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(American Journal of Botany. 2006;93:319-325.)
© 2006 Botanical Society of America, Inc.

Brief Communication

Molecular confirmation of the hybrid origin of Eupatorium godfreyanum (Asteraceae)1

Kunsiri Chaw Siripun and Edward E Schilling4

2Kasetsart University, Kamphaengsaen Campus, Nakhon Pathom, 73140 Thailand; and 3Department of Ecology & Evolutionary Biology, University of Tennessee, Knoxville, Tennessee 37996 USA

Received for publication March 19, 2005. Accepted for publication November 23, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Analysis of nuclear ribosomal ITS sequence data was used to assess the relationships of Eupatorium godfreyanum, an agamospermous polyploid species of putative hybrid origin. A data set of ITS sequences that included representatives of all but two of the North American species of Eupatorium was compiled from a combination of previously published and newly obtained results. Assessment of the data showed that each species was relatively distinctive, although the results from parsimony analysis suggested that there was little phylogenetic structure within the data beyond a basal split between members of the dog fennel group ("Traganthes") and the remainder of the genus. Cloning was required to obtain readable ITS sequence from E. godfreyanum, and analysis of individual clones produced sequences that matched closely those of either E. rotundifolium or E. sessilifolium. The ITS sequence data thus supported the hypothesis that Eupatorium godfreyanum is of hybrid origin from a combination of E. rotundifolium and E. sessilifolium.

Key Words: Asteraceae • Eupatorieae • Eupatorium • ITS • hybridization • molecular phylogeny


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Hybridization and agamospermy have combined to produce a bewildering maze of morphological variability within Eupatorium L. that defies simple classification. Interspecific hybridization has been well documented among various species combinations (Sullivan, 1978 ; Jordan, 1991 ; Sullivan et al., 1991 ). Phenotypes that are generally intermediate between parental species may be stabilized and transmitted intact through apomictic reproduction, in some cases achieving geographical distributions beyond either original parent (Sullivan, 1976 ). This may produce a blurring of the otherwise relatively distinct morphological boundaries between species. The presence of natural variability for many morphological traits and the common occurrence in hybrids of character states that are not simply intermediate between parents make it difficult to be certain that in fact hybridization rather than some other phenomenon has produced a given pattern of variation. Molecular markers, however, provide another set of tools to evaluate proposed hypotheses of hybridization. Here we present confirmatory evidence from the molecular level of the proposed hybrid origin of a polyploid apomict in Eupatorium, E. godfreyanum Cronquist.

Eupatorium in its current delimitation (King and Robinson, 1970 , 1987 ) is a temperate genus of about 43 species with its greatest diversity in eastern North America, but it is also represented in eastern Asia (Kawahara et al., 1989 ) and by a single species in Europe. A striking feature in eastern North America is the presence of species that are represented by a mixture of sexual diploids and agamospermous polyploids, the latter usually having much more extensive geographic ranges than the former (Grant, 1953 ; Sullivan, 1976 ). Efforts to assess whether the apomicts within such species are of auto- or alloploid origin have produced varying and sometimes inconclusive results to date (Sullivan, 1972 ; Yahara and Sullivan, 1986 ; Yahara, 1990 ; Yahara et al., 1991 ). There are also taxa that are entirely diploid and sexual, as well as others that are composed entirely of agamospermous, polyploid populations. Because the agamospermous taxa differ in morphology from known sexual diploids and may appear to be intermediate between them, a number of them have been proposed to be of hybrid origin.

Eupatorium godfreyanum is an entirely agamospermous species that has been proposed to combine genomes from E. sessilifolium L. and E. rotundifolium L. (Cronquist, 1985 ). This taxon was long recognized under the name E. vaseyi Porter (E. sessilifolium L. var. vaseyi [Porter] Fernald & Griscom), but Cronquist (1985) has documented that the type specimen of E. vaseyi is at variance with this usage and rather represents a likely hybrid derivative of E. album L. and E. sessilifolium. Eupatorium godfreyanum has a scattered geographic distribution, ranging from New Jersey to North Carolina and west to southern Ohio and West Virginia (Fig. 1). In both of its putative parents, mixed assemblages of sexual diploids and agamospermous polyploids, the polypoloids have achieved a widespread geographic distribution in eastern North America (Figs. 1, 2). In contrast, the sexual diploids of both are relatively restricted, with those of E. sessilifolium known from only a limited area in the southern Appalachian Mountains, whereas those of E. rotundifolium are confined to a relatively narrow band in southern Georgia and northern Florida (Figs. 1, 2; Sullivan, 1976 ).


