Page  105 ï~~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 jpringle@rbg.ca plaureto@grcc.edu 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 apparently 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 morphological features of the two species are compared and their phylogenetic relationship discussed. Chromosome counts indicate S. vossii is octoploid (2n = 8x = 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 Solidago L. are often difficult to identify to species. There are approximately 100 species of goldenrod in North America, with the greatest number of these occurring in eastern North America (Semple and Cook 2006), where they are an important 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 goldenrod), 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 either "Threatened" or "Endangered" (USFWS 1988a; USFWS 1988b; USFWS 1985b; USFWS 1985a respectively). Semple and Cook (2006) presented a taxonomic overview of the genus Solidago 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

Page  106 ï~~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 morphological 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 hypotheses regarding the hybrid origin of entities called Solidago houghtonii including 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 (2n = 6x = 54) species of hybrid origin (Laureto 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 characters are compared with those of S. houghtonii (Table 1). Morphological data were calculated as follows: Each examined specimen represents one evaluation of the number of heads and all non-numerical 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, Western 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 protocol of L. Michael Hill (2009), root tips were removed from plants and pre-treated in 0.002 M 8 -hydroxyquinoline for 5 hours at 150C and then fixed for 15 minutes in acetic acid-alcohol (1:3) which was warmed to 600C. The fixed root tips were then hydrolyzed in 1 N HCl for 30 minutes at 600C 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

Page  107 ï~~2010 THE MICHIGAN BOTANIST 107 2010 THE MICHIGAN BOTANIST 107 aebnnc 2 1.Telcto4fS osi nsuhr rwodad aksacute sot 404 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 passing it through the following five solutions: 1:1 ethyl alcohol-glacial acetic acid, 3:1 ethyl alcoholglacial acetic acid, 9:1 ethyl alcohol-glacial acetic acid, 95% ethyl alcohol, 95% ethyl alcohol (second 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 1000x 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 biology, 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 individuals 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

Page  108 ï~~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. The analyses were done by Paul Kron in the lab of Dr. Brian Husband, Department of Integrative Biology, University of Guelph. For each sample, approximately 2 x 2 cm of leaf tissue from S. vossii and an internal standard, Glycine max 'Polanka', were co-chopped with a new razor blade in 0.5 ml ice-cold modified de Laat buffer (Kron and Husband 2009) with 50 g/ml propidium iodide, a DNA-specific fluorochrome, and 50 g/ml RNAse. After 20-90 minutes of staining time the suspension of released nuclei from each sample was filtered through a 30 m filter into a labeled test tube. The nuclear DNA content of the internal standard Glycine max 'Polanka' is reported to be 2.5 pg/2C (Dolezel et al. 1994; Dolezel et al. 2007), where pg represents the amount of nuclear DNA expressed in the mass units picograms and 2C (2C-value) refers to the complete somatic DNA content regardless of ploidy (Kron et al. 2007). Samples were run on a BD FACSCalibur flow cytometer (Becton Dickinson) at low or medium pressure (depending on nuclei number) for up to 5 minutes. Data were acquired using CellQuest Pro software (BD Biosciences). In FCM the measured parameter is the fluorescence of isolated nuclei. The fluorescence intensity of the nuclei is recorded as peaks on a histogram. When the genome size of the internal standard is known, the absolute DNA content, in picograms, for the test nuclei can be estimated from the relative fluorescence values (Kron et al. 2007). The mean fluorescence of the Solidago peak was divided by the mean fluorescence of the Glycine max peak, multiplied by 2.5 pg/2C, and expressed in DNA pg/2C. All samples were analyzed with a 256 channel scale using Modfit software (Verity Software) to estimate the fluorescence ratio, the coefficients of variation (CV), and the number of nuclei present. According to Greilhuber et al. (2007) a CV 5 is recommended for genome size estimation whereas a CV 7 is regarded as acceptable for ploidy determination. The number of nuclei present is recommended to be 1300 for genome size estimations but much lower numbers are generally considered to be acceptable for ploidy determinations. The DNA pg/2C values for the 23 Solidago vossii samples were compared to the mean DNA pg/2C value of Solidago hispida Muhlenberg ex Willdenow, a known diploid goldenrod (Jess Peirson, unpublished data). RESULTS AND DISCUSSION Diagnosis Solidago vossii J. S. Pringle et P. J. Laureto, sp. nov. Solidagini houghtonii similis sed chromosomatum numero 2n=8x=72 foliis

