Genetic structure of three Endangered Plants of the Santa Rosa Plain: Burke's goldfields (Lasthenia burkei), Sonoma sunshine (Blennosperma bakeri), and Sebastopol meadowfoam (Limnanthes vinculans)

Burke's goldfields (Lasthenia burkei), Sonoma sunshine (Blennosperma bakeri), and Sebastopol meadowfoam (Limnanthes vinculans) are herbaceous annual plants that inhabit vernal pool and swale habitats mainly on the Santa Rosa Plain of Sonoma County, California. All are Federallyand State-listed as endangered. The goal of our research was to determine the genetic variation between populations in order to inform conservation efforts as to the distinctiveness of populations for seed banking and ex situ conservation, and to infer possible explanations for genetic subdivisions to aid in management decisions. Using 15 microsatellite markers we genetically surveyed 21 populations of L. vinculans, genotyping 577 individuals. Our results indicate a substantial divergence and limited gene flow between Santa Rosa Plain (SRP) populations and an outlying Napa county population (mean FST = 0.180). Among SRP populations genetic variation is significantly lower (FIT = 0.052), with most (82%) of the overall SRP population variation explained by within population genetic variation. L. vinculans shows relatively high genetic diversity at all sites (average Hexp = 0.65 ± 0.19, average Hobs = 0.53 ± 0.23, average number of alleles per locus = 7.419 ± 3.748). We examined 307 L. burkei plants from 15 populations. We used 42 inter-simple-sequence-repeats (ISSR) and random amplified polymorphic DNA (RAPD) nuclear DNA markers to measure genetic characteristics. Species wide, we found that geographically separate populations of L. burkei were genetically distinct (FST = 0.22), using Analysis of Molecular Variance (AMOVA). Half of this variation (FST = 0.11) was due to the difference between Lake and Sonoma County populations; the other half was due to variation within county populations (also FST = 0.11). We were able to examine only 64 individuals of B. bakeri, scattered among five populations, due to unforeseen extraction problems resulting in the poor quality of 80% of our DNA samples. Using 27 ISSR and RAPD markers, our preliminary assessment was that regional and local patterns of genetic variation exist in this species that merit further study in 2008 and are of conservation concern. Our troubleshooting of the extraction protocols has suggested areas where we can improve our methods so as to improve DNA quality in the 2008 survey. Genetic Structure of Three Endangered Plants 2 INTRODUCTION Population decline in most species is due to the degradation and loss of their habitats, often including diverse microhabitats (Foin et al 1998). Many species of plants are associated with distinct microhabitats (Maliakal-Witt et al 2005). Due to both adaptation and genetic drift, changes in habitat are often accompanied by genetic changes in plant populations (Hufford and Mazer, 2003, Soule and Mills 1998). Where microhabitats occur as discrete and small-scale areas, they create an opportunity for small-scale genetic divergence (Frankel and Soule 1980). The differential distribution of genetic variation in plant populations (genetic structure) develops over time due to adaptation, genetic drift, gene flow and natural selection (Slatkin 1987, Futuyama 1986). Gene flow is determined primarily by the dispersal of pollen and seed. When gene flow is reduced, isolated populations diverge due to natural selection and the chance effects of genetic drift. If gene flow is a common occurrence, the divergence of genetic structure is reduced. By analyzing differences in genetic structure between populations of plants using selectively neutral markers, taking into account their breeding system and observations of potential causes of gene flow, we can determine the degree of genetic isolation between populations and assess the potential for microevolution of the species. If genetic data are assessed relative to potential influences on genetic structure, results can be used to inform conservation efforts to maintain or simulate ecological phenomena that have played a role in the microevolutionary development of the species. Further, analysis of the genetic structure of populations can inform our conservation decisions with respect to possible translocation of individuals (i.e., seeds or plants) from one area to another, appropriate establishment of new populations, and design of possible seed collection scenarios for ex situ seed storage. Lasthenia burkei, Blennosperma bakeri, and Limnanthes vinculans are herbaceous annual plants that inhabit vernal pool and swale habitats mainly on the Santa Rosa Plain. They are all Federally and State listed as endangered. Populations of the three listed plants are fragmented, with many populations persisting in small habitat fragments that are vulnerable to off-site impacts and inappropriate site management. The remaining populations occur on privately owned sites, on recently established preserves, or on mitigation banks in