1. INTRODUCTION Carolina hemlock (Tsuga caroliniana Engelmann) (Figure 1) is a rare conifer endemic to the highlands of the southeastern United States, where it is experiencing significant decline as a result of infestation by the hemlock woolly adelgid (HWA, Adelges tsugae Annand). Since 2003, Camcore (an international tree breeding and conservation program at NC State University) and the USDA Forest Service have cooperated to conserve the genetic resources of Carolina hemlock from across the species’ geographic range (Jetton et al. 2008). The goal of this ex situ conservation effort is to plant small field conservation banks and establish seed reserves that will serve as a source of genetic material to be utilized for breeding and restoration efforts once effective adelgid management strategies are in place. A key factor to the success of gene conservation programs is that seed collections capture a representative number of alleles to adequately protect the gene pools of the species in question. To do this we must understand the population genetic structure of a species and how its genetic diversity varies across the landscape. Camcore and the USDA Forest Service addressed this issue by employing amplified fragment length polymorphism (AFLP) molecular markers to: (1) compare the genetic variation of a seed sample collected from nine populations in 2003 for ex situ gene conservation to that present in a broader foliage sample collected in 2005 from 15 natural stands, and (2) investigate the genetic relationships among the populations sampled. Ex situ Seed Collection Represents Genetic Variation Present in Natural Stands of Carolina Hemlock (Tsuga caroliniana), a Southern Appalachian Endemic Threatened by the Hemlock Woolly Adelgid (Adelges tsugae) Robert M. Jetton 1, Kevin M. Potter 2, William S. Dvorak 1, W. Andrew Whittier 1, and James “Rusty” Rhea 3 1 Camcore, Dept. of Forestry & Environmental Resources, NC State University, Raleigh, NC 2 Dept. of Forestry & Environmental Resources, NC State University & USDA Forest Service EFETAC, RTP, NC 3 USDA Forest Service, Forest Health Protection, Region 8, Asheville, NC PopulationTrees (#) Populations (#) Polymorphic Loci # % Expected Heterozygosity Foliage (natural stands)1441510180.80.302 Seed (ex situ)110910390.40.331 PopulationTrees (#) % Polymorphic Loci Expected Heterozygosity Bluff Mountain892.40.3309 Caesar's Head597.10.3244 Carolina Hemlocks1092.40.3113 Crabtree692.30.3291 Cradle of Forestry895.80.3302 Hanging Rock51000.3801 Linville Falls1077.40.2655 Table Rock31000.3145 Wildcat1091.60.3289 PopulationBMCH/TRCHCGCTCoFLFWCEH Bluff Mountain.0.0110.0130.0070.0040.0300.0000.107 Caesar's Head/Table Rock0.029.0.0000.0490.0000.0130.0120.154 Carolina Hemlocks0.0350.003.0.0560.0080.0430.0060.128 Crabtree0.0210.0950.118.0.0610.0700.0300.087 Cradle of Forestry0.0150.0000.0300.118.0.0180.0010.154 Linville Falls0.0720.0420.0990.1410.044.0.0160.198 Wildcat0.0000.0350.0310.0610.0160.044.0.131 Eastern hemlock0.1460.1900.1810.1310.1950.2600.175. RESULTS & DISCUSSION  Genetic differentiation between the foliage sample and the seed sample was relatively small (F ST = 0.037). Nei’s genetic distance between the two was small (0.018), and differences in the expected heterozygosity were minimal, although the percent of polymorphic loci was higher in the seed sample (Table 1).  Analysis of the seed sample showed moderate genetic differentiation among the populations represented (F ST = 0.0584). The populations themselves showed relatively similar levels of genetic variation (Table 2).  Population pairwise genetic distance and genetic differentiation values tended to be smaller for populations of closer proximity (Table 3). Genetic distance and genetic differentiation between the Cradle of Forestry and Caesar’s Head/Table Rock populations was 0, as they were between the Wildcat and Bluff Mountain provenances.  A consensus neighbor-joining dendrogram based on Nei’s genetic distance supports clustering of the Caesar’s Head/Table Rock, Cradle of Forestry, Carolina Hemlocks, Wildcat, and Bluff Mountain populations in a south-to-north trend (Figure 3).  The data show that some of the populations with the highest levels of genetic diversity are located along the eastern and southern edges of the species’ distribution. This suggests that the Pleistocene glacial refuge for Carolina hemlock may have been located southeast of the Appalachian Mountains, similar to the pattern we previously described for eastern hemlock (Potter et al. 2008). Implications for Gene Conservation: The results of this study demonstrated little genetic differentiation between the ex situ seed sample and the broader foliage sample from natural stands, indicating that seed collections have successfully represented the genetic variation present in the natural stands. Furthermore, molecular marker studies such as we present here are instructive to the design and success of current and future gene conservation strategies for Carolina and eastern hemlock. The data on the genetic structure and variation among populations help us to understand how best to manage ex situ conservation plantings and seed reserves so that a broad and adaptive genetic base is maintained for breeding and restoration efforts. We can also use the data to better identify those populations that should receive priority for in situ protection (chemical and biological controls) so that high levels of genetic diversity are maintained in the surviving populations that will be critical to the restoration of the species. MATERIALS & METHODS Provenance Seed and Foliage Collections: This study included 15 populations of Carolina hemlock in the Southern Appalachian