Page  109 ï~~2010 THE MICHIGAN BOTANIST 109 basalibus serratulis capitulis phyllariis pluribus ligulisque latioribus et acheniis majoribus pappi setis inaequalibus non clavatis differens. Type Holotype: U.S.A., Michigan, Crawford Co., N shore of Howe's Lake, ca. 6 km W of Grayling, Sec. 32, T27N R4W, Voss 11008, 14 August 1992, MICH; Isotypes: BLH, MSC, TRT, UMBS; Paratypes (all from the same locality): Somerville 1933, 1 September 1933, WUD-the earliest collection; Pringle 1365, 9 Aug 1972, HAM; Laureto 1119, 3 August 2001, WMU (Fig. 2). Description Perennial herb. Stems 4-7+ dm tall, erect, arising singly from a branching caudex. Proximal to mid-stem glabrous, becoming increasingly strigillose distally. Basal leaves tapering to a long, winged frimbriate petiole. Petiole bases marcescent (attached to old stems for more than one season). Blades ovate, 10-24 mm wide, 3-nerved (2 prominent lateral nerves arising proximally and running alongside midnerve for some distance before diverging), surfaces glabrous, margins ciliate, sparsely to regularly serrulate, each tooth appressed and ending in a gland, apices acute. Proximal cauline leaves tapering to winged petiole-like bases partially clasping stems, linear-oblanceolate, 150-180 mm long, 10-24 mm wide, glabrous, 3-nerved, margins ciliate, entire, apices acute. Mid-cauline to distal leaves sessile, blades linear-lanceolate, mid often obscurely 3-nerved, distal 1-nerved, 60-100 mm long, 5-10 mm wide, reduced distally. Heads (15-) 20-60 (-100+), not secund, in corymbiform arrays. Branches and peduncles moderately to densely strigillose, peduncles 6-12 mm long with 0-3 linear bracteoles. Involucres campanulate, 6-9 mm long. Phyllaries 18-24 in 3-4 series, unequal, linear to oblanceolate, 3-nerved, margins ciliate, apices obtuse to rounded. Ray florets 6-8, bright yellow, laminae 6.5-7.5 mm long and 1.2-2.0 mm wide. Disc florets 9-14, bright yellow, corollas 4.0-4.5 mm, lobes 1.0-1.4 mm. Achene obconic, 1.8-2.5 mm, glabrous, ribbed (ribs sometimes dark). Pappus of bristles of varying length, the longer ones 4.0-5.5 mm, not clavate. Etymology The specific epithet honors Dr. Edward G. Voss, a world-renowned botanist from Michigan who wrote the manual on Michigan's flora-Michigan Flora Volumes I-III (Voss 1972; Voss 1985; Voss 1996). Dr. Voss taught at the University of Michigan for decades, served on the editorial committee for the International Code of Botanical Nomenclature from 1969 to 1993, chaired the General Committee on Botanical Nomenclature from 1999 to 2005, founded The Michigan Botanist, and was its editor from 1962 to 1976. Ploidy Determination Observations of root tip meristem cells at metaphase of mitosis revealed that the chromosome number of Solidago vossii is 2n = 8x = 72. Figure 5 presents a S. vossii root-tip cell at the metaphase stage of mitosis. The term "ploidy" refers to the number of sets of chromosomes that an indi

