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Understanding the factors that drive the evolution of pathogenic fungi is central to revealing the mechanisms of virulence and host preference, as well as developing effective disease control measures. Prerequisite to these pursuits is the accurate delimitation of species boundaries. Colletotrichum gloeosporioides s.l. is a species complex of plant pathogens and endophytic fungi for which reliable species recognition has only recently become possible through a multi-locus phylogenetic approach. By adopting an intensive regional sampling strategy encompassing multiple hosts within and beyond agricultural zones associated with cranberry (Vaccinium macrocarpon Aiton), we have integrated North America strains of Colletotrichum gloeosporioides s.l. from these habitats into a broader phylogenetic framework. We delimit species on the basis of genealogical concordance phylogenetic species recognition (GCPSR) and quantitatively assess the monophyly of delimited species at each of four nuclear loci and in the combined data set with the genealogical sorting index (gsi). Our analysis resolved two principal lineages within the species complex. Strains isolated from cranberry and sympatric host plants are distributed across both of these lineages and belong to seven distinct species or terminal clades. Strains isolated from V. macrocarpon in commercial cranberry beds belong to four species, three of which are described here as new. Another species, C. rhexiae Ellis & Everh., is epitypified. Intensive regional sampling has revealed a combination of factors, including the host species from which a strain has been isolated, the host organ of origin, and the habitat of the host species, as useful indicators of species identity in the sampled regions. We have identified three broadly distributed temperate species, C. fructivorum, C. rhexiae, and C. nupharicola, that could be useful for understanding the microevolutionary forces that may lead to species divergence in this important complex of endophytes and plant pathogens.

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Citation: Doyle VP, Oudemans PV, Rehner SA, Litt A (2013) Habitat and Host Indicate Lineage Identity in Colletotrichum gloeosporioides s.l. from Wild and Agricultural Landscapes in North America. PLoS ONE 8(5): e62394.

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Funding: Financial support from the National Science Foundation [www.nsf.gov] (DBI 0749751 and DEB 1011120 - Doctoral Dissertation Improvement Grant), the Torrey Botanical Society Graduate Research Fellowship [www.torreybotanical.org], and The New York Botanical Garden [www.nybg.org] helped to fund this research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All necessary permits were obtained for the described field studies from the Delaware Division of Parks and Recreation, the National Forest Service, and The Nature Conservancy. All other samples collected in this study were on private land and did not require permits. There were no endangered or protected species collected for this study.

Sequences were automatically assembled into contigs, and edited manually in Sequencher version 4.9 (GeneCodes Corp., Ann Arbor, Michigan). Alignments were carried out with the online version of the sequence alignment program MAFFT version 6 [41], [42] using the iterative refinement option G-INS-i for each locus independently. GenBank accession numbers for sequences generated for this study are provided in Table S1.

Microscopic observations of conidia, phialides, and ascospores were made from specimens mounted in water. Hyphal appressoria were observed using slide cultures; a 10 mm2 block of CMA was placed on a CMA plate, each of the four corners of the block was inoculated, covered with a sterile coverslip, and incubated at 25C. Microscopic observation of perithecial development on cranberry fruit was made by surface sterilizing symptomatic field collected fruit, cutting in half transversely, placing face down on V8 agar, and incubating at room temperature (22C) for approximately 3 weeks. The fruit was removed from the plate after perithecial development and fixed in FAA (3.7% formaldehyde, 5% glacial acetic acid, and 50% ethanol) before dehydration in an alcohol-xylene dehydration series and embedded in Paraplast X-TRA (Leica Microsystems, Buffalo Grove, IL, USA). The fruit was sectioned at 8 µm and stained with acid fuchsin and Cotton blue in lactic acid and mounted in Permount (Fisher Scientific, Pittsburgh, PA, USA).

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In order to test the hypothesis that multiple sympatric lineages within the C. gloeosporioides species complex infect cranberry in the field, North American isolates need to be placed in a broader phylogenetic context. Therefore, sequence data generated in this study were combined with an earlier study of isolates of C. gloeosporioides s.l. from the New World tropics [14]. Outgroup sampling was expanded from the aforementioned study to include two isolates of C. aff. acutatum. Individual locus data were analyzed separately to assess the topological congruence among datasets and the utility of each to resolve terminal lineages with robust statistical support for sister group relationships within the C. gloeosporioides species complex. Strain data are summarized in Table 1 and character and tree statistic data are summarized in Table 2.

Sequence data from nrITS has been widely used in fungal phylogenetic studies and has been proposed as a barcode locus for fungi [68]. However, our analysis indicates it neither provides adequate resolution for reliable species assignment, nor does it reliably assess phylogenetic relationships within the C. gloeosporioides species complex, as has been reported in previous studies of Colletotrichum [14], [23], [69]. Despite the low phylogenetic resolution inferred from nrITS data, six nodes within the species complex were supported in more than 75% of the parsimony bootstrapped datasets (Figure S1) and eight nodes in more than 70% of the maximum likelihood bootstrapped datasets (Figure S5). In addition, C. sp. indet. D (4766, 3386, 4801) was determined to be closely related but peripheral to the C. gloeosporioides species complex, indicating this is a suitable outgroup, as previously suggested by Rojas et al. [14].

Phylogenetic analysis of partial tub2 sequence data largely supported the inferences made from nrITS data, but provided further resolution within the species complex, recovering 20 well-supported nodes in both MP and ML analyses (Figures S2 and S6). Isolates from cranberry (V. macrocarpon and V. oxycoccos) were distributed among 5 clades, including lineages originally described from tropical regions.

Phylogenetic analysis of the apn2 locus provided greater resolution for terminal lineages than that achieved with either tub2 or nrITS. Bootstrap analyses provided significant statistical support for 23 nodes and 20 nodes for MP (Figure S3) and ML (Figure S7) analyses, respectively, with six clades in both analyses containing isolates from V. macrocarpon and V. oxycoccos. While support for terminal lineages from apn2 sequence data was greater than that from partial tub2, support for sister group relationships was not as robust.

A similar topology was recovered from the analysis of nucleotide data from apn2/matIGS, the intergenic spacer bridging the apn2 and mating-type locus, with respect to the nrITS, tub2, and apn2 gene trees while providing greater resolution than the other three datasets. Bootstrap analyses of apn2/matIGS recovered strong branch support for 25 and 29 nodes for MP (Figure S4) and ML (Figure S8), respectively. In agreement with the apn2 analysis, six lineages include isolates from V. macrocarpon and V. oxycoccos. In addition to increased resolution of terminal clades within the species complex, compared with apn2, support for sister group relationships within the species complex is stronger than inferences based on partial tub2.

The primary concordance (PC) tree estimated with Bayesian concordance analysis from the four nuclear markers remained unchanged regardless of the value of the discordance parameter (α). The PC tree (Figure 2) is consistent with the assignment of individual isolates to terminal lineages on the basis of the concatenated analysis. Concordance factors are reported for all nodes above the species level. The low concordance factors (below 0.5) for several species, including C. fructicola, C. asianum, C. siamense, C. kahawae, C. rhexiae, and C. fructivorum appear to be due to the lack of resolution provided by individual markers rather than topological discordance. Similar to the analysis of the combined data, two principal lineages were resolved in the PC tree. However, relationships among a few species within these two lineages are distinctive from the combined analysis. For example, Colletotrichum theobromicola is placed within the principal lineage that includes C. gloeosporioides, and C. asianum is no longer sister to C. tropicale. 041b061a72