Genetic variation in Artemisia campestris: do distinct genetic varieties exist within the species? United States Department of Agriculture Lab Report NFGEL Project #319 Forest Service Project submitted by Andrew Bower, Area Geneticist Mark Skinner, Regional Botanist US Forest Service US Forest Service 1835 Black Lake Blvd. SW Region 6 Regional Office Olympia, WA 98512 1220 SW 3rd Avenue p: 360-956-2405 Portland, OR 97204 Washington Office [email protected] p: 503-808-2150 Forest Management [email protected] Theodore B. Thomas, Ecologist Pacific Region, Washington Fish and Wildlife Office Branch of Listing, Critical Habitat 510 Desmond Drive SE, suite 102 Lacey, WA 98501 p: 360-753-4327 National Forest [email protected] Genetics Laboratory (NFGEL) This work was conducted under the direction of Valerie Hipkins, PhD……...…………………………..NFGEL Director Report prepared by Barbara Wilson, PhD. Carex Working Group, Corvallis, Oregon 2480 Carson Rd Placerville, CA Laboratory technical work conducted by 95667 Andrew Jackson, Keenan Raleigh, Valerie Hipkins, 530-622-1609 Tissue Preparation Ed Sprigg, Amanda Cutler, Jennifer DeWoody, Jacob (phone) Snelling Ploidy Analysis Keenan Raleigh, Amanda Cutler, Valerie Hipkins 530-622-2633 (fax) Randy Meyer, Amanda Cutler, Jian Alsarraj, Valerie Isozyme Analysis [email protected] Hipkins Valerie Hipkins May 7, 2016 NFGEL Artemisia Report INTRODUCTION The pattern of variation in certain herbaceous Artemisia campestris along the Columbia River is complex and does not lend itself to simple taxonomic classification. In the Flora of the Pacific Northwest, Artemisia borealis is treated as a subspecies of A. campestris, and A. campestris is described as “A highly polymorphic species, divisible into a number of rather diverse but apparently wholly confluent subordinate taxa” (Hitchcock et al. 1955, p. 60). The taxonomic history of Artemisia campestris and A. borealis is more like a braided stream than the expected tree (Arnett 2012) and taxonomists disagree about how to treat the taxa (Table 1). To add to the confusion, both A. borealis and A. campestris are variable. Two forms of A. borealis and/or A. campestris occur along the Columbia River in Washington and, historically, in Oregon. One form, “wormskioldii,” is typically biennial and is short, with hairier leaves and stems, slightly broader leaves, and more compact inflorescences with bigger flowers. It blooms in April and May with occasional stragglers blooming into later summer. The other form, “scouleriana,” is usually perennial and is much taller, though heights overlap, with less hairy leaves and stems, narrower leaves, and long, open inflorescences with smaller flowers. It blooms in August and September. The wormskioldii plants resemble arctic/alpine A. borealis (Figures 1 and 2). The scouleriana plants resemble widespread low elevation A. campestris. Intermediates occur, and seeds collected from wormskioldii plants occasionally produce an offspring with the scouleriana phenotype (K. Amsberry, per. comm.). The wormskioldii form lives on sand dunes and similar habitats along the Columbia. This habitat has been mostly destroyed as a consequence of dam-building, and two wormskioldii populations remain, at Beverly and at Miller Island. The scouleriana form is widespread in the Pacific Northwest. It is fairly common at several sites along the Columbia River, including Beverly and Miller Island. The wormskioldii form is a listed rare plant; critically imperiled globally, endangered in Washington, and a candidate species with the USFWS (Washington Natural Heritage Program 2014). It is heavily managed and new populations have been established. However, the mixed populations at Beverly and Miller Island, and the existence of morphologically intermediate plants raise the question of whether wormskioldii is a real taxon or a set of extreme phenotypes in variable scouleriana populations. If wormskioldii is a not a real taxon, then it could be delisted and resources used to preserve it could be re-directed to manage other rare plants. This isozyme study was initiated to assess the genetic variation within and among wormskioldii and scouleriana occurrences to determine if these forms are genetically distinct. The evolutionary relationships of both of these forms were also compared to A. borealis samples. For purposes of this report, the three putative taxa tested are identified as A. borealis, A. campestris var. scouleriana, and A. campestris var. wormskioldii. 