Page  110 ï~~110 THE MICHIGAN BOTANIST Vol. 49 A"A D. la Cl danD. it /loho Im'f t st itc FIGURE 2. Solidago vossii J. S. Pringle and P. J. Laureto (drawn by P. J. Laureto). A. habit. B. Basal leaf. C. Head with only some florets drawn. D. Phyllary with chlorophyllous zone dark. B. Achene, mature, with disc floret and pappus still attached. vidual possesses and it is notated by an "x". An individual with two sets of chromosomes is referred to as a diploid (2x), three sets would be a triploid (3x), and so on with tetraploid (4x), pentaploid (5x), etc. It is also important to identify if one is referring to the reduced number of chromosomes found in egg and sperm cells (denoted as "n") or the number of chromosomes in non-reduced tissue (denoted as "2n"). The base number of chromosomes in the genus Solidago is reported to be x = 9 (Semple et al. 1984; Semple and Cook 2006). Since S. vossii has eight times that amount it is an octoploid species, the only known octoploid species of goldenrod. The nuclear DNA content of 23 Solidago vossii individuals was estimated by FCM. Figure 6 shows typical histograms for the octoploid S. vossii and a diploid

Page  111 ï~~2010 THE MICHIGAN BOTANIST 111 2010 THE MICHIGAN BOTANIST 111 FIGURE 3. Photo of Solidago vossii leaf showing black fungal spots. Note the sparsely toothed margin. (Photo by J. S. Pringle) Solidago, S. hispida, as analyzed by FCM. The mean DNA content of 24 diploid Solidago hispida samples was 2.086 pg per nucleus (Jess Peirson, unpublished data). The nuclear DNA content of all accessions of S. vossii ranged from 7.334 to 7.759 pg/2C nucleus (Table 2). The difference between the sample with the lowest (Arrowhead Rd. 07-7.334 pg per nucleus) and the highest value (Howes Lake 01-7.759 pg per nucleus) was 0.425 pg (= 1.058-fold) with a mean DNA content of 7.507 pg per nucleus. The mean nuclear DNA content of S. vossii is 3.599-fold greater than the diploid S. hispida, indicating that S. vossii is octoploid throughout its range. Relationships Solidago vossii belongs to Solidago sect. Ptarmicoidei because of its corymboid inflorescence, its rosette of large basal leaves with marcescent petiole

Page  112 ï~~112 THE MICHIGAN BOTANIST Vol. 49 112 THE MICHIGAN BOTANIST Vol. 49 FIGURE 4. A. Habitat of Solidago vossii along the northeast shore of Howes Lake during peak flowering. B. Habitat at Portage Creek during late fall showing numerous seed heads. C. Solidago vossii growing in the gravel at the edge of Arrowhead Road. (Photos by P. J. Laureto) C.

Page  113 ï~~2010 THE MICHIGAN BOTANIST 113 2010 THE MICHIGAN BOTANIST 113 FIGURE 5. Micrograph of Solidago vossii root tip cell at metaphase showing 2n = 8x = 72 chromosomes. bases, and its oligoneurate phyllaries (Semple and Gandhi 2004). Morphologically, the closest species to S. vossii is S. houghtonii. Both species have a corymboid inflorescence of large, bright yellow heads, hairs in the inflorescence, a basal rosette of strap-shaped leaves arising from a branched caudex, and a similar 3-nerved pattern of leaf venation. However, S. vossii can be easily distinguished from S. houghtonii. Solidago vossii is generally a larger plant. Its ray florets are much wider than those of S. houghtonii, its distal stem and the branches and pedicles of its inflorescence have many more hairs than those of S. houghtonii, and most notably, its basal leaves have gland-tipped teeth, which are lacking in S. houghtonii. Solidago vossii and S. houghtonii are compared morphologically in Table 1. Laureto's (2010; Laureto and Barkman, in press) phylogenetic study on the hybrid origin of Solidago houghtonii found that the octoploid population, recognized here as the new species S. vossii, shares the same parental taxa as true S. houghtonii. This suggests that the octoploid S. vossii may have been derived from the hexaploid S. houghtonii; although diploid x polyploid crosses are highly unlikely (J. Semple pers. comm.). Alternatively, S. vossii may have arisen through independent hybridization events involving the same parental taxa as S. houghtonii. While Solidago vossii is morphologically similar to and shares the same parental taxa as S. houghtonii its octoploid genome size would prevent it from