remnant natural pools or where they have been seeded into newly created pools. Most remaining natural populations are now more geographically isolated from each other than they were historically, potentially beyond the reach of pollinators or seed dispersal mechanisms. The goals of our research were to Genetic Structure of Three Endangered Plants 3 determine the genetic variation between populations in order to inform conservation efforts as to the distinctiveness of populations for seed banking and ex situ conservation, and to infer possible natural explanations for genetic subdivisions to aid in management decisions, such as the movement of seeds from remnant natural site to inoculate created mitigation pools. To accomplish these goals we developed molecular genetic techniques to characterize and compare individual populations of the three listed plants. We adapted microsatellite markers recently developed for Limnanthes alba by Kishore et al. (2004) to examine fine scale and rangewide genetic structure of L. vinculans. We developed Inter-Simple-Sequence Repeats (ISSRs) and Random Amplified Polymorphic DNAs (RAPDs) markers to examine the amount and distribution of genetic variation throughout the range of Lasthenia burkei and Blennosperma bakeri. METHODS Study System The Santa Rosa Plain extends west of Santa Rosa, north of Cotati, south of Windsor and east of Sebastopol (Figure 1). Historically, the Santa Rosa Plain was a contiguous and vast oak savannah, with vernal pools and swales spread throughout. These wetlands, which support a characteristic community of now largely endangered or threatened endemic plants and animals, become visible each spring as a succession of showy annual plants flower. Vernal pools and swales today occur in the Santa Rosa Plain as remnant habitat in a matrix of agriculture, urbanization, and fragmented remains of valley oak (Quercus lobata) savannah, grassland, and persistent wetland vegetation. The Santa Rosa Plain is the primary habitat of three endemic endangered vernal pool plants: Lasthenia burkei, Blennosperma bakeri, and Limnanthes vinculans, all State and Federally listed endangered plants. The future recovery of these three species is addressed in the Santa Rosa Plain Conservation Strategy (2005) that aims to create a long-term program to conserve their sensitive habitats and mitigate potential adverse effects due to future development on the Santa Rosa Plain. (http://www.fws.gov/sacramento/es/santa_rosa_conservation.html). Housing development, agriculture, reclaimed water irrigation, and the cessation of livestock grazing on preserved sites have contributed to the severe decline and demise of a Genetic Structure of Three Endangered Plants 4 majority of the area's pool fauna and flora within the last 50 years (CH2MHILL 1995). Altered hydrology, increased refuse, and foot traffic have contributed to site quality reduction in some areas. Remnant populations on private sites may constitute some of the few remnant high quality vernal pools on the Santa Rosa Plain, but these are currently not properly protected in the longterm, and quality may decline if management (e.g. horse or goat grazing) is changed over time by changing private owners. Intensive agricultural practices (including disking, vineyards, overgrazing, and orchards) have destroyed or damaged other vernal pool areas. In some cases pools spared in development projects in the Santa Rosa Plain area are declining in viability (D. Cook, pers. com). The floristic quality of the remaining pools has declined within the last 10-15 years in other, larger preserved areas due to the abrupt change of the historic site-specific grazing regimes to no grazing, which has greatly favored the establishment of exotic plant competitors (G. Cooley, pers. com.). Marty (2005) showed cattle grazing of exotic annual grasses in Central Valley vernal pools enhanced native plant diversity there. Further scientific study into the effectiveness of grazing regimes for Santa Rosa Plain vernal pool ecosystem viability is warranted for successful recovery of these listed plants. Much of the original pool area in the Santa Rosa Plain has been destroyed either by intensive agriculture or by urban development (CH2MHILL 1995). The Santa Rosa Plain Conservation Strategy has identified areas for conservation. A reserve system is being established by the Santa Rosa Plain Conservation Strategy Implementation Committee that includes historic (yet largely degraded) vernal pool preserves and sites where artificial pools have been created and sown with listed plants within the past 10 years (S. Talley pers. com.). Mitigation and preservation banks currently include a mixture of natural and created pools/swales of variable habitat quality. Study Species Burke’s goldfields (Lasthenia burkei) Lasthenia burkei is a spring-flowering herbaceous annual plant in the Aster family (Asteraceae) that inhabits vernal pools and swales on the Santa Rosa Plain in Sonoma County, California and in Lake County, California (CPC, 2007). It is known from about 20 extant p