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(American Journal of Botany. 2008;95:1096-1108.) doi: 10.3732/ajb.0800024 © 2008 Botanical Society of America, Inc. |
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Mycology |
2 Department of Forest Resources, University of Idaho, Moscow, Idaho 83844-1133 USA 3 Center for Research on Invasive Species and Small Populations, University of Idaho, Moscow, Idaho 83844-1133 USA 4 Department of Fish and Wildlife Resources, University of Idaho, Moscow, Idaho 83844-1133 USA
Received for publication 19 January 2008. Accepted for publication 7 July 2008.
ABSTRACT
Fungal endophytes are important in plant ecology and common in plants. We attempted to test cointroduction and host-jumping hypotheses on a community basis by comparing endophytes isolated from invasive spotted knapweed (Centaurea stoebe, Asteraceae) in its native and invaded ranges. Of 92 combined, sequence-based haplotypes representing eight classes of Fungi, 78 occurred in only one of the two ranges. In the native range of C. stoebe, one haplotype of Alternaria alternata was clearly dominant, whereas in the invaded range, no haplotype was dominant. Many haplotypes were closely related to one another and novel. For example, six putative, new species of Botrytis were discovered as endophytes of C. stoebe, which has never been reported to have Botrytis spp.. Apparent differences between the two communities of endophytes were significant according to an analysis of similarity, but phylogenetic community structure did not differ significantly between the ranges. Both host-jumping and cointroduction of fungal endophytes likely took place during the spotted knapweed invasion.
Key Words: Asteraceae • Centaurea stoebe • Botrytis phylogeny • diversity • endophytes • geographic origins of fungi • native and invaded ranges • spotted knapweed
Endophytic fungi grow inside plant tissue without causing any obvious symptoms and are an important though widely neglected component of plant communities. Endophytes have recently been shown to be key elements in plant symbiosis, affecting host tolerance to stressful conditions (Redman et al., 2002
; Rodriguez et al., 2004; Marquez et al., 2007), plant defense (Omacini et al., 2001; Bailey et al., 2006), plant growth (Ernst et al., 2003), and plant community biodiversity (Clay and Holah, 1999). However, the diversity, geographic distribution, and host specificity of endophytes remain largely unknown (Arnold et al., 2001; Otero et al., 2002, 2007; Ganley et al., 2004; van Bael et al., 2005; Arnold and Lutzoni, 2007; Higgins et al., 2007). Recent estimates of fungal diversity (Hawksworth, 1991, 2001; Cannon, 1997; Schmit and Mueller, 2007) suggest that more than 90% of fungal species are not described, and there are no specific estimates of the number of existing endophytes (Hyde et al., 2007).
The geographic distributions of specific fungi are under recent study (Taylor et al., 2006). For example, Gladieux et al. (2008) showed that Venturia inaequalis, causal agent of apple scab, is likely native to the same area in central Asia where apple itself was domesticated. Similarly, an Asian origin for the dry rot fungus Serpula lacrymans has been inferred from a study of its genetic variation (Kauserud et al., 2007). Intense interest in the origins of the amphibian chytrid fungus, Batrachochytrium dendrobatidis, has resulted in a hypothesis that it originated in South Africa (Weldon et al., 2004). The human dermatophyte Trichophyton rubrum appears likely to have evolved in Africa, given that its genotypic diversity was highest there (Gräser et al., 2007). Fungal species endemic to Antarctica have been described recently (De Hoog et al., 2005). However, even phytopathogenic fungi, such as the Ophiostoma species that have caused global pandemics of Dutch elm disease in the past century, remain of uncertain geographic origin (Brasier and Buck, 2001). This uncertainty is surprising given the potentially devastating consequences of introducing a fungal pathogen to a new geographic range (e.g., the introduction into North America of the chestnut blight pathogen). In general, without baseline mycofloras from early in the Age of Exploration (15th century), it is possible that the current geographic distributions of fungi may reflect both recent homogenization events and original ranges. This possibility is especially true for endophytes that could have been dispersed during plant introductions, both deliberate and inadvertent (Cox, 2004).
