Epichloe Novae-Zelandiae, a New Endophyte from the Endemic New Zealand Grass Poa Matthewsii Adrian Leuchtmann A, Carolyn A

NEW ZEALAND JOURNAL OF BOTANY https://doi.org/10.1080/0028825X.2019.1651344

RESEARCH ARTICLE Epichloe novae-zelandiae, a new endophyte from the endemic New Zealand grass Poa matthewsii Adrian Leuchtmann a, Carolyn A. Young b, Alan V. Stewartc, Wayne R. Simpsond, David E. Humed and Barry Scott e aPlant Ecological Genetics, Institute of Integrative Biology, ETH Zurich, Zürich, Switzerland; bNoble Research Institute, LLC., Ardmore, OK, USA; cPGG Wrightson Seeds Ltd., Lincoln, New Zealand; dGrasslands Research Centre, AgResearch Ltd., Palmerston North, New Zealand; eGenetics Group, School of Fundamental Sciences, Massey University, Palmerston North, New Zealand

ABSTRACT ARTICLE HISTORY Epichloe endophytes (Clavicipitaceae) infect pooid grass genera Received 10 June 2019 worldwide but predominantly in the Northern Hemisphere, but Accepted 29 July 2019 appear to be rare in native grasses of the Southern Hemisphere. First published online 15 Because of benefits that hosts may receive from the symbiosis, August 2019 Epichloe endophytes have been extensively studied and are HANDLING EDITOR considered important components of sustainable agriculture. Eric McKenzie There are only a few studies available on the incidence of endophyte infection in grasses of the Southern Hemisphere and KEYWORDS most grass species have never been examined. Here we report on Alkaloid genotype; a survey of native grasses of New Zealand including 25 endemic Dichelachne micrantha; or indigenous species. We sampled up to 10 plants per species at endemic New Zealand different sites from both the North and South Island of New grasses; endophyte infection; Epichloe novae-zelandiae; Zealand and examined tissues microscopically for endophyte interspecific hybrid; Poa infection. Overall, only two species were found to be infected, Poa matthewsii matthewsii (Matthew’s bluegrass) and Dichelachne micrantha (short-hair plume grass). Based on analyses of tefA and tubB genes, both endophytes were found to be interspecific hybrids. The endophyte of the new host D. micrantha was previously described as Epichloe australiensis, while the endophyte of P. matthewsii is a new species named here E. novae-zelandiae. The new species is a hybrid derived from E. amarillans, E. bromicola and E. typhina subsp. poae. Alkaloid analyses in planta suggested that E. novae-zelandiae can produce small amounts of peramine, early pathway indole-diterpenes and ergot alkaloids, but no lolines or lolitrems. Target specific primers suggested the presence of genes for ergot alkaloids and peramine, but genes of only early pathway steps for the other alkaloids. Furthermore, genes for both mating type idiomorphs (MTA and MTB) were present, a single copy of MTA and two copies of MTB. Endophytes of native grasses may provide a genetic resource that could be exploited for developing pasture grass cultivars with improved performance.

CONTACT Adrian Leuchtmann [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/0028825X.2019.1651344 © 2019 The Royal Society of New Zealand 2 A. LEUCHTMANN ET AL.