Figure 1
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  		Fig. 1 Geographic distribution of Eupatorium rotundifolium (diagonal shading, range of diploid cytotype) and E. godfreyanum (dotted shading) in the eastern USA

 

Figure 2
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  		Fig. 2 Geographic distribution of Eupatorium sessilifolium (diagonal shading, range of diploid cytotype) in the eastern USA

 
This project was conducted as part of a larger general study of the genetic and phylogenetic aspects of agamospermy in Eupatorium (Siripun, 2004 ). The goal of the current project was to evaluate the hypothesis that E. godfreyanum is an alloploid derivative of E. rotundifolium and E. sessilifolium. DNA sequence data from the nuclear ITS region, which is biparentally inherited, was obtained for samples of all three species, as well as from other taxa of Eupatorium and of Eutrochium Raf. (=Eupatoriadelphus R. King and H. Rob.; Lamont, 2004 ), which was used as the outgroup. The presence in E. godfreyanum of DNA sequences characteristic of both E. rotundifolium and E. sessilifolium was expected to provide evidence of its hybrid origin.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
Taxon sampling
Samples of Eupatorium (Appendix) were collected in the field and either extracted fresh or stored at –80°C until ready for DNA extraction. Herbarium material was utilized as the sole source for two species and for confirmatory sequences for three others. For both E. rotundifolium and E. sessilifolium, sampling included three populations that were inferred to be sexual diploids (for each species labeled D1, D2, D3) based on the production of viable pollen and three that were inferred to be agamospermous polyploids (P1, P2, P3) based on the lack of pollen. Eutrochium was chosen as the outgroup based on previously published molecular results that show it to be the sister group to Eupatorium (Schilling et al., 1999 ; Ito et al., 2000 ; Schmidt and Schilling, 2000 ). ITS sequences for Eutrochium and for some additional species of Eupatorium were obtained from GenBank (Appendix).


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  		Appendix Voucher information for collections of Eupatorium and Eutrochium (Eupatoriadelphus R.M. King & H. Rob.) used in this study. Vouchers are at TENN.

 
Molecular methods
Preparations of total DNA were made from fresh (0.1–2.0 g) or frozen (1–3 g) leaves generally following the procedure of Doyle and Doyle (1987) . Extractions from herbarium specimens were performed with the DNeasy Plant Minikit (Qiagen, Valencia, California, USA) and utilized a portion of a single, dried (ca 0.1 g) leaf. The crude DNA extracts of some samples required further purification using the Wizard Kit protocol (Promega, Madison, Wisconsin, USA). ITS amplifications were performed in 20-µL reactions using 10–20 ng of genomic DNA, 10x PCR buffer (Promega), 1.8–2.25 mM MgCl2, 0.2 mM each dNTP, 1.25 units of Taq polymerase, and 0.2 µM each primer. Primers used were "ITS-4" (5'-TCCTCCGCTTATTGATATGC-3') and "ITS-5" (5'-GGAAGTAAAAGTCGTAACAAGG-3'; White et al., 1990 ). PCR was performed with the following protocol: 94°C for 2 min; 40 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min; and a final extension of 72°C for 3 min. All PCR products were checked by agarose gel electrophoresis and purified by the Wizard Kit protocol. Sequencing was done at the University of Tennessee Automated Sequencing Facility, utilizing the ABI Prism Dye Terminator Cycle Sequencing reaction kit on an ABI 373 DNA sequencer (Perkin-Elmer, Foster City, California, USA). The amplification primers were used as the sequencing primers. The initial sequence data text files were edited after comparison with the same data displayed in four-color electropherograms before they were analyzed further. Sequence alignment was performed using the Clustal X program (version 1.6, Thompson et al., 1994 ). GenBank accession numbers are provided in the Appendix.