In general, an exotic, invasive plant can either colonize its invaded range accompanied by fungi from its native range (i.e., the cointroduction hypothesis), or it can leave its native associates behind and acquire new associates (i.e., the host-jumping hypothesis). The ecological implications of these two scenarios for plant invasions are unknown. Invasive plants are less competitive and less abundant in their native ranges than in their invaded ranges (Broennimann et al., 2007), and endophytes could contribute to the greater competitiveness that is seen in the invaded ranges. For example, in the host’s invaded range, cointroduced endophytes could act as novel weapons (Bais et al., 2003; Callaway and Ridenour, 2004; Cappuccino and Arnason, 2006) that would increase the competitiveness of their hosts by producing novel allelochemicals inhibitory to evolutionarily naive plants. Alternatively, endophytes acquired in the invaded range through host-jumping might enhance the competitiveness of invasive plants if those endophytes were mutualists (i.e., the enhanced mutualism hypothesis: Richardson et al., 2000; Klironomos, 2002; Reinhart and Callaway, 2006). However, in spite of their importance as common symbionts of plants and potential contributors to invasiveness, the endophyte status of invasive plants is rarely determined, and the geographic origins of endophytes have never been conclusively investigated.
Centaurea stoebe L. s. l. (= C. maculosa, C. pseudomaculosa, C. biebersteinii, C. micrantha, Asteraceae), or spotted knapweed, is an established model system in invasion biology research (Ochsmann, 2000; Cox, 2004; Inderjit, 2005; DiTomaso and Healy, 2006; Nentwig, 2007). This Eurasian plant has a wide distribution in its native range: from western France to northwestern Kazakhstan, across the Ural Mountains, and extending into southwestern Siberia (Klokov, 1963; Czerepanov, 1994; Ochsmann, 2000, 2001). Its invasion in North America started in the late 1890s, and the plant now infests millions of hectares in North America causing more than 150 million U.S. dollars of economic damage annually (Pimentel et al., 2000; Story et al., 2006).
Our experiments to date (A. Shipunov, G. Newcombe, and A. Raghavendra, unpublished data) have indicated that endophytes can affect the competitiveness of C. stoebe. As both cointroduction and host-jumping of endophytes align with hypotheses of plant invasion that are based on enhanced competitiveness, we sought here to compare endophyte communities from the two ranges of C. stoebe and to test these hypotheses with traditional comparative and phylogenetic methods. In addition, we tried to shed some light on the extent of taxonomic novelty in the endophyte communities of one invasive plant species.
MATERIALS AND METHODS
Sampling
In 2003–2006, we collected Centaurea stoebe from sites in both its native and invaded ranges. We sampled a total of 53 populations of knapweed in the invaded range, primarily from the northwestern United States (i.e., Montana, Idaho, Washington, and Oregon), where knapweed infestation is common (Fig. 1), with additional sites in Canada (British Columbia and Ontario) and the eastern United States (Michigan and New York). In three cases, we repeated sampling from the same knapweed populations (all in the Palouse region of northern Idaho) in 2004 and 2006. We sampled a total of 49 populations in the native range, along a broad transect, from western Europe through the Ural Mountains and from Moscow (central Russia) to Dagestan in northeastern Caucasus.
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), we used a standard identification key (Ochsmann, 2000) for all determinations and photographed plants and flowering heads with bracts for most samples in situ. From each individual plant, 10–20 flowering heads with ripe achenes were collected. All collected seed heads were refrigerated to prevent loss or "storage succession" (Dugan and Lupien, 2002) of fungal symbionts.
Isolation and cultivation of fungi
Endophytes were isolated from achenes after ethanol–bleach surface sterilization of plant tissues (Schulz et al., 1993; method II: 30 s in 96% ethanol, 3 min in 15% NaOCl solution, and 30 s in ethanol), and cultured on potato dextrose agar (PDA). In all, 8763 achenes were used for isolation. Most of the endophyte isolates began to grow within the first 10 d of plating. The cultures were kept for one month or until it became clear that no more fungal isolates would appear. Each isolate received its own cultivation identification number (CID). After isolation, cultures were classified morphologically (microscopic and culture characters were used), and selected isolates (one per group) were subcultured. DNA from all pure isolates was sequenced (described next) to assist in species identification. All fungi were grown at 19°C; "blue" (near-ultraviolet) light was used to induce sporulation (Dahlberg and Van Etten, 1982).
