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Characterization of 14 novel microsatellite loci in the Argentine black and white tegu (Salvator merianae) via 454 pyrosequencing

In: Amphibia-Reptilia
Authors:
Jared P. Wood 1Department of Biology, Southwestern Adventist University, Keene, Texas 76059, USA

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Todd S. Campbell 2Department of Biology, University of Tampa, Tampa, 401 W. Kennedy Blvd, Florida 33606, USA

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Robert B. Page 3Department of Biology, College of St. Benedict & St. John’s University, Collegeville, Minnesota 56321, USA

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We have developed 14 novel microsatellite loci for the Argentine black and white tegu (Salvator merianae), using samples from invasive populations from the US state of Florida. Pyrosequencing was used to identify 3154 potentially amplifiable microsatellite loci and to subsequently develop 14 informative markers. These 14 markers were screened for variation in 40 individuals from Miami-Dade County, Florida. All loci were polymorphic and contained between 2 and 4 alleles per locus (mean ± SE = 2.71 ± 0.24), with observed heterozygosity ranging from 0.00 to 0.88 (mean ± SE = 0.38 ± 0.07). Four of the loci (Teg4, Teg5, Teg17, Teg19) significantly deviated from Hardy-Weinberg proportions and three of these loci (Teg4, Teg5, Teg19) showed evidence of null alleles. In addition, there was statistical evidence for genotypic disequilibrium between Teg14 and Teg19. BLASTn searches of NCBI’s ‘nr/nt’ database using microsatellite containing 454 fragments as queries were largely uninformative; however, it is likely that some of these markers will be of utility in S. merinae’s native range.

After habitat destruction, invasive species are the next greatest threat to global biodiversity (Wilcove et al., 1998). Florida is especially susceptible to invasion by nonnative herpetofauna because of its numerous ports of entry, subtropical climate, and disturbed habitats (Pernas et al., 2012; Mazzotti et al., 2015). The Argentine black-and-white tegu (Salvator merianae) is one of the four largest non-native lizards currently breeding in Florida (Engeman et al., 2011). They are also one of the largest lizards in the New World, reaching sizes of up to 145 cm total length and 8 kg (Lopes and Abe, 1999; Duarte Varela and Cabrera, 2000). S. merianae is native to South America (Luxmoore, Groombridge and Broads, 1988). However, a breeding population of S. merinae was documented in portions of Hillsborough and Polk Counties in 2006 (Engeman et al., 2011) and the existence of this population has since been attributed to activities associated with the exotic pet industry (Engeman et al., 2011). S. merinae has already been documented depredating American alligator (Alligator mississippiensis) and red-bellied cooter (Pseudemys nelson) nests in Florida (Mazzotti et al., 2015). Thus, S. merianae is currently viewed as a direct threat to Florida’s sensitive fossorial wildlife (e.g., sea turtles, gopher tortoise (Gopherus polyphemus), eastern indigo snake (Drymarchon couperi), American crocodile (Crocodylus acutus), Cape Sable seaside sparrow (Ammodramus maritimus mirabilis), and Key Largo woodrat (Neotoma floridana smalli); Mazzotti et al., 2015).

Since S. merinae’s initial introduction to Hillsborough and Polk Counties, a new breeding population has been documented approximately 330 km away in southern Miami-Dade County (Pernas et al., 2012). It is unclear whether this recent establishment is the result of dispersal or the consequence of secondary human-mediated introduction. However, to prevent further spread of S. merianae throughout Florida, it is essential for managers to know how this new population became established. Microsatellite-based population genetic approaches have considerable potential to provide perspective on this question, but as of now, such genetic resources are not available for S. merinae. To facilitate such endeavors, we developed 14 novel microsatellite markers from S. merianae that will be used to examine the introduction histories of and degree of differentiation and connectivity between Florida’s invasive S. merinae populations.