Cloning of the ITS region from samples of E. godfreyanum was necessary because data obtained from direct sequencing of the PCR product were not satisfactory. Purified PCR products were ligated into pGEM-T (Promega) according to the manufacturer's instructions. Competent Top10 F' (Invitrogen, San Diego, California, USA) cells were transformed via electroporation, and the resulting colonies were screened for plasmids with inserts by PCR using the original amplification primers. Plasmids were isolated from a single recombinant colony using an alkaline lysis/PEG precipitation protocol (Sambrook et al., 1989 ).

Data analysis
Phylogenetic relationships were analyzed using the maximum parsimony approach, implemented with the computer program PAUP* 4.0b10 (Swofford, 2003 ). A heuristic search with 1000 random addition replicates and with tree-bisection-reconnection (TBR) branch swapping was used, with gaps treated as missing data. Bootstrap analysis (Felsenstein, 1985 ) was performed with 1000 replicates using a heuristic search strategy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
ITS sequences
The ITS sequences obtained for Eupatorium were consistent with previous reports for the genus in approximate length of the ITS-1, ITS-2, and 5.8S regions. The 11-bp deletion in ITS-2 relative to Eutrochium and most other members of the tribe was observed in all newly sampled species of Eupatorium. Pairwise divergence between samples representing different species of Eupatorium varied between 7 and 38 bp differences (1–6% divergence), including substitutions and in some cases single base-pair indels. Where multiple samples were examined, pairwise differences within species were 0–3 bp (0–0.5% divergence). For E. rotundifolium, there was little difference between sexual and agamospermous populations, and there was no variation among populations of E. sessilifolium.

Samples of E. rotundifolium and E. sessilifolium differed consistently in ITS sequence by a minimum of 13 single base-pair substitutions and two single base-pair indels; because of the indels the ITS region was 2 bp longer in E. rotundifolium. The ITS sequences that were obtained for individual clones showed that the samples of E. godfreyanum each had at least two different ITS sequence motifs, one that matched closely those of E. rotundifolium and one that matched exactly that of E. sessilifolium. A clone from sample 823 produced a sequence that matched E. sessilifolium for the nine distinctive positions of the ITS-1 region but matched E. rotundifolium for the six distinctive positions of the ITS-2 region; it is likely that this sequence represents a PCR recombinant. The presence of ITS sequences of different lengths within each sample of E. godfreyanum explained why direct sequencing of PCR products produced unsatisfactory results.

Phylogenetic analyses
The data set that was used for parsimony analysis included 60 samples representing 28 species, and 118 potentially informative variable base positions as well as an additional 23 were variable but not parsimony informative. Parsimony analysis yielded 23583 minimum length trees of 243 steps with a consistency index of 0.71 and a retention index of 0.88. The consensus tree had almost identical topology to the bootstrap tree (Fig. 3). Samples of Eupatorium are separated from the outgroup, Eutrochium, with 100% bootstrap support. Within Eupatorium, a basal split separated samples of the dog fennel group ("Traganthes group") from the remainder of the data set. Above this basal split was a large polytomy, with species or groups of species forming strongly supported clades. A clade formed by the Eurasian species had weak (55%) bootstrap support, but the European (E. cannabinum) and Asiatic samples were each placed in clades that were individually well supported. For the North American samples, each species with multiple samples formed a strongly supported clade, but the only species grouping that received more than 50% bootstrap support was that of E. album and E. petaloideum. Note that the sample identified by Ito et al. (2000) as E. sessilifolium was placed with our sample of E. semiserratum, and conversely their sample of E. semiserratum was placed with our samples of E. sessilifolium; our interpretation is that their data for the two species are reversed. The only species in the analysis that was not monophyletic was E. godfreyanum, and individual clones of this species were placed with either E. rotundifolium or E. sessilifolium (Fig. 3).


