TAXON 61 (1) • February 2012: 197–210 Peirson & al. • Cytogeography of Solidago subsect. Humiles

Polyploidy, infraspecific cytotype variation, and speciation in Goldenrods: The cytogeography of Solidago subsect. Humiles (Asteraceae) in North America

Jess A. Peirson,1,2 Anton A. Reznicek2 & John C. Semple3

1 Department of Ecology and Evolutionary Biology, The University of Michigan, Ann Arbor, Michigan 48109-1048, U.S.A. 2 The University of Michigan Herbarium, 3600 Varsity Drive, Ann Arbor, Michigan 48108-2228, U.S.A. 3 Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada Author for correspondence: Jess A. Peirson, [email protected]

Abstract Polyploidy is an important evolutionary mechanism in plants, and in some genera (e.g., Solidago in Asteraceae) it is particularly widespread and is hypothesized to have played a major role in diversification. Goldenrods are notorious for their ploidy variation, with roughly 14% and 32% of recognized North American species being polyploid or including multiple cytotypes, respectively. We used traditional chromosome counts and flow cytometry to examine cytogeographic patterns, biogeographic and evolutionary hypotheses, and species boundaries in S. subsect. Humiles. Chromosome numbers and DNA ploidy determinations are reported for 337 individuals, including 148 new reports. Cytotypes show significant geographic struc- turing. Solidago simplex and S. spathulata were uniformly diploid (2n = 18) in western North America, while cytogeographic patterns in eastern North America were regionally complex and included 2n, 4n, and 6n cytotypes. Cytotypes within S. simplex were ecogeographically segregated and mixed-ploidy populations were rare. Data from this study and additional biosystematic data indicate that cytotypes in S. simplex fulfill the requirements of multiple species concepts and should best be treated as distinct species. Polyploid cytotypes possibly formed recurrently, however, and evolution and species boundaries within poly- ploid S. simplex will require additional study. Results from this study and accumulated data from other studies suggest that biological species diversity in Solidago is considerably higher than currently recognized taxonomically.

Keywords cytogeography; flow cytometry; genome size; North America; polyploidy; Solidago

Supplementary Material Tables S1 and S2 are available in the free Electronic Supplement to the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

Introduction ploidy data suggest that 30%–70% of flowering plants have some incidence of lineage-specific polyploidy in their histo- Polyploidy has long been recognized as an important ries (Stebbins, 1950; Grant, 1981; Masterson, 1994). The most- mechanism in plant evolution (Muntzing, 1936; Stebbins, conservative models have estimated that between 2%–4% of 1971; Lewis, 1980). Results from early field and greenhouse speciation events in angiosperms have involved a change in studies demonstrated that polyploid plant species were often ploidy (Otto & Whitton, 2000; Coyne & Orr, 2004). Wood & al. ecologically distinct from their diploid relatives and often oc- (2009) suggest, however, that the actual incidence of polyploid cupied at least partially separate geographic ranges (Muntzing, speciation in flowering plants may be closer to 15%, represent- 1936; Stebbins, 1942; Löve & Löve, 1943; Clausen & al., 1945; ing a four-fold increase over the earlier estimates. Wood & al. Haskell, 1951). Yet while our knowledge of the prevalence of also found that approximately 12%–13% of angiosperm species polyploidy and the molecular genetics of polyploid function harbored some level of infraspecific ploidy variation (i.e., they have greatly expanded over the last century (Ehrendorfer, 1980; comprised multiple ploidy races). Lewis, 1980; Soltis & Soltis, 2000; Wendel, 2000; Soltis & al., Soltis & al. (2007) argued that while infraspecific ploidy 2004; Parisod & al., 2010), our understanding of the interplay variation in many groups has been ignored taxonomically, of polyploidy and ecology and evolution remains considerably chromosomal races within many species actually fulfill the more limited (Soltis & al., 2010). Despite these limitations, criteria of multiple species concepts. This widespread inclu- polyploidization is often regarded as a major driver of plant sion of multiple chromosomal races within broadly defined speciation and as probably the prime example of how sympatric species has the potential to mask significant amounts of speciation in plants can occur because it can confer instanta- biological species diversity and to obscure our understand- neous reproductive isolation (Coyne & Orr, 2004; Rieseberg ing of evolution and speciation in many groups (Soltis & al., & Willis, 2007; Soltis & al., 2007). 2007). Concerns have also been raised that unrecognized in- Genomic data suggest that one or more whole-genome fraspecific ploidy variation could pose major hurdles to the duplications occurred early in the evolution of the angiosperms conservation of rare species and to ecological restoration ef- (reviewed by Soltis & al., 2009), and broad-scale surveys of forts (Soltis & al., 2007; Severns & Liston, 2008). Soltis & al.

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(2007) surmised that the reluctance of botanists to recognize the cytogeography of S. simplex within the region had not been infraspecific chromosomal races as distinct entities was due adequately characterized. in part to a long botanical tradition of including multiple cyto­ This study uses chromosome counts and flow cytometry types in single species and to the practicality of relying on a data in Solidago subsect. Humiles to address the following largely phenetic species concept. This in turn raises questions main questions: (1) With our increased taxon and population for genera with substantial amounts of taxonomically unrec- sampling, what are the distributions of cytotypes in Solidago ognized cytotypic variation. How is this cytotypic variation subsect. Humiles across its North American range, and how are partitioned within species complexes? Are cytotypes ecologi- those cytotype distributions different from the ones presented cally, geographically, or morphologically distinct? Is it likely in Ringius (1986) and Ringius & Semple (1987)? (2) What does that polyploid cytotypes formed recurrently? Are cytotypes our expanded knowledge of cytogeographic patterns tell us largely reproductively isolated? In what cases should infraspe- about the biogeographic and evolutionary history of Solidago cific cytotypes be recognized as good biological species? How subsect. Humiles (e.g., the Holocene biogeography of S. sim- significantly have we underestimated the amount of biological plex and single vs. recurrent origin of polyploidy)? (3) Using a species diversity in these groups? framework similar to that described by Soltis & al. (2007), what In some groups in particular (e.g., genera like Packera do cytogeographic patterns and additional biosystematic data Á. Löve & D. Löve, Solidago L., and Symphyotrichum Nees tell us about potential species boundaries between cytotypes in Asteraceae), polyploidy and infraspecific ploidy variation of S. simplex, and how does this compare to patterns found in are abundant and appear to have played important roles in other Solidago species complexes with similar ploidy variation. diversification and speciation (Brouillet & al., 2006; Semple & Cook, 2006; Trock, 2006; Semple & Watanabe, 2009). Goldenrods (Solidago spp.) have long been notorious for their Materials and methods complex patterns of morphological and ploidy variation. The genus contains approximately 100 recognized species, with Study group. — Solidago subsect. Humiles is composed of 77 of those native to the United States and Canada (Semple five species: S. arenicola, S. kralii, S. plumosa, S. spathulata & Cook, 2006). Fourteen percent (11/77) of these species are DC., and S. simplex (following Semple & Cook, 2006). The strictly polyploid (i.e., they exist only at the tetraploid level or goldenrods in S. subsect. Humiles have resinous glands on the above). An additional 32% (25/77) of North American species foliage and involucral bracts that cause all members of the harbor some level of infraspecific cytotype variation. Cyto- group to be glutinous. In addition, all species in the subsection logical data extracted from Semple & Cook (2006) indicate have virgate to paniculiform arrays with non-secund capitula that chromosome numbers in the genus range from diploid (2n (Semple & Cook, 2006). = 18) to 14x (14n = 126). While these statistics indicate that Solidago subsect. Humiles is endemic to North America approximately 46% of Solidago species show some direct inci- and transcontinental in distribution. The broadly circumscribed dence of polyploidy in their histories, cytotypes have typically Solidago simplex is widespread and transcontinental in dis- been circumscribed at infraspecific rank or simply included tribution but absent from the center of the continent. Other in broadly defined taxa. species are much more restricted. Solidago arenicola, S. kralii, Solidago subsect. Humiles (Rydb.) Semple presents an in- and S. plumosa are narrowly distributed endemics in the south- teresting system in which to examine patterns of polyploidy and eastern United States (Fig. 1). Solidago arenicola is restricted infraspecific cytotype variation. The subsection is composed to rocky or sandy riverbanks and floodplains in the Cumber- of one widespread, taxonomically and cytologically complex land Plateau region of northern Alabama and Tennessee. Soli- species, Solidago simplex Kunth, and four narrowly endemic dago kralii is confined to sand hills along the Coastal Plain fall species. Solidago simplex was previously shown to be diploid line in a small area of Georgia and South Carolina. Solidago throughout its range in western North America but to include plumosa is known from a single population on mafic rocks diploid, tetraploid, and hexaploid populations in eastern North along the Yadkin River in Stanley Co., North Carolina. Soli- America. Ringius (1986) and Ringius & Semple (1987) pro- dago spathulata inhabits sand dunes along the Pacific coast posed that polyploid populations of S. simplex (treated as S. glu- from central California to northern Oregon (Fig. 1). tinosa Nutt. at the time) in eastern North America evolved from Ringius (1986) divided Solidago simplex into diploid S. sim- a single migration of diploid S. simplex from western North plex subsp. simplex (2n = 18) and polyploid S. simplex subsp. America and subsequent polyploidization. The recent descrip- randii (4n = 36, 6n = 54). Diploid subsp. simplex is widespread tion of two closely related species, the tetraploid S. arenicola yet patchily distributed in montane and alpine habitats through- Keener & Kral (2003) and diploid S. kralii Semple (2003), and out the western cordillera from Alaska to Mexico (Fig. 1; var. rediscovery of S. plumosa Small (ploidy unknown, thought to simplex and var. nana (A. Gray) Ringius). Disjunct, eastern have been extinct, A. Weakley pers. comm.) in the southeast- diploid populations in the northern Great Lakes region and ern United States, however, have raised questions concerning Gaspé, Quebec have also been placed in subsp. simplex (Fig. 1; cytogeographic patterns within the complex and the previ- var. simplex and var. chlorolepis (Fern.) Ringius, respectively). ous hypothesis of a single origin of polyploidy in S. subsect. Polyploid subsp. randii is restricted to the Great Lakes region Humiles. In addition, our inclusion of previously unstudied and Appalachian Mountains in eastern North America (Fig. 1), populations in the glaciated Great Lakes region showed that and four varieties are currently recognized in the subspecies.

