Landscape genetic analysis and population structure of three sympatric amphibians in managed redwood forest of northwestern California

Transcription

1 1 Landscape genetic analysis and population structure of three sympatric amphibians in managed redwood forest of northwestern California Cindy Castaneda, CSU Los Angeles February 10, February 10, 2015 Dr. Andres Aguilar, CSU Los Angeles February 10, 2015

2 2 TABLE OF CONTENTS Acknowledgements 3 Executive Summary...4 Project Objectives.. 4 Project Approach 5-8 Project Outcomes 9 Conclusions.9 References..10

3 3 ACKNOWLEDGEMENTS This project was supported by the Agriculture and Food Research Initiative Competitive Grant no from the USDA National Institute of Food and Agriculture. I am grateful to Dr. Andres Aguilar for his support and guidance with this research. I thank Dr. Elizabeth Torres and Dr. Hong-lie Qiu for their constructive suggestions. I also greatly thank the graduate studies and research office at CSU, Los Angeles for the university fund Research, Scholarship, and Creative Activity (RSCA) award, which helped fund sequencing.

4 4 Executive Summary In this research, the relationship between geographic distance and genetic distance is being studied in a collection of three endemic redwood forest amphibians: southern torrent salamander, coastal giant salamander, and the tailed frog. Southern torrent salamanders (Rhyacotriton variegatus), tailed frogs (Ascaphus truei), and coastal giant salamanders (Dicamptodon tenebrosus) inhabit heavily logged redwood forests, making these species of conservation concern. Globally amphibians as a group are considered endangered due to their high environmental sensitivity. Understanding animal dispersal capabilities and limitations in disturbed habitats will assist with conservation efforts. Mitochondrial data is currently being used to examine the relationship between population genetic structure and landscape features among these taxa. Mitochondrial data provides valuable insight into patterns of genetic structure and identifies threats to the persistence of these amphibians. I am employing a landscape genetics approach to reveal landscape features of the redwood forest that influence the population genetic structure among these three species. I am currently analyzing genetic and landscape data to characterize population genetic patterns and to determine relationships between landscape features and gene flow. Project Objectives I initially proposed to study the landscape genetics of the arboreal salamander (Aneides lugubris) within the San Gabriel Mountains, CA; however, due to a lack of rain in fieldwork resulted in unsuccessful sampling. Therefore, genetic structure in the coastal tailed frog, the southern torrent salamander, and the coastal giant salamander is being studied using specimens previously collected by Aguilar et al. (2013). Since whole genomic DNA was previously extracted from tail clips of these taxa I did not perform DNA extraction as proposed. Genetic data analysis is being performed on mitochondrial data versus microsatellite data because mitochondrial markers are the most widely used markers in population genetics studies (Martinez-Solano and Gonzalez, 2008). As proposed, GIS is being used for landscape analysis of genetic data. My goals with this research are to (1) determine landscape features which limit migration and gene flow in a disturbed landscape and (2) compare the similarities and differences in landscape features and gene flow among the three species. Genetic and landscape data provide valuable insight into patterns of genetic structure and can identify threats to the persistence of these amphibians. In understanding how species respond to changes in forest and stream structure, that are due to land management, I wish to offer insight for management practices. In highlighting threats to the evolutionary heritage of species related to habitat fragmentation I hope to decrease the risk of localized extinction. Among my potential career paths is a career with the USDA Forest Service (FS). Ecologist and biologist within Forest Service protect and manage grasslands and nationals forest and as a future conservation biologist my interest is to tackle issues regarding the conservation, research, and restoration of the environment, which goes in hand with the Forest Service s mission.