Sequence-based identification
More than 200 (288) pure cultures were selected for DNA extraction, amplification, and sequencing. Because most of our cultures did not adequately express diagnostic morphological characters, we used ITS sequence data to assist in species identification, specifically the region amplified with universal fungal primers ITS1 and ITS4 (White et al., 1990). PCR was carried out as follows: the reaction mixture in a total volume of 25 µL contained a final concentration of 1.5 mM MgCl2, 0.5 mM dNTPs (Applied Biosystems, Foster City, California, USA), 10 pmol of each primer, 1.5 U AmpliTaq DNA polymerase (Applied Biosystems), and 30–60 ng genomic DNA. Samples were incubated in a thermal cycler at 94°C for 4 min, followed by 35 cycles of 94° for 1 min, 54° for 1 min, 72° for 1 min; and finally 72° for 3 min. Single-band PCR products were purified using ExoSAP-IT (USB, Cleveland, Ohio, USA) according to the manufacturer’s instructions. Purified PCR products were then sequenced in both directions with the ITS1 and ITS4 primers. Sequencing reaction products were then cleaned using differential precipitation with ethanol. DNA sequences were determined by an ABI 3130xl automated sequencer (Applied Biosystems). Sequences were then aligned to other sequences obtained from the GenBank database using the program Sequencher 4.5 (Gene Codes, Ann Arbor, Michigan, USA).
To improve the quality of identification for Alternaria and Ulocladium (Pleosporaceae), for which ITS sequences do not provide sufficient resolution, we chose the Alt a 1 gene (Alternaria allergen gene). This gene aids resolution among species of these genera (Hong et al., 2005). The primers are described in Hong et al. (2005). PCR conditions for Alt a1 amplification were 3.5 mM MgCl2, 0.5 mM dNTPs (Applied Biosystems), 2 pmol of each primer, 1.5 U AmpliTaq DNA polymerase (Applied Biosystems), and 30–60 ng genomic DNA, in a 25-µL reaction volume. Samples were incubated in a thermal cycler at 94°C for 4 min, followed by 35 cycles of 94° for 1 min, 55° for 1 min, 72° for 1 min; and finally 72° for 3 min. For better identification and subsequent phylogenetic analysis of fungal genus Botrytis (Helotiaceae) samples, we amplified the gene for heat-shock protein 60 (HSP60), which has given the best resolution in phylogenetic analyses of this genus (Staats et al., 2005). Primers and PCR conditions for HSP60 are described elsewhere (Staats et al., 2005). For DNA-based identification, we used the standard NCBI GenBank (Bethesda, Maryland, USA) nucleotide BLAST search with unmodified options. Then we checked the results with 100% identity and used them as species designations for our endophytes. If sequence similarity was less than 100%, we used the most similar taxa for generic determination only. We did not use a 95% cutoff value as a species boundary (Arnold and Lutzoni, 2007) because in some instances fungi with just a single nucleotide difference could satisfy some species criteria (discussed later). Instead, we used the neutral term haplotype for all of our distinct sequences (Table 1). The name of each haplotype contains its generic abbreviation, CID number, and letter(s) for "Alt a 1" sequence, such as "alt002b" (Alternaria CID 002, "b" type of Alt a 1. Sequences for all unique haplotypes were deposited in GenBank (122 sequences, accessions EF589828-EF589908, EF682095–EF682126 and EU386596–EU386605, see also Table 1).
