2010 THE MICHIGAN BOTANIST 105 SOLIDAGO VOSSII (ASTERACEAE), A NEW SPECIES OF GOLDENROD FROM NORTHERN MICHIGAN Pamela J. Laureto James S. Pringle Department of Biological Sciences Royal Botanical Gardens Grand Rapids Community College P.O. Box 399, Hamilton, ON 143 Bostwick NE Canada L8N 3H8 Grand Rapids, Michigan 49503 [email protected] [email protected] ABSTRACT A new species of Asteraceae from Michigan U.S.A., Solidago vossii J. S. Pringle & P. J. Laureto, is described based on morphology, habitat, native range, and genome size. Solidago vossii is appar - ently endangered as it is known only from a northern wet prairie/pine barrens complex within a 2.25 square mile area near the border of southern Crawford and Kalkaska counties in northern Michigan. Morphologically, S. vossii is most similar to S. houghtonii , but differs in several features. The mor - phological features of the two species are compared and their phylogenetic relationship discussed. Chromosome counts indicate S. vossii is octoploid (2 n = 8 x = 72) and flow cytometry data support an octoploid genome size for plants from throughout the known range. KEY WORDS: Asteraceae, chromosome counts, flow cytometry, new species, Solidago vossii INTRODUCTION For botanists, both professional and amateur, goldenrods in the genus Sol - idago L. are often difficult to identify to species. There are approximately 100 species of goldenrod in North America, with the greatest number of these occur - ring in eastern North America (Semple and Cook 2006), where they are an im - portant component of the autumn flora. Some species, such as S. altissima L. (late goldenrod) and S. nemoralis Aiton (gray goldenrod), are ubiquitous and often occur in such dense populations that they are commonly described as “weeds”. However, many other species are sparsely distributed throughout their native range, and four species, S. albopilosa E. L. Braun (white-haired golden - rod), S. houghtonii A. Gray (Houghton’s goldenrod), S. shortii Torrey & A. Gray (Short’s goldenrod), and S. spithamaea M A. Curtis ex A. Gray (Blue Ridge goldenrod), have such restricted distributions that they are federally listed as ei - ther “Threatened” or “Endangered” (USFWS 1988a; USFWS 1988b; USFWS 1985b; USFWS 1985a respectively). Semple and Cook (2006) presented a taxonomic overview of the genus Sol - idago in which they recognized two sections, section Solidago , comprising 11 subsections, and section Ptarmicoidei (House) Semple and Gandhi. Section Ptarmicoidei currently includes six species that are easily distinguished from the other goldenrods by their corymboid (flat-topped) inflorescences. This section has its center of distribution in the Great Lakes region, with five of its six species 106 THE MICHIGAN BOTANIST Vol. 49 (S. houghtonii , S. ohioensis Riddell, S. ptarmicoides (Torrey & A. Gray) B. Boivin , S. riddellii Frank , and S. rigida L.) occurring in the state of Michigan. Although a specimen had been collected at the site as early as 1933, Edward G. Voss first noted in the 1960s that a goldenrod population on the sandy shores of the tiny Howes Lake in Crawford County, Michigan, resembled Solidago houghtonii and reported it as that species (in Guire and Voss 1963). James S. Pringle visited this site and observed these plants in the early 1970s. From mor - phological observations and a chromosome count he believed that the Howes Lake plants were distinct from true S. houghtonii , and suggested these plants might have a hybrid origin different from that of S. houghtonii . Since Pringle’s observations were made, additional occurrences have been discovered, and the population is now believed to be contiguous within an area of approximately 2.25 square miles (Fig. 1). Recently, Laureto (2010; Laureto and Barkman, in press) tested Pringle’s hy - potheses regarding the hybrid origin of entities called Solidago houghtonii in - cluding plants from throughout the geographic range of the species (Genesee Co. NY, Bruce Peninsula ON Canada, Mackinaw Co. MI, and Crawford Co. MI). True S. houghtonii is a hexaploid (2 n = 6 x = 54) species of hybrid origin (Lau - reto 2010; Laureto and Barkman, in press). Laureto’s molecular phylogenetic analysis of the hybrid origin of plants called S. houghtonii confirmed that the Crawford County plants are of hybrid origin and suggested that these plants were likely derived from the hexaploid S. houghtonii because they share the same parental taxa with true S. houghtonii . Chromosome count and flow cytometry data presented here indicate the Crawford County plants are nevertheless distinct from S. houghtonii . They are described here as a new species, Solidago vossii J. S. Pringle and P. J. Laureto. MATERIALS AND METHODS Plant Material Thirty-five specimens of Solidago vossii were used for the description of the new species. Thirty plants were observed in the field and five were herbarium specimens. The primary diagnostic charac - ters are compared