Introduction Epichloe endophytes form natural associations with many pooid grass genera predominantly in the Northern Hemisphere (Schardl 2010). They systemically infect above ground parts of the grasses and mostly remain symptomless. Some species, however, form external fruiting structures thus causing choke disease, and impairing flowering and seed set. Diversification of symptomless, asexual species has frequently occurred by interspecific hybridisation resulting in allopolyploid species with genomes from two or three parental species (Moon et al. 2004; Campbell et al. 2017). The more widespread asexual endophytes are extensively studied because hosts may receive substantial benefits from the symbiosis including increased resistance to drought, herbivores and pathogens (West 1994; Bazely et al. 1997; Clarke et al. 2006), and are therefore considered important components of sustainable agriculture (Kauppinen et al. 2016). Since the early twentieth century, Epichloe endophytes have been also reported from grasses native to South America, South Africa and Australia (Gibbs Russel and Ellis 1982; Miles et al. 1998; Cabral et al. 1999). Several of these grasses including species of Festuca, Poa, Melica and Echinopogon have been associated with toxicoses in grazing animals causing staggers or drunken-like behaviour (Everist 1981; Pomilio et al. 1989). However, com- pared to the Northern Hemisphere, endophyte infection appears to be much less common in native grasses of the Southern Hemisphere, although introduced grasses are frequently infected in agricultural settings of Southern Hemisphere countries. After separation of New Zealand from Australia with the opening of the Tasman Sea some 80 million years ago, a very rich endemic flora developed from descendants of the Gondwana biota (Molnar et al. 1975; Fleming 1979). According to the ‘Flora of New Zealand’ (Edgar and Connor 2000), 157 grass species are endemic to New Zealand and 31 additional species are indigenous with a shared distribution in Australia and other Pacific islands. There is little knowledge on incidence of endophyte infection in native New Zealand grasses since few of these species have been examined (Davis and Guy 2001; Rolston et al. 2002). So far, two grass species have been found to be infected by Epi- chloe endophytes, namely Echinopogon ovatus (G. Forst.) P. Beauv. (Miles et al. 1998) and Poa matthewsii Petrie (Stewart et al. 2004). Echinopogon ovatus is indigenous to Australia and New Zealand and host of two endophytes. Epichloe aotearoae (C.D. Moon, C.O. Miles &Schardl)Leuchtm.&Schardl,representingaunique,apparentlynon-hybridlineage within the Epichloe genus was isolated from Australian and New Zealand populations. The other species, E. australiensis (C.D. Moon & Schardl) Leuchtm., an interspecific hybrid that involved ancestral lineages closely related to extant E. festucae Leuchtm., Schardl & M.R. Siegel and E. typhina (Pers.) Tul. & C. Tul., was identified from Australian populations only (Moon et al. 2002). The endophyte of the endemic P. matthewsii is unde- scribed, although its morphology and cultural characteristics clearly allowed identification as Epichloe (Stewart et al. 2004). In a previous survey, Rolston et al. (2002) collected grass seeds from 24 native New Zealand grasses (19 of which were endemic), and examined seedlings for endophyte infection using an immunoblot technique and morphological examination. No endophyte infections were detected in any of the grasses analysed, including samples from seven native species of Poa and two native species of Festuca. Likewise, incidental examination of 18 endemic grasses showed no endophyte infection (Davis and Guy 2001). This NEW ZEALAND JOURNAL OF BOTANY 3 contrasts with the common occurrence of endophytes in other species of Poa and Festuca in the Northern Hemisphere and South America. Rolston et al. (2002) suggest that colo- nisation of these grasses by endophytes occurred more recently after the isolation of New Zealand biota from continental populations involving Epichloe endophytes that have evolved in the Northern Hemisphere. However, there is one endophyte that diverged early in the evolution of Epichloe and appears to be unique to the Southern Hemisphere (Moon et al. 2002). Furthermore, introduced species of Festuca and Lolium, including the agriculturally important species Lolium perenne L. (perennial rygrass) and Schedonorus arundinaceus (Schreb.) Dumort. (tall fescue), are found to be commonly infected at roadsides in New Zealand (Rolston et al. 2002). Since most native grasses of New Zealand are unexamined for the presence of Epichloe (approximately 120 species), and as endophytes have been detected in some New Zealand grasses previously, it seemed likely that more grass species may harbour Epichloe endophytes. In the present study, we report results from a recent survey of native New Zealand grasses for detecting endophyte infections. We describe a new species of Epichloe from the endemic New Zealand grass, P. matthewsii, and report a new host of the previously described E. australiensis. In addition, we conducted PCR-based genetic analyses of mating type genes and genes involved in alkaloid synthesis from the new endophyte of P. matthewsii and from E. australiensis, which can predict potential alkaloid profiles of infected host grasses.

Materials and methods Collection and screening for endophyte infection Collection of plant material was conducted from October 2017 to January 2018 at sites on the North Island and South Island of New Zealand (Table 1). One tiller per plant was sampled from up to 10 different plants at a site, stored in plastic bags and taken to the laboratory. Endophyte infection was evaluated microscopically in leaf sheath tissues stained for fungal hyphae with aniline blue (Clark et al. 1983). From all plants that tested positive, endophytes were isolated in pure culture for morphological confirmation and DNA sequencing. Plant species were identified from pressed voucher specimens collected in the field using the keys provided by Edgar and Connor (2000). Further samples from P. matthewsii had been collected in previous years from Port Hills near Christchurch and from 25 natural populations occurring in native forest rem- nants on Banks Peninsula. These populations were screened for endophyte infection by immunoblotting (Simpson et al. 2012).

Endophyte isolation Isolations were made from surface-disinfected plant tissues placed on supplemented malt extract-agar (SMA) containing 1% malt extract, 1% glucose, 0.25% bacto peptone, 0.25% yeast extract, 1.5% bacto agar and 0.005% oxytetracycline (Pﬁzer, New York, NY) in Petri dishes (Leuchtmann 1994). Endophyte colonies growing out from tissues were transferred to fresh media and pure cultures preserved in tubes under mineral oil for long-term storage. An isolate from P. matthewsii (AL0725/2) is deposited at CBS culture collection (Utrecht, The Netherlands) and an isolate from Dichelachne micrantha (Cav.) Domin 4 A. LEUCHTMANN ET AL.