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) to calculate the distance matrix between communities based on phylogenetic distances from a given phylogenetic tree. This tree (based on aligned ITS data) was calculated using the program PAUP* version 4.0b10 (Swofford, 2003) under maximum parsimony criterion with heuristic search. The search used the stepwise addition option and was repeated 1000 times from different starting points with tree-bisection-reconnection (TBR) branch swapping; all characters were equally weighted and unordered, and alignment gaps were treated as missing data. Finally, the 50% majority rule consensus tree rooted with the muc285 haplotype was constructed. The resulting tree (not shown) coincided well with a recently published phylogeny of fungi (Hibbett et al., 2007).
To understand the level of taxonomic novelty in our endophytes, we used data from our Botrytis haplotypes. Botrytis is an ideal taxon to use because all 22 recognized species of this genus have been sequenced. In addition to our sequences, we used GenBank data for 21 species (excluding Botrytis xallii) (Staats et al., 2005). In total, we used 61 operational taxonomic units (OTUs). All sequences were aligned with the program CLUSTAL_X (Thompson et al., 1997) using gap opening cost = 15, gap extension cost = 6.66, and IUB weight matrix, then corrected by eye. The most parsimonious trees based upon HSP60 DNA sequences were obtained by heuristic search using TBR branch swapping on starting trees generated by random sequence addition (1000 replications) using PAUP* 4.0.10b (Swofford, 2003). Bootstrap values were evaluated by 500 replications using a heuristic search with simple sequence addition, TBR branch swapping, and MULTREES. The HSP60 sequences of Monilinia fructigena and Sclerotinia sclerotiniorum were used as outgroups. The best-fit models of evolution for the data sets were chosen through a likelihood ratio test using the program MrModeltest version 2.2 (Nylander, 2004). Models obtained were used for Bayesian analyses using the Markov chain Monte Carlo method (MCMC). MCMC chains were run for 2000000 generations, sampling every 100th generation resulting in 20000 trees. The first 5000 trees were discarded as burn-in, and the remaining 12000 trees were summed to calculate the posterior probabilities. Functional characters were mapped onto the molecular phylogeny using the program MacClade version 4.06b (Maddison and Maddison, 2000).
For most statistical data analysis, the R statistical environment (R Development Core Team, 2007) and specifically its "vegan" package (Oksanen et al., 2007) were used. Haplotype-accumulation curves were generated for endophytes from each range of knapweed using the program EstimateS 8.0 (Colwell, 2006). Web GIS Google Earth (Google) was used to determine precise geographical coordinates for the collection sites of samples.
RESULTS
Endophyte diversity in native and invaded ranges
Some knapweed achenes did not yield any endophyte isolates. A few gave rise to multiple isolates, as can occur in endophyte studies (Sahashi et al., 1999). Overall, the numbers of isolates per plant and per achene were 8.1 and 0.26, respectively. Isolation frequencies per plant and per achene were higher in the native than in the invaded range of C. stoebe: 0.35 and 13.71 vs. 0.19 and 5.21, respectively. In all, 2291 fungal isolates and 92 combined (ITS + Alt a 1 + HSP60) haplotypes were obtained (Table 1). On average, each site revealed five different haplotypes. Endophyte isolates belonged to five different classes of Pezizomycotina (Hibbett et al., 2007), as well as to Saccharomycetes (Eremothecium spp.), Agaricomycetes (Coprinus sp.) and Zygomycetes (Mucor sp.). Most of the endophytes (i.e., 88.7% of isolates from the native range and 83.8% from the invaded range) belonged to Dothideomycetes, in particular Pleosporales. Some haplotypes belonged to groups that are not commonly isolated as plant endophytes: Coprinus sp., Mucor sp., and Pezizales (Arnold and Lutzoni, 2007; Higgins et al., 2007). The most frequent genera of endophytes were Alternaria, Botrytis, and Fusarium (Table 1). Plant-based haplotype accumulation curves were not asymptotic, and the numbers of predicted haplotypes (Chao index: 82 ± 15 for the native range, and 135 ± 47 for the invaded range) were significantly greater than the number of observed haplotypes (52 and 50, respectively), especially for the invaded range (fig. 2).