with those of S. houghtonii (Table 1). Morphological data were calculated as fol - lows: Each examined specimen represents one evaluation of the number of heads and all non-numer - ical characters and five measurements for all numerical characters (i.e. florets, leaves, phyllaries). A total of eight individual Solidago vossii plants were collected, including achenes, from areas throughout the 2.25 square mile range of this species. These were used for determination of ploidy number by chromosome counting and flow cytometry methods described below. Plants and achenes were collected under Michigan Department of Natural Resources Threatened and Endangered Species permits #1855 and #1928. Voucher specimens are deposited in the Hanes Herbarium, West - ern Michigan University (WMU). Chromosome Counts Chromosomes counts were made from root-tip meristems of four field-collected Solidago vossii plants which were grown in the greenhouse at Grand Rapids Community College. Following the pro - tocol of L. Michael Hill (2009), root tips were removed from plants and pre-treated in 0.002 M 8- hydroxyquinoline for 5 hours at 15°C and then fixed for 15 minutes in acetic acid-alcohol (1:3) which was warmed to 60°C. The fixed root tips were then hydrolyzed in 1 N HCl for 30 minutes at 60°C and stained with 1% aceto-orcein for 5 minutes. Mayer’s albumen was applied to a cover-slip, which was placed over the preparation, and the root tips were squashed in order to separate the cells 2010 THE MICHIGAN BOTANIST 107 FIGURE 1. a. Map of Michigan showing location of Solidago vossii population. Map modified from Benbennick 2007. b. The location of S. vossii in southern Crawford and Kalkaska counties is out - lined. A small colony of a few individuals occurs on the east side of Lake Margrethe at the entrance to Camp Grayling (not shown). Map modified from U.S. Census Bureau 2010. and make the chromosomes more visible. After removing the cover-slip, the tissue was dried by pass - ing it through the following five solutions: 1:1 ethyl alcohol-glacial acetic acid, 3:1 ethyl alcohol- glacial acetic acid, 9:1 ethyl alcohol-glacial acetic acid, 95% ethyl alcohol, 95% ethyl alcohol (sec - ond change). The cover-slip/root-tip squash was permanently fixed to a clean slide using a drop of Euparal. From one or more slides, five metaphase cells were inspected for each plant. Chromosome counts were performed at 1000× magnification using a Nikon Microphot FXA microscope (Nikon Corp.) with differential interference contrast optics in place. Flow Cytometry Flow cytometry (FCM) is a rapid and robust analytical tool that allows accurate determination of DNA content for a large number of nuclei (Kron et al. 2007). Originally it was used to identify and characterize cancerous cells, but today it is revolutionizing such fields as ecology, evolutionary biol - ogy, and systematics (Kron et al. 2007). Since the method allows for the estimation of the nuclear DNA content of a large number of nuclei, it is being used to provide information about the ploidy level of individuals and the organismal composition of populations (Suda et al. 2007). The gathering of such information has historically been time-consuming which meant that the DNA content of in - dividuals within a population was estimated from just a few individuals. FCM has made it possible to gather information on genome size and ploidy level for large numbers of individuals in a relatively short amount of time. FCM data provide strong support for the determination of ploidy level within a population especially when combined with data from chromosome counts (Suda et al. 2007). FCM quantifies the intensity of light signals by individual nuclei that have been stained with a DNA-specific fluorochrome and excited by a laser beam. As the nuclei pass single-file at high speed through the illuminating beam they fluoresce and scatter light. These light signals are used to provide an estimate of the amount of DNA in the nuclei which is then quantified using known standards (Kron et al. 2007). FCM analysis was performed using fresh young leaves from 23 Solidago vossii seedlings which 108 THE MICHIGAN BOTANIST Vol. 49 TABLE 1. Comparison of Solidago vossii and S. houghtonii diagnostic characters. Features Solidago vossii Solidago houghtonii Growth habit single single or cespitose Stems 4–7+ dm tall 3–5 dm tall Basal leaves sparsely to regularly serrulate entire Number of heads 20 to 60 5 to 50 Involucre 6.0–9.0 mm long; 5.0–8.0 mm long; 18 to 24 phyllaries 15 to 18 phyllaries Ray florets 6 to 8; lamina 1.2–2.0 mm wide 6 to 12; lamina 0.5–0.6 mm wide Achene 1.8–2.5 mm 1.4–1.8 mm Pappus bristles of various lengths; not clavate blistles of equal length; clavate Chromosome number 2n = 72; octoploid 2n = 54; hexaploid Distribution localized in wet prairie in shorelines of northern Lakes Crawford and Kalkaska counties Michigan and Huron had been grown in the Grand Rapids Community College greenhouse.