Table 1. Native pooid grasses examined for infection with number of plants, collection sites in New Zealand, and status of grass species (endemic or indigenous). Number of plants Grass species Examined Infected Regiona Collection site Status Chionochloa 5 0 Wellington Tararua Range, Herepai Hut endemic cheesemanii 3 0 Manawatu- Ruahine Range, Rangiwahia Hut Wanganui Chionochloa sp. 1 10 0 Taranaki Mt. Taranaki, Fanthams Peak endemic Chionochloa sp. 2 10 0 Canterbury Mt. Cook, Hooker Valley endemic Cortaderia richardii 5 0 Wellington Tararua Range, Herepai Hut endemic Deyeuxia aucklandica 5 0 West Coast Arthur’s Pass, Otira Valley endemic Deyeuxia avenoides 5 0 Canterbury Korowai-Torlesse Tussocklands Park endemic 6 0 Canterbury Mt. Cook, Hooker Valley 5 0 Canterbury Arthur’s Pass, Scott’s Track 5 0 Canterbury Porters Pass, Bealey Valley Dichelachne crinita 6 0 Canterbury Banks Peninsula, Kaituna Valley Scenic indigenous Reserve 5 0 Canterbury Korowai-Torlesse Tussocklands Park Dichelachne 6 5 Northland Hokianga, Arai-Te-Uru Recreation indigenous micrantha Reserve Elymus solandri 5 0 Canterbury Porters Pass, Bealey Valley endemic Festuca contracta 6 0 Canterbury Banks Peninsula, Site 2 endemic 5 0 Canterbury Mt. Cook, Hooker Valley Festuca mathewsii 5 0 West Coast Arthur’s Pass, Otira Valley endemic Hierochloe cuprea 10 0 Taranaki Mt. Taranaki, Dawson Falls endemic 3 0 Taranaki Mt. Taranaki, Manganui 5 0 Wellington Tararua Range, Herepai Hut 5 0 Waikato Tongariro National Park, Tama Lakes 5 0 Canterbury Mt. Cook, Sealy Tarns 5 0 West Coast Arthur’s Pass, Otira Valley 5 0 Canterbury Korowai-Torlesse Tussocklands Park Koeleria 5 0 Canterbury Mt. Cook, Sealy Tarns endemic novozelandica Lachnagrostis lyallii 1 0 West Coast Franz Josef endemic 5 0 Canterbury Banks Peninsula, Site 2 5 0 Canterbury Mt. Cook, Hooker Valley 5 0 West Coast Arthur’s Pass, Otira Valley Poa buchananii 5 0 Canterbury Banks Penninsula, Site 2 endemic Poa cita 5 0 West Coast Arthur’s Pass, Otira Valley endemic Poa colensoi 10 0 Taranaki Mt. Taranaki, Fanthams Peak endemic 5 0 Taranaki Mt. Taranaki, Manganui 5 0 Canterbury Mt. Cook, Hooker Valley 10 0 Canterbury Mt. Cook, Sealy Tarns 5 0 West Coast Arthur’s Pass, Otira Valley Poa mathewsii 10 7 Canterbury Banks Peninsula, Kaituna Valley Scenic endemic Reserve 5 1 Canterbury Banks Peninsula, Site 2 3 2 Canterbury Banks Peninsula, Site 3 Poa novae-zelandiae 5 0 West Coast Arthur’s Pass, Otira Valley endemic 5 0 Canterbury Mt. Cook, Sealy Tarns 1 0 West Coast Franz Josef Glacier Poa sp. 1 9 0 Taranaki Mt. Taranaki, Dawson Falls endemic Rytidosperma 5 0 Canterbury Mt. Cook, Hooker Valley endemic clavatum Rytidosperma gracile 10 0 West Coast Arthur’s Pass, Otira Valley indigenous 5 0 Canterbury Porters Pass, Bealey Valley 5 0 Manawatu- Woodville, Coppermine Trail Wanganui Rytidosperma 2 0 West Coast Arthur’s Pass, Otira Valley endemic nigricans

(Continued) NEW ZEALAND JOURNAL OF BOTANY 5

Table 1. Continued. Number of plants Grass species Examined Infected Regiona Collection site Status Rytidosperma 5 0 Waikato Tongariro National Park, Tama Lakes endemic pulchrum 5 0 Canterbury Porters Pass, Bealey Valley Rytidosperma 5 0 Canterbury Mt. Cook, Sealy Tarns endemic setifolium aRegional council boundaries.

(AL1759) in the International Collection of Microorganisms from Plants (ICMP, Auck- land, New Zealand). For DNA extraction isolates were grown on sterilised cellophane above potato dextrose agar (PDA) (Cassago et al. 2002), or on liquid V-8 medium on a rotary shaker for up to four weeks (Leuchtmann 1994).

Morphological examination Colony growth and morphology were examined and photographed from cultures grown on PDA in Petri dishes at 24°C in the dark. Microscopic observations of conidiogenous cells and conidia were made from mycelium mounted in lactic acid. Measurements were taken with an ocular micrometer at 1000×using an Olympus BH-2 microscope (Olympus Corp., Tokyo, Japan) with phase contrast optics. For each fungal structure 20 measurements per isolate were taken, and range and mean (in brackets) are given for conidia size.

