125 Oak Ecosystem Restoration on Santa Catalina Island, California

125 POPULATION STRUCTURE AND GENETIC VARIATION OF ISLAND OAK, QUERCUS TOMENTELLA ENGELMANN ON SANTA CATALINA ISLAND Mary V. Ashley1*, Saji Abraham1, Laura C. Kindsvater2, Denise A. Knapp3 and Kathleen Craft4 1Department of Biological Sciences, M/C 066 University of Illinois Chicago 845 W. Taylor St. Chicago, IL 60607 2Save-the-Redwoods League 114 Sansome Street, Suite 1200 San Francisco, CA 94104-3823 3 Department of Ecology, Evolution, and Marine Biology University of California, Santa Barbara Santa Barbara, CA 93106 4Department of Plant Biology University of Minnesota College of Biological Sciences 250 Biological Sciences Center 1445 Gortner Ave. St. Paul, MN 55108 *Corresponding author: Phone: 312-413-9700 FAX: 312-996-9462 E-mail: [email protected] ABSTRACT: The island oak, Quercus tomentella Engelmann, is an island endemic, found only on the California Channel Islands and Guadalupe Island, Mexico. It is among the rarest oaks and represents a distinct component of California Island biodiversity. Despite its important role in island ecosystems and its conservation vulnerability, few studies have addressed its evolution and conservation status. Here we report a population genetic study of Q. tomentella on Santa Catalina Island. This is the first application of DNA microsatellite markers for a species belonging to the oak section Protobalanus, and we show that microsatellite loci developed for American, European, and Asian white oaks amplify successfully in a member of Protobalanus. Genotypes of eight microsatellite loci were used to examine levels of genetic variation in 68 samples of Santa Catalina Q. tomentella, and we assess how existing variation is structured among different groves on Santa Catalina. Levels of genetic variation are quite high, with gene diversity estimates ranging from 0.658 to 0.909 and five to 19 alleles per locus. The variation, however, is not distributed evenly on Santa Catalina, with some stands harboring much more genetic diversity than others. We found evidence for predominantly clonal reproduction in two groves. Fourteen trees sampled at one of these sites had only two genotypes. In contrast, trees sampled from other groves were genetically distinct and thus likely recruited from acorns. The genetic data from this study can assist restoration efforts by identifying genetically diverse seed sources, and conversely, areas where genetic supplementation might be beneficial. KEYWORDS: Quercus tomentella, microsatellites, California Islands, conservation genetics, clonal reproduction Oak ecosystem restoration on Santa Catalina Island, California: Proceedings of an on-island workshop, February 2-4, 2007. Edited by D.A. Knapp. 2010. Catalina Island Conservancy, Avalon, CA. Population Structure and Genetic Variation of Q. tomentella 126 INTRODUCTION Developing effective plans for the protection and management of rare or threatened species requires detailed information that may be ecological, demographic, or genetic. While ecological and demographic information may be the most pressing need for a short-term protection or restoration plan, information on genetic variability, genetic structure, and inbreeding is often required for effective long-term management. Furthermore, genetic data can provide information on whether populations of threatened species have been historically isolated and are thus genetically distinct, or whether gene flow has maintained connectivity. Genetic data can also be used to identify genetically diverse or evolutionarily unique populations or individuals to target for restoration or protection. Here we apply DNA microsatellite analysis to address questions regarding the population and conservation genetics of the island oak, Quercus tomentella, on Santa Catalina Island. Q. tomentella has a highly restricted distribution, found on only five California Channel Islands (Anacapa, Santa Rosa, Santa Cruz, Santa Catalina and San Clemente) and on Guadalupe Island, Baja California, Mexico. On Catalina, it is restricted to eight locations, with two locations supporting large metapopulations (Orizaba/Fern Canyon and Gallagher’s Canyon), and the remainder forming smaller, isolated groves (Figure 1). Most populations of island oak have suffered from human land use practices on the islands, including overgrazing by livestock and deer, and increased erosion. This is true on Santa Catalina, where nonnative livestock, mule deer, and bison either occur currently or have in the past. Ecosystem managers for Santa Catalina are especially interested in the distribution of genetic diversity for Q. tomentella given a 2007 wildfire that burned in an area which supports the second largest grove of the trees on the island (Gallagher’s Canyon). Resprouts of the burned trees are at risk from browsing by introduced mule deer, as are any seedlings found in unburned groves. DNA microsatellites are useful markers in population and conservation genetics because of characteristics including high levels of variability and codominant inheritance (Ashley and Dow 1994; Chase et al. 1996; LeFort et al. 1999). They have been successfully used for a