region (Figure 2). The ex situ seed sample consisted of seed collected in August and September 2003 from 64 mother trees representing nine of these populations located in North and South Carolina. Seed was collected from 3 to 10 mother trees per population. The natural stand foliage samples were made in all 15 populations in May 2005, covering much of the geographic range of Carolina hemlock and encompassing 144 trees. Each tree sampled for seed and/or foliage within a population was located at least 100 m from the next. For the ex situ seed sample, two seedlings from each of 55 mother trees were grown from seed in the greenhouse and needles were harvested. For the natural stand sample, needles were harvested from each of the 144 foliage samples collected. Genomic DNA for all samples was extracted from needle samples using the DNeasy Plant Mini Kit (Qiagen, Chatsworth, California, USA). AFLP marker generation: Amplified fragment length polymorphisms (AFLPs) (Vos et al. 1995) are reproducible, require no previous knowledge of genomic sequences, and generate a large amount of information. For this project, the restriction digestion, adapter ligation, pre-amplification, and selective amplification polymerase chain reaction (PCR) steps generally followed the Myburg and Remington (2000) protocol. Pre-amplification was conducted using EcoRI+AC and MseI+CC primers, and selective amplification was performed using EcoRI+3/ MseI+4 primers. Twenty-four selective primer combinations were screened across samples from nine Carolina hemlock and six eastern hemlock trees, with the combinations of EcoRI +ACC/MseI+CCTG, EcoRI +ACG/MseI+CCTG and EcoRI+ACT/MseI+CCTG chosen for their high levels of polymorphism. Each EcoRI primer was fluorescently labeled with a different label for multiplex fragment separation and detection on an ABI Prism 3100 capillary sequencer at the Iowa State University DNA Laboratory (Ames, Iowa). Data analyses: The program AFLP-SURV version 1.0 generated genetic diversity and population genetic structure estimates using the average expected heterozygosity of the marker loci as a measure of genetic diversity. This approach estimates allelic frequencies at each marker locus in each population, assuming they are dominant and have only two alleles. These allele frequencies were generated by applying a Bayesian method with non-uniform prior distribution of allele frequencies. Hardy-Weinberg genotypic proportions were assumed, and 1,000 permutations were run in each analysis of population genetic structure (F ST ) and genetic distance. A neighbor-joining (NJ) dendrogram was generated to visualize the relationships among provenances using the seed sample (ex situ) populations, including one of the two seedlings from each mother tree. The seed sample populations were used rather than the natural stands because of more consistent fragment amplification across provenances. The Hanging Rock provenance was not included because of an insufficient sample size, while the adjacent Table Rock and Caesar’s Head provenances were combined into a single population. The dendrogram was constructed using the NEIGHBOR and CONSENSE components of PHYLIP 3.6. Table 1: AFLP genetic diversity present within the foliage sample (natural stand) and the seed sample (ex situ conservation) populations. Table 2: AFLP genetic diversity present within the seed sample populations. Table 3: Pairwise differences among Carolina hemlock seed sample populations. Upper diagonal: pairwise Nei’s genetic distance. Lower diagonal: pairwise F ST estimates. ACKNOWLEDGEMENTS The authors would like to thank Michael Tighe for coordinating the collection of foliage and seed samples and processing, sowing, and growing seedlings from the ex situ seed collection; Jianfeng Li for initial testing of the AFLP primer combinations; and Ross Whetten, Saul Garcia, Mollie LeBude, and Emily Heaton for helpful discussions and suggestions regarding laboratory procedures and data analysis. This research was supported by Camcore and the Christmas Tree Genetics program at North Carolina State University, and in part through Research Joint Venture Agreement 09-JV-11330146-087 between the USDA Forest Service and North Carolina State University, and Grant Agreement 03-DG-11083150-850 between the USDA Forest Service Forest Health Protection and Camcore, NC State University. REFERENCES Jetton, R.M.; Dvorak, W.S.; Whittier, W.A. 2008. Ecological and genetic factors that define the natural distribution of Carolina hemlock in the southeastern United States and their role in ex situ conservation. Forest Ecology and Management. 255: 3212-3221. Myburg, A.A.; Remington, D.L. 2000. Protocol for high-throughput AFLP analysis using LI-COR IR2 automated sequencers. Raleigh, North Carolina: North Carolina State University Forest Biotechnology Group. 45. Potter, K.M.; Dvorak, W.S.; Crane, B.S. [and others]. 2008. Allozyme variation and recent evolutionary history of eastern hemlock (Tsuga canadensis) in the southeastern United States. New Forests. 35: 131-145. Vos, P.; Hogers, R.; Bleeker, M. [and others]. 1995. AFLP: A new technique for DNA-fingerprinting. Nucleic Acids Research. 23: 4407-4414. Figure 3: Consensus neighbor-joining dendrogram depicting Nei’s genetic distances among the Carolina hemlock seed collection (ex situ) populations, with eastern hemlock used as an outgroup. The values represent the percent bootstrap support for the nodes from 1000 replicates; those above 50 percent are considered well-supported. Figure 2: The geographic range of Carolina hemlock and the provenances sampled for the molecular marker analysis. Figure 1: Carolina hemlock at Mount Jefferson, N.C.; foliage sample (inset).