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), endophyte floras of knapweed plants in the native and invaded ranges differed in the abundance of their most frequent haplotypes (Fig. 3). One haplotype, alt002b, dominated the community of the native range at 43.54% relative abundance (Table 1); no other haplotype in the native range exceeded 12% relative abundance. Alt002c and alt002a were the second and third most common haplotypes of the native range at 11.7% and 6.46%, respectively. In striking contrast, the invaded range lacked any dominant haplotype equivalent to alt002b in the native range. Instead, the invaded range was characterized by a number of more or less equal haplotypes, none of which exceeded 16% relative abundance: alt002a, epi066, cla063, alt002b, and aub061 were the top five, in descending order (Table 1). Most of the 92 haplotypes were relatively rare; 78 of the 92 haplotypes, or 85%, occurred in only one of the two ranges. The most abundant of the haplotypes that were restricted to one range only was alt002m at 3.8% abundance in the native range. Of the 78 one-range haplotypes, 69 were present in that one range at less than 1% abundance. Haplotypes such as bot079, ple015p, and epi497 that were present in both ranges at low frequency did not have a range preference, according to a one-sample proportion test (Fig. 3).
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). These indices take into account "unseen shared species" and thus reduce undersampling bias (Unterseher et al., 2008). They were used for calculating distances between endophyte communities. The matrix of distances were then used as the source of principal coordinates analysis, and the results were plotted on the two-dimensional subspace of the first two coordinates (Fig. 4A). In this plot, endophyte communities from the native and invaded ranges were more or less clearly separated (ANOSIM [ANalysis Of [dis]SIMilarity; Oksanen et al., 2007] values based on 1000 permutations: R = 0.2303, P < 0.01).
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). In this case (Fig. 4B), ANOSIM differences were not significant (R = –0.063, P = 0.56). Three samples from eastern North America were located among Eurasian samples in both plots. This finding was supported with ANOSIM; differences between endophyte communities from the knapweed native range and the communities from eastern North America were not significant (R = 0.014, P = 0.44).
In three sites, our sampling was repeated after an interval of two years (2004 and 2006). Isolation rates in the repeated samples differed substantially (a more than twofold difference) between years. The positions of points belonging to these repeated samplings on the first ordination plot (Fig. 4A, points marked with italic numbers) suggest that the magnitude of difference between samples from various years is at least comparable with the magnitude of differences between samples from various localities. Nonparametric MANOVAs ("adonis" analysis: Oksanen et al., 2007) based on 1000 permutations and Chao dissimilarities indicated that community structure differed between years (F = 2.557, P < 0.001), but not among localities (F = 1.004, P = 0.668) nor for years nested in localities (F = 3.6850e+15, P = 0.171), suggesting that the difference between years is greater than the difference between samples.
Novelty among endophytes of Centaurea stoebe: the case of Botrytis
Of the ITS sequences, 32.8% were identical to sequences in GenBank, 29.9% had 99% identity, and 37.3% had 90–98% identity. For Alt a 1 sequences, only 21.2% had identical sequences in GenBank, and 30.3%, 45.5%, and 0.03% sequences had 99%, 90–98%, or <90% identity, respectively. In some cases, even haplotypes with 99% similarity in ITS sequences demonstrated different distributions. As an example, Cladosporium haplotypes cla063 and cla307 were distributed differently in the two ranges of C. stoebe (Fig. 5).
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fig. 2). Most branches had strong bootstrap and Bayesian support, with the exception of the branch leading to bot360 and bot361 (Fig. 6). In many cases, branches leading to endophytes as terminal taxa (bot360 and bot361; bot079 and bot378) or branches supporting groups of related haplotypes (bot080, bot093 and bot109; bot095 and bot1093) were longer or at least comparable with branches leading to the recognized species of Botrytis. On the basis of relative branch lengths and node support, we identified six groups of novel Botrytis endophytes that should be regarded as undescribed species: (1) bot079, (2) bot378, (3) bot095 + bot1093, (4) bot080 + bot093 + bot109, (5) bot360 and (6) bot361. In contrast, the bot017 haplotype belonged to the B. cinerea group.