Figure 3
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  		Fig. 3 Strict consensus of 23583 minimum length trees obtained from parsimony analysis of Eupatorium ITS sequence data (CI = 0.71; RI = 0.88). Bootstrap values (1000 replicates) are above branches; dashed lines are branches not receiving at least 50% bootstrap support. Outgroup species (Eutrochium) are in uppercase. Origin of samples and identification codes shown in the Appendix ; for E. rotundifolium and E. sessilifolium, D and P designate samples from inferred diploid and polyploid populations, respectively

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The major result of this study is that the newly reported molecular evidence supports the hypothesis that Eupatorium godfreyanum is of hybrid origin from a combination of E. rotundifolium and E. sessilifolium. A survey of the genus showed that, in general, the ITS sequences from different species were relatively distinctive (Fig. 3), with sampling of all species except for the polyploid E. anomalum Nash and the rare E. resinosum Torr. Both copies of the distinctive ITS sequences from the two putative parents were detectable in samples of E. godfreyanum (Fig. 3). The ITS data provided no evidence that any other species was involved in the origin of E. godfreyanum. This is particularly notable for E. pilosum, which has sometimes been included within E. rotundifolium (as E. rotundifolium var. saundersii; Cronquist, 1985 ) and has also been considered to be a possible parent of E. godfreyanum. It is, however, clearly distinctive in its ITS sequence from both E. rotundifolium and E. godfreyanum (Fig. 3).

The molecular data thus are consistent with the supposed intermediacy in morphological features, which has been cited to support the hybrid origin of E. godfreyanum (Cronquist, 1985 ). As has been noted to be the case for many hybrids and hybrid derivatives (Rieseberg, 1995 ), the morphology of E. godfreyanum is not strictly intermediate between its putative parental species, but rather consists of a mixture of qualititative characters that match one or the other parental species as well as intermediacy in quantitative ones (Table 1).


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  		Table 1 Morphological comparison of Eupatorium godfreyanum and its putative progenitors, E. rotundifolium and E. sessilifolium

 
Knowledge of its origin does not completely clarify how E. godfreyanum should be treated taxonomically. As noted by Cronquist (1985) , taxonomic treatment in complexes that include apomictic polyploids has a large arbitrary component. Within Eupatorium, many of the apomictic taxa have been reported to be triploid (Sullivan, 1976 ); thus even if they are of alloploid origin, they may be genetically and morphologically closer to one parent than the other and thus arguably treated at the varietal rather than the species level. In the case of E. godfreyanum, because it differs consistently from either parent and seems to persist where it occurs, we recommend its recognition at the species level.

Although the molecular data for the genus was presented primarily to form a backdrop for analysis of E. godfreyanum, some notable aspects of the overall phylogenetic patterns in Eupatorium are evident. One is that the ITS data provide a perspective on the generic-level phylogeny, suggesting a basal split between the species with paniculate inflorescences and highly dissected leaves that have been recognized as the "Traganthes group" (the dog-fennels) and the remainder of the genus, a result that is fairly consistent with results of karyotype analyses (Watanabe et al., 1990 ). Within the remainder of the genus, there was, however, little phylogenetic resolution based on ITS data. The lack of resolution suggests that divergence within the genus between Eurasia and North America occurred in a relatively short period, at approximately the same time as the species-level divergence in North America. A second notable aspect is the contrast between the levels of divergence of species for ITS in Eupatorium and those that have been reported for other genera of Asteraceae from eastern North America. The relatively large divergence in the ITS region between E. rotundifolium and E. sessilifolium (15 of 636 changes or a divergence of 3%) contrasts to near uniformity in ITS sequences between morphologically distinctive species such as Helianthus microcephalus/H. divaricatus (Schilling et al., 1998 ), Liatris cylindracea/L. oligocephala (Hardig et al., 2005) and Solidago shortii/S. discoidea (Beck et al., 2004 ). One potential explanation is that species of Eupatorium actually have relatively small population sizes (note that based on isozyme data, this has been suggested for E. altissimum, Yahara et al., 1991 ), potentially subject to periodic bottlenecks, and thus sequence changes may undergo fixation at a relatively high rate.


   FOOTNOTES
 
1

 The authors thank E. Lickey and E. Grand for help with fieldwork and G. Beattie, P. Heise, J. Miller, and R. Small for advice and technical support. Financial support was provided by the H. R. De Selm and L. H. Hesler Funds and the Department of Botany, University of Tennessee. Back

4Author for correspondence (e-mail: eschilling{at}utk.edu ) Back


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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