DNA sequencing DNA was extracted from freeze-dried mycelia with the Quick-DNA Fungal/Bacterial MiniPrep Kit (Zymo Research, Irvine, California) or NucleoSpin Plant II Kit (Macherey-Nagel, Düren, Germany) as described previously (Oberhofer and Leuchtmann 2012). Amplifications of intron rich portions of translation elongation factor 1-alpha (tefA) were performed using primer pairs 5′-GGG TAA GGA CGA AAA GAC TCA-3′ and 5′-CGG CAG CGA TAA TCA GGA TAG-3′, and portions of β-tubulin (tubB) primer pairs 5′-TGG TCA ACC AGC TCA GCA CC-3′ and 5′-TGG TCA ACC AGC TCA GCA CC-3′ (Craven et al. 2001). Reactions were performed in a final volume of 25 µl containing 5 μl of commercial GoTaq reaction buffer, 400 nM MgCl2, 125 nM dNTPs, 400 nM primers and 0.05 u/μl GoTaq (Promega Corporation, Madison, Wiscon- sin). Amplification was performed as described by Oberhofer and Leuchtmann (2012) with about 10 ng of genomic DNA in a standard touchdown cycle and products were purified with GenElute™ PCR Clean-Up Kit (Sigma-Aldrich, St. Louis, Missouri). Label- ling reactions were performed in a final volume of 10 μl using BigDye® Terminator v3.1 (Applied Biosystems™, Foster City, California) according to manufacturer’s instructions. Products were cleaned with Sephadex™ G-50 (GE Healthcare, Buckinghamshire, United Kingdom), before obtaining sequences on a 3130xl Genetic Analyzer (Applied Biosys- tems™, Foster City, California). For separating different alleles of hybrid isolates, PCR products were cloned into bacterial plasmids. Purified PCR products (25 ng) of tubB and tefA genes were ligated into 6 A. LEUCHTMANN ET AL.

M13-plasmid-vectors employing the pGEM®-T Easy Vector System (Promega Corpor- ation, Madison, Wisconsin) as described previously (Oberhofer and Leuchtmann 2012). PCR products of expected size were puriﬁed from excess primers and dNTPs and then used for BigDye reaction as described above. All haplotypes of both genes were sequenced and manually edited with Sequencher 4.8 (Gene Codes Corporation, Ann Arbor, MI). Sequences are available from GenBank (National Center for Biotechnology Information, Bethesda, Maryland; http://www.ncbi.nlm.nih.gov/) under accession numbers MN013153–MN013158 (E. novae-zelandiae) and MN150703–MN150706 (E. australiensis).

Phylogenetic analyses New sequences and sequences downloaded from GenBank were aligned with MUSCLE implemented in Geneious 9.1.7 (Biomatters, Auckland, New Zealand). Alignments of partial sequences of the tubB gene included introns 1–3, and of the tefA gene introns 1–4. Phylogenetic trees were inferred by maximum likelihood (ML) with likelihood settings from best-ﬁt models selected by hLRT in Modeltest 3.7 (Posada and Crandall 1998) and performed with PAUP* 4.0a for Macintosh. The ML trees were generated in a heuristic search with gaps treated as missing information and random sequence additions. Bootstrap support values were estimated from 100 ML replications with random number of seed and stepwise sequence addition. Phylogenetic placement of the mating type genes mtAC and mtBA were inferred using Phylogeny.fr (Dereeper et al. 2008, 2010). The alignments were performed by MUSCLE (Edgar 2004) with Gblocks curation (Castresana 2000). Phylogenetic trees were inferred by maximum likelihood (ML) with likelihood substitution model HKY85.

Alkaloid analyses Alkaloids were extracted from freeze-dried and ground herbage and seed samples of P. matthewsii. Lolitrem B, peramine and ergovaline analyses were performed using standard high-performance liquid chromatography (HPLC) methods (Gallagher et al. 1984; Tapper et al. 1989; Spiering et al. 2002). Lolines were measured by a modiﬁed gas chro- matographic (GC) method described in Miles et al. (1998), and a subset of samples was also analysed by ELISA for the indole-diterpenes paxilline and lolitrems, and for peramine and ergot alkaloids (Garthwaite et al. 1994).

Multiplex PCR and alkaloid gene detection Multiplex PCR using target-specific primers for alkaloid and mating type genes was performed as previously described (Charlton et al. 2014). For a complete primer list see Sup- plementary material S1. DNA was extracted from fungal mycelium using the Quick-DNA fungal/bacterial kit (Zymo, Irvine, California). Genomic DNA was diluted to 1 ng/μland 3ngofDNAwasusedina 25μl PCR reaction with 5 μl commercial GoTaq reaction buffer containing MgCl2, 1 U GoTaq DNA polymerase (Promega Corp., Madison, Wiscon- sin), 0.2 mM each dNTP and 1 μM target-specific primers. The cycling parameters were 94°C for 1 min, followed by 30 cycles of 94°C for 15 s, 56°C for 30 s and 72°C for 45 s, then 72°C for NEW ZEALAND JOURNAL OF BOTANY 7

10 min. Amplicons were analysed by gel electrophoresis with 1.5% agarose gels in Tris-boric- EDTA buﬀer, and DNA fragments visualised with ethidium bromide UV transillumination.