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, Fig. 6 B). This effort showed that the evolution of endophytism from parasitism has occurred at least twice in Botrytis (Fig. 7A). The transition from eudicots to monocots as hosts of Botrytis appeared to have evolved independently three times (i.e., at the nodes leading to B. porrii, the B. sp.-B. squamosa group, and the B. aclada group, see Fig. 7B).
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Endophyte communities of plants are typically dominated by a few species or haplotypes. For example, Venturia ditricha was the dominant endophyte of Betula pubescens subsp. czerepanovii in Finland (Ahlholm et al., 2002), whereas a haplotype of Lophodermium s. l. dominated the endophyte community of Pinus monticola in its native range (Ganley and Newcombe, 2006). The endophyte community of C. stoebe in its native range followed this rule with a dominant member, the alt002b haplotype of Alternaria alternata, at 44% abundance, and 53 other haplotypes, all at less than 11.7% abundance including 14 singletons at 0.08% (Table 1). In contrast, the endophyte community of C. stoebe in its invaded range lacked a dominant member equivalent to alt002b, although the latter was present in the invaded range at 10.39% abundance and consisted of many rare haplotypes including 13 singletons at 0.11%. The lack of a dominant member of the community in the invaded range does not clearly support either cointroduction or host-jumping.
Despite robust sampling, our accumulation curves were not asymptotic, suggesting that we have not exhaustively sampled endophyte species or haplotype diversity in C. stoebe. Further sampling would likely reveal more diversity, especially for the invaded range where twice the number of observed haplotypes was predicted by the Chao index of extrapolated species richness. Our preliminary environmental sequences (Arnold et al., 2007) based on the DNA extracted from a single plant (A. Shipunov, G. Newcombe, and A. Raghavendra, unpublished data) show that even plants with 0% isolation frequency from achenes contain DNA of some common endophyte haplotypes (like cla063) in vegetative parts. Thus, endophytes may be absent from the achenes of a plant that is otherwise colonized by endophytes, so further sampling of leaves, stems, or roots of C. stoebe might be more efficient than further sampling of achenes.
A low overall isolation frequency of 0.26 endophyte isolates per achene (i.e., 2291 isolates from 8863 achenes) suggested that competition among isolates within the small (i.e., 3–4 mm3) achenes did not exacerbate undersampling. Repeated sampling from the same host populations is consistent with previous investigations of the seasonal fluctuations of endophyte communities (Sahashi et al., 1999; Gao et al., 2005).
Comparisons of communities like ours, with many rare members, may be misleading due to a bias in undersampling. Most recently, a new statistical approach to reduce undersampling bias, the Chao similarity index (Chao et al., 2005), has been developed. Using this approach (Fig. 4A), we found that the endophyte communities of the two ranges are distinct. The difference is greater than expected by chance, providing support for host-jumping. Host-jumping was also suggested by the high number of haplotypes that were found only in the invaded range (Table 1). At the same time, we cannot rule out cointroduction because 13 of 20 haplotypes frequent in the native range also occurred in the invaded range (Fig. 3).
In the phylogenetic ordination analysis (Fig. 4B), the difference between endophyte communities of the two ranges disappeared. In other words, phylogenetic patterns support the similarity between fungal communities from native and invaded ranges. This similarity is likely a result of both cointroduction of the most frequent endophytes (as discussed earlier) and host-jumping from a phylogenetically similar suite of species. The most outstanding example of the latter is five haplotypes of Ulocladium that were isolated from C. stoebe only in the invaded range (with individual abundances from 0.21 to 2.25). Phylogenetically, Ulocladium is a part of Alternaria s. l. (Hong et al., 2005), and these haplotypes were also isolated from plants native to the Pacific Northwest (A. Shipunov, G. Newcombe, and A. Raghavendra, unpublished data).
Interestingly, western North America and Europe can be distinguished by climatic niche spaces for C. stoebe (Broennimann et al., 2007). Although a minor focus in this study, the similarity of endophyte communities of C. stoebe in eastern North America and the native range in both ordinations is intriguing in this context. Climate matching has not been attempted for eastern North America and the native range of C. stoebe, but it may still be interesting to speculate on the role of climate in relation to abundance shifts in endophyte communities.