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Solidago simplex subsp. randii var. racemosa (Greene) Ringius Field sampling. — Our sampling scheme filled in gaps inhabits rocky riverbanks throughout the Appalachian Moun- in coverage (taxonomic and geographic) in previously under- tains, from West Virginia to New Brunswick, while S. simplex represented regions (e.g., the southeastern United States) and subsp. randii var. monticola (Porter) Ringius is confined to greatly increased coverage in regions with potentially com- barrens and serpentine soils in New England and southern Que- plex cytogeographic patterns (e.g., the Great Lakes region). We bec. Solidago simplex subsp. randii var. ontarioensis (Ringius) sampled taxa from across their ranges in the Great Lakes region Ringius and S. simplex subsp. randii var. gillmanii (A. Gray) and the southeastern United States, with several additions from Ringius are endemic to the Great Lakes region, inhabiting eastern and western North America (Table 1). At each site, we rocky shores from the Bruce Peninsula, Ontario to southern harvested rhizome cuttings from 1–9 widely spaced individuals and eastern Lake Superior and active dune systems along the (clones spaced > 3 m apart). We then transplanted those cuttings shores of lakes Huron and Michigan, respectively. to Matthaei Botanical Gardens at the University of Michigan

60° S-S

50° S-S

S-S

40° S-R SP S-R

S-R

AR PL 30°

KR

>6 individuals 20° 3–5 individuals 1–2 individuals S-S diploid (2n = 18) 0 500 1000 km tetraploid (4n = 36) mixed (2n and 4n) hexaploid (6n = 54)

120° 110° 100° 90° 80° 70° Fig. 1. Distribution of cytotypes within Solidago simplex and the other species of Solidago subsect. Humiles in North America based on data from this study and from literature reports. Generalized distributions of each species are indicated by dashed lines except for S. plumosa, which is indicated by a single point. Taxa are labeled as follows: AR, S. arenicola; KR, S. kralii; PL, S. plumosa; S-R, S. simplex subsp. randii; S-S, S. simplex var. simplex; SP, S. spathulata. Symbols are scaled for sample sizes at each population. Mixed-ploidy population samples consisted of four to six diploid individuals with a single tetraploid individual (see text).

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Table 1. Locality information for 40 populations of Solidago subsect. Humiles sampled in this study. Nomenclature follows Semple & Cook (2006). Countries are designated as follows: Can, Canada; U.S.A., United States. Collector abbreviations are as follows: JP, J. Peirson; S, J. Semple; S&S, J. Semple & B. Semple; Voss, E.G. Voss; Hr&St, Hrusa & G.L. Stebbins. JP and Voss vouchers are deposited at MICH; S, S&S, and Hr&St vouchers are deposited at WAT. The majority of ploidy determinations (2n, 4n, 6n) were inferred from flow cytometry analysis. Direct chromosome counts are indicated by an asterisk (*). No. individuals Country State County Lat. Long. 2n 4n 6n Voucher(s) Solidago arenicola Locust Creek at Rte 231 U.S.A. Alabama Blount 34.02 −86.57 – 2 – JP 608 Swann Bridge U.S.A. Alabama Blount 34.00 −86.60 – 1+1* – JP 609, S&S 11196 Lily Bridge U.S.A. Tennessee Morgan 36.10 −84.72 – 4 – JP 610

Solidago kralii Vaucluse U.S.A. South Carolina Aiken 33.61 −81.82 1 –– JP 605 I-20 north of Graniteville U.S.A. South Carolina Aiken 33.62 −81.83 1* – – S&S 11218 Bowens Mill 1 U.S.A. Georgia Ben Hill 31.84 −83.21 1* – – S&S 11212 Bowens Mill 2 U.S.A. Georgia Ben Hill 31.84 −83.21 1* – – S&S 11216-B Hartford U.S.A. Georgia Pulaski 32.25 −83.40 1* – – S&S 11208

Solidago plumosa Yadkin River U.S.A. North Carolina Stanley 35.41 −80.09 3 – – JP 604

Solidago simplex subsp. randii var. gillmanii West of Detour Village U.S.A. Michigan Chippewa 45.97 −84.06 – 7 – Voss 16893 West of Manistique U.S.A. Michigan Schoolcraft 45.91 −86.32 – 8 – JP 590 Silver Lake State Park U.S.A. Michigan Oceana 43.65 −86.54 – 2 – JP 595 Wilderness State Park at Sturgeon Bay U.S.A. Michigan Emmet 45.71 −84.95 – 7 – JP 531 Thompson’s Harbor State Park U.S.A. Michigan Presque Isle 45.35 −83.57 – 5 – JP 789 Warren Dunes State Park U.S.A. Michigan Berrien 41.91 −86.60 – 4 – JP 517

Solidago simplex subsp. randii var. monticola Falls of Lana U.S.A. Vermont Addison 43.90 −73.06 – 2 – JP 581

Solidago simplex subsp. randii var. ontarioensis West of Sault St. Marie, Gros Cap Can Ontario Algoma 46.53 −84.59 – 1* – S 11086 Tobermory, Big Tub Lighthouse Can Ontario Bruce 45.26 −81.67 – 5 – JP 475 Georgian Bay Can Ontario Bruce 45.25 −81.52 – 4 – JP 560 Fort Wilkins State Park U.S.A. Michigan Keweenaw 47.47 −87.86 – 5 – JP 625 Tobermory, Elgin Street Can Ontario Bruce 45.26 −81.64 – 6 – JP 562 Government Dock Can Ontario Algoma 47.94 −84.85 – 6 – JP 557 South of Tobermory, Hay Bay Road Can Ontario Bruce 45.24 −81.68 – 6 – JP 563 Sandy Beach Can Ontario Algoma 47.96 −84.86 – 1 – JP 555 Seul Choix Point U.S.A. Michigan Schoolcraft 45.92 −85.91 – 9 – JP 467

Solidago simplex subsp. randii var. racemosa Middle Fork River at Audra U.S.A. West Virginia Barbour 39.04 −80.07 – 4 – JP 598 Carnifex Ferry U.S.A. West Virginia Nicholas 38.21 −80.94 – 3 – JP 603

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Table 1. Continued. No. individuals Country State County Lat. Long. 2n 4n 6n Voucher(s) Holton Dam U.S.A. Pennsylvania York 39.81 −76.33 – 4 – JP 585 Valley Falls U.S.A. West Virginia Marion 39.39 −80.09 – – 5 JP 597

Solidago simplex subsp. simplex var. simplex Rte 612 and Deward Road U.S.A. Michigan Kalkaska 44.77 −84.85 3 – – JP 464 I-75 south of Gaylord U.S.A. Michigan Otsego 44.97 −84.67 4 – – JP 647 Fletcher Road U.S.A. Michigan Kalkaska 44.57 −85.06 1 –– JP 541 Big Creek Road U.S.A. Michigan Oscoda 44.67 −84.28 3 – – JP 542 North of St. Helena U.S.A. Michigan Roscommon 44.40 −84.41 4 1 – JP 463 Rte 612 and I-75 U.S.A. Michigan Crawford 44.78 −84.72 7 –– JP 538 Staley Lake Road U.S.A. Michigan Crawford 44.65 −84.64 4 1 – JP 535 Nahanni National Park Reserve Can NW Territories – 61.42 −126.84 1* – – S 11156 Terrace Bay Can Ontario Thunder Bay 48.77 −87.11 5 1 – JP 550

Solidago spathulata Gearhart/Seaside U.S.A. Oregon Clatsop 46.02 −123.93 2 – – JP 636 Cavedale Rd east of Hwy-12 U.S.A. California Sonoma 38.38 −122.46 1* – – Hr&St 11428

or to the University of Waterloo North Campus Greenhouses. removed the supernatant. We then resuspended the pellet in 50 For each population, we deposited a single population voucher µg/ml propidium iodide and incubated it at room temperature in MICH or WAT. We potted rhizome/rootstock cuttings in for 20–45 minutes. We ran samples on a BD FACSCalibur standard potting soil and watered them weekly. flow cytometer in the Department of Integrative Biology at Chromosome number and DNA ploidy determination. the University of Guelph. — We made meiotic counts from pollen mother cells dis- We analyzed most samples (128/140) using Modfit sected from field-prepared buds fixed in acetic ethanol (3 : 1/ v.3.0 software (Verity Software) to estimate peak means, co- EtOH : glacial acetic acid) and mitotic counts from root tip efficients of variation (CV), and nuclei number. For twelve preparations following protocols outlined in Semple & Cook samples in which the Solidago peak was very close to the Gly- (2004). We prepared permanent slides for some samples fol- cine max peak, we used CellQuest Pro v.4.0 software, manually lowing protocols in Semple & al. (1981). gating peaks. We calculated DNA content as: We determined DNA ploidy (sensu Hiddeman & al., 1984) Solidago mean using flow cytometry after calibrating the relative DNA content DNA Content = 2.5 × (from flow cytometry) with previously determined chromo- Glycine max mean some numbers from a subset of populations in the study. We used at least one calibration/standardization for each recovered where 2.5 equals the standardized mean genome size of Gly- DNA ploidy level (2x, 4x, 6x). Other studies have used similar cine max (in pg/2c) and the other mean values represent the methodologies for a number of plant species, including two experimentally determined values for each sample. other species of Solidago (Halverson & al., 2008; Schlaepfer Literature review and mapping. — We compiled pub- & al., 2008a). lished chromosome counts through literature searches and We harvested fresh Solidago leaf material from green- through cross-referencing with Ringius & Semple (1987). We house-grown plants and stored it in cool conditions for up to listed population and cytovoucher data for literature reports one week. For each sample, we chopped approximately one accepted in this study in Table S1 (Electronic Supplement). At half of a young leaf in 0.8 ml ice-cold LB01 buffer (Dolezel least one of the authors examined cytovouchers for nearly all & al., 1989) with 50 µg/ml propidium iodide and added 50 µg/ literature reports to confirm species determinations. We geore- ml RNAse. We used an approximately equal amount of fresh ferenced populations and pooled the literature counts with data leaf from Glycine max (L.) Merr. ‘Polanka’ as an internal DNA from this study to create cytogeographic maps representing content standard (2.5 pg/2c; cited in Dolezel & al., 1994, 2007). all taxa in Solidago subsect. Humiles. We considered reports After chopping, we filtered each sample through a 30 µm filter from the same locality (populations < 1 km apart when geore- into a microcentrifuge tube. We centrifuged each sample and ferenced) as intrapopulation samples for mapping purposes.

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Results diploid except for one tetraploid count of S. simplex var. simplex from the Yukon Territory, Canada (see below). Patterns in east- Chromosome counts, flow cytometry, and literature re- ern North America were more complex at a regional level and ports. — Chromosome numbers and DNA ploidy determina- included diploid, tetraploid, and hexaploid populations. In gen- tions are reported for five species and 337 individuals, includ- eral, however, cytotypes in eastern North America had region- ing 148 new reports (Table 2). This compares to two species ally allopatric distributions. A single West Virginia population and ca. 130 reports in Ringius & Semple (1987). Of the 148 of S. simplex var. racemosa was found to be hexaploid. Our new reports, 140 were DNA ploidy determinations from flow diploid DNA ploidy determination represents the first report cytometry and 8 were direct counts (Tables 1–2). Flow cytom- for S. plumosa Small and is consistent with an unpublished etry recovered three non-overlapping DNA ploidy groups that direct count (G. Nesom, pers. comm. to J. Semple). correspond to 2x, 4x, and 6x counts (Table 3; Fig. 2). These Although multiple ploidy levels were found in eastern data were consistent with literature reports and indicated that North America, almost no within-population variation was only three ploidy levels have been found in S. subsect. Humiles: observed. Three mixed-ploidy populations were discovered diploid (2n = 18), tetraploid (4n = 36), and hexaploid (6n = 54). in this study (mean number of intrapopulation samples = 5.33, No odd-ploidy individuals (e.g., triploid with 3n = 27 or penta- minimum = 5, maximum = 6). In each case, a single, cryp- ploid with 5n = 45) have been detected in S. subsect. Humiles. tic tetraploid individual was recovered from an otherwise Cytogeographic patterns. — Cytotypes within S. subsect. diploid population in the northern Great Lakes region. Be- Humiles show significant geographic and taxonomic structur- cause the number of individuals sampled per population was ing (Figs. 1, 3). All counts from western North America were small (5–6 individuals in the mixed-ploidy populations), our

Table 2. Summary of chromosome counts and DNA ploidy determinations for Solidago subsect. Humiles from this study and from literature reports. Rare exceptions to the general patterns within S. simplex are enclosed in parentheses. Hexa- ploid determinations from Valley Falls, West Virginia are indicated by an asterisk (*). Detailed information on study sites and literature reports is presented in Tables 1 and S1, respectively. Somatic No. of dets. No. of dets. Total no. Taxon chromosome no. DNA ploidy from literature this study of counts S. arenicola 2n = 36 4n 1 8 9 S. kralii 2n = 18 2n 2 5 7 S. plumosa 2n = 18 2n – 3 3 S. simplex 2n = 18, 36, 54 2n, 4n, 6n 174 129 303 subsp. randii 2n = 36 (54) 4n (6n) 74 (6) 89 (5) 163 (11) var. gillmanii 2n = 36 4n 15 33 48 var. monticola 2n = 36 4n 15 2 17 var. ontarioensis 2n = 36 4n 23 43 66 var. racemosa 2n = 36 (54*) 4n (6n*) 21 (6*) 11 (5*) 32 (11*) subsp. simplex 2n = 18 (36) 2n (4n) 93 (1) 32 (3) 125 (4) var. chlorolepis 2n = 18 – 7 – 7 var. nana 2n = 18 – 1 – 1 var. simplex 2n = 18 (36) 2n (4n) 85 (1) 32 (3) 117 (4) S. spathulata 2n = 18 2n 12 3 15 Totals 189 148 337

Table 3. Sample frequencies and relative DNA content as determined by flow cytometry analysis of fresh leaf tissue from species in Solidago subsect. Humiles. Populations with both 2n and 4n cytotypes were counted twice. Relative DNA content Ploidy level No. populations No. individuals Mean (± SD) Min. Max. Diploid 11 37 2.30 (0.06) 2.21 2.50 Tetraploid 24 98 4.36 (0.11) 4.07 4.58 Hexaploid 1 5 6.08 (0.08) 5.96 6.17

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ability to detect rare cytotypes at the population level was Discussion also low. Beaudry (1969) reported the only other mixed-ploidy counts for subsect. Humiles from Lac Laberge, Yukon Ter- Cytogeography of Solidago subsect. Humiles. — This ritory, Canada. His single tetraploid and four diploid counts, study confirmed many of the broad-scale patterns found by however, were all from seedlings germinated from a single previous cytogeographic work on S. simplex and S. spathu- maternal individual. lata (Ringius & Semple, 1987), but our increased taxon and Rejected reports. — Sixteen literature reports were re- population coverage provided greater resolution and revealed jected due to misidentifications (Table S2 in the Electronic additional patterns not previously documented (especially in Supplement). the Great Lakes region and the southeastern United States).

A Solidago simplex var. simplex B Solidago simplex var. ontarioensis C Solidago simplex var. racemosa 100 100 150 * 80 80 120 * 60 60 90

40 40 60 *

Number of nuclei

20 20 30

0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Relative fluorescence Relative fluorescence Relative fluorescence Fig. 2. Fluorescence histograms of stained nuclei isolated during flow cytometry analyses of fresh tissue of Solidago simplex and the internal standard (Glycine max ‘Polanka’). The Solidago peak is indicated with an asterisk. A, diploid S. simplex var. simplex from Oscoda County, Michi- gan; B, tetraploid S. simplex var. ontarioensis from Tobermory, Ontario; C, hexaploid S. simplex var. racemosa from Valley Falls, West Virginia.

Fig. 3. Distribution of cyto- types within Solidago simplex in the North American Great Lakes region: diploid S. simplex var. simplex (black circles); mixed diploid and tetraploid S. simplex var. simplex (black stars); tetraploid S. simplex var. gillmanii (shaded squares); tetraploid S. simplex var. ontari- oensis (shaded triangles). Mixed ploidy S. simplex var. simplex population samples consisted of five or six diploid individuals with a single, cryptic tetraploid individual (see text).

var. simplex (2n)

var. simplex (mixed 2n and 4n)

var. gillmanii (4n)

var. ontarioensis (4n) 0 250 500 km

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Patterns in western North America remain unchanged from var. simplex in northern Lower Michigan. These diploid popu- earlier studies; the subsection is known almost entirely at the lations were restricted to xeric, sandy soils in jack pine (Pinus diploid level throughout its range from Alaska and northern banksiana Lamb.) barrens. Zimmerman (1956) first noted the Canada south through the Rocky Mountains and along the occurrence of these inland populations, but neither Ringius Pacific coast. Disjunct populations in the mountains of north- (1986) nor Ringius & Semple (1987) included them in their ern Mexico are also presumed to be diploid but have not yet biosystematic and cytogeographic studies. Voss (1996) sur- been sampled for cytogeographic work. Cytogeographic pat- mised that these plants might be allied with western North terns in eastern North America from Maryland north and east American S. simplex var. simplex based on their rather numer- through New England, Quebec, and New Brunswick remain ous, small capitula (ca. 4–5 mm) but was unsure of their ploidy. essentially unchanged from earlier studies. Solidago simplex Morphologically, plants in these populations are strikingly sim- var. monticola and S. simplex var. racemosa are tetraploid ilar to diploid S. simplex populations from Wyoming, Montana, throughout their scattered, disjunct ranges in this region. A and southwestern Canada. In Michigan, these goldenrods occur recent literature report confirmed the highly restricted dis- in pine barren communities with several other disjunct western tribution of diploid S. simplex var. chlorolepis on serpentine North American species (e.g., Agoseris glauca (Pursh) D. Dietr. outcrops in Gaspé, Quebec (Gervais & al., 1999). Patterns in and Festuca altaica Trin.), which are also absent from similar the Great Lakes region, the southern Appalachians, and the pine barren communities in Michigan’s Upper Peninsula and southeastern United States, however, are considerably more northern Wisconsin (Johnston, 1958; Mustard, 1982). complex than previously shown. Previous cytogeographic studies did not examine popula- In the Great Lakes region, cytogeographic patterns in S. sim- tions of Solidago simplex from the southeastern United States plex var. gillmanii and S. simplex var. ontarioensis broadly (south and west of Maryland) or include any of the currently conformed to patterns found in previous studies (Fig. 3). Tetra- recognized southeastern endemic species. Our inclusion ploid S. simplex var. gillmanii is restricted to active coastal sand of S. arenicola, S. kralii, S. plumosa, and southern popula- dunes along the shores of Lake Michigan and Lake Huron (the tions of S. simplex var. racemosa in West Virginia greatly in- other varieties of S. simplex do not occur in this habitat type). creased our understanding of cytogeographic patterns in the Tetraploid S. simplex var. ontarioensis is restricted to limestone/ region. Both S. kralii and S. plumosa are diploid throughout dolomite shorelines and outcrops along the Niagara Escarpment their highly restricted ranges and represent the only diploid in Ontario and Michigan and to granite/basalt outcrops along the members of S. subsect. Humiles in the southeastern United eastern shore of Lake Superior and the Keweenaw Peninsula. States. Solidago arenicola is tetraploid throughout its scat- Observation of plants in the common garden suggests that S. sim- tered, disjunct range along river systems of the Cumberland plex var. ontarioensis is composed of two phenotypically distinct Plateau in Alabama and Tennessee. The Tennessee plants were sets of populations (Peirson, 2010). Larger-statured plants are only tentatively included in S. arenicola by Semple & Cook restricted to dolomite shores along the boundary of the Niagara (2006), but their wide-spatulate rosette leaves, few-headed Escarpment, while smaller-statured plants occur predominantly arrays with large capitula (ca. 8–12 mm), and occurrence in on the granite/basalt outcrops around Lake Superior. This pheno­ deep sandy alluvium along river floodplains clearly unite them typic differentiation in S. simplex var. ontarioensis raises the with S. arenicola from northern Alabama. possibility that there are two lineages of tetraploid rock outcrop Prior to this study, the ploidy of disjunct, southern popula- plants in the Great Lakes region. tions of S. simplex var. racemosa in the Appalachian Mountains Solidago simplex var. simplex is composed of two allopat- of West Virginia was unclear. A set of hexaploid counts from ric, ecologically distinct sets of populations in the Great Lakes Valley Falls, West Virginia in the 1950s (Beaudry, 1963) raised region, a distribution that is considerably different from what the possibility that robust, large-headed plants along the New earlier studies proposed (Fig. 3). While we confirmed previous and Gauley Rivers and their tributaries represented a distinct reports of diploid S. simplex var. simplex along the northern group of hexaploid populations. We examined three popula- shore of Lake Superior (east to Terrace Bay, Ontario), our re- tions of S. simplex var. racemosa from West Virginia, including sults indicated that it does not extend southeastward to the east- the Valley Falls population. All chromosome counts and flow ern shore of Lake Superior or to the Bruce Peninsula of Lake cytometry determinations from Valley Falls were hexaploid. Huron. Ringius & Semple (1987) concluded that diploid and Ploidy determinations from the other West Virginia populations tetraploid S. simplex occurred sympatrically in the Great Lakes indicated that plants in those populations were tetraploid. These region because diploid counts from the eastern shore of Lake results indicate that the variety is likely tetraploid throughout Superior and from Lake Huron had previously been assigned most of the Appalachian Mountains; the Valley Falls site re- to S. simplex var. simplex (Morton, 1981; Semple & al., 1981; mains the only location where hexaploid individuals of S. sub- Ringius & Semple, 1987). Examination of cytovouchers from sect. Humiles have been recorded. these studies and plants transplanted to the Matthaei Botanical Biogeography and evolution in Solidago simplex. — The Gardens for this study, however, indicated that these diploid, complex cytogeographic patterns revealed by this study and rock outcrop plants are not S. simplex. They appear most similar much recent work on the evolution of polyploid plant species to the sand dune endemic S. hispida var. huronenis Semple. suggest that the original biogeographic and evolutionary hypoth- In addition to the Lake Superior shoreline populations, we eses proposed by Ringius (1986) and Ringius & Semple (1987) documented disjunct inland occurrences of diploid S. simplex for S. simplex were likely too simple. Their cytogeographic work

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identified disjunct occurrences of diploid S. simplex subsp. sim- this study were in the recently glaciated Great Lakes region. plex at the northern extreme of the distribution in eastern North This raises the potential that the endemic polyploid varieties America. They hypothesized that these rare diploids represented of S. simplex in the Great Lakes region had postglacial origins either relicts from a previously more widespread distribution from diploids in the region. (e.g., var. chlorolepis in Gaspé, Quebec) or postglacial migrants The probable recurrent evolution of higher ploidy lineages from an Alaskan-Beringian refugium (e.g., var. simplex along in S. simplex is consistent with conclusions from other golden- the northern shore of Lake Superior). Our increased sampling rod species. Schlaepfer & al. (2008b) determined that multiple in the Great Lakes region revealed a third, more southerly, polyploidization events were significantly more likely than a disjunct group of diploid populations. Do these populations single origin of tetraploids in S. gigantea. They inferred as represent an additional migration of diploid S. simplex from many as seven independent origins in eastern North America. western North America? Or do all three disjunct occurrences Similarly, Halverson & al. (2008) concluded that the evolution of diploid S. simplex represent remnants of a previously more of polyploidy in S. altissima was complex, and they rejected widespread distribution in northeastern North America? The the hypothesis that polyploid cytotypes had a single, ancient occurrence of diploids throughout glaciated western Canada origin. Together, these results from Solidago are consistent and at the northern extreme of the distribution in eastern North with numerous molecular phylogenetic studies that have dem- America runs counter to the long-standing hypothesis that poly- onstrated that the recurrent formation of polyploid lineages is ploid plants were better colonizers than diploids following the the norm in many groups (Segraves & al., 1999; Soltis & Soltis, last glaciation (Brochmann & al., 2004; Ehrendorfer, 1980). 1999; Soltis & al., 2004; Rebernig & al., 2010; Symonds & al., In addition to diploids at the northern extreme of the dis- 2010; Artyukova & al., 2011). tribution, we also documented diploids at the southern extreme Cytotype variation and species boundaries. — Cytogeo- of the distribution in the southeastern United States (S. kralii graphic patterns in Solidago simplex reveal a widely distrib- and S. plumosa). This is significant because diploid S. plumosa uted species composed of two cytologically distinct, essentially is ecologically and morphologically similar to populations of allo­patric subspecies that are each in turn composed of several tetraploid S. simplex var. racemosa from the southern Appala- ecologically, geographically, and morphologically separable chians. Semple & Cook (2006) noted this similarity and sug- taxa. At some point, the packaging of this vast amount of bio- gested that S. plumosa might be conspecific with S. simplex. logical diversity into a single species raises the question: How This raises the possibility that S. plumosa (or a closely related many biological species are actually represented by S. simplex? diploid ancestor) was involved in the origin of S. simplex var. Below we examine the more focused question of whether the racemosa in the southeastern United States. Greene (1898) cytotypes within S. simplex (i.e., diploid subsp. simplex and segregated these southern populations as the specifically dis- polyploid subsp. randii) actually represent distinct species. tinct S. racemosa Greene. Thus the presence of ecologically We use an approach similar to the one taken by Soltis & al. and morphologically distinct endemic species in the southeast- (2007) to examine species boundaries between cytotypes of ern United States (especially the diploid S. plumosa) suggests Solidago simplex and compare those results to patterns found a more complicated evolutionary history within S. subsect. in several other polyploid complexes in Solidago (data summa- Humiles than previously proposed. rized in Tables 4–5). We adopt de Queiroz’s (1998, 2007) uni- While Ringius & Semple (1987) formally proposed that fied concept that considers species to be segments of separately tetraploid Solidago simplex subsp. randii was a monophyletic evolving metapopulation lineages that can be separated by a lineage that had a single origin from diploid S. simplex, Ringius variety of operational species criteria. We apply four general- (1986) cited the great amount of morphological variation in ized species criteria to cytotypic variation in S. simplex. Biologi- the polyploid subspecies as evidence that its origins may have cal: does gene flow or interbreeding occur between cytotypes? been polyphyletic. Phylogeographic data suggests that poly- Evolutionary/ecological: do cytotypes represent lineages with ploids formed multiple times and do not represent an ancient differing distributions/ecologies and evolutionary fates? Phylo- monophyletic lineage (Peirson, in prep.). Polyploid individu- genetic: do cytotypes represent monophyletic lineages united by als harbored 24 distinct chloroplast haplotypes that did not shared, derived characters? Taxonomic/phenetic: do cytotypes form a single clade; eight haplotypes were shared with diploids form morphologically separable clusters of individuals? while 16 were found in polyploids only. A single origin of poly- • Biological. – Polyploidization has often been cited as a ploidy would result in one ancestral chloroplast haplotype. Ei- prime example of how sympatric speciation can occur because ther polyploids evolved multiple times, or extensive gene flow it confers instantaneous reproductive isolation between the dip- and chloroplast capture from other polyploid Solidago species loid parent and polyploid derivative. This appears to be the case have resulted in a complex pattern of haplotype diversity. The in Solidago simplex. Pollination studies in S. simplex and other plausibility of the recurrent origin of polyploids in S. simplex goldenrod species indicate that there is strong triploid block is further supported by the rare occurrence of cryptic tetra- in the genus and that interploidy crosses are overwhelmingly ploids in otherwise diploid populations. While these data do (almost entirely) unsuccessful (e.g., Melville & Morton, 1982; not indicate extensive recurrent polyploidy, they do show that Ringius, 1986). The extensive body of traditional cytological polyploid formation and potential speciation in S. simplex can work in the genus also suggests that there are strong intrin- potentially occur on contemporary ecological timescales. Inter- sic barriers to intercytotype gene flow and that the formation estingly, the three mixed-ploidy populations discovered during and establishment of odd ploidy individuals (i.e., triploid or

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penta­ploid) are extremely rare events. Semple (1992) found that mirrored patterns in other Solidago species, and the data sum- only 0.12% (8 of 6908 records) of North American chromo- marized in Table 5 indicate that infraspecific cytotypes in five some counts for asters and goldenrods were from odd ploidy of the other species show some degree of geographic separa- individuals. Recent cytogeographic studies utilizing flow cy- tion. Interestingly, the pattern in S. gigantea and S. nemoralis tometry (with substantially larger sample sizes than traditional is the opposite of the pattern in S. simplex; in those species studies) have reached the same conclusion (Halverson & al., the polyploid cytotypes have western distributions (Brammall 2008; Schlaepfer & al., 2008a), suggesting that there is no major & Semple, 1990; Schlaepfer & al., 2008a). triploid or pentaploid bridge between cytotypes. This contrasts In the Great Lakes region, the main area where both cyto­ with some other species where significantly higher levels of types of S. simplex occur, they show nearly complete ecogeo- odd-ploidy individuals have been reported within populations graphic separation (Fig. 3). This type of regional ecogeographic (e.g., Galax urceolata, Burton & Husband, 1999). Halverson segregation is evident within some Solidago species but ap- & al.’s (2008) work on S. altissima suggested, however, that parently absent in others (e.g., S. altissima in Halverson & al., gene flow between diploid and hexaploid cytotypes may be 2008). Tetraploid populations of S. uliginosa in the Great Lakes facilitated by tetraploid plants where the cytotypes co-occur. region, while not widely geographically segregated from dip- It is unclear if this pattern will turn out to be common in other loids, occupy a distinct habitat type. Chmielewski & al. (1987) goldenrods that exhibit apparently high levels of cytotype co- found that tetraploid S. uliginosa was restricted to alvar (lime- occurrence (e.g., in S. curtisii ; Cook & Semple, 2008). stone pavement) habitats, while diploids were apparently absent • Evolutionary/ecological. – Ecological differentiation from this habitat. Laureto & Pringle (2010) recently described and/or geographic separation of polyploid plants from their the octoploid Solidago vossii J.S. Pringle & P.J. Laureto as a diploid relatives have been reported for many species (e.g., Ga- distinct species. This narrow polyploid endemic is restricted to lax urcoleata, Johnson & al., 2003; Tolmiea, Judd & al., 2007), inland, mesic sand prairies in a 6 km² area of northern Michi- and recent work on polyploidy in Achillea borealis has for the gan. Its presumed hexaploid progenitor, the U.S. federally first time demonstrated that polyploidy itself can mediate eco- threatened S. houghtonii A. Gray, is restricted almost entirely logical divergence among cytotypes through changes in fitness to calcareous Great Lakes shorelines in northern Michigan (Ramsey, 2011). Cytotypes within S. simplex display strong and Ontario. geographic structuring with the diploid cytotype widespread • Phylogenetic. – While higher ploidies may superficially in western North America and the tetraploid cytotype confined seem to be diagnosable by chromosome number (sensu Cracraft, to eastern North America. Ringius & Semple (1987) pointed 1983), the apparent frequency of recurrent polyploidization in out that the geographic segregation of cytotypes in S. simplex plants suggests that little phylogenetic weight can be placed

Table 4. Species criteria applied to variation in Solidago simplex. Species criteria Taxon Biological Evolutionary/ecological Phylogenetic Phenetic/taxonomic Between Yes, interploidy crosses Yes, appear to be distinct No, morphological variation Yes, diploids typically subsp. randii unsuccessful; 3n and 5n lineages, largely distinct and cpDNA suggest mul- have smaller capitula and and individuals not reported; geographic ranges, eco- tiple origins of polyploid pollen grains, shorter disk subsp. simplex phenological separation logical separation in regions subspecies corolla lobes, less pubescent between cytotypes in the where cytotypes co-occur achenes, and less acute leaf Great Lakes region apices; but sometimes dif- ficult to distinguish

Within 4n Yes (in part), infra-sub- Yes, appear to be distinct No, morphological variation Yes (in part), var. gillmanii subsp. randii species crosses involving lineages, largely distinct and cpDNA suggest mul- distinct from rest; other vari- var. gillmanii are largely geographic ranges, ecologi- tiple origins of polyploid eties differ in leaf shape and unsuccessful; phenological cal separation in regions of varieties; likely more margin serration, but all three separation between var. gill- co-occurrence; but vars. than four distinct lineages polymorphic and sometimes manii and var. ontarioensis monticola, ontarioensis, present difficult to distinguish in the Great Lakes region and racemosa all occupy rock substrate habitats

Within 2n Unknown Yes, var. chlorolepis Unclear, cpDNA does not Yes (in part), varieties differ subsp. simplex disjunct to eastern Quebec resolve relationships; in leaf shape, leaf apex shape, and restricted to serpentine vars. chlorolepis and nana and disk corolla lobe length; soils; but possible recurrent not sampled but vars. chlorolepis and sim- ecotypic formation of var. plex difficult to distinguish nana on different mountain summits Data were summarized from the following: Ringius (1986), Ringius & Semple (1987), Semple & Cook (2006), and Peirson (2010).

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on ploidy alone (Soltis & Soltis, 1999; Soltis & al., 2004). As populations approximately 75% of the time; thus phenetic gaps Soltis & al. (2007) pointed out, this likely recurrent formation between cytotypes should in theory often correlate with reduc- is one of the main arguments against recognizing infraspecific tions in gene flow and at least partial speciation. The subspecies polyploids as distinct species. Phylogeographic cpDNA data of Solidago simplex are phenetically separable and differ from support a possible recurrent evolution of polyploids in S. simplex each other by not only quantitative, ploidy-related morpho- (Peirson, in prep.), and this is consistent with general conclusions logical traits such as capitula size, phyllary length, and pol- from some other studied goldenrod species as well (Halverson len grain diameter but also by leaf and pubescence characters & al., 2008; Schlaepfer & al., 2008b). Laureto & Barkman (2011) (Table 4). Interestingly though, presumed incipient S. simplex hypothesized a single origin of the hexaploid S. houghtonii, tetraploids in otherwise diploid populations are cryptic with but their data also revealed three phylogeographic clusters that their diploid progenitors (Peirson, pers. obs.). Infraspecific could alternatively be interpreted as three independent origins. cyto­types in some other studied Solidago species are sepa- Robust phylogenetic data is lacking for Solidago (see discus- rable by a mix of presumably ploidy-related and non-ploidy- sion in Beck & al., 2004), but recurrent formation of polyploid related traits (Table 5). The pattern in S. nemoralis is similar cytotypes seems likely in a number of species (e.g., S. curtisii to the pattern in S. simplex. Diploid and tetraploid subspecies and S. nemoralis). It is unclear what effects recurrent formation differ in capitula, floral, and achene characters, but appar- has had on intercytotype gene flow and/or lineage divergence, ent incipient tetraploids in the otherwise diploid S. nemoralis and additional molecular phylogenetic and population genetic subsp. nemoralis differ from their progenitors only in their studies will be needed to test these hypotheses. slightly larger capitula. Infraspecific cytotypes in several spe- • Phenetic/taxonomic. – A review by Rieseberg & Wil- cies (e.g., S. curtissii and S. gigantea) are apparently essentially lis (2007) demonstrated that diagnosable phenotypic clus- indistinguishable from each other (Melville & Morton, 1982; ters in plants corresponded to reproductively isolated sets of Cook & Semple, 2008; Schlaepfer & al., 2008a).

Table 5. Species criteria applied to patterns of cytotype variation in six species of Solidago. Species Species criteria Biological Evolutionary/ecological Phylogenetic Phenetic/taxonomic Solidago Yes, interploidy crosses Yes, distinct lineages with Yes (in part), 2n and 6n Yes (in part), capitula size altissima unsuccessful; 3n and 5n in- distinct geographic ranges cytotypes are distinct lin- differs between 2n and 6n dividuals rare, but 4n plants at continental scale, but eages, but mixed-population cytotypes; tetraploids in zone may form a bridge between local co-occurrence where cytotypes are more closely of sympatry obscure pattern 2n and 6n cytotypes in cytotype ranges overlap related to each other (e.g., sympatry lineage recombination?)

Solidago Unknown, extensive recur- No, within population varia- Unknown No, cytotypes apparently not curtisii rent polyploidy may act as tion, overlapping ranges, distinguishable a bridge between cytotypes probable recurrent origins of polyploids Solidago Yes, 3n individuals very Yes, distinct lineages with Unknown Yes, capitula size differs be- flexicaulis rare distinct geographic ranges tween 2n and 4n cytotypes

Solidago Yes, interploidy crosses Yes, appear to be distinct No, cpDNA suggests mul- No, cytotypes apparently not gigantea unsuccessful; 3n and 5n lineages, largely distinct tiple origins of polyploids distinguishable individuals rare geographic ranges

Solidago Yes, 3n individuals not Yes, 2n and 4n subspecies Unknown, but presumably Yes, capitula, floral, and nemoralis reported are distinct lineages with recurrent formation of 4n achene characters differ distinct geographic ranges, plants within primarily 2n between 2n and 4n subspe- but sporadic formation of subsp. nemoralis cies; but cytotypes within 4n plants within 2n subspe- subsp. nemoralis not readily cies distinguishable

Solidago Yes, 3n individuals not Yes, distinct lineages with Unknown Yes, vegetative and phyllary rigida reported largely distinct geographic characters differ between ranges cytotypes, but sometimes difficult to distinguish In addition to Semple & Cook (2006), data were summarized from the following: S. altissima (Melville & Morton, 1982; Halverson & al., 2008), S. curtissii (Cook & Semple, 2008; Cook & al., 2009), S. flexicaulis (Chmielewski & Semple, 1985; Cook & Semple, 2008; Cook & al., 2009), S. gigantea (Melville & Morton, 1982; Schlaepfer & al., 2008a, b), S. nemoralis (Brammall & Semple, 1990; Semple & al., 1990), and S. rigida (Heard & Semple, 1988).

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Conclusion and leaf margin serration, but they are all phenotypically variable and lack striking adaptations like those found in var. This study of Solidago simplex and cumulative cytological gillmanii. From an evolutionary standpoint, they are equally data from studies of other Solidago species strongly support as complicated. Not only does phylogeographic data suggest the idea that polyploidy has been an important factor in the multiple origins, but common garden and morphological data diversification of the genus and that infraspecific chromosomal also suggest that S. simplex var. ontarioensis and var. racemosa races in a number of well-studied species represent divergent, each comprise at least two allopatric, morphologically distinct reproductively isolated lineages. Soltis & al. (2007) surmised lineages (Peirson, 2010; Ringius, 1986). In fact, Ringius re- that the predominant use of traditional morphological species ferred to S. simplex var. racemosa as a “complex assemblage concepts, the practicability (or lack thereof) of describing often of morphotypes”, and Greene (1898) recognized the southern cryptic polyploid species, and longstanding botanical tradi- populations as the distinct S. racemosa. How then should these tion have all contributed to the hesitancy of many systematists remaining three polyploid varieties be treated? to recognize infraspecific cytotypes as distinct species. This All of the available evidence indicates that they should seems to be the case in S. simplex as well. From the species not be subsumed into a broadly defined Solidago simplex, but criteria used above, however, there appear to be few biological until additional data is gathered and the evolutionary history reasons for recognizing the entities that comprise S. simplex of this complex more thoroughly resolved, taxonomic deci- subsp. simplex and S. simplex subsp. randii as one single spe- sions will remain preliminary hypotheses. Recent advances in cies (Table 4). Cytotypes in S. simplex likely cannot interbreed next-generation sequencing that have facilitated the gathering because of intrinsic barriers to intercytotype gene flow (bio- of genomic-level genetic data for non-model organisms hold logical species), have almost completely separate geographic significant promise for systems like this (Hudson, 2008; Emer- ranges and occupy different habitats where they co-occur re- son & al., 2010). An approach similar to the one used by Griffin gionally (ecological/evolutionary species), and are phenetically & al. (2011) to examine the evolution of polyploid Australian separable not only by ploidy-related morphological traits like alpine grasses will be essential to untangling the complicated capitula size and phyllary length but also by leaf shape and evolution of polyploidy in Solidago simplex and other polyploid pubescence characters (phenetic/taxonomic species). complexes in Solidago. While a complete picture of the evolu- At the same time, however, the case of Solidago simplex tion of polyploidy in Solidago is for now still out of reach, it is also highlights some of the difficulties in trying to parse tax- clear that effective biological species diversity in the genus is onomically complicated polyploid complexes. As currently considerably higher than currently recognized taxonomically. circumscribed, polyploid S. simplex subsp. randii comprises four varieties. Phylogeographic data suggest that the subspe- cies may be an assemblage of independently derived polyploid Acknowledgements lineages (Peirson, in prep.). A significant part of the taxonomic difficulty then centers on determining which and how many We thank P. Kron and B. Husband (University of Guelph) for in- polyploid species to recognize. From a morphological and valuable assistance with the flow cytometry and G. Nesom (Botanical ecological perspective, S. simplex var. gillmanii clearly war- Research Institute of Texas) for providing an unpublished chromosome rants recognition as a distinct species (the correct name would count for S. plumosa. We thank two anonymous reviewers for their be S. gillmanii (A. Gray) E.S. Steele). The species is adapted to critical evaluations of the manuscript. Funds for this project were active shoreline dune systems in the Great Lakes region and is provided by grants to J. Peirson from the National Science Foundation strikingly distinct from the other polyploid entities in S. sim- (Doctoral Dissertation Improvement Grant), Hanes Memorial Fund, plex. It possesses elongate vertical rhizomes that facilitate sur- Michigan Botanical Foundation, and the University of Michigan and vival from sand burial; the Pacific coastal endemic Solidago by Discovery Grants to J. Semple from the Natural Sciences and En- spathulata is the only other member of S. subsect. Humiles gineering Research Council of Canada. that is similarly adapted to active sand dunes. But even with its distinct morphology, is it possible that tetraploid S. gillmanii itself had multiple, independent origins? And if so, have the Literature Cited common selective regime of the sand dune environment and the connectivity of dune systems along the shores of Lakes Artyukova, E.V., Kozyrenko, M.M., Kholina, A.B. & Zhuravlev, Huron and Michigan been strong enough forces to shape an Y.N. 2011. High chloroplast haplotype diversity in the endemic legume Oxytropis chankaensis may result from independent poly- assemblage of independent lineages into a single, well-defined ploidization events. Genetica 139: 221–232. species? The apparent convergent evolution of other sand dune Beaudry, J.R. 1963. Studies on Solidago L. VI: Additional chromo- endemic goldenrods in the Great Lakes region certainly sug- some numbers of taxa of the genus Solidago. Canad. J. Genet. gests that dune systems in the region exert strong selective Cytol. 5: 150–174. pressures. These and other questions regarding the evolution Beaudry, J.R. 1969. Études sur les Solidago L. IX: Une troisième liste of S. gillmanii remain to be tested. de nombres chromosomiques des taxons du genre Solidago et de certains genres voisins. Naturaliste Canad. 96: 103–122. The three other polyploid varieties of Solidago simplex Beaudry, J.R. & Chabot, D.L. 1959. Studies on Solidago L. IV. 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210 Vol. 61 TAXON 61 (1) • February 2012: Electronic Supplement, 1 pp. Peirson & al. • Cytogeography of Solidago subsect. Humiles

International Journal of Taxonomy, Phylogeny and Evolution

Electronic Supplement to

Polyploidy, infraspecific cytotype variation, and speciation in Goldenrods: The cytogeography of Solidago subsect. Humiles (Asteraceae) in North America Jess A. Peirson, Anton A. Reznicek & John C. Semple

Taxon 

1 TAXON — Article version: 13 Jan 2012: Electr. Suppl., 6 pp. Peirson & al. • Cytogeography of Solidago subsect. Humiles

Table S. Locality and cytovoucher information for 189 published chromosome counts from Solidago subsect. Humiles. Nomenclature follows Semple & Cook (2006). Countries are designated as follows: Can, Canada; U.S.A., United States. References for counts are indicated by superscripts as follows: a, Beaudry & Chabot (1959); b, Raven & al. (1960); c, Beaudry (1963); d, Beaudry (1969); e, Löve & al. (1971); f, Mulligan & al. (1972); g, Hartman (1977); h, Taylor & Taylor (1977); i, Keil & Pinkava (1979); j, Morton (1981); k, Semple & al. (1981); l, Ward & Spellenberg (1986); m, Ringius & Semple (1987); n, Semple & al. (1989); o, Semple & al. (1992); p, Gervais & al. (1999); q, Semple & al. (2001); r, Semple (2003); s, Semple & Cook (2004). Species/location Country State/prov. County Lat. Long. Count Herb. Voucher(s) Solidago arenicola NW of Cleveland, woods near U.S.A. Alabama Blount 34.02 −86.57 36s WAT Semple & Semple Locust Creek of Warrior 11191 River

Solidago kralii S of Hartford, GA-230 1 km U.S.A. Georgia Pulaski 32.28 −83.45 18(9II)r WAT Cook & al. 701 SE of US-341/GA-27 US-1, NE of Blythe, S of Ellis U.S.A. Georgia Richmond 33.33 −82.18 18(9II)r WAT Semple & Semple Pond. 11217

