228 2788295 230 201 F Mid-body ribs

All follicles less than 10mm dia., oviducts thin, flat, opaque.

55 SS149 22-Aug-04 Mahogany Hammock, Main Park Rd. 517998 2804656 307.5 270 F

Mid-body ribs


56 SS157 18-Oct-04 Nine Mile Pond, Main Park Rd. 520299 2793097 172 151 M Mid-body ribs Testes not developed

57 SS165 23-Oct-04 S.R. 9336, just outside ENP 543362 2809295 182 160 F Mid-body ribs

Oviducts thin, no follicle development evident

58 SS166 17-Oct-04 Main Park Road, ENP 527147 2812977 3'-4' est. Tail To FLMNH

59 SS168 31-Oct-04 Mrazek Pond, Main Park Rd. 511150 2786109 255.5 224 F Mid-body ribs


60 SS172 17-Nov-04 Hole-in-the-Donut, spoil mound, ENP 533777 2805491 396 355 F

Mid-body ribs

Follicles 10-12mm dia or less, oviducts thick, opaque.

61 SS174 12-Nov-04 Main Park Road, Mahogany Ham., ENP 518878 2802594

3'-4' est. Tail To FLMNH

62 SS176 23-Nov-04 Flamingo, Main Park Rd, ENP 507772 2781437 427 382.5 F Mid-body ribs

Follicles most 8-10mm, all less than 15mm, oviducts muscular.

63 SS178 27-Nov-04 Taylor Slough, 50 yds from airboat 540574 2807622 294.5 259.5 F Mid- All foliclles 5 mm dia. or less,

25

ramp, ENP body ribs oviducts thin, flat, opaque.

64 SS182 2-Dec-04 Flamingo, between lodge and cottages, ENP 506014 2780110 267.5 237 F Tail

All foliclles less than 5 mm dia., oviducts very thin, flat, not at all muscular.

65 SS183 5-Dec-04 On road to Pa-hay-okee, ENP 522324 2812858 231 201 M Tail Testes small, but turgid

66 SS187 8-Dec-04 Hole-in-the-Donut, spoil mound, ENP 530813 2807216

Est. 6' + n.d. n.d.

Mid-body ribs

Hit by flat mower. Head and pieces collected.

67 SS188 8-Dec-04 Hole-in-the-Donut, spoil mound, ENP 530779 2807129 n.d. n.d. n.d. Tail

Hit by flat mower. Only tail section recovered.

68 SS190 16-Dec-04 L-67 Ext. Canal Levee 532979 2841175 276 241 M Tail Testes turgid

69 SS195 17-Dec-04 L-67 Ext. Canal Levee 532728 2848082 347 306.5 M Mid-body ribs Testes turgid

70 SS196 17-Dec-04 L-67 Ext. Canal Levee 532773 2846836 290 251.5 M Mid-body ribs Testes turgid

71 SS200 22-Dec-04 On road to Royal Palm, ENP 538550 2809364 235.5 208 F Tail

All follicles less than 10 mm in dia., ducts thin, transparent, not muscular.

72 SS201 29-Dec-04 On road to Pa-hay-okee, ENP 522824 2812569 253.5 223 M Tail Only right testis enlarged and turgid

73 SS202 31-Dec-04 Shark Valley Tower, ENP 523434 2837846 203.5 177 M Tail Both testes flacid, not enlarged or turgid

74 SS206 6-Jan-05 L-67 Ext. Canal Levee 532882 2844671 262.5 233 F Tail

All follices less than 5 mm dia., ducts thin, transparent, non-muscular.

75 SS208 13-Dec-04 Shark Valley Tram Road, ENP 523351 2847353 250.0

+ 216.0

+ F Tail

Headless carcass. All follicles less than 10 mm in dia.(most less than 5), ducts thin, transparent, not muscular.

76 SS209 20-Dec-04 Shark Valley Tram Road, ENP 523351 2847353 293 257 M Tail Testes somewhat enlarged, only moderatley turgid.

77 SS210 Early Dec 2004

Highway 41, near Shark Valley, ENP 524316 2849283 184 164 F Tail

D.O.R. No follicle development observed.

78 SS217 25-Jan-05 L-67 Ext. Canal Levee 532798 2846910 240 208.5 M Tail Testes enlarged, turgid

79 SS220 31-Jan-05 L-67 Ext. Canal Levee 532836 2840685 235.5 204.5 M Tail Testes both somewhat enlarged, only moderately turgid.

26

80 SS221 31-Jan-05 L-67 Ext. Canal Levee 532801 2846750 291 254.5 M Tail Testes enlarged, turgid

81 SS222 2-Feb-05 Main Park Road, SW of Pa-hay-okee turnoff, ENP 519532 2807950 272.5 237 M Tail

Testes both somewhat enlarged, only moderately turgid.