Page  114 ï~~114 THE MICHIGAN BOTANIST Vol. 49 114 THE MICHIGAN BOTANIST Vol. 49 20 Soidago vossn (G1yc w mx A. Fluorescence I Fluorescence FIGURE 6. Integrated fluorescence histograms of nuclei isolated during FCM analysis. A. The octoploid Solidago vossii (sample 01 from Howes Lake) and B. a diploid Solidago species (S. hispida). Each were isolated, stained and analyzed simultaneously with the internal reference standard Glycine max 'Polanka' (2C = 2.50 pg DNA). The ratio of the Glycine max peak means that S. vossii was equal to 3.104 and hence the 2C DNA amount of S. vossii was estimated as 7.759 pg indicating the plant is octoploid. For comparison, the ratio of the Glycine max peak for the diploid S. hispida was 0.834 indicating a 2C DNA amount of 2.086 pg. Note that the y-axis scale varies between samples. Fluorescence was measured with a 585/42 (FL2) photodetector. Note that the y-axis scale varies between samples (A. 0-120, B. 0-90). interbreeding with the hexaploid S. houghtonii. This supports its recognition as a new species. An unusual characteristic of S. vossii plants, observed in the field by Pringle in 1972 and by Laureto in 2002 and in 2010, is that the plants seem regularly to be infected with a leaf fungus (Fig. 3). The fungus forms blackish, circular spots 4-10 mm in diameter. Neither Laureto nor Pringle have observed leaf fungi on any plants of S. houghtonii. These leaf fungi may serve as an unusual diagnostic character for S. vossii. Distribution and Habitat This new species is endemic and apparently rare in the state of Michigan, U.S.A. Its primary distribution is an area of approximately 2.25 square miles in southern Crawford and Kalkaska counties (Fig. 1). All occurrences are located on State land within the Hanson State Game Refuge and the Camp Grayling Military Reservation, a National Guard training facility that is managed by the Michigan Department of Military Affairs. In addition to the primary distribution, there is one small population located along the entrance road to Camp Grayling on the east side of Lake Margrethe. As noted above, the first collection of this new species was made in 1933 at Howes Lake (the type locality). According to our field observations and reports prepared for the Camp Grayling Military Reservation (Higman et al. 1994), the plants occur in abundance from the northeast shore of Howes Lake (Fig. 1; Fig. 4a) southwest to Portage Creek (Fig. 1; Fig. 4b). They can even be found grow

Page  115 ï~~2010 THE MICHIGAN BOTANIST 115 TABLE 2. Assessment of the ploidy level of 23 Solidago vossii individuals using the nuclear DNA content (pg/2C) determined through flow cytometry. All S. vossii samples have a coefficient of variation (C.V.) value less than 7 indicating a relatively small and acceptable variation between the florescence peaks of the sample as compared to the internal standard. The average S. hispida nuclear DNA content is provided for comparison (Jess Peirson, unpublished data). Species Solidago vossii Population/Sample Arrowhead Road 01 Arrowhead Road 02 Arrowhead Road 03 Arrowhead Road 04 Arrowhead Road 05 Arrowhead Road 06 Arrowhead Road 07 Arrowhead Road 08 Arrowhead Road 09 Arrowhead Road 10 Howes Lake 01 Portage Creek 01 Portage Creek 02 Portage Creek 03 Portage Creek 04 Portage Creek 05 Portage Creek 06 Portage Creek 07 Portage Creek 08 Portage Creek 09 Portage Creek 10 Portage Creek 11 Portage Creek 12 DNA pg / 2C 7.601 7.452 7.497 7.566 7.512 7.462 7.334 7.474 7.665 7.401 7.759 7.421 7.548 7.627 7.383 7.466 7.421 7.581 7.426 7.343 7.532 7.431 7.657 AVG = 2.086 S. vossii C.V. 2.1 2.6 2.7 3.4 2.1 3.1 3.1 2.8 3.1 2.7 2.5 2.2 2.6 2.3 2.2 2 2.6 2.2 2.7 3 3.1 2.9 3 Interpretation of ploidy level 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x 8x Solidago hispida 2x ing in the gravel along the edges of roads that wind through the area (Fig. 4c). The primary site is about 5.5 km (3 miles) long and 1.2 km (0.75 miles) wide. Higman et al. 1994 described this site as a sandy glacial outwash plain with low dune ridges. The dune ridges represent the former shorelines of postglacial Lake Margrethe. The vegetation type of this new species' habitat is fen to northern wet prairie within a jack pine barrens/wet prairie complex (Higman et al. 1994). Here the plants occur in the alkaline soils of intermittent wetlands and old interdunal depressions along with several other species that are characteristic of Great Lakes interdunal wetlands. In addition, there are many species characteristic of mesic sand prairies. Plant associates include such species as Pinus banksiana (jack pine), Andropogon gerardii (big bluestem), Hypericum kalmianum (Kalm's St. Johnswort), Potentilla fruiticosa (shrubby cinquefoil), Prunus pumila (sand cherry), Lobelia spicata (lobelia), Castilleja coccinea (Indian paintbrush), Houstonia longifolia (long-leaved bluets), Eleocharis elliptica (spikerush), and Carex conoidea and C. flava (sedges). Several other rare plant species are located within this unique wet prairie complex including the special concern Trichophorum clintonii (Clinton's bulrush) and Sporobolus heterolepis