DNA from a single S. merianae captured in Miami-Dade County, Florida, USA (25°26′0.70″N, 80°30′5.77″W) was submitted to the University of Georgia Genomics Facility (GGF), where this isolate was pooled with DNA from two other species that were differentiated by terminal barcodes (Meyer et al., 2007). Genomic DNA was obtained from liver tissue using the Wizard Genomic DNA Purification Kit (Promega) according to the manufacturer’s instructions. A library of single stranded template DNA fragments was then produced using the GS FLX Titanium General Library Preparation Kit (Roche). Initial sequencing employed the 454 GS FLX Titanium Sequencing Kit XLR70 (Roche) run on 1/4 70 × 75 mm picotiter plate and additional sequencing employed the 454 GS FLX Titanium Sequencing Kit XL+ (Roche) run on 1/2 70 × 75 mm picotiter plate. The GGF also performed basic data processing, such as base calling and filtering.

These sequencing efforts yielded a total of 127 343 751 bp across 300 675 reads (see online Supplementary Data File 1). Of these reads, 90 457 were generated using the XLR70 kit (mean length = 275.8 bp, std. dev. = 155.5 bp) and 210 218 were generated using the XL+ kit (mean length = 487.1 bp, std. dev. = 199.1 bp). We then used MSATCOMMANDER 0.8.2 (Faircloth, 2008) to scan these pyrosequencing reads for dinucleotide microsatellites with ⩾eight tandem repeats and tri-pentanucleotide microsatellites with ⩾six tandem repeats. In total, MSATCOMMANDER identified 3154 presumptively non-redundant potentially amplifiable loci (PALs). Finally, we used PRIMER3 (Rozen and Skaletsky, 2000) to design primers targeting these potentially amplifiable loci (PALs) via batch processing of repeat containing 454 fragments (see online Supplementary Data File 2).

Twelve dinucleotide, four trinucleotide, and four tetranucleotide loci whose corresponding 454 fragments contained at least ten, nine, and seven tandem repeats respectively were manually selected for marker development. An M13(-21) sequence was fused to the 5′ end of either the forward or reverse primer of each primer pair in order to facilitate fluorescent labeling with 6-FAM via the nested PCR approach described by Schuelke (2000). These 20 loci were then screened for polymorphism and scoring reliability using DNA isolated from muscle tissue of 11 individuals sampled from Miami-Dade County. All reactions had a final volume of 25 μl and contained 2 μl of template (DNA concentration between 10 and 100 ng/μl), 5 μl of 5× buffer, 1.5 mM MgCl2, 0.2 mM of each dNTP, 0.8 μM of non-M13(-21)-twinned primer, 0.8 μM 0f 6-FAM labeled M13(-21) primer, 0.2 μM of M13(-21)-twinned primer, and 0.625 units of GoTaq polymerase (Promega). Reaction conditions were as follows: 2 min at 94°C followed by 25 cycles of (1) 94°C for 30 s, (2) 62°C for 30 s decreasing by 0.3°C per cycle, and (3) 72°C for 40 s, followed by eight cycles of (1) 94°C for 30 s, (2) 53°C for 30 s, and (3) 72°C for 40 s, followed by a final cleanup step of 30 min at 72°C. Genotyping reaction products were visually inspected by gel electrophoresis by loading 5 μl of PCR product in 2% agarose gels. Products from successful reactions were shipped to the Arizona State University DNA Lab, where fragment analysis was performed using an Applied Biosystems 3730. Of the 20 loci that were screened, 14 were polymorphic and straightforward to score. Thus, we genotyped additional individuals at these 14 loci for a total of 40 individuals from the Miami-Dade County population. Locus-specific primers, as well as their melting temperatures, size ranges, and summary statistics are presented in table 1. All loci were scored manually using PEAK SCANNER 1.0 (Applied Biosystems). Allelic bins were determined by graphically examining the rank-ordered fragment size distributions of each locus, so that we could identify breaks in the amplicon sizes (Guichoux et al., 2011). We then wrote functions in Microsoft EXCEL to bin the data from each locus into discrete classes that were defined by each allele’s empirically determined size range.

Table 1.

Characterization of 14 microsatellite loci genotyped in S. merianae. Samples collected from Miami-Dade County, Florida, USA.

Table 1.
Table 2.

Results of the BLASTn searches of NCBI’s ‘nucleotide collection (nr/nt)’ database using microsatellite containing 454 fragments as queries.

Table 2.