2 NFGEL Artemisia Report METHODS SAMPLE COLLECTION A total of 310 samples of Artemisia spp. were submitted for this project, with 31 plants collected from each of 10 populations, all in Washington (Table 2; Figure 3). Two populations from the Columbia River were identified as A. campestris var. wormskioldii, two populations from the Olympic Mountains were identified as A. borealis, and the remaining populations were identified as A. campestris var. scouleriana. Most of the scouleriana populations were collected on the Columbia River from Beverly to Miller Islands, inclusive, and two came from Whidbey Island in Puget Sound. Samples were collected into individual plastic bags and kept cool until shipment to NFGEL using overnight delivery. PLOIDY ANALYSIS Flow cytometry was used to compare relative DNA content among a subset of samples from each population. An approximately 1.0 cm2 piece of leaf tissue was diced in 0.4 mL of Partec® CyStain extraction buffer using a double-edge blade for approximately 30 seconds. Immediately following, 1.6 ml of the CyStain staining buffer was added to the sample, which was then placed through a 10 µm filter and immediately analyzed on a Partec PA Ploidy Analysis system using the parameters: gain = 380, LL = 30, speed = 3, linear scale. ISOZYME ANALYSIS All samples were prepared for isozyme analysis by grinding an approximately 2 cm2 piece of leaf tissue with a mortar and pestle under liquid nitrogen until it was a fine powder, then adding around 400 µL (12-15 drops with a plastic pipette) of Tris based extraction buffer (Cheliak and Pitel 1984) and allowing the slurry to freeze to the mortar. When the slurry thawed, 100 µL was transferred to each of three flat-bottom 96-well plates, which were then frozen at -80°C until analysis. Starch gel (11% v/v) electrophoresis was used to examine isozyme variation. Three buffer combinations from Conkle et al. (1982) were used to examine 16 enzyme stains using recipes adapted from Wendel & Weeden (1989), revealing a total of 9 loci used for analyses. Five loci were examined in a lithium borate electrode buffer-tris citrate gel buffer combination (system LB): fluorescent esterase (FEST; EC 3.1.1.-), leucine aminopeptidase (LAP; EC 3.1.11.1), phosphoglucomutase (PGM; EC 5.4.22.-), malic enzyme (ME7; EC 1.1.1.40), and aconitase (ACO; EC 4.2.1.3). Seven loci were examined in a sodium borate electrode buffer-tris citrate gel buffer combination (system SB): aspartate catalase (CAT; EC 1.11.1.6), triose-phosphate isomerase (TPI; EC 5.3.1.1), uridine diphosphoglucose pyrophosphorylase (UGPP; EC 2.7.7.9), phosphoglucose isomerase (PGI; EC 5.3.1.9), aminotransferase (AAT; EC 2.6.1.1), glycerol dehydrogenase (GLYDH; EC 1.1.1.6), and 6-phosphogluconic dehydrogenase (6-phospho-D- gluconate) (6pgd; EC 1.1.1.44). Five loci were examined on a morpholine citrate electrode and gel buffer (system MC6): malate dehydrogenase (MDH; EC 1.1.1.37), diaphorase (DIA; EC 1.6.99.-), isocitrate dehydrogenase (NADP form) (IDH; EC 1.1.1.42), shikimate dehydrogenase 3 NFGEL Artemisia Report (SKD; EC 1.1.1.25), and 6-phosphogluconic dehydrogenase (6-phospho-D-gluconate) (6pgd; EC 1.1.1.44). Three of these enzyme stains were run and found to be monomorphic for all samples (FEST, CAT, AAT-1 and AAT-2); three other stains were found to have insufficient activity among samples (LAP, IDH, and MDH); and one stain (UGPP) had no activity in A. campestris samples but contained two segregating alleles in the A. borealis samples. 6PGD was run on both the SB and MC6 buffer systems, but only data from the SB system was used in the analysis. Data from nine isozyme loci were used in the final analysis. DATA ANALYSIS Isozyme data were examined for standard measures of genetic diversity within populations (alleles per locus, effective alleles per locus, observed heterozygosity, and fixation) and for differentiation among populations using GenAlEx v. 6 (Peakall and Smouse 2006). If the two putative subtaxa (wormskioldii and scouleriana) represent distinct subspecies or varieties, we predicted that populations of each subtaxa would be more genetically similar to each other than they were to populations from another subtaxa or species. Put another way, two putative taxa are expected to show greater differentiation than individual populations within each taxa. Genetic differentiation among populations was examined using analysis of molecular variance (AMOVA) and principal coordinates analyses (PCoA), both implemented in GenAlEx v. 6. In addition, Nei’s (1972) unbiased genetic distance between all pairs of populations was visualized using UPGMA to build a population phenogram using Mega v. 5.2 (Tamura et al. 2011). Admixture analyses and population assignment tests were conducted in Structure v. 2.3.4 (Falush et al. 2003, 2007; Pritchard et al. 2000), estimating likelihoods for 500,000 replicates following a 50,000 burn-in period, with parameters set to assume independent allele frequencies among populations, with other parameters set to the default. The number of genetic clusters tested (K) was defined as K={2:8}, and each K was examined five times. The results of the admixture analyses were examined using two methods. First, the mean and variance of the log- likelihood of each K (over the five replicates) was examined to identify the K with the greatest likelihood and low variance. Second, the delta-K method of Earl & vonHoldt (2012) was used to examine the decrease in the rate of change of log-likelihood over each K as a method to choose the most likely number of genetic clusters, as employed by Structure Harvester v.