Results Endophyte infection Samples were collected from 25 pooid grass species (Table 1). Of these, 22 are endemic and three indigenous to New Zealand. In total, 281 plants were examined. However, only indi- viduals of two species were found to be infected by endophytes, Poa matthewsii (Matthew’s bluegrass) collected from Banks Peninsula near Christchurch and Dichelanchne micrantha (short-hair plume grass). While P. matthewsii has been reported to be infected before (Stewart et al. 2004), D. micrantha has never been found to be infected and represents a new host for an Epichloe endophyte discovered in New Zealand. This grass species is indigenous to New Zealand from North Cape to Auckland, but is also found in Australia, Norfolk Island, Easter Island and New Guinea. Plants may occur in scrub, at roadsides or banks usually not far from the coast in the lowland zone (Edgar and Connor 2000). Our samples were collected along a walking track near the coast in Hokianga (Northland). Colonies of the endophyte of P. matthewsii were very slow growing on PDA (10– 12 mm diameter in 10 weeks) and formed a white, sparse aerial mycelium when young, that later became light brown and convoluted (Figure 1A). Conidiogenous cells were sparse and produced few obovate to navicular conidia, 3.3–5.7 ×1.8–3.1 µm in size (Figure 1B–D). The endophyte of D. micrantha formed a white, dense, slow-growing colony on PDA that became yellowish brown and submerged at the margin (Figure 1E, F). Conidia, 4.5–7×2.5–3.5 µm in size, were formed sparsely on the typically, laterally arranged conidiophores. Screening based on immunoblotting of endophyte infection of P. matthewsii in 25 natural populations occurring on Banks Peninsula showed that infection is common, aver- aging 60% infection rate overall plants tested. Of the sites surveyed approximately one third had 90%–100% infection rate, another third 50%–83% infection rate and the remain- ing third less than 10% infection rate. There was no correlation between infection rate and ecological observations made at these sites, such as degree of shade, degree of drought, size of the forest remnant or forest species present. Besides Banks Peninsula, infected plants were also found at sites on Otago Peninsula (10% infection rate), and in the North Island at Taihape (86% infection rate) and at Coppermine Creek near Palmerston North (100% infection rate). Strains from each of these sites appear to diﬀer genetically based on Simple Sequence Repeat (SSR) patterns (unpublished data, M.J. Faville, pers. comm.), which may be expected from long standing endophyte populations that are sep- arated by large geographical distance and do not interbreed. Symptoms of choke have never been observed on infected P. matthewsii.

Phylogenetic analyses Sequence analysis of tefA and tubB from the P. matthewsii endophyte (isolate AL0725) indicated that it is a complex hybrid. Three diﬀerent gene copies each of tefA and tubB were identiﬁed. Maximum likelihood phylogenetic reconstruction placed copies in 8 A. LEUCHTMANN ET AL.

Figure 1. Morphology of Epichloe endophytes in culture. A-D, E. novae-zelandiae; A, Colony grown for 6 weeks on PDA at 24°C; B, Conidiogenous cell; C-D, Conidia. E-F, E. australiensis; E, Colony grown from infected grass tissue; F, Endophytic hyphae stained with aniline blue. diﬀerent subclades within the Epichloe trees (Figures 2 and 3). Copy 1 of tefA was most closely related to sequences of E. amarillans J.F. White, copy 2 to sequences of E. bromicola Leuchtm. & Schardl and copy 3 to E. typhina (Pers.) Tul. & C. Tul (Figure 2). The third copy clustered at the base of a subclade containing E. typhina genotypes from Poa pratensis L. and P. nemoralis L., which are referred to as E. typhina subsp. poae (Leuchtmann et al. 2014). Diﬀerent gene copies of tubB group with the same subclades as in tefA, copy 1 with E. amarillans genotypes, copy 2 with E. bromicola genotypes and copy 3 with E. typhina subsp. poae genotypes, again at the base of the subclade containing genotypes of Poa hosts (Figure 3). The endophyte of D. micrantha (isolate AL1759) has two gene copies, one clustered in a subclade with E. festucae Leuchtm., Schardl & M.R. Siegel and one in a subclade with NEW ZEALAND JOURNAL OF BOTANY 9

Figure 2. Phylogeny derived from maximum likelihood (substitution model TrN+G) analysis of partial tefA gene sequences including introns 1–4 of representative Epichloe species and alleles obtained from E. novae-zelandiae and from E. australiensis of the unreported host Dichelachne micrantha (Dm). The tree is midpoint rooted at the left edge. Numbers at branches are ML bootstrap support percentages from 100 replications. Sequences are designated by the taxon name including host abbreviation in par- enthesis, followed by gene copy number (for hybrid taxa) and GenBank accession number.Host abbreviations: Ac = Agrostis capillaris, Ah = A. hyemalis, Ast = A. stolonifera, Ae = Achnatherum eminens, Agr = Agropyron repens, Amm = Ammophila breviligulata, Be = Bromus erectus, Bee = Brachye- lytrum erectum, Bp = Brachypodium pinnatum, Bs = B. sylvaticum, Ca = Cinna arundinacea, Dg = Dactylis glomerata, Dm = Dichelachne micrantha, Ec = Elymus canadensis, Er = Elymus repens, Ev = E. virginicus, Eo = Echinopogon ovatus, Fa = Festuca arundinacea, Fr = F. rubra, Frc = F. rubra subsp. commutata, Gs = Glyceria striata, Hb = Hordeum brevisubulatum, Hl = Holcus lanatus, Hm = H. mollis, Lc = Leymus chinen- sis, Lp = Lolium perenne, Pm = Poa mathewsii, Pn = Poa nemoralis, Pp = P. pratensis, Psj = P. secunda subsp. juncifolia, Rnj = Roegneria kamoji, Sph = Sphenopholis obtusata. 10 A. LEUCHTMANN ET AL.

