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Genetic Diversity and Distinctiveness of Plectrohyla Guatemalensis (Anura: Hylidae) in Guatemala

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Genetic Diversity and Distinctiveness of Plectrohyla Guatemalensis (Anura: Hylidae) in Guatemala Genetic diversity and distinctiveness of Plectrohyla guatemalensis (Anura: Hylidae) in Guatemala A thesis submitted to The University of Manchester for the degree of Mphil Environmental Biology in the Faculty of Life Sciences 2014 Olga Alejandra Zamora Jerez Contents Contents ............................................................................................................................................ 2 List of Tables .................................................................................................................................... 3 List of Figures ................................................................................................................................... 3 Abstract ............................................................................................................................................ 4 Declaration ....................................................................................................................................... 5 Copyright .......................................................................................................................................... 5 Dedication ......................................................................................................................................... 6 Acknowledgements........................................................................................................................... 6 Introduction ...................................................................................................................................... 8 Amphibians and Ecosystem Services .............................................................................................. 9 Amphibians and regulation functions....................................................................................... 10 Amphibians and Production Functions .................................................................................... 12 Amphibians and Information Functions .................................................................................. 14 Conservation Genetics of Amphibians and Microsatellites ........................................................ 15 Amphibians of Guatemala: Plectrohyla guatemalensis ................................................................ 17 Aims of the project ......................................................................................................................... 21 Methods .......................................................................................................................................... 22 Field survey ................................................................................................................................ 22 DNA extraction ........................................................................................................................... 25 Microsatellite Primer development ........................................................................................... 26 DNA amplification ..................................................................................................................... 27 Microsatellite Genotyping ......................................................................................................... 28 Data Analysis .............................................................................................................................. 28 Results ............................................................................................................................................. 31 Field Surveys and population status ......................................................................................... 31 Microsatellite development ........................................................................................................ 31 Population genetics of Plectrohyla guatemalensis ..................................................................... 39 Discussion ....................................................................................................................................... 42 Population status of Plectrohyla guatemalensis ........................................................................ 42 Microsatellite development ........................................................................................................ 44 Preliminary Population Genetics Analysis of Plectrohyla guatemalensis ............................... 47 Conservation and the future ...................................................................................................... 48 References ....................................................................................................................................... 51 Appendices ...................................................................................................................................... 60 2 List of Tables Table 1 Localities where P. guatemalensis surveys were conducted ............................................... 25 Table 2 Multiplex PCR tested for the P. guatemalensis samples ..................................................... 30 Table 3 Sample size, Number of different alleles, Allele range and null allele frequency for the three populations analyzed ............................................................................................................... 33 Table 4 Observed heterozygosity, expected heterozygosity , Inbreeding index, P-value for HWE and allelic richness for the three populations analyzed .................................................................... 40 Table 5 Complete set of primers tested for in this project ............................................................... 60 Table 6 STRand results for the nine loci ......................................................................................... 62 Table 7 P-values for the Hardy-Weinberg Analysis ........................................................................ 66 Table 8 AMOVA results per loci for the three populations ............................................................. 66 Table 9 Allele range for each locus ................................................................................................. 66 Table 10 Localities, coordinates and samples collected................................................................... 67 List of Figures Figure 1 Map of the endemism zones proposed by Schuster et al. (2000). ...................................... 18 Figure 2 Map of the distribution of P. guatemalensis in Guatemala................................................ 19 Figure 3 Plectrohyla guatemalensis collected in the forests of Laj Chimel, Quiche. ...................... 20 Figure 4 Map of the localities where P. guatemalensis surveys were done. .................................... 24 Figure 5 Allele frequency distribution for each locus. .................................................................... 38 Figure 6 Allelic patterns across the three populations ..................................................................... 39 Figure 7 STRUCTURE bar plots for the whole data set (K=3). ..................................................... 41 Figure 8 Investigation license extended by CONAP ....................................................................... 80 Figure 9 Collection license extended by CONAP ........................................................................... 81 Figure 10 Exportation license extended by CONAP ....................................................................... 82 Word count: 23,284 3 Abstract The University of Manchester Olga Alejandra Zamora Jerez MPhil Environmental Biology Genetic diversity and distinctiveness of Plectrohyla guatemalensis (Anura: Hylidae) in Guatemala 2014 Microsatellite markers are acquiring more attention for the study and conservation of populations. They can help scientists understand and ask a variety of questions about populations. Next Generation Sequencing technologies (NGS) are becoming the preferred method for scientists to develop microsatellites since they can produce large amounts of data in less time than conventional cloning. P. guatemalensis is a hylid frog that inhabits forests of Guatemala, México and Honduras. In this project microsatellite markers were developed using both methods to do a first assessment on the genetic status of P. guatemalensis in Guatemala. Microsatellites primers were developed using both conventional cloning techniques at Sheffield University (SHF) and NGS. Twenty one pairs of primers were developed using data generated from an Illumina Miseq sequencer while 16 primers were developed using cloning methods in Sheffield. In total a set of eight polymorphic loci were used to evaluate the population genetics of P. guatemalensis.This study shows the use of NGS technologies and bioinformatics tools to be a quick and effective way of obtaining Possible Amplifiable Loci (PALs). In this project only a few loci could be tested and more experiments are needed before being able to determine if one method is advantageous over the other. Results showed that the observed heterozygosity was higher than expected for seventeen loci across all populations, and divergence from Hardy Weinberg Equilibrium was found in seven loci in Laj Chimel and Tatasiriré populations and four loci from Trifinio. The inbreeding coefficient (FIS) varied from -0.578 to 0.633 across populations. The negative value for some loci suggests possible
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