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Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546-0312
Jean-Jacques Guillaumin
Catherine Ravel
INRA, UMR 1095, 234 Avenue du Brézet, F-63100 Clermont-Ferrand, France
Chunjie Li
College of Pastoral Agriculture Science and Technology, Lanzhou University, Gansu Grassland Ecological Research Institute, Key Laboratory of Grassland Argo-Ecosystem, Ministry of Agriculture, Lanzhou 730020, China
Kelly D. Craven 2
Christopher L. Schardl 3
Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546-0312
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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Several species of Achnatherum (grass tribe Stipeae) and Melica (tribe Meliceae) typically are infected by nonpathogenic, seed-transmissible fungi with characteristics of Neotyphodium species (anamorphic Clavicipitaceae). Molecular phylogenetic studies clearly have distinguished the endophytes from Achnatherum inebrians (from Xinjiang Province, China), A. robustum and A. eminens (both from North America) and indicate that the A. inebrians endophyte comprises a unique nonhybrid lineage within the Epichloë and Neotyphodium phylogeny, whereas the endophytes of A. robustum, and A. eminens are hybrids with multiple Epichloë species (holomorphic Clavicipitaceae) as ancestors. Likewise distinct hybrid origins are indicated for Neotyphodium species from the European Melica species, M. ciliata and M. transsilvanica, the South African species M. decumbens and M. racemosa, and the South American species M. stuckertii. Neotyphodium species have been described from A. inebrians from Gansu Province, China, (N. gansuense), A. eminens (N. chisosum), M. stuckertii (N. tembladerae) and the South African Melica species (N. melicicola). However the endophytes from A. robustum and the European Melica species have not been described and the phylogenetic relationships of N. gansuense have not been investigated. Here we report a comprehensive study of morphological features and phylogenetic analyses of β-tubulin and actin gene sequences on an expanded collection of endophytes from the Stipeae and Meliceae. These data provide a firm foundation for the description of two new Neotyphodium species, N. guerinii from M. ciliata and M. transsilvanica, and N. funkii from A. robustum. We also propose the new variety, N. gansuense var. inebrians for endophytes of A. inebrians from Xinjiang Province, which are morphologically and phylogenetically distinct from, yet clearly related to, N. gansuense from Gansu Province.
Key words: Achnatherum, Clavicipitaceae, epichloë endophytes, evolution, grasses, hybridization, Melica, Poaceae, symbiosis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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). The Neotyphodium species generally reside in grass apoplasts, where they maintain perennial asymptomatic infections and are dispersed asexually through the seed. Some epichloë endophytes produce ergot or indolediterpenoid alkaloids, causing toxic syndromes in grazing livestock (Thompson and Stuedemann 1993). Endophytes also may synthesize lolines (exo-1-aminopyrrolizidines) (Blankenship et al 2001) or the pyrrolopyrazine, peramine (Tanaka et al 2005), which have potent insect-deterring or insecticidal activities (Riedell et al 1991, Rowan and Gaynor 1986). The vast majority of Neotyphodium and Epichloë species described to date inhabit grasses in tribes Poeae, Aveneae, Bromeae or Triticeae (Moon et al 2004), which represent a crown group relative to other pooid tribes such as Stipeae and Meliceae (Soreng and Davis 1998). Nevertheless, several described and undescribed Neotyphodium species also have been identified in the Stipeae and Meliceae (Moon et al 2004, Schardl and Leuchtmann 2005), so the characteristics and evolutionary relationships of these Neotyphodium species are of special interest.
It long has been known that some members of grass tribes Stipeae and Meliceae can have debilitating effects on grazing animals due to alkaloids produced by their endophytes. For example, it was reported more than a century ago that animals that grazed Achnatherum inebrians (Hance) Keng (tribe Stipeae), a range grass of Mongolia and northwestern China, could become intoxicated to the point of barely being able to stand, generally recovering within a few days (Bruehl et al 1994, Hance 1876). Aptly, A. inebrians is commonly referred to as drunken horse grass (
). Neotyphodium gansuense Li et Nan has been described from A. inebrians plants in Gansu Province, China (Li et al 2004), and the authors noted that this relatively fast growing endophyte is morphologically distinct from the slow growing isolates from the same host species in Xinjiang Province (Bruehl et al 1994, Miles et al 1996).
In southwestern USA and northwestern Mexico, Achnatherum robustum (Vasey) Barkworth (=Stipa robusta [Vasey] Scribn.), also is commonly infected with endophyte (Kaiser et al 1996). The narcosis-inducing properties associated with endophyte infection of A. robustum near Cloudcroft, New Mexico, and the nearby Sacramento Mountains and Sierra Blanca have earned it the common name sleepy grass (Bailey 1903, Faeth et al 2006, Kingsbury 1964). Like A. inebrians, its toxicity is attributed to the presence of ergot alkaloids (Petroski et al 1992). Achnatherum eminens (Cav.) Barkworth (=Stipa eminens Cav.) is also native to the southwestern United States and is host to the endophyte Neotyphodium chisosum (White et Morgan-Jones) Glenn et al (White and Morgan-Jones 1987), although no reports of toxic activity have been associated with A. eminens.
Grasses of tribe Meliceae also are known to be infected with epichloë endophytes. Neotyphodium melicicola Moon et Schardl infects Melica decumbens Thunb. and Melica racemosa Thunb. of South Africa (Moon et al 2002) and can cause drunken behavior in grazing animals. In Europe and temperate parts of Asia, Melica ciliata L. and Melica transsilvanica Schur are widespread and known to be infected with endophytes (Baltisberger and Leuchtmann 1991, White 1987), although neither has been associated definitively with toxic activity.
Our understanding of the evolution of the epichloë endophytes has been advanced greatly by the application of molecular phylogenetic methods, revealing that many Neotyphodium species have arisen by interspecific hybridization among Epichloë species or between Epichloë and Neotyphodium species (Craven et al 2001b, Gentile et al 2005, Moon et al 2000, 2002, 2004, Schardl et al 1994, Tsai et al 1994). Phylogenetic analyses of genes encoding β-tubulin (tubB) and translation elongation factor 1-
(tefA) have been conducted on Neotyphodium species from a broad taxonomic range of host grasses worldwide (Moon et al 2004). Endophyte isolates from A. inebrians, tentatively designated ‘Neotyphodium inebrians’, represent a unique nonhybrid lineage within the Epichloë and Neotyphodium phylogeny. An undescribed endophyte of A. robustum is an interspecific hybrid of likely Epichloë elymi Schardl et Leuchtm. and Epichloë festucae Leuchtm. et al ancestry. The A. eminens endophyte N. chisosum is a hybrid of three ancestors most closely related to extant Epichloë bromicola Leuchtm. et Schardl, Epichloë amarillans J.F. White and Epichloë typhina (Pers. : Fr.) Tul. (Moon et al 2004). The endophyte isolates from M. ciliata and M. transsilvanica are related and also have an apparent hybrid origin (Moon et al 2004). In the present study phylogenetic analyses of tubB and actin gene (actG) sequences, along with morphological examinations, were undertaken on an expanded set of isolates from A. inebrians, A. robustum and M. ciliata and support the formal description of two new Neotyphodium species and one new variety.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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) from infected