Figure 3. Phylogeny derived from maximum likelihood (substitution model K80+G) analysis of partial tubB gene sequences including introns 1–3 of representative Epichloe species and alleles obtained from E. novae-zelandiae and from E. australiensis of the unreported host Dichelachne micrantha (Dm). The tree is midpoint rooted at the left edge. Numbers at branches are ML bootstrap support percentages from 100 replications. Sequences are designated by the taxon name including host abbreviation in par- enthesis, followed by gene copy number (for hybrid taxa) and GenBank accession number. For host abbreviations see Figure 2. NEW ZEALAND JOURNAL OF BOTANY 11

E. typhina subsp. poae (Figures 2 and 3). These gene copies were identical or nearly identical with alleles previously found in the interspeciﬁc hybrid E. australiensis from Ec. ovatus. The endophyte infecting D. micrantha is therefore recognised as E. australiensis.

Alkaloids in planta Seed and herbage from several infected P. matthewsii plants were analysed for the presence of alkaloids. Loline alkaloids (NANL, NAL, NFL) and lolitrem B (an indole-diterpene) were not detected in any of the samples from herbage or seed. Lolitrems were also undetectable in the ELISA assay while early pathway indole-diterpenes were detected by the paxilline ELISA. Peramine was found in three out of 20 samples at low concentrations (≤3.9 ppm) using HPLC, but not with the ELISA assay. The ergot alkaloid ergovaline was present in herbage at low concentrations (≤0.35 ppm) while the ELISA assay also detected ergot alkaloids. Furthermore, in a laboratory assay testing resistance to Argentine stem weevil (Listronotus bonariensis) infected plants were found to be more resistant than uninfected plants (unpublished results, A.J. Popay, pers. comm.).

Predicted alkaloids and mating types from genetic analyses Data from diagnostic PCR using DNA isolated from E. australiensis (AL1759) and E. novae-zelandiae (AL0725/1, AL0725/2, AL0725/3) showed variation based on the presence of marker genes involved in the biosynthesis pathway of ergot alkaloids, indole-diterpenes, lolines and peramine, as well as the mating type genes (Figure 4). The E. australiensis isolate AL1759 only had markers present for the perA gene required for peramine biosynthesis, as well as the perA-ΔR allele that lacks the reductase domain. A similar marker profile has been observed for E. australiensis in Ec. ovatus (C.A. Young and C.L. Schardl, unpublished results). The E. novae-zelandiae isolates AL0725/1, AL0725/2, and AL0725/3 showed identical banding patterns with markers present for all alkaloid classes. The PCR with the perA markers were identical to E. australiensis AL1759, with both the perA and perA-ΔR alleles present. Some markers for the loline biosynthesis genes were present, but it is uncertain if lolines would be produced, as genes for the later pathway steps (lolO and lolP) were absent. Ergot alkaloid biosynthesis genes were present, but of the markers tested cloA was not detected. Endophytes with this profile would not be expected to produce ergovaline, but provided the early pathway biosynthesis genes encode functional proteins, we predict that chanoclavine or agroclavine could be produced. Based on the indole-diterpene genes that were detected, we predict that 13-des- oxypaxilline could be produced. Epichloe novae-zelandiae and E. australiensis have different contributions of the mating type idiomorphs. E. australiensis only amplified with the mtAC primers, which indicates that both progenitors were mating type A (MTA). Epichloe novae-zelandiae contained both mating type markers. Sequence verification of the mating type genes showed that MTA is present as a single copy from E. typhina subsp. poae genotypes, whereas MTB has two copies, one each from the E. amarillans and E. bromicola genotypes (Supplemen- tary material S2). The genotype groupings of the mating type genes are consistent with the phylogenies represented by tefA and tubB. The two triparental hybrids E. novae-zelandiae and E. chisosa (J.F. White & Morgan-Jones) Schardl have different parental contributions 12 A. LEUCHTMANN ET AL.

Figure 4. Diagnostic multiplex PCR with genomic DNA from Epichloe australiensis AL1759 and E. novae- zelandiae isolates (AL0425/1, AL0425/2, AL0425/3). House-keeping gene (HK), mating type (MT) genes and predicted genes of the alkaloid pathways of peramine (PER), lolines (LOL), ergot alkaloids (EAS) and indole-diterpenes (IDT) are shown. Controls are E. festucae isolates E2368 and Fl1, and E. coenophiala e19. The 1 kb+ ladder (Invitrogen, Carlsbad, California) was used to size the fragments. of the mating type idiomorphs, although they descended from the same ancestral species (Supplementary material S2).