5 5 Project Approach Study Area Genetic structure in the coastal tailed frog, the southern torrent salamander, and the coastal giant salamander is being studied using specimens previously collected by Aguilar et al. (2013). These species were collected in the North Coast physiographic providence of Mendocino County, California (Figures 1-3). Sampling Area and DNA Extraction I performed Polymerase Chain Reactions (PCR s) with a series of mitochondrial primers on previously extracted DNA (Aguilar et al, 2013). Amplified PCR products were quantified by agarose gel electrophoresis on a 1.5% agarose gel. PCR products were sent to Laragen, Inc. for sequencing. Complete cytb sequences were aligned using Geneious R7 ( Kearse et al., 2012). Population Genetic Analysis To estimate genetic differentiation among sites pairwise F ST is currently being calculated. F ST is a measure of population differentiation due to genetic structure (Wright 1951,1965). Sequence variation will be assessed by calculating haplotype diversity (h) and nucleotide diversity (π). Haplotype diversity measures the number and frequency of different alleles within a population (Hallldorsson et al., 2004). Nucleotide diversity is the mean divergence between sequences and measures the number of base pair differences between individuals in a population. (Nei and Lei, 1979) Landscape Analysis GIS is currently being used to examine the relationship between landscape variables and genetic connectivity. Euclidean distance and least-cost paths (LCP) are being calculated using Arc GIS. These dispersal paths will represent potential gene flow routes between sites and should predict genetic distance. LCP models will determine whether particular landscape variables such as vegetation, streams, solar radiation, wetness, and elevation are more informative predictors of genetic connectivity (Aguilar et al, 2013). Linear regressions will be used to identify the strength of relationships between genetic distance and geographical distance from various LCPs.

6 Figure 1. Sample collection locations of coastal giant salamander in Mendocino County, CA (United States). 6

7 Figure 2. Sample collection locations of southern torrent salamander in Mendocino County, CA (United States). 7

8 Figure 3. Sample collection locations of coastal tailed frog in Mendocino County, CA (United States). 8

9 9 Project Outcomes I am currently analyzing genetic and landscape data to characterize population genetic patterns and to determine relationships between landscape features and gene flow. This research aims to predict why amphibian populations are declining. These predictions will serve as incentives for conservation actions and as hypotheses for future research. This research will inform management plans for the conservation of amphibian diversity in coastal redwood forest. Conclusions Little has been written about the genetic structure of the coastal redwood forest fauna; thus, in comparing coastal giant salamanders, with southern torrent salamander, and tailed frogs I will contribute greatly to the field. Future research should look at gene flow among the three species using mtdna and nuclear data. In combining mitochondrial and nuclear markers it increases the total number of characters available for analysis; thus, to fully characterize population genetic patterns within and across taxa both mtdna and nuclear markers of the genome should be investigated (Makowsy and Chesser, 2009). In addition, spatial analysis and molecular genetic techniques used in this study may be applied to other organismal systems. The Watershed Management Internship Program, in collaboration with the USDA, was an invaluable opportunity for me as I gained real-world research experience. Through my participation I got hands on experience in the laboratory where I learned techniques in molecular genetics and in modeling dispersal routes I refined my GIS skills of manipulating, analyzing and presenting spatial and geographical information. Also, this internship gave me the opportunity to refine my fieldwork skills and learn management and conservation strategies. Furthermore, my involvement in the internship gave me confidence in my abilities and allowed me to network with professionals in my field. Ultimately, this internship has prepared me for a future career with the Forest Service and enhanced my success as a conservation biologist.

10 10 References Aguilar A, Douglas RB, Gordon E, Baumsteiger J, Goldsworthy MO Elevated genetic structure in the coastal tailed frog (Ascaphus truei) in managed redwood forests. Heredity 104: Drummond, AJ, Ashton, B, Buxton S, Cheung M, Cooper A, Duran C, Field M, Heled J, Kearse M, Markowitz S, Moir L, Stones-Havas S, Sturrock S, Thierer T, Wilson A Geneious v7.1.1 < Halldorsson BV, Bafina V, Edwards N, Lippert R, Yooseph S, Istrail S A survey of computational methods for determining haplotypes. Lecture Notes in Computer Science 2983: Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 12: Makowsky R, Chesser J A striking lack of genetic diversity across wide-ranging amphibian Gastrophryne carolinenisis (Anura: Microhylidae). Genetica 15: Martinez-Solano I, Gonzalez EG Patterns of gene flow and source-sink dynamics in high altitude populations of the common toad Bufo bufo (Anura: Bufonidae). Biological Journal of Linean Society 95: Nei M, Li WH Mathematical model for studying genetic variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences 76: Wright S The genetical structure of populations. Annals of Eugenics 15: Wright S The interpretation of population structure by F- statistics with special regard to system of mating. Evolution 19: