Plant Syst Evol DOI 10.1007/s00606-009-0155-y ORIGINAL ARTICLE Evidence for multiple, autoploid origins of agamospermous populations in Eupatorium sessilifolium (Asteraceae) Kunsiri C. Grubbs Æ Randall L. Small Æ Edward E. Schilling Received: 14 October 2008 / Accepted: 25 January 2009 Ó Springer-Verlag 2009 Abstract Comparative analyses were made of agamo- uncommon suggest that monitoring of populations might spermous populations of Eupatorium sessilifolium, which be warranted to evaluate whether measures are needed to have previously been documented to be polyploid, to enhance their continued survival. determine whether they are alloploid or autoploid in origin and to assess the possibility that they have arisen more than Keywords Eupatorium Á Asteraceae Á Autoploid Á once. There was no variability in ITS sequences among Polyploidy Á ITS Á ISSR seven agamospermous and eight sexual diploid populations of E. sessilifolium, which is consistent with morphological observations in suggesting that the agamospermous popu- Introduction lations were autoploids. The ITS sequence characteristic of E. sessilifolium differs from all other North American A distinctive aspect of North American Eupatorium L. is species by a minimum of 15 changes, and heterogeneity or the widespread presence of agamospermy, which in this polymorphism would be expected if the agamospermous genus is always associated with polyploidy (Sullivan populations were alloploids. Analysis of the chloroplast- 1972). In some cases, there are groups of polyploid pop- based trnC-psbM spacer region showed variability among ulations that are morphologically distinct from any diploid, both sexual diploid and agamospermous populations of and have been recognized as taxonomically distinct. E. sessilifolium, which suggested that the agamospermous Examples for which genomic relationships have been populations stem from multiple origins. Analysis of ISSR established include E. godfreyanum Cronquist (Siripun and data revealed considerable intraspecific variability within Schilling 2006a) and E. novae-angliae (Fernald) V. I. E. sessilifolium, and the distribution of variability, with Sullivan ex A. Haines and Sorrie (Schilling et al. 2007). agamospermous populations showing variability from one Other groups of polyploid populations are morphologically another, added further evidence for multiple origins of indistinguishable from associated sexual diploid popula- agamospermous populations. The results in conjunction tions and have been considered to be conspecific with with distributional evidence that the sexual diploid popu- them. Examples include E. altissimum L., E. rotundifolium lations of E. sessilifolium are geographically restricted and L., and E. sessilifolium L. (Sullivan 1976). There are, however, still questions regarding whether the polyploids within a species are auto- or alloploid in origin, and whe- K. C. Grubbs ther they have single or multiple origins. Previous work Department of Science, Kasetsart University, that attempted to address these questions using flavonoids Kamphangsaen Campus, Kamphangsaen, Nakhon Pathom 73140, Thailand or isozymes (e.g. Sullivan 1972; Yahara and Sullivan 1986; Yahara et al. 1991) utilized markers that were not capable R. L. Small Á E. E. Schilling (&) of decisively resolving among the alternatives. Department of Ecology and Evolutionary Biology, Independent origins of polyploidy have been docu- University of Tennessee, Knoxville, TN 37996, USA mented in various groups, and in fact form a recurrent e-mail: [email protected] theme where this has been investigated closely (Soltis et al. 123 K. C. Grubbs et al. 2003). For apomicts, though, the contrasting situation has geographical area in the southern Blue Ridge Mountains, sometimes been documented in which large numbers and the species is represented by agamospermous, poly- of populations may form essentially a single clone ploid (triploid where chromosome counts are available, (Hollingsworth and Bailey 2000; Poulin et al. 2005;Xu Grant 1953; Montgomery and Fairbrothers 1970; Sullivan et al. 2003). The generally low levels of variation for DNA 1976) populations across the rest of its range. Other than sequence data have suggested that divergence in Eupato- the differences in presence or absence of viable pollen, the rium in eastern North America may be relatively recent polyploids are indistinguishable both morphologically and (Siripun and Schilling 2006a), so it is possible that apo- by flavonoid chemistry (Sullivan 1972) from the diploids. mictic populations within species of Eupatorium may be This information suggests that the agamospermous popu- relatively recent invaders of disturbed habitats that could lations of E. sessilifolium are autopolyploid. Hybridization stem from single origins. within Eupatorium is frequent, however, and hybrids or Eupatorium sessilifolium is a morphologically uniform hybrid-derived apomicts are known between E. sessilifo- species that occupies a relatively wide range in eastern lium and several other species, including E. rotundifolium North America (Sullivan 1972; Siripun and Schilling (Siripun and Schilling 2006a), E. perfoliatum L. (Sullivan 2006b; Fig. 1). It is distinguished from other members of 1972), and E. petaloideum Britton, and E. hyssopifolium L. the genus by its glabrous stems, and leaves that are sessile, (Schilling, unpublished data). Thus, it is also possible that truncate, relatively long (7–15 cm), and pinnately nerved. the polyploid populations of E. sessilifolium were derived Sullivan (1972; 1976) documented that diploid, sexual via hybridization and are allopolyploid without this being populations are found only in a relatively restricted reflected in the morphology or chemistry. Fig. 1 Map of eastern North America showing the distribution range approximate sources of populations sampled for molecular data, of Eupatorium sessilifolium (solid line) with the documented range numbered as in Table 1 (circles sexual diploid populations, triangles of the diploid populations (stippled areas); enlargement shows agamospermous polyploid populations) 123 Evidence for multiple, autoploid origins of agamospermous populations In this study we employed three different molecular Materials and methods approaches to examine the relationships of diploid and polyploid populations of E. sessilifolium. Two primary Sources of plant material questions were addressed: (1) are the polyploid populations of E. sessilifolium allo- or autoploid in origin, and (2) are Samples of E. sessilifolium were collected from field the polyploid populations the result of a single or multiple populations in the southeastern USA in the area where both origins? Each of the three data sets provides unique sexual diploid and agamospermous polyploid populations insights into these questions. have been documented previously (Table 1; Fig. 1). At Previous studies of ITS sequence variation in Eupato- each site, multiple individuals were sampled. Ploidy level rium have shown that each diploid species has a unique ITS and type of reproduction of populations was inferred from sequence type and that little intraspecific variation exists the presence or absence of pollen coupled with the pres- (Siripun and Schilling 2006a; Schilling et al. 2007). ence of filled cypselae. Sullivan (1972, 1976) demonstrated Further, studies of allopolyploid species and hybrids in the absolute correlation within Eupatorium between male Eupatorium have shown that ITS sequence types repre- sterility and polyploidy, and the further correlation between sentative of all parents are maintained in allopolyploid and polyploidy and agamospermy, and the presence of uni- hybrid individuals (Siripun and Schilling 2006a; Schilling formly filled cypselae in heads in which the flowers lacked et al. 2007). We used ITS sequence data to determine not only pollen but also anthers provided further evidence whether polyploid E. sessilifolium populations were of of their agamospermous nature. Measurements were made allo- or autoploid origin. If polyploid E. sessilifolium of a number of characters (leaf length and width, phyllary populations are of alloploid origin then we expected to length and width; corolla length, cypsela and pappus detect multiple different ITS sequence types within indi- length) for comparisons. viduals. Alternatively, if E. sessilifolium populations are of autoploid origin then we expected to obtain a single ITS Isolation of DNA sequence type. Because chloroplast DNA is maternally inherited DNA samples were extracted principally from young (Sullivan et al. 1991), phylogenetic analyses of cpDNA leaves with the CTAB protocol of Doyle and Doyle (1987). sequences are expected to reflect only maternal lineages To decrease the interference from phenolic compounds, and can be used to address the issue of single versus DNA extractions were cleaned up using the Wizard Kit multiple origins of polyploids. If polyploid E. sessilifoli- protocol (Promega, Madison, WI, USA). The purified DNA um originated once then we expect all polyploid extracts were quantified and the concentrations were populations to have a single cpDNA sequence type (if no adjusted to 25–100 ng/ll. mutations have occurred subsequent to polyploid forma- tion), or to form a monophyletic group relative to sampled Molecular methods diploid populations. Alternatively, if polyploid E. sessi- lifolium populations have arisen through multiple origins For ITS sequence studies, only one individual from each then we expect different polyploid populations to have population