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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 , CA 94104-3823

3 Department of Ecology, Evolution, and Marine Biology University of , 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, . 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 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).

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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. This was followed by a final extension at 72ºC for five minutes. The length of the amplified sequence was measured running an aliquot of 1.5 µL of each PCR product on a sequencing machine (Applied Biosystems 3730) along with LIZ500 ladder (Applied Biosystems). All microsatellite genotypes were scored using Gene Mapper software Version 3.7 (Applied Biosystems).

Genetic analysis

Descriptive statistics for the eight microsatellite loci, including allele frequencies, observed and expected heterozygosity, allelic richness, and the inbreeding coefficient, FIS, were estimated using the software FSTAT v2.9.3 (Goudet 2001). Genotypes were inspected by eye to identify genetically identical individuals (putative clones). We also used the ‘identity analysis’ function in CERVUS 3.0 (Marshall et al. 1998) to identify exact matches. CERVUS also calculates the individual probabilities of identity based on population allele frequencies. This value represents the probability given the genotype of one individual that a second individual will have the same genotype. For the three trees in Mt. Orizaba where multiple stems were sampled (such as the one shown in Figure 2), only one stem sample was included in the analyses. However, when we found exact genetic matches for trees that were growing separately (see below), those trees were left in the analyses.

For population analyses, we grouped together samples from four sites in the central region of Santa Catalina surrounding Mt. Orizaba (Upper Mt. Orizaba, Cape Canyon, Sweetwater, and Lower Fern Canyon). These groves are closer in proximity to each other than the other groves and some of these sites had inadequate sample sizes to analyze independently. However, population substructure may occur within this pooled sample. Calculations of pairwise FST (a measure of population differentiation) were obtained using FSTAT, and using GenePop, a Fisher exact test was performed for all pairs of populations for all loci to test for differences in allelic distributions (Raymond and Rousset 1995).

RESULTS

Genetic Diversity and Structure

We were able to test and optimize eight microsatellite primer pairs developed for white oak species to amplify presumably homologous loci in Q. tomentella. All eight loci were polymorphic. For the 68 individual samples, we scored over 93% of the loci. The microsatellite variability for Santa Catalina Q. tomentella was high and similar to other studies of oaks, with five to 19 alleles per locus (mean 10.13) and gene diversity of 0.658 to 0.909 per locus (mean 0.7617) (Table 2). With all samples combined, observed heterozygosities were generally lower than expected and therefore the inbreeding coefficient, FIS, was positive and significant (P < 0.01) for all loci except QpZAG15 and QpZAG9.

Levels of genetic variation were not distributed evenly across the sampled stands (Table 3). The unevenness of sample sizes makes direct comparisons difficult, but some pairwise comparisons are noteworthy. A sample of six trees at Renton Mine had a total of 12 alleles, one of which was found only at that site. A similarly small sample of five trees at Twin Rocks harbored 21 alleles, four of which were unique to that site. Similarly, 14 individuals sampled at Lone Tree Grove had a total of 14 alleles, while 12 trees from Gallagher’s Canyon had 32 alleles. The pooled samples from the Mt. Orizaba area were highly diverse and harbored many (28) alleles that were not found elsewhere on the island. FIS values by

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Table 2. Descriptive statistics for eight microsatellite loci. Sample size (N), number of alleles, inbreeding coefficient (FIS), observed heterozygosity (Ho), and gene diversity (HE).

Locus N # Alleles FIS HO HE QpZAG110 65 19 0.103* 0.547 0.909 QrZAG58 58 6 0.880* 0.076 0.708 QpZAG11 63 16 0.278* 0.557 0.880 QpZAG15 63 11 -0.081 0.705 0.764 QpZAG1/5 67 6 0.251* 0.484 0.658 MSQ4 68 8 0.309* 0.526 0.660 QM69-2M1 69 5 0.269* 0.434 0.697 QpZAG9 65 10 0.057 0.823 0.832

*significant at P<0.01

stand (Table 3) ranged from large negative values (heterozygote excess, Renton Mine and Lone Tree Grove) to large positive values (heterozygote deficiency, Mt. Orizaba). Heterozygote excess at Renton Mine and Lone Tree Grove is likely due to the inclusion of genetically identical clones in the analysis. The heterozygote deficiency for the Mt. Orizaba sample may be due to population substructure (Wahlund effect) among the different stands pooled for the analysis.

