Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass

Hettiarachchige et al. BMC Evolutionary Biology (2015) 15:72 DOI 10.1186/s12862-015-0349-6 RESEARCH ARTICLE Open Access Phylogenomics of asexual Epichloë fungal endophytes forming associations with perennial ryegrass Inoka K Hettiarachchige1,2,4, Piyumi N Ekanayake1,3,4, Ross C Mann1,4, Kathryn M Guthridge1,3,4, Timothy I Sawbridge1,3,4, German C Spangenberg1,2,3,4 and John W Forster1,2,3,4* Abstract Background: Perennial ryegrass (Lolium perenne L.) is one of the most important species for temperate pastoral agriculture, forming associations with genetically diverse groups of mutualistic fungal endophytes. However, only two taxonomic groups (E. festucae var. lolii and LpTG-2) have so far been described. In addition to these two well-characterised taxa, a third distinct group of previously unclassified perennial ryegrass-associated endophytes was identified as belonging to a putative novel taxon (or taxa) (PNT) in a previous analysis based on simple sequence repeat (SSR) marker diversity. As well as genotypic differences, distinctive alkaloid production profiles were observed for members of the PNT group. Results: A detailed phylogenetic analysis of perennial ryegrass-associated endophytes using components of whole genome sequence data was performed using complete sequences of 7 nuclear protein-encoding genes. Three independently selected genes (encoding a DEAD/DEAH box helicase [Sbp4], a glycosyl hydrolase [family 92 protein] and a MEAB protein), none of which have been previously used for taxonomic studies of endophytes, were selected together with the frequently used ‘house-keeping’ genes tefA and tubB (encoding translation elongation factor 1-α and β-tubulin, respectively). In addition, an endophyte-specific gene (perA for peramine biosynthesis) and the fungal-specific MT genes for mating-type control were included. The results supported previous phylogenomic inferences for the known species, but revealed distinctive patterns of diversity for the previously unclassified endophyte strains, which were further proposed to belong to not one but two distinct novel taxa. Potential progenitor genomes for the asexual endophytes among contemporary teleomorphic (sexual Epichloë) species were also identified from the phylogenetic analysis. Conclusions: Unique taxonomic status for the PNT was confirmed through comparison of multiple nuclear gene sequences, and also supported by evidence from chemotypic diversity. Analysis of MT gene idiomorphs further supported a predicted independent origin of two distinct perennial ryegrass-associated novel taxa, designated LpTG-3 and LpTG-4, from different members of a similar founder population related to contemporary E. festucae. The analysis also provided higher resolution to the known progenitor contributions of previously characterised perennial ryegrass-associated endophyte taxa. Keywords: Pasture grass, Whole genome sequencing, Taxonomy, Nuclear gene, Mating type, Progenitor * Correspondence: 1 Department of Economic Development, Jobs, Transport and Resources, Biosciences Research Division, AgriBio, Centre for AgriBioscience, Bundoora, Melbourne, Victoria 3083, Australia 2 School of Applied Systems Biology, La Trobe University, Bundoora, Melbourne, Victoria 3086, Australia Full list of author information is available at the end of the article © 2015 Hettiarachchige et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hettiarachchige et al. BMC Evolutionary Biology (2015) 15:72 Background The genus Lolium, which belongs to the sub-family Pooideae (cool-season grasses) of the grass and cereal family Poaceae, includes several important forage and turf species [1,2]. Perennial ryegrass (Lolium perenne L.) is extensively cultivated for pasture production on a global basis [3,4]. Like other cool-season grasses, perennial ryegrass is often infected with clavicipitaceous fungal endophytes that include both sexual and asexual taxa [5-7]. The asexual (anamorphic) taxa were previously assigned to a separate genus (Neotyphodium), but in accord with general recommendations for fungal taxonomy, have been recently been combined with the sexual (teleomorphic) taxa within a single genus, designated Epichloë [8]. Asexual Epichloë endophytes colonise the intercellular spaces of leaf sheaths, culms, and rhizomes, and infrequently the surface of leaf blades, without inducing obvious pathological symptoms [9]. These asexual endophytes do not produce stromata, and rely solely on the host plant for transmission [10,11]. The vegetative phase of growth for sexual is similar to that of asexual Epichloë species [12], but in the sexual stage, stromata are formed around the developing inflorescences and prevent emergence of the floral meristem [7,12]. Asexual Epichloë species form mutualistic associations with their hosts [13,14]. Benefits for the host related to abiotic stress tolerance are obtained through enhanced growth, increased seedling vigour and persistence, particularly under water stress and nutrient deficiency [15,16]. The endophyte also confers biotic stress tolerance to the host grass, through production of several classes of biologically active alkaloids. Peramine and loline produced by the endophyte are active against insect pests, while lolitrem B and ergovaline are toxic to mammalian herbivores [17-19]. Conversely, as part of the symbiosis, the plant provides certain benefits to the endophyte such as shelter, nutrition, reproduction and distribution [14,20,21]. Perennial ryegrass has been found to be host to two distinct fungal endophyte taxa: Epichloë festucae var. lolii (Latch, Christensen and Samuels) Bacon and Schardl syn. N. lolii (Latch, Christensen and Samuels) Glenn, Bacon and Hanlin [22] and Lolium perenne taxonomic group 2 (LpTG-2) [23]. Distinct taxonomic groups of asexual Epichloë endophytes are proposed to have evolved either by direct evolution from a single teleomorphic species, probably due to loss of the sexual state, or through interspecific hybridisation events between either sexual species or distinct sexual and asexual lineages, the latter generating heteroploid genetic constitutions [24-27]. The haploid taxon E. festucae var. lolii has been identified as a direct derivative of Epichloë festucae Leuchtm., Schardl & M. R. Siegel while the heteroploid LpTG-2 arose as an interspecific hybrid between E. festucae var. lolii and E. typhina (Pers.) Tul. & C. Tul. [24,26]. Page 2 of 14 Previous phylogenetic characterisation studies of both sexual and asexual Epichloë species have largely been based on the use of par (...truncated)