In sum, because both cointroduction and host-jumping were suggested by our data, endophytes of the invaded range in western North America may be a mixture of Eurasian endophytes that could function as novel weapons and North American endophytes that could enhance mutualism. Other studies of cointroduction vs. host-jumping of fungi have been similarly inconclusive (Tedersoo et al., 2007; White and Backhouse, 2007). Had our study revealed endophytes with known geographic ranges in either Eurasia or North America, we could then have drawn conclusions on cointroduction or host-jumping for individual haplotypes even if the community analyses were problematic due to undersampling bias. Although we did obtain endophytes (i.e., the asterisked haplotypes of Table 1) that matched named taxa in GenBank, none of these taxa could be linked to particular geographic ranges. The undescribed species that we isolated from achenes of C. stoebe (e.g., the six haplotypes of Botrytis) represent an outstanding example of species that, by definition, do not have known geographic ranges.
One third of the haplotypes from our study had less than 98% identity with their closest relatives from GenBank; this may be evidence that we have isolated more than 30 new species of fungi from C. stoebe. Species of fungi can be distinguished by 1-bp differences in their ITS sequences, particularly if there is also evidence for more rapid evolution of other genes (Schubert et al., 2007). Our phylogenetic analysis of the Alternaria-Ulocladium group suggests at least nine new lineages belonging to these genera (A. Shipunov, G. Newcombe, and A. Raghavendra, unpublished data). However, this conclusion is preliminary because sequence data exists for only a small fraction (< 25%) of the species described in this group (Pryor and Gilbertson, 2000; Hong et al., 2005). Therefore, we could have obtained endophytes that have been described but not yet sequenced.
In contrast, Botrytis represents a completely different situation. All 22 recognized species have been sequenced (Staats et al., 2005). Remarkably, we found six groups of novel Botrytis endophytes for which there appears to be phylogenetic evidence that they represent undescribed species (Fig. 6). These findings are especially noteworthy in light of the complete absence of any records of Botrytis as a pathogen of C. stoebe (Farr et al., 2008); Botrytis cinerea is the only Botrytis sp. that has ever been reported on Centaurea, and only on C. cyanus and C. moschata. This latter point is tempered somewhat by the fact that all the other 30 fungal genera reported in this study are "first reports" for C. stoebe globally (Farr et al., 2008). Interestingly, the addition of our endophytes to the Botrytis phylogenetic tree provided some new insights into the evolution of host specificity and lifestyle (Fig. 7). Contrary to Staats et al. (2005), it seems likely that there were no secondary transitions of Botrytis from monocots to eudicots, because eudicots appeared to be the primary and ancestral hosts for the genus. According to the character mapping, Botrytis endophytes appear to have evolved from parasites, which is not surprising (Carroll, 1988; Saikkonen et al., 1998).
Because the host ranges of knapweed endophytes are not known, it is impossible to estimate the ratio of endophyte to host species. However, our results proved that a thorough investigation of endophyte diversity from one plant species may substantially increase the number of known fungal species (e.g., six new species added to a genus, Botrytis, of 22 known species). Estimates of 1.5 million species (Hawksworth, 2001) or even 9.9 million species (Cannon, 1997) of fungi look feasible, given that there are approximately 250000–420000 species of vascular plants (Govaerts, 2001; Thorne, 2002).
FOOTNOTES
1 Funding was provided by the Center for Research on Invasive Species and Small Populations of the University of Idaho. The authors thank T. Prather, L. Wilson, M. Baynes, C. Bennett, P. Haefliger, H. Mueller-Schaerer, L. Abramova, A. Laktionov, V. Popov, R. Hufbauer, A. Blair, D. Naumoff, V. Saveljev, P. Kulikov, P. Koeniger, T. Heekin, E. Nikolaeva, S. Majorov, S. Beer, A. Serebryanaya, I. Zhemchugova, G. Klinkova, and I. Schanzer for additional seedhead collections and S. Lassen, M. Crawford, and R. Ganley for technical help. 
5 Author for correspondence (e-mail: dactylorhiza{at}gmail.com)
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