Table 3. Sample size, number of distinct genotypes, and allele information for groves sampled. Private alleles are those found only in a single grove. FIS (inbreeding coefficient) is also shown by grove.

# Trees # Genotypes # Alleles # Private FIS Grove Genotyped Alleles Renton Mine 6 3 12 1 -0.57 Gallagher’s 12 10 32 2 -0.12 Canyon Mt. Orizaba* 31 29 71 28 0.26 Lone Tree 14 2 14 1 -0.91 Twin Rocks 5 5 21 4 0.16 *Samples from Upper Mt. Orizaba/Lower Fern Canyon, Cape Canyon, and Sweetwater were combined.

The different groves showed strong genetic differentiation. All pairwise comparisons of allelic differentiation were highly significant (Fisher exact tests). FST values (Table 4) were very high relative to other oak microsatellite studies, ranging from 0.1005 to 0.5257. These values should be considered cautiously because of the small sample sizes, particularly for Twin Rocks and Renton Mine Grove.

Clonal Growth

We tested whether trees that appeared to be growing clonally (Figure 2) were indeed clones. Leaves from five stems, two stems, and three stems for trees 8-1, 8-15 and 8-20, respectively, all had identical multi- locus genotypes. Only one copy of their genotype was included in subsequent

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Table 4. Pairwise FST values for Quercus tomentella groves, Catalina Island, California.

Gallagher’s Mt. Orizaba* Lone Tree Grove Twin Rocks Canyon Renton Mine 0.4233 0.2156 0.6922 0.4299 Gallagher’s Canyon 0.1005 0.3649 0.1878 Mt. Orizaba* 0.3363 0.1145 Lone Tree Grove 0.5257

*Samples from Upper Mt. Orizaba, Cape Canyon, Sweetwater, and Lower Fern Canyon were combined

analysis. However, we found many other trees with identical genotypes. Of the total possible 2,278 pairwise comparisons between the 68 sampled trees, 60 pairs of trees had identical genotypes. All matching trees occurred at the same site. Because of the high variability of the microsatellite loci, the probability that two individuals would share an identical multi-locus genotype by chance is exceedingly small. Indeed, for all exact genotype matches, the probability of identity ranged from 1.32 X 10-7 to 9.13 X 10-10 based on the identity analysis implemented in CERVUS. Therefore individuals that share the same genotype are almost certainly the result of asexual reproduction through clonal growth such as root sprouting. This was particularly striking in two groves, Lone Tree and Renton Mine (Table 3). At the Renton Mine site, four trees shared one genotype and the other two sampled were genetically distinct. At the Lone Tree grove, only two genotypes were found among the 14 trees sampled: nine trees shared one genotype and five shared the other. In contrast, other sites showed no or only low levels of clonal growth. At Twin Rocks, all individuals were genetically distinct. The combined Mt. Orizaba samples and the Gallagher’s Canyon samples were mostly comprised of genetically distinct individuals, with two and three genotypes shared by two trees, respectively.

DISCUSSION

Quercus tomentella is a rare island endemic belonging to a small, enigmatic group of oaks, the intermediate or golden oaks (section Protobalanus). Q. tomentella is an important component of the oak woodland communities on several islands and is threatened by anthropogenic activities and habitat alterations throughout its range. In this study, we characterize the population genetics of island oak on Santa Catalina Island by sampling trees representing many of the existing stands on the island and analyzing their microsatellite genotypes.