82 SS224 14-Feb-05 L-67 Ext. Canal Levee 532929 2842524 336 289 M Tail Testes enlarged, turgid

83 SS274 9-Sep-05 US-41, E. of L-67 Ext. *532758 *2849300 176 164.5 F Tail D.O.R. 25 degrees 45 39.4 80 degrees 40 03.3

84 SS278 9-Aug-05 AeroJet Rd, 1 mile S. of SR 9336 *544167 *2807685 68.5 60 M Tail D.O.R.

85 SS284 10-Aug-05 Main Park Road, Snake Bight, ENP 512568 2787156 367 326 F

Tail and rib

No fat reserves, gut empty. Numerous oviduct pockets.

86 SS287 30-Aug-05 HID spoil mound, ENP *533777 *2805491 M Mid-body rib

Hit by mower, No head. No tail. 75 cm of carcass.

87 SS289 13-Sep-05 Main Park Road, near Entrance, ENP *543162 *2809295 78 69 Tail D.O.R.

88 SS293 28-Sep-05 Main Park Road, Rowdy Bend, ENP 509655 2784430

*195.5 F Tail

*No head. All follicles less than 10 mm dia. Tail 25.0 cm

89 SS294 29-Sep-05 Main Park Road, near Entrance, ENP 541333 2808655 100.5 88.5 Tail D.O.R.

90 SS295 25-Oct-05 HID spoil mound, ENP *533777 *2805491 M Tail Hit by mower. 122 cm of carcass. Tail 19.0 cm

91 SS296 7-Oct-05 S-332 C *541253 *2811716 325 287 F Tail Hit by mower. 25 deg. 30 54.7 80 deg. 33 40.2

92 SS300 20-Oct-05 Frog Pond 542931 2810116 254 221.5 M Tail D.I.F. hit and killed by farm equipment.

93 SS304 20-Oct-05 Frog Pond *542931 *2810116 246.5 214.5 M Tail D.I.F. 25 deg. 26 11.4 80 deg. 33 36.6

94 SS305 20-Oct-05 Frog Pond *542931 *2810116 208 178 M Tail D.I.F. 25 deg. 26 13.1 80 deg. 33 40.7

95 SS306 20-Oct-05 Frog Pond *542931 *2810116 197.5 170 M Tail D.I.F. 25 deg. 25 59.0 80 deg. 33 47.0

96 SS308 4-Nov-05 Frog Pond *543598 *2812228 378 334 F Tail D.I.F. 25 deg. 25 56.8 80 deg. 33 51.7

97 SS309 6-Nov-05 Main Park Road, ENP 512851 2787362 200 175 F Tail Half-way between Mrazek Pond and West Lake.

98 SS310 7-Nov-05 Main Park Rd., Mrazek Pond *511150 *2786109 220 193.5 F Tail 99 SS311 22-Oct-05 Frog Pond *543598 *2812228 231.5 201.5 M Tail D.I.F. hit and killed by farm

27

equipment.

100 SS312 22-Oct-05 Frog Pond *543598 *2812228 247 216 F Tail D.I.F. hit and killed by farm equipment.

101 SS313 22-Oct-05 Frog Pond *543598 *2812228 279.5 247.5 F Tail D.I.F. hit and killed by farm equipment.

102 SS314 22-Oct-05 Frog Pond *543598 *2812228 287.5 254 F Tail D.I.F. hit and killed by farm equipment.

103 SS325 13-Dec-05 S-332 C *541253 *2811716 n.d. n.d. M Tail Est. tail length 29 cm 104 SS327 14-Dec-05 L-67 Ext. Canal Levee *532899 *2840896 278 243 M Tail 25 deg. 41.015 80 deg. 40.367 105 SS333 20-Dec-05 L-67 Ext. Canal Levee *532899 *2840896 282 248 M Tail 25 deg. 43 43.8 80 deg. 40 21.3