Taxonomy Epichloe novae-zelandiae Leuchtm. & A.V. Stewart, sp. nov. (Figure 1A–D) MycoBank MB832060 Typiﬁcation. New Zealand. Christchurch, Port Hills, culms of Poa matthewsii, 25 March 2007, A. Stewart and A. Leuchtmann AL0725/2, holotype Myc60051 (ZT); ex type culture CBS 145797; GenBank accession nos. MN013153–MN013155 (tubB) and MN013156–MN013158 (tefA). Hybrid with genetic relationships to E. amarillans J.F. White, E. bromicola Leuchtm. & Schardl and E. typhina (Pers.) Tul. & C. Tul. subsp. poae. Etymology. Referring to New Zealand (Latin Nova Zelandia), the origin of the endemic host species. Colonies emerging from nodes of infected, surface sterilised plant segments after 24 days of incubation, white on PDA forming a sparse aerial mycelium when young, later NEW ZEALAND JOURNAL OF BOTANY 13 becoming light brown and raised with convoluted, brain-like structure, margin irregularly lobed, reverse greyish brown; slow growing, attaining 10–12 mm diameter in 10 weeks at 24°C. Conidiogenous cells sparse, arising laterally from hyphae, cylindrical at base tapering toward the apex, mostly lacking septum near the base, 14–40 µm long, 1.6–2.5 µm wide at the base. Conidia obovate to navicular asymmetric, hyaline, aseptate, 3.3–5.7 (5.0) ×1.8– 3.1 (2.5) µm. Sexual morph not observed.

Discussion In the present study on endophyte infection of endemic and indigenous grasses of New Zealand, two species, P. matthewsii and D. micrantha, were found to be infected. The low incidence of infection is consistent with previous surveys suggesting that endophytes in native grasses are rare in New Zealand (Davis and Guy 2001; Rolston et al. 2002). Overall, including unpublished records, 68 (36%) of the 188 native grasses have so far been examined and only three species were found to be infected (Supplementary material S3). A hypothesis that could possibly explain this finding is that Epichloe endophytes have evolved on the Eurasian continent after separation of land masses from Gondwanaland some 80 million years ago, which later became New Zealand. Thus, radiation of grass species in New Zealand may have occurred mostly in the absence of endophytes. Using molecular clock estimates, divergence date of the endophytic Epichloe clade from the ancestral Claviceps lineage was estimated at 58.5 million years ago suggesting that Epichloe and its endophytic lifestyle evolved after the split of New Zealand from Gondwanaland (Ambrose et al. 2014). The New Zealand endemic grasses currently known to be infected, i.e. Ec. ovatus and P. matthewsii, may have reached New Zealand later together with the endophyte by long distance dispersal and subsequent speciation. Supporting this view, a genetic and biogeographic study suggests that species of the genus Poa are likely to have radiated from North America to Australasia (Soreng 1990). Moreover, Poa species typically have a tuft of hair (on the lemma) attached to their diaspores, which evidently is very effective for dispersal by wind or animals (Gillespie and Soreng 2005). Other potential mechanisms that would allow transoceanic dispersal may involve migratory birds or transport by rafting. The presence of multiple gene copies of tefA and tubB suggests that E. novae-zelandiae, the endophyte of P. matthewsii, is an interspecific hybrid involving ancestral genotypes of three different sexual Epichloe species (E. typhina and E. bromicola native to Eurasia, and E. amarillans native to North America). It is interesting to note that the same ancestral species are also involved in the triparental hybrid E. chisosa, a rare species infecting Ach- natherum eminens (Cav.) Barkworth in Texas (Moon et al. 2004; Figures 2 and 3), but the ancestral genotypes have contributed different mating type idiomorphs. The mechanisms leading to interspecific hybridisations remain speculative, but most likely involve superin- fection of an already infected host grass, followed by parasexual processes with hyphal fusion and karyogamy between resident and immigrant endophytes (Schardl et al. 1994). Over time, redundant chromosomes or chromosomal segments of an initially allopolyploid genome may become lost giving rise to incomplete subgenomes. As three copies were identified for each of the two genes analysed in E. novae-zelandiae, it cannot be deter- mined whether its genome is incomplete. However, different copy numbers for different 14 A. LEUCHTMANN ET AL. genes have been found in other species, e.g. the biparental hybrids E. australiensis, E. melicicola (C.D. Moon & Schardl) Schardl and E. uncinata (W. Gams, Petrini & D. Schmidt) Leuchtm. & Schardl, and the triparental hybrid E. coenophiala (Morgan- Jones & W. Gams) C.W. Bacon & Schardl (Tsai et al. 1994; Moon et al. 2002). A hypothetical scenario of the origin of the hybrid endophyte of P. matthewsii leading to the current triparental hybrid status would involve two hybridisation events possibly on different continents followed by long distance dispersal. It can be envisioned that a Poa host on the Eurasian continent became infected simultaneously by ancestral lineages of E. typhina and E. bromicola, two sexual endophytes of Eurasian origin that can horizon- tally disperse by ascospores (Leuchtmann and Schardl 1998), and upon which hybridisation occurred. Subsequently, the Poa host with its hybrid endophyte may have moved to North America along the connecting land masses of Bering Strait, where it became superinfected by an ancestral lineage of E. amarillans, a sexual endophyte native to North America (White 1994), which resulted in a triparental hybrid. Then, either by movement via South America and Australia or by long distance dispersal, the Poa host reached New Zealand, where it became established and evolved into P. matthewsii. Although the genus Poa has diversified within the last 4.3 million years in Australasia (Birch et al. 2014), in all other endemic Poa species that have been analysed in this and a previous study no endophyte was found (Rolston et al. 2002). This may suggest that the biogeographic origin of these Poa species is different from that of P. matthewsii, or alternatively that the endophytes of other Poa hosts have been lost. Besides being rare, most endophytes of indigenous grasses in the Southern Hemisphere have genotypes that are genetically closely related to lineages found in the Northern Hemi- sphere. There is only one endophyte lineage known, that is believed to have originated in the Southern Hemisphere (Moon et al. 2002). Gene phylogenies place this lineage at the base of a clade with E. typhina [see Moon et al. (2002) and Figures 2 and 3] suggesting that it diverged relatively early in the evolution of Epichloe endophytes. A representative of this lineage is the non-hybrid E. aotearoae (C.D. Moon, C.O. Miles & Schardl) Leuchtm. & Schardl that occurs in Australia and New Zealand infecting indigenous Ec. ovatus. This species possibly gave rise to the E. aotearoae-like genes of E. melicicola, a hybrid species found on Melica in South Africa (Moon et al. 2002). The second gene copy of E. melicicola is closely related to an endophyte lineage found in the Northern Hemisphere, as it is the case with genotypes of all other hybrid Southern Hemisphere species known to date, i.e. E. australiensis (Moon et al. 2002), E. tembladerae (Cabral & J.F. White) Iannone & Schardl (Cabral et al. 1999), E. pampeana (Iannone & Cabral) Iannone & Schardl (Iannone et al. 2009), E. cabralii Iannone, M.S. Rossi & Schardl (McCargo et al. 2014) and E. novae-zelandiae of the current study. These findings suggest that movements of endophytes across continents and oceans must have occurred repeatedly, although precise mechanisms of dispersal are unknown. Endophyte taxa putatively originating from interspecific hybridisations are very common among the asexual species of Epichloe (Moon et al. 2004). In fact, of the 25 previously analysed asexual species, 22 appeared to be of hybrid origin as indicated by the presence of two or more copies of tubB, tefA or actB genes that are related to different ancestral species (Leuchtmann et al. 2014; Campbell et al. 2017; Shymanovich et al. 2017). Hybrid endophytes have been found in grasses from all continents (except Antarctica) and in most grass tribes known to host Epichloe fungi. This suggests that the hybrid status of NEW ZEALAND JOURNAL OF BOTANY 15 asexual species may be advantageous for the endophyte and its host. Unlike sexual species, asexual seed-borne endophytes cannot recombine, which would allow for diversification and adaptation to a changing environment or to stress. Hybridisation may offer alternative means to acquire beneficial genes that have evolved in other Epichloe symbioses and that may increase stress tolerance. From an applied perspective, endophyte genes that confer increased resistance to the host against herbivory or plant disease would be most useful. For example, multiple copies of genes coding for insect deterrent alkaloids such as peramine and loline may protect hosts from a much wider range of potential herbivores.