Santa Catalina Q. tomentella harbor relatively high levels of nuclear DNA variability. Allelic diversity and gene diversity are high and similar to microsatellite studies of mainland species of oaks (e.g. Craft et al. 2002; Craft and Ashley 2007). Thus it appears that the populations on Santa Catalina have not experienced severe and prolonged population bottlenecks. Q. tomentella has maintained diversity despite island endemism and recent anthropogenic disturbances. However, there is a striking level of population structure on the island that is generally not observed in mainland populations of oaks. Our observed FST values, shown in Table 4, greatly exceed values reported for other oaks over comparable distances. For example, a study of remnant bur oak populations, Q. macrocarpa, spanning all of northeastern Illinois, found pairwise FST values that are approximately an order of magnitude lower (Craft and Ashley 2007). These FST values must be interpreted cautiously because of small sample sizes and the inclusion of clones. However, pairwise comparisons for stands with no or few clones (e.g. Twin Rocks and Gallagher’s Canyon) also had high FST values. Other indicators, such as the number of private alleles confined to single stands, also provide support for high between-stand differentiation. Studies on other oaks suggest

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that their wind-dispersed pollen travels great distances, and often isolated stands receive more than half their pollen from outside the stand. High levels of pollen flow are believed to keep oaks reproductively connected over large areas (Dow and Ashley 1998b; Streiff et al. 1999; Nakanishi et al. 2004; Craft and Ashley 2007). Whether pollen flow is much more limited among Q. tomentella stands, and if this explains the genetic differentiation between stands, is something that deserves further investigation. Paternity studies of acorns can be used to infer pollination distances and patterns (Dow and Ashley 1998b; Streiff et al. 1999), and genetic analysis of seedlings can also characterize seed dispersal distances (Dow and Ashley 1996). Studies of flowering phenology could also determine if temporal isolation might be occurring through flowering asynchrony.

Some stands of Q. tomentella were much more genetically diverse than others. In particular, groves in Gallagher’s Canyon and groves localized to Mt. Orizaba are quite rich in genetic diversity. At the other extreme, the stand at Lone Tree Grove is comprised of a small number of genetically identical clones, and harbors very limited diversity. Our results suggest that clonal reproduction may be quite common in this species, and that it will not always be straightforward to distinguish between asexual and sexual reproduction. Genetic identification of oak clones has been very limited to date (Ainsworth et al. 2003). The tree shown in Figure 2 has stems that appear to be connected at their base, and its growth structure suggests that it may be a single individual, as we confirmed here. In contrast, trees sampled in other stands were not suspected to be connected through their roots, and only through genetic analysis was their clonal relationship identified.

The extensive clonal growth at Lone Tree Grove may be limiting sexual reproduction and seed set, and we recommend monitoring acorn production on Santa Catalina. Although oaks are not reported to have a genetic self-incompatibility, mating studies have shown that selfing is rare or nonexistent (Dow and Ashley 1998b; Fernandez and Sork 2005), and hand-crosses with self pollen reportedly lead to little or no seed set in some species (Yacine and Bouras 1997). Thus pollen limitation may occur in stands with many identical genotypes, low genetic diversity and high clonal reproduction. We would also suggest that flowering phenology be monitored, as this could be limiting pollen flow among stands if different stands flower at different times.

This study represents the first stages of characterizing the genetic and evolutionary status of this important species. Future studies will include analysis of trees on all the islands where Q. tomentella occurs. Spatial patterns of genetic structure within and between islands will be examined. We also plan to look for evidence of hybridization with another oak species, Q. chrysolepis, that occurs in mixed stands of Q. tomentella on some of the islands, including Santa Catalina, where hybrids have been reported. It is possible that hybrids were included in our study, although all samples used were identified in the field as Q. tomentella. These studies should provide an important contribution to the study of the biodiversity of the California Islands.

ACKNOWLEDGMENTS

Research and Collecting Permit number 06-06 was issued by the Santa Catalina Island Conservancy for this research. Many people assisted with sample collections, including John Knapp and Lauren Danner. We thank Kevin Feldheim and the Pritkzer Lab at the Field Museum. Crystal Guzman provided laboratory assistance.

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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.