Page  116 ï~~116 THE MICHIGAN BOTANIST Vol. 49 (prairie dropseed), and the state threatened Juncus vaseyi (Vasey's rush), and Viola novae-angliae (New England violet) (USFWS 1997). Conservation Status Like many rare plants, Solidago vossii is vulnerable to extirpation because of its small population size; all but a few individuals occur within a 2.25 square mile area. The population is further at risk due to disturbance from frequent illegal off-road-vehicle (ORV) use (personal observation; USFWS 1997). Currently the Department of Military Affair's environmental staff is managing the Federal and State listed species within the Camp Grayling Reserve (Larry Jacobs, Michigan Department of Military Affair's, pers. comm. 2010). Barricades have been erected in many areas to prevent ORV traffic and other areas have signage warning of protected species. Recognizing that the plants from Crawford and Kalkaska counties are not Solidago houghtonii affects the population in a substantial way; it loses its federal protected status as part of a threatened species. Identifying these plants as a new species means that they are even rarer than S. houghtonii, and we recommend them for consideration for future federal listing and encourage the State of Michigan to award them immediate state protection. ACKNOWLEDGMENTS The authors wish to thank Jess Peirson for help in obtaining the FCM data and for sharing the nuclear DNA content of Solidago hispida. We also thank Mike Penskar for helpful discussions, for making available the reports, data, and EO records of the Michigan Natural Features Inventory, and for making contacts with the MI DNR and Camp Grayling personnel on our behalf. We are grateful to Anton Reznicek for helpful discussions during the preparation of this manuscript. And lastly, we express our sincere gratitude to Todd Barkman, Mike Penskar, and John Semple for reviewing this manuscript and providing constructive recommendations. LITERATURE CITED Benbennick, D. (2007). Map of Michigan highlighting Crawford County. http://familypedia.wikia. com/wiki/File:Map-ofMichiganhighlightingCrawfordCounty.svg. Accessed 11/30/2010. Dolezel, J., M. Dolezelova, and F. J. Novak. (1994). Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata and M. balbisiana). Biologia Plantarum 36: 351-357. Dolezel, J., J. Greilhuber, and J. Suda. (2007). Estimation of nuclear DNA content in plants using flow cytometry. Nature Protocols 2: 2233-2244. Greilhuber, J., E. M. Temsch, and J. C. M. Loureiro. (2007). Nuclear DNA Content Measurement. Pp. 67-101 in: Doleful, J., J. Greilhuber, and J. Suda, editors. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Wiley-VCH, Weinheim, Germany. Guire, K. E. and E. G. Voss. (1963). Distribution of distinctive shoreline plants in the Great Lakes region. The Michigan Botanist 2: 99-114. Higman, P. J., P. J. Comer, M. R. Penskar, M. L. Rabe, D. A. Albert, J. T. Legge, T. R. Leibfried, L. J. Scrimger, and M. B. Austin. (1994). Final Report for a Floristic and Natural Features Inventory of Camp Grayling Military Reservation, Grayling, Michigan. Michigan Natural Features Inventory, Lansing, MI. 112 pp. + appendices and maps. Hill, L. M. Step-by-step instructions for staining and studying plant chromosomes. http://people. bridgewater.edu/~1hill/chromtechnique.htm. Accessed 3/20/2009. Kron, P., J. Suda, and B. C. Husband. (2007). Applications of flow cytometry to evolutionary and population biology. Annual Review of Ecology, Evolution, and Systematics. 38: 847-876.