106 SS337 3-Jan-06 Main Park Rd., 3.5 mi. n. of Flamingo *521544 *2819349 347 305 M Tail D.O.R.

107 SS339 6-Jan-06 Main Park Rd., Dwarf Cypress Sign 521897 2810756 218 189 F Tail D.O.R.

108 SS340 9-Jan-06 L-67 Ext. Canal Levee *532899 *2840896 290 255 M Tail 25 deg. 42 33.2 80 deg. 40 19.0 (Hill, SFWMD)

109 SS341 9-Jan-06 L-67 Ext. Canal Levee *532899 *2840896 304 266.5 M Tail 25 deg. 42 24.1 80 deg. 40 18.5 (Hill, SFWMD)

110 SS342 9-Jan-06 L-67 Ext. Canal Levee *532899 *2840896 263 231 M Tail 25 deg. 41 59.2 80 deg. 40 17.5 (Hill, SFWMD)

111 SS347 12-Jan-06 Shark Valley Tram Rd., ENP *523351 *2847353 328 286 M Tail about 3 miles south of parking lot

112 SS348 13-Jan-06 Old Tamiami Canal, E. of Shark Valley 523815 2849244 267.5 235 M Tail Dead in canal

113 SS350 18-Jan-06 W. of Ficus Pond w/"Beatrice", ENP 517342 2806499 256.5 223 M Tail

114 SS351 18-Jan-06 W. of Ficus Pond w/"Beatrice", ENP 517342 2806499 282 247 M Tail

115 SS352 19-Jan-06 L-67 Ext. Canal Levee *532899 *2840896 263 230 M Tail 25 deg. 41 00.7 80 deg. 40 22.0 116 SS354 25-Jan-06 Pay-Hay-Okee Hammock 521570 2813641 462 414 F Tail 117 SS355 1-Feb-06 L-67 Ext. Canal Levee *532899 *2840896 370 327 F Tail 25 deg. 43 31.7 80 deg. 40 20.9

118 SS360 17-Feb-06 W. of Ficus Pond w/"Beatrice", ENP 517423 2806458 277 241 M Tail

119 SS361 24-Feb-06 W. of Ficus Pond w/"Beatrice", ENP 517397 2806545 250 219 M Tail

28

120 SS362 1-Mar-06 W. of Ficus Pond w/"Beatrice", ENP 517867 2806364 245 215 M Tail

121 SS363 17-Mar-06 Pay-Hay-Okee Hammock 521544 2813714 301 261 M Tail 122 SS364 17-Mar-06 Pay-Hay-Okee Hammock 521544 2813714 221.3 191.5 M Tail 123 SS365 17-Mar-06 Pay-Hay-Okee Hammock 521544 2813714 192.5 168 M Tail 124 SS366 17-Mar-06 Pay-Hay-Okee Hammock 521544 2813714 271 233 M Tail 125 SS367 17-Mar-06 Pay-Hay-Okee Hammock 521544 2813714 419.5 368 M Tail 126 SS368 17-Mar-06 Pay-Hay-Okee Hammock 521544 2813714 332 295 F Tail 127 SS369 10-May-06 Coptic Hammock, ENP 242 209 F Tail

128 SS372 350 309 M Tail D.O.R. US41 ? (from Tamiami R.S. freezer)

129 SS373 204 179 M Tail (from Tamiami R.S. freezer) 130 SS374 25-Apr-06 Shark Valley Tram Rd., ENP *523351 *2847353 160 139.5 M Tail (from Tamiami R.S. freezer)

131 SS375 137.5 118.5 F Tail D.O.R. US41 ? (from Tamiami R.S. freezer)

132 SS376 29-May-06 Main Park Rd., by Pineland sign 532295 2811728 177.5 156 F Tail 133 SS377 30-May-06 Main Park Rd. 515562 2789186 173 152.5 F Tail 134 SS379 9-Jun-06 S-12C *527368 *2849401 225 195.5 M Tail 135 SS380 1-Aug-05 HID spoil mound, ENP *533777 *2805491 136 SS384 19-Jul-06 Main Park Rd., Rock Reef Pass 525846 2812907

137 SS388 21-Jul-06 Frog Pond 544150 2811324 167 M Tail D.I.F. hit and killed by farm equipment.

138 SS389 21-Jul-06 Frog Pond 543663 2809750 180 n.d. Tail D.I.F. hit and killed by farm equipment.



141 SS397 27-Jul-06 Frog Pond 543212 2810126 245 215 M Tail D.I.F. hit and killed by farm equipment.

142 SS399 28-Jul-06 Frog Pond 544258 2812807 n.d. n.d. Tail D.I.F. hit and killed by farm equipment.

143 SS400 28-Jul-06 Frog Pond 544228 2812958 260 F Tail D.I.F. hit and killed by farm

29

equipment.

144 SS401 28-Jul-06 Frog Pond 544044 2812953 226 197 F Tail D.I.F. hit and killed by farm equipment.

145 SS407 4-Aug-06 Frog Pond 544183 2811093 175 F Tail D.I.F. hit and killed by farm equipment.

146 SS408 4-Aug-06 Frog Pond 544264 2811138 220 M Tail D.I.F. hit and killed by farm equipment.



149 SS422 10-Aug-06 Frog Pond 543657 2810761 220 194 F Tail D.I.F. hit and killed by farm equipment.





154 SS429 10-Aug-06 Frog Pond 544067 2813760 257 222 M Tail D.I.F. hit and killed by farm equipment.


156 SS432 11-Aug-06 Frog Pond 543218 2813406 361 n.d. F Tail D.I.F. hit and killed by farm equipment.

157 Store01 8-Apr-05 Sunset Reptiles, 9761 Sw 72nd St, Miami, FL 33173 *564964 *2842805

cast-off skin

158 Vietnam01 17-Oct-05 Vietnam *681245 *1188992 cast-off skin



30











final report genetic characterization of populations of the

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