Solidago simplex subsp. randii var. gillmanii Great Duck Island, Lake Can Ontario Manitoulin 45.69 −82.94 36 j WAT Morton & Venn 9493 Huron Mackinaw City U.S.A. Michigan Emmet 45.78 −84.74 36c MT Voss 58-280, Voss 58-281 Rogers City U.S.A. Michigan Presque Isle 45.42 −83.80 36 j WAT Morton & Venn 10911 Indiana Dunes State Park U.S.A. Indiana Porter 41.68 −87.02 36m WAT Ringius 1511, 1512, 1513, 1516, 1517, 1521 Huron Beach U.S.A. Michigan Presque Isle 45.57 −84.14 36m WAT Ringius 1818, 1819 Manistique U.S.A. Michigan Schoolcraft 45.91 −86.32 36m WAT Ringius 1840 Baileys Harbor U.S.A. Wisconsin Door 45.07 −87.11 36m WAT Ringius 1845, 1847

Solidago simplex subsp. randii var. monticola Black Lake Can Quebec Megantic 46.03 −71.40 36a MT Beaudry & Cinq- Mars 56-433 Thetford Mines Can Quebec Megantic 46.07 −71.30 36m WAT Ringius 1805, 1806, 1808, 1809 Whiteface Mountain U.S.A. New York Essex 44.35 −73.86 36c MT Parker 58-19-2 Mount Mansfield U.S.A. Vermont Chittenden 44.54 −72.81 36c MT Beaudry 58-246 Mount Desert Island U.S.A. Maine Hancock 44.31 −68.34 36m WAT Ringius 1743, 1754, 1757, 1759, 1762, 1763, 1768, 1769

Solidago simplex subsp. randii var. ontarioensis Old Woman Bay Can Ontario Algoma Distr. 47.79 −84.90 36d MT Purchase 62-5-1, 62- 5-2, 62-5-3, 62-5-4, 62-5-5 Gros Cap Can Ontario Algoma Distr. 46.53 −84.59 36m WAT/MT Ringius 1448 Michipigoten Harbor Can Ontario Algoma Distr. 47.94 −84.85 36m WAT Ringius 1987, 1988, 1989, 1990 Old Woman Bay Can Ontario Algoma Distr. 47.79 −84.90 36m WAT Ringius 2005, 2006 Georgian Bay, near Cypress Can Ontario Bruce 45.25 −81.52 36m WAT Ringius 2066 Lake Provincial Park

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Table S. Continued. Species/location Country State/prov. County Lat. Long. Count Herb. Voucher(s) Killarney Can Ontario Manitoulin 45.98 −81.46 36m WAT/MT Ringius 2041, 2042, 2043, 2045, 2047 Tobermory, end of Elgin Street Can Ontario Bruce 45.25 −81.68 36k WAT/MO Semple & Brammall 2788 Tobermory, shore of Lake Can Ontario Bruce 45.25 −81.68 36k WAT Semple 2437 Huron Tobermory, along Bay Street Can Ontario Bruce 45.25 −81.68 36(18II)k WAT/MO Semple & Brammall 2787 Tobermory, along Front Street Can Ontario Bruce 45.26 −81.64 36(18II)k WAT/MO Semple & Brammall 2780 Copper Harbor U.S.A. Michigan Keweenaw 47.47 −87.86 36m WAT Ringius 1887

Solidago simplex subsp. randii var. racemosa Aroostook River Can New Brunswick Victoria 46.81 −67.77 36m WAT Ringius 1631, 1632, 1633, 1635, 1637, 1639, 1640, 1642, 1643, 1646 Plaster Rock on Tobique River Can New Brunswick Victoria 46.91 −67.39 36m WAT Ringius 1780, 1786, 1787 Drummondville, St. Francis Can Quebec Drummond 45.89 −72.48 36m WAT Ringius 1658, 1659, River 1661, Semple & Brouillet 3407 Drummondville, St. Francis Can Quebec Drummond 45.89 −72.48 36k WAT Semple & Brouillet River 3407 Valley Falls State Park U.S.A. West Virginia Marion 39.39 −80.09 54c MT Core 57-661, 57-662, 57-663, 57-664, 57- 667, 57-668 Great Falls on the Potomac U.S.A. Maryland Montgomery 39.00 −77.25 36m WAT Semple & Ringius River 7663 Arnold Pond U.S.A. Maine Franklin 45.39 −70.79 36m WAT Semple & Keir 4621 PA 372 at Susquehanna River U.S.A. Pennsylvania York 39.81 −76.33 36m WAT Semple & Ringius 7612

Solidago simplex subsp. simplex var. chlorolepis Mount St. Anne Can Quebec Gaspe 49.00 −66.04 18p QFA Boudreau 96-175, 96-176 Mount Albert, Gaspe Penin- Can Quebec Gaspe 48.91 −66.18 18m WAT Ringius 1691 sula, near headwaters of Ruisseau de Diable Mount Albert, Vallee du diable Can Quebec Gaspe 48.91 −66.18 18p QFA Lavoie & al. 94-5 Mount Albert, Gaspe Penin- Can Quebec Gaspe 48.91 −66.18 18m WAT Ringius 1690, 1700, sula 1702

Solidago simplex subsp. simplex var. nana Mt. Hood, Cooper Spur Trail U.S.A. Oregon Hood River 45.38 −121.65 18q WAT Semple & Xiang below Eliot Glacier 10270

Solidago simplex subsp. simplex var. simplex Lac Laberge (seedling) Can Yukon – 60.99 −135.13 18d MT Marsh 63-6-1, 63-6- 3, 63-6-4, 63-6-5 Lac Laberge (seedling) Can Yukon – 60.99 −135.13 36d MT Marsh 63-6-2

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Table S. Continued. Species/location Country State/prov. County Lat. Long. Count Herb. Voucher(s) Lake Jasper, Jasper National Can Alberta – 53.11 −117.99 18 j WAT Morton NA4600 Park Lavoy, East of Vegreville on Can Alberta – 53.47 −111.90 18 j WAT Morton & Venn Hwy. 16 NA14175 Muncho Lake Provincial Park Can British Columbia – 58.95 −125.77 18i WAT Morton & Venn NA14135 Saskatoon Can Saskatchewan – 52.22 −106.75 18 j WAT Morton NA2234 Watson Lake Can Yukon – 60.06 −128.69 18 j WAT Morton NA2005 Keele River Can NW Territories Mackenzie 64.37 −124.90 18(9II)f DAO Cody 19936 Distr. Jasper National Park, Miette Can Alberta – 53.13 −117.78 18m WAT Semple & Brouillet Hot Springs 4332 West of Mannville Can Alberta – 53.33 −111.26 18m WAT Semple & Brouillet 4283 East of Obed Lake Can Alberta – 53.54 −117.12 18m WAT Semple & Brouillet 4315 Kamloops Can British Columbia – 50.68 −120.27 18m WAT Chinnappa 615A North of Kinaskan Lake Can British Columbia – 57.69 −130.04 18m WAT Chinnappa 2667 Windermere Lake Can British Columbia – 50.46 −115.99 18m WAT Semple & Brouillet 4362 Terrace Bay Can Ontario Thunder Bay 48.77 −87.11 18m WAT Ringius 1970, 1972, Distr. 1974, 1977, 1979 3 km west of the Ross River on Can Yukon – 61.99 −132.61 18m WAT Chinnappa 2307 Campbell Hwy West of Marshall Can Saskatchewan – 53.22 −109.82 18m WAT Semple & Brouillet 4267 Springside Can Saskatchewan – 51.34 −102.74 18m WAT Semple & Brouillet 4218 Dawson City Can Yukon – 64.07 −139.42 18m WAT Chinnappa 2472 42 km west of Dawson City Can Yukon – 64.23 −140.23 18m Chinnappa 2469 7 km south of McQuesten Can Yukon – 63.54 −137.28 18m WAT Chinnappa 2662 River on Klondike Loop 18 km SW of Ross River on Can Yukon – 61.86 −132.36 18m WAT Chinnappa 2281 Campbell Hwy 80 km SE of Ross River on Can Yukon – 61.75 −131.16 18m WAT Chinnappa 2265 Campbell Hwy Klondike Loop, 12 km S of Can Yukon – 62.73 −136.69 18n WAT Chinnappa 2665 Pelly Crossing near the 2nd lake Alaska Hwy, KP600, Testa Can British Columbia – 58.79 −122.66 18o WAT Chmielewski & al. River flats, just N of Testa 3773 service station Alaska Hwy, 21 km SE of Can British Columbia – 59.61 −126.79 18o WAT Chmielewski & al. Fireside 3783 Cassiar Hwy, 6 km N of Can British Columbia – 59.42 −129.18 18o WAT Chmielewski & al. Boya Lake Provincial Park 4720 turnoff Cassiar Hwy, KP729.8, at Can British Columbia – 60.00 −129.05 18o WAT Chmielewski & al. Yukon border 4709 12 km N of Iskut just N of Can British Columbia – 57.93 −130.05 18o WAT Chmielewski & al. Tsasbye Creek 4752