Page  117 ï~~2010 THE MICHIGAN BOTANIST 117 Kron, P. and B. C. Husband. (2009). Hybridization and the reproductive pathways mediating gene flow between native Malus coronaria and domestic apple, M. domestica. Botany 87:864-74. Laureto, P. J. (2010). The allopolyploid origin and population genetics of the rare Solidago houghtonii (Asteraceae). Ph.D. dissertation, Kalamazoo, Michigan, U.S.A.: Western Michigan University. Laureto, P. J. and T. J. Barkman. (In press). Nuclear and chloroplast DNA suggest a complex single origin for the threatened allopolyploid Solidago houghtonii Torrey & A. Gray ex Gray (Asteraceae) involving reticulate evolution and introgression. Systematic Botany. Semple, J. C. and R. E. Cook. (2006). Solidago. Pp. 107-166 in: Flora of North America Editorial Committee, editors. Flora of North America North of Mexico, vol.20, Asteraceae. Part 2. Astereae and Senecioneae. Oxford University Press, New York, NY. Semple, J. C. and K. N. Gandhi. (2004). Solidago sect. Ptarmicoidei, a new combination to replace a "rankless" name used by Torrey and A. Gray (Asteraceae: Astereae). Sida 21: 755-757. Semple, J. C., G. S. Ringius, C. Leeder, and G. Morton. (1984). Chromosome numbers of goldenrods, Euthamia and Solidago (Compositae: Astereae). II. Additional counts with comments on cytogeography. Brittonia 36: 280-292. Suda, J., P. Kron, B. C. Husband, and P. Trivni ek. (2007). Flow cytometry and ploidy: applications in plant systematics, ecology and evolutionary biology. Pp. 103-130 in: Doleful, J., J. Greilhuber, and J. Suda, editors. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Wiley-VCH, Weinheim, Germany. Unites State Census Bureau. (2010). http.//factfinder.census.gov/home/saff/main.html? _Lang=en. Accessed 11/30/2010 United States Fish and Wildlife Service (USFWS). (1985a). Endangered and threatened wildlife and plants; determination of threatened status for Solidago spithamaea (Blue Ridge goldenrod). Federal Register 50(60): 12306-12309. United States Fish and Wildlife Service (USFWS). (1985b). Endangered and threatened wildlife and plants; determination of threatened status for Solidago shortii (Short's goldenrod). Federal Register 50(172): 36085-36089. United States Fish and Wildlife Service (USFWS). (1988a). Endangered and threatened wildlife and plants; determination of threatened status for Solidago albopilosa (white-haired goldenrod). Federal Register 53(67): 11612-11615. United States Fish and Wildlife Service (USFWS). (1988b). Endangered and threatened wildlife and plants; determination of threatened status for Solidago houghtonii (Houghton's goldenrod). Federal Register 53(137): 27134-27137. United States Fish and Wildlife Service (USFWS). (1997). Recovery Plan for Houghton's Goldenrod (Solidago houghtonii A. Gray). Fort Snelling, MN. Vii + 58pp. Voss, E. G. (1972, 1985, 1996). Michigan Flora: A guide to the Identification and Occurrence of the Native and Naturalized Seed-Plants of the State. Part I: Gymnosperms and Monocots (1972); Part II: Dicots (Saururaceae-Cornaceae) (1985); Part III: Dicots (Pyrolaceae-Compositae) (1996). University of Michigan Herbarium, Ann Arbor, MI; Cranbrook Institute of Science Bulletins 55, 59, 61.