Discord between morphological and phylogenetic species boundaries: incomplete lineage sorting and recombination results in fuzzy species boundaries in an asexual fungal pathogen

Abstract

BackgroundTraditional morphological and biological species concepts are difficult to apply to closely related, asexual taxa because of the lack of an active sexual phase and paucity of morphological characters. Phylogenetic species concepts such as genealogical concordance phylogenetic species recognition (GCPSR) have been extensively used; however, methods that incorporate gene tree uncertainty into species recognition may more accurately and objectively delineate species. Using a worldwide sample of Alternaria alternata sensu lato, causal agent of citrus brown spot, the evolutionary histories of four nuclear loci including an endo-polygalacturonase gene, two anonymous loci, and one microsatellite flanking region were estimated using the coalescent. Species boundaries were estimated using several approaches including those that incorporate uncertainty in gene genealogies when lineage sorting and non-reciprocal monophyly of gene trees is common.ResultsCoalescent analyses revealed three phylogenetic lineages strongly influenced by incomplete lineage sorting and recombination. Divergence of the citrus 2 lineage from the citrus 1 and citrus 3 lineages was supported at most loci. A consensus of species tree estimation methods supported two species of Alternaria causing citrus brown spot worldwide. Based on substitution rates at the endo-polygalacturonase locus, divergence of the citrus 2 and the 1 and 3 lineages was estimated to have occurred at least 5, 400 years before present, predating the human-mediated movement of citrus and associated pathogens out of SE Asia.ConclusionsThe number of Alternaria species identified as causing brown spot of citrus worldwide using morphological criteria has been overestimated. Little support was found for most of these morphospecies using quantitative species recognition approaches. Correct species delimitation of plant-pathogenic fungi is critical for understanding the evolution of pathogenicity, introductions of pathogens to new areas, and for regulating the movement of pathogens to enforce quarantines. This research shows that multilocus phylogenetic methods that allow for recombination and incomplete lineage sorting can be useful for the quantitative delimitation of asexual species that are morphologically indistinguishable. Two phylogenetic species of Alternaria were identified as causing citrus brown spot worldwide. Further research is needed to determine how these species were introduced worldwide, how they differ phenotypically and how these species are maintained.

A new high‐resolution melting assay for genotyping Alternaria species causing citrus brown spot

“…24 In particular, the SCAR marker OPA1-3 proved to be highly efficient in generating discriminant sequences. 6,9,20 In the present study, the alignment of the OPA1-3 sequences, belonging to Alternaria reference strains of the species/morphotypes, more commonly reported on citrus, showed evidence of a high degree of variability. Different informative SNPs were found that encouraged the screening of the entire Alternaria collection available by an HRM approach, since this technique allows the detection of even single base variations among samples.…”

“…Concerning the first one, several genetic regions with higher sequence variation, compared to more classical barcoding regions such as ITS or endopolygalacturonase, were identified in Alternaria genus, and used to estimate phylogenies among small‐spored species . In particular, the SCAR marker OPA1–3 proved to be highly efficient in generating discriminant sequences . In the present study, the alignment of the OPA1–3 sequences, belonging to Alternaria reference strains of the species/morphotypes, more commonly reported on citrus, showed evidence of a high degree of variability.…”

“…19 The specific SCAR marker OPA1-3 has been employed successfully in the delineation of closely related species of the genus Alternaria, since it contains enough SNPs to differentiate species within the genus. 6,20 As such, this marker was used in the present investigation to set up an HRM assay able to genotype an Alternaria spp. population from citrus fruit and leaves collected in different geographical areas of southern Europe.…”

Contrasting species boundaries between sections Alternaria and Porri of the genus Alternaria

“…Stewart et al . () found a few putative recombination events in two anonymous DNA regions (Flank 3 and OPA1.3) for citrus brown pathogen A. alternata populations. Alternaria alternata / A. tenuissima populations from Pistacia spp.…”

“…Recently, Stewart et al . () revealed phylogenetically distinct lineages within the citrus brown pathogen A. alternata from a worldwide collection of isolates, and Huang et al . () presented a novel phylogenetic reconstruction for the same pathogen in China.…”