Conclusions Endophytes are increasingly viewed as important components of sustainable agriculture, because of their potential to relieve or protect host plants from various biotic and abiotic stresses. Grass endophytes have been, and still are, of vital importance to New Zealand agriculture, where managed pastures largely depend on the beneﬁts of endophytes (Johnson et al. 2013). Notably, the endophyte of perennial ryegrass (E. festucae var. lolii) provides protection against Argentine stem weevil and a range of other invertebrate pests (Popay and Hume 2011). Therefore, selected and bred cultivars of perennial ryegrass that contain an endophyte account for a large proportion of seed used for pastures in New Zealand, and most old ryegrass pastures are highly infected with endophytes (Latch and Christensen 1982; Widdup and Ryan 1992). Furthermore, toxins produced by the endophyte may deter grazing ruminants, so that infected grasses are grazed less intensively than uninfected grasses, which protects swards from over-grazing (Edwards et al. 1993). Endo- phytes from native grasses, such as E. novae-zelandiae infecting P. matthewsii may provide a genetic resource that could be exploited for developing new grass cultivars with improved performance.

Acknowledgements We thank New Zealand Department of Conservation (DOC) for permission to remove samples from public conservation land, Neil Fowke and Kerry Ford for advice on permit application or collection sites, Arvina Ram and Bea Arnold for laboratory assistance, and Niki Mindars for help with photographs. Delaney Medcalf, Amy Flanagan, Nikki Charlton, Will Hendricks and Ellen Hume are acknowledged for diagnostic evaluation of alkaloid and mating type proﬁles of isolates and seed samples. We also thank Brian Tapper and Jan Sprosen for in planta alkaloid analyses, and Artemis Treindl for reading and improving the manuscript. We acknowledge the Genetic Diversity Centre of ETH Zurich (GDC), where part of the sequence data were generated, and ETH Zurich for ﬁnancial support to AL during sabbatical leave.

Disclosure statement No potential conﬂict of interest was reported by the authors.

ORCID Adrian Leuchtmann http://orcid.org/0000-0002-9070-0902 Carolyn A. Young http://orcid.org/0000-0003-0406-8398 Barry Scott http://orcid.org/0000-0002-2224-2946 16 A. LEUCHTMANN ET AL.

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