We used GENALEX 6.5 (Peakall and Smouse, 2012) to calculate several summary statistics including: number of alleles, effective number of alleles, observed heterozygosity, and expected heterozygosity. We used GENEPOP 4.3 (Rousset, 2008) to test for departures from Hardy-Weinberg proportions, departures from genotypic equilibrium, and to calculate the Weir and Cockerham (1984) estimator of FIS. M ratios (Garza and Williamson, 2001) were calculated in EXCEL using output from GENALEX. We also used MICRO-CHECKER 2.2.3 (Van Oosterhout et al., 2004) to examine each locus for evidence of null alleles, large allele dropout, and scoring errors (table 1).

In order to give readers a feel for the level of sequence conservation in genomic regions immediately surrounding each locus, we conducted BLASTn searches of NCBI’s ‘nucleotide collection (nr/nt)’ database using the 454 fragments that primers were designed from as queries. These searches were performed using NCBI’s default settings for BLASTn and a critical E-value of 10−7 – a somewhat stringent threshold designed to filter out alignments that only, or overwhelmingly, correspond to microsatellite repeat regions.

The number of alleles (k), number of genotypes (N), observed heterozygosity (HO), expected heterozygosity (HE), Weir & Cockerham estimator of FIS, number of effective alleles, and M-ratio for each locus are given in table 1. In addition, table 1 gives the mean for each of these population genetic parameters across all 14 loci, as well as the standard error of the mean. Upon performing Holm’s (1979) correction for multiple testing, four loci (Teg4, Teg5, Teg17, Teg19) showed significant deviations from Hardy-Weinberg expectations, with Teg4, Teg5, and Teg19 exhibiting homozygote excess (table 1). Therefore, it was not surprising that MICRO-CHECKER detected evidence of null alleles at these three loci. After correcting for multiple testing (Holm, 1979), there was also statistical evidence for genotypic disequilibrium between Teg14 and Teg19. The mean M-ratio across the 14 loci (mean ± SE = 0.68 ± 0.09) was very close to the critical value of 0.68 suggested by Garza and Williamson (2001). This result is not surprising given that the Miami-Dade population was recently established and is consistent with the notion that the founding event involved a limited number of individuals.

Herein, we have described the development of 14 novel microsatellite loci from S. merianae. The resources we have developed will serve to assess the degree of gene flow between the two invasive populations currently established in Florida and gather insights into their introduction histories. Although there is limited allelic richness across these 14 loci (38 alleles total), preliminary analyses are suggesting that differentiation between the Hillsborough-Polk and Miami-Dade populations is pronounced (GST = 0.170; GST=0.545). Thus, at present, it seems likely that these markers will provide sufficient resolution for obtaining a general understanding of S. merinae population genetic dynamics in Florida. Unfortunately, our BLASTn searches were largely non-informative. However, a portion of the 454 fragment that Teg19 was identified from, including the repeat containing region, exhibited moderate sequence similarity with a microsatellite containing region of the Anolis carolinensis genome (table 2). As such, Teg19 should receive priority among researchers seeking to extend these resources to populations where amplification success may be an issue, such as within S. merinae’s native range or in other teiid species.

Acknowledgements

JPW and RBP would like to thank their late friend and mentor, D.H. Reed, for his advice and support during the earliest phases of this project. We also gratefully acknowledge J. Ketterlin Eckles, C. Hughes, K. Krysko, K. Enge, G. Klowden, F. Mazzotti, and Balm Boyette Scrub Preserve for providing samples, field resources and assistance, logistics, and site access. JPW was supported, in part, by the Wallace Endowment to the University of Louisville, Department of Biology. Funding for field work was provided by Charlotte Harbor National Estuary Program, the National Fish and Wildlife Foundation, the Institute for Biological Invasions at the University of Tennessee, and the University of Tampa Dana grant.

Footnotes

Supplementary Data File 1: Zip archive containing FASTA files of the 454 sequences generated during the course of our study.

Supplementary Data File 2: Excel spreadsheet containing the primers associated with the potentially amplifiable loci identified during the course of our study.

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Footnotes

Associate Editor: Sylvain Ursenbacher.

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