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Table S. Continued. Species/location Country State/prov. County Lat. Long. Count Herb. Voucher(s) Telegraph Creek Rd., 1 km NE Can British Columbia – 57.91 −131.12 18o WAT Chmielewski & al. of Telegraph Creek 4738 Telegraph Creek Rd., 40 km S Can British Columbia – 58.26 −130.62 18o WAT Chmielewski & al. of Dease Lake 4735 Telegraph Creek Rd., 5 km S Can British Columbia – 58.41 −130.08 18o WAT Chmielewski & al. of Dease Lake 4731 Campbell Hwy, KP570, 21 km Can Yukon – 62.04 −135.90 18o WAT Chmielewski & al. SE of Carmacks Junction 4606 Campbell Hwy, KP560, just N Can Yukon – 62.08 −135.65 18o WAT Chmielewski & al. of turnoff to Frenchman L. 4608 Gov’t Campground Campbell Hwy, KP539, N of Can Yukon – 62.10 −135.30 18o WAT Chmielewski & al. turnoff to Frenchman L. 4610 Gov’t Campground Campbell Hwy, KP410, 10 km Can Yukon – 62.20 −133.52 18o WAT Chmielewski & al. SE of Faro Junction 4614 S of Whitehorse, Klondike Can Yukon – 60.59 −134.87 18o WAT Chmielewski & al. Loop 2, KP108.3 3883 Klondike Loop 2, 2 km N of Can Yukon – 62.41 −136.59 18o WAT Chmielewski & al. MacGregor Creek, KP406- 4604 407 5 km S of Pelly Crossing, Can Yukon – 62.78 −136.58 18o WAT Chmielewski & al. Klondike Loop, KP460 4600 Dempster Hwy, KP12.2 Can Yukon – 64.03 −138.58 18o WAT Chmielewski & al. 4561 Silver Trail Hwy, NE of Stew- Can Yukon – 63.38 −136.65 18o WAT Chmielewski & al. art Crossing, KP19 4563 Cassiar Hwy. (Hwy-37) 80 km Can British Columbia – 59.45 −129.22 18q WAT Semple & Semple S of Alaska Hwy, ca. 5 km 10631 N of Boya Lake Alaska Hwy. just N of Haines Can Yukon – 60.76 −137.54 18q WAT Semple & Semple Junction 10623 Klondike Loop (YT-2), Can Yukon – 61.30 −135.55 18q WAT Semple & Semple KP270, N of Fox Lake 10626 Tweedsmuir Park, near lodge Can British Columbia – 53.10 −125.88 18(9II)h UBC Turner 843 Snowy Range, 4 miles NW of U.S.A. Wyoming Albany 41.35 −106.20 18g RM Hartman 3043 Centennial Inner Basin, San Francisco U.S.A. Arizona Coconino 35.34 −111.66 18i ASU Keil 11724, 11723, Peaks Reeves & Keil K11576 Slopes of Mt Fairplay, Taylor U.S.A. Alaska – 63.68 −142.26 18 j WAT Morton NA2095 Hwy Sante Fe Basin U.S.A. New Mexico Sante Fe 35.76 −105.82 18 j WAT Morton & Venn NA7105 2 km west of Shaw Creek on U.S.A. Alaska – 64.26 −146.15 18m WAT Chinnappa 2442 Alaska Hwy 55 km northeast of Slana on U.S.A. Alaska – 63.12 −143.29 18m WAT Chinnappa 2545 Glenn Hwy Nederland U.S.A. Colorado Boulder 39.96 −105.52 18m WAT Semple 6548 US-50 west of Poncha Springs U.S.A. Colorado Chaffee 38.54 −106.22 18m WAT Semple 7743 Echo Lake U.S.A. Colorado Clear Creek 39.66 −105.60 18m WAT Semple 6600 Mount Evans U.S.A. Colorado Clear Creek 39.59 −105.65 18m WAT Semple 6596, 6597

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Table S. Continued. Species/location Country State/prov. County Lat. Long. Count Herb. Voucher(s) South of Idaho Springs U.S.A. Colorado Clear Creek 39.73 −105.53 18m WAT Semple 6604 South of Grand Mesa on U.S.A. Colorado Delta 39.01 −107.99 18m WAT Semple 7790 CO-65 West of Monarch Pass U.S.A. Colorado Gunnison 38.50 −106.33 18m WAT Semple 7747 RM National Park, Trail Ridge U.S.A. Colorado Larimer 40.40 −105.72 18e COLO Snyder L11077 Road Keystone U.S.A. Colorado Summit 39.61 −105.97 18m WAT Semple 6556 Florissant U.S.A. Colorado Teller 38.95 −105.28 18m WAT Semple & Brouillet 7251 Pike’s Peak U.S.A. Colorado Teller 38.84 −105.04 18m WAT Semple 7722 Wheeler Peak U.S.A. Nevada White Pine 38.98 −114.31 18m WAT Semple 5745 North of Gearhart U.S.A. Oregon Clatsop 46.02 −123.93 18m WAT Semple & Brouillet 7113 Powder River Pass U.S.A. Wyoming Johnson 44.16 −107.10 18m WAT Semple & Brouillet 4452 Alaska Hwy (MP1368), NW of U.S.A. Alaska – 63.70 −144.32 18n WAT Chinnappa 2456 Tok, 12 km NW of Dot Lake North of Dot Lake, Alaska U.S.A. Alaska – 63.67 −144.10 18o WAT Chmielewski & al. Hwy 4470 Glenn Hwy, 1 km E of Tolsona U.S.A. Alaska – 62.10 −146.02 18o WAT Chmielewski & al. Lake Rd. 4102 CO-82 16 km E of Aspen, E of U.S.A. Colorado Pitkin 39.13 −106.68 18q WAT Semple & Zhang FR-106 10450 County line, SE of San Mateo, U.S.A. New Mexico McKinley/ 35.30 −107.60 18q WAT Semple, Suripto & San Mateo Rd. Valencia Ahmed 9368 N of Grants, San Mateo Spring U.S.A. New Mexico McKinley/ 35.32 −107.62 18q WAT Semple, Suripto & Canyon Valencia Ahmed 9370, 9372 Cebolleta Mts., Mt. Taylor U.S.A. New Mexico Valencia 35.25 −107.59 18q WAT Semple, Suripto & Ahmed 9379 Cebolleta Mts., Mt. Taylor U.S.A. New Mexico Valencia 35.25 −107.59 18q WAT Semple, Suripto & Ahmed 9380 W of Buffalo, US-16 between U.S.A. Wyoming Johnson 44.19 −106.92 18q WAT Semple & Xiang Pole Creek & Caribou Creek 10211 Wheeler Peak U.S.A. New Mexico Taos 36.56 −105.42 18l NMC Spellenberg 5822

Solidago spathulata Point Reyes U.S.A. California Marin 38.00 −123.00 18d MT Nobs 60-235-2, Rousi 60-25 Twin Peaks U.S.A. California San Francisco 37.75 −122.45 18d MT Beaudry & al. 59-211, 59-211-1, 59-211-3 Lake Merced U.S.A. California San Francisco 37.72 −122.50 18d MT Beaudry & al. 59- 210-1, 59-218-1 Point Reyes U.S.A. California Marin 38.00 −123.00 18(9II)b JEPS Raven 13694 Pebble Beach U.S.A. California San Mateo 37.24 −122.41 18m WAT Ringius 1482 North of Sand Lake U.S.A. Oregon Tillamook 45.33 −123.97 18m WAT Semple & Brouillet 7119 CA-1, just N of rd. to Point U.S.A. California Mendocino 38.96 −123.73 18n WAT Semple & Heard Arena Lighthouse 8542 CA-1, N of Anchor Bay, ca. 11 U.S.A. California Mendocino 38.84 −123.63 18(9II)n WAT Semple & Heard km N of Sonoma Co. line 8548

S5 TAXON — Article version: 13 Jan 2012: Electr. Suppl., 6 pp. Peirson & al. • Cytogeography of Solidago subsect. Humiles

Table S. Locality and cytovoucher information for 16 published chromosome counts that were rejected in this study. Nomenclature follows Semple & Cook (2006). Countries are designated as follows: Can, Canada; U.S.A., United States. Citations for counts are indicated by the same superscripts as in Table S1. Identification Location Country State/Prov. County Count Herbarium Voucher(s) S. cf. missouriensis Fort Saskatchewan Can Alberta – 18a MT G.H. Turner 55-276-3, 55-276-4, 55-276-5

S. cf. hispida Perce Mt, Gaspe Can Quebec Gaspe 18 j WAT Morton NA4142 Peninsula

Undetermined 1 km NE of U.S.A. New Mexico Lincoln 18(9II)l NMC/MO/ASU Spellenberg 1493 Capitan Peak

S. speciosa Black Hills U.S.A. South Dakota Custer 18m WAT Semple & Brouillet 4476

S. hispida 18.8 km N of Pancake Can Ontario Algoma Distr. 18k,m WAT/MT/MO Semple & Brouillet Bay Provincial Park 2862

S. hispida 18.6 km S of Montreal Can Ontario Algoma Distr. 18(9II)k WAT/MO Semple & Brammall River Harbor, rocky 2860 shore of Lake Superior S. hispida North of Agawa Bay Can Ontario Algoma Distr. 18m WAT Ringius 2009, 2012

S. hispida Mica Bay Can Ontario Algoma Distr. 18m WAT Ringius 2105, 2016, 2018

S. hispida Pancake Bay Can Ontario Algoma Distr. 18m WAT/MT Ringius 1435

S. hispida Lake Superior, west of Can Ontario Algoma Distr. 18 j WAT Morton NA2311 Pancake Bay

S. missouriensis Bow Falls, Banff Can Alberta – 36 j WAT Morton NA3412

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