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CALIFORNIA STATE UNIVERSITY, NORTHRIDGE GENETIC VARIATION AND GENE FLOW IN PENTACHAETA LYONII (ASTERACEAE) AT SEVEN SITES IN SOUTHERN CALIFORNIA A thesis submitted in partial fulfillment of the requirements for the degree of Masters of Science in Biology By Lisa L. Zung August 2012 The thesis of Lisa L. Zung is approved: ____________________________________ __________________ Dr. David A. Gray Date ____________________________________ __________________ Dr. Paula M. Schiffman Date ____________________________________ __________________ Dr. Jennifer A. Matos, Chair Date California State University, Northridge ii ACKNOWLEDGEMENTS This project would not have been possible without the help and support of the following individuals. I wish to thanks my fellow graduate students Taylor Anderson-McGill, Christopher Bowman-Prideaux, Beck Wherle and Christina McNeal for their advice, and support. I thank Mark Harris and Chris Chabot for their help in troubleshooting my lab protocols, Nikki Osborne for letting me use her lab equipment and her assistance with Arlequin, and Pavel Lieb for sequencing my samples. I am grateful to Jocelyn Holt and Tarja Sagar for showing me collecting sites and aiding in my field work. Thank you Dr. Christy Brigham for your assistance in coordinating with the National Park Service, I would not have been able to collect samples without your help. Thank you Tracy Valentovitch for your aid in making maps and all other GIS related things. I also wish to thank the CSUN Graduate Studies and Biology Department for financial assistance. Many thanks to Dr. Dave Gray and Dr. Paula Schiffman for their guidance, advice and editing of this text. I would like to especially thank my mentor Dr. Jennifer Matos for understanding and being patient with me. Finally, I would like to thank my family and friends for their encouragement and patience with the unexpectedly long process this research has been, I truly could not have completed this without their support and love. iii TABLE OF CONTENTS SIGNATURE PAGE .......................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii ABSTRACT .........................................................................................................................v INTRODUCTION ...............................................................................................................1 METHODS ..........................................................................................................................6 RESULTS ............................................................................................................................9 DISCUSSION ....................................................................................................................11 LITERATURE CITED ......................................................................................................17 APPENDIX ........................................................................................................................23 iv ABSTRACT GENETIC VARIATION AND GENE FLOW IN PENTACHAETA LYONII (ASTERACEAE) AT SEVEN SITES IN SOUTHERN CALIFORNIA By: Lisa L. Zung Masters of Science in Biology The reduction of suitable habitat due to urbanization is an increasing threat to many native plant populations. As a result, plant populations that were once continuous have become fragmented into smaller isolated populations. These resulting small populations face reduced gene flow and increased genetic drift, decreasing genetic variability. Populations with reduced genetic variability are more susceptible to disease and environmental change. Southern California is a region where the encroachment of urban development has taken a heavy toll on native plant populations. In particular, Pentachaeta lyonii, a native federally listed endangered plant that was once found in many parts of Los Angeles County and southeastern Ventura County now persists in a few locations in the Santa Monica Mountains and Simi Hills. The objective of this study was to determine the population structure and estimate gene flow between seven sites where P. lyonii occurred. To accomplish this, DNA was extracted from whole plants collected at each site, and the nuclear genetic markers internal transcribed spacer 1 and 2 (ITS1 and ITS2) were amplified and sequenced for each sample. The resulting sequences were then compared using an analysis of molecular variance (AMOVA), and used to v estimate levels of gene flow. A high level of genetic variation was found among sites (90.82%) with very little variation found within them (9.18%). Gene flow was highly restricted (estimated Nem = 0.03) and there was a high level