variety of studies in oaks, including investigations of gene flow (Dow and Ashley 1998b; Dutech et al. 2005; Craft and Ashley 2007) mating systems (Dow and Ashley 1998a; Lexer et al. 1999; Sork et al. 2002) and hybridization between species (Muir et al. 2000; Craft et al. 2002; Muir and Schlotterer 2005). Microsatellites are also extremely useful for discriminating individual genotypes and thus revealing clonal structure of populations (Schilder et al. 1999; Reusch et al. 2000; Ainsworth et al. 2003). Previous studies have developed microsatellite markers for species either in the Section Quercus, the white oaks (Dow et al. 1995; Steinkellner et al. 1995; Isagi and Suhandono 1997) or species in Section Lobatae, the red or black oaks (Aldrich et al. 2002). Q. tomentella belongs to Section Protobalanus, the intermediate or golden oaks, a small group of oaks restricted to the southwestern United States and northwestern Mexico. The evolutionary affinities of Section Protobalanus are uncertain, but they may be more closely related to the white oaks (Manos et al. 1999; Manos et al. 2001). Therefore we tested, optimized and scored microsatellite genotypes in Q. tomentella using loci that were developed in white oak species. Our objectives were to use genetic analysis to assess levels of genetic variability in Catalina Island Q. tomentella, examine fine-scale population structure on the island, and to evaluate levels of clonal versus sexual reproduction. In the future we plan to place this study of Catalina Island Q. tomentella within a larger framework of island oak diversity across the entire species range and use our results to better understand the biology, evolution, and conservation potential of this rare oak species. Population Structure and Genetic Variation of Q. tomentella 127 METHODS Study sites and sampling Leaves were collected in 2006 and 2007 from 68 representative trees located in eight Santa Catalina Island groves (Table 1). Collection locations are shown in relation to all of the island groves in Figure 1. For three trees in Upper Mt. Orizaba (8-1, 8-15 and 8-20) that had multiple stems (ramets) appearing to be connected at the ground, leaves from multiple stems were intentionally sampled to determine whether these were indeed clones. An example of this (Tree 8-1) is shown in Figure 2. For all other sampling, care was taken to sample trees with clearly distinct stems, to avoid multiple sampling of trees likely to be single clones (genets). Trees were sampled from representative locations within each stand, taking into account grove density and configuration, likely clones, and accessibility. At two of the smaller groves (Twin Rocks and Lone Tree), all trees were sampled. Leaf samples collected were labeled and transferred to the lab at University of Illinois at Chicago, in paper envelopes with silica gel. On reaching the lab, the samples were frozen at -75ºC. Table 1. Number of trees existing and sampled for each grove of Quercus tomentella, Catalina Island, California Grove # Stems # Apparent Total Area # Trees (ramets)1 Individuals (m2) 1 Genotyped (genets) 1 Renton Mine 23 10 500 6 Gallagher’s Canyon 960 392 30,219 12 Cape Canyon2 393 189 6,375 5 Sweetwater Canyon2 78 49 625 3 Mt. Orizaba/Fern Canyon2 1,723 869 42,488 23 Lone Tree 24 14 1,078 14 Twin Rocks ~5 5 2,500 5 1Census and survey performed in 2003 and 2004 (McCune 2005) 2Samples from Upper Mt. Orizaba/Lower Fern Canyon, Cape Canyon, and Sweetwater were combined for analysis. Not sampled: Swain’s Canyon and one of two small groves at Twin Rocks. Microsatellite genotyping From the collected leaf samples, 0.003g of the frozen leaf material was ground to a fine powder using liquid nitrogen. DNA extraction was carried out using DNeasy Plant Mini Kit (Qiagen Inc., Valencia California). The extracted DNA was stored in TE buffer at 4ºC. Eight microsatellite loci were used in this study: QpZAG1/5, QpZag 110 and QpZag 9 developed in the European oak Q. petraea (Steinkellner et al. 1997), MsQ4 developed in the North American Q. macrocarpa (Dow et al. 1995; Dow and Ashley 1996), QpZAG11, QpZAG15, QrZAG58 in Q. robur (Kampfer et al. 1998) and QM69-2M1 developed in the Asian Q. myrsinifolia (Isagi and Suhandono 1997). Population Structure and Genetic Variation of Q. tomentella 128 Figure 1. Map of Quercus tomentella sampling sites. Figure 2. Multiple stems of Tree 8-1 from Upper Mt. Orizaba. Leaf samples collected from five different stems were genetically identical. Population Structure and Genetic Variation of Q. tomentella 129 Polymerase Chain Reaction (PCR) was carried out using 0.2 -0.4µg genomic DNA with a PCR mix of 5X PCR buffer (Promega), 500µM dNTP, 0.04 µM of the forward primer with the universal fluorescent- labeled M13 (-21) sequence appended at its 5’ end, 0.6 µM reverse primer, 1.5-3.0 mM MgCl2, 1.0 µg/µl bovine serum albumin, and 0.25 U Taq (Promega). DNA amplification was performed as per the optimized PCR conditions with an initial preheat at 94ºC for five minutes followed by 38 cycles of a denaturation at 94ºC for 30 seconds, 52-56ºC annealing for 30 seconds and extension at 72ºC for 30 seconds.

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