of genetic differentiation (FST = 0.91 and GST = 0.89) clustering sites into what seems to be larger northern and southern groups. Because only one set of nuclear molecular markers was assessed, and within these the sequence divergence was less than 1%, additional genetic analyses should be undertaken to verify this difference before developing restoration plans that would involve moving seeds and/or pollen from one area to another. Until more is known about the population genetics of P. lyonii, conservation of its habitat should be a priority. vi INTRODUCTION As human populations grow, urban centers are expanding and encroaching upon surrounding natural areas. Land that was once covered by large expanses of native vegetation has become a fragmented patchwork with an increasingly urban mix (Saunders et al. 1991, Collinge 1996, Jaeger 2000, McKinney 2002, Fischer and Lindenmayer 2007). Species that once had relatively large continuous or patchy distributions have become less abundant and more isolated (Wilcove et al. 1986, Saunders et al. 1991, McKinney 2002, Riley et al. 2003, Lienert 2004). In small isolated populations stochastic processes, such as genetic drift, have much greater effects (Shaffer 1987, Ellstrand and Elam 1993, Lienert 2004). Genetic drift is the random change in the allele frequencies from one generation to the next (Hendricks 2005). In large populations the chance that allele frequencies will change significantly due to drift is small, but in small populations the proportional impact of genetic drift can be large, possibly leading to the eventual fixation or loss of alleles (Wright 1949, Lacy 1987, Ellstrand and Elam 1993, Hendrick 2005). As a result, there can be increased differentiation between populations and reduced genetic variability within a population (Ellstrand and Elam 1993, Hendrick 2005). In small populations, the probability that fixed alleles will be slightly deleterious is greater than in large populations (Lienert 2004). This is because selection is a greater factor than genetic drift in larger populations, so deleterious alleles are more likely to be selected out (Lacy 1987). However, in small populations genetic drift can be a factor that is just as strong as or stronger than selection. Therefore, alleles that might have been eliminated by selection could become fixed even if they are somewhat deleterious (Ellstrand and Elam 1993). Fixation of these alleles can 1 reduce fitness and the consequent loss of genetic variability may hinder the capacity of a population to adapt to a changing environment (Lacy 1987, Ellstrand and Elam 1993, Young et al. 1996, Lienert 2004, Aguilar et al. 2008). The effects of genetic drift may be counterbalanced by gene flow. Gene flow is the successful movement of alleles from one population to another through dispersal of individuals or gametes. Gene flow decreases differentiation between populations and can increase genetic variation within populations. Therefore, if there are sufficient levels of gene flow, the negative effects of genetic drift can be reduced (Lienert 2004, Aguilar et al. 2008). In plants, gene flow happens via seeds or pollen and can occur over great distances or can be restricted (Loveless and Hamrick 1984). Seed dispersal is usually more limited than pollen dispersal, so many studies use pollen as a measure of dispersal ability (Ellstrand 1992b, Ouborg et al. 1999). In wind pollinated species, gene flow may occur over great distances and at significant rates resulting in populations with little genetic differentiation. Examples of this include fragmented populations of Acer saccharum in southeastern Canada (Young et al. 1993) and populations of the tropical tree Milicia excelsa in Cameroon (Bizoux et al. 2009). Even though gene flow is less restricted in wind-pollinated trees, small populations that have a history of fragmentation can have a high degree of differentiation such as in stands of European beech (Fagus sylvatica) in Spain (Jump and Peñuelas 2006) and junipers (Juniperus communis) in Ireland (Provan et al. 2008) Gene flow is more complicated in species that have animal pollinators, where factors such as foraging distance, may have an effect (Ellstrand and Elam 1993, Steffan- Dewenter and Tscharntke 1999, Lienert 2004). Pollinators with short foraging distances 2 may have a restricting influence on gene flow resulting in populations with a higher degree of genetic differentiation (Loveless and Hamrick 1984). Examples of this include insect pollinated populations of Salvia pratensis, Scabiosa columbaria, and Silene latifolia in the Netherlands (Van Treuren et al. 1991, Barluenga et al. 2011), Pulsatilla vulgaris in Germany (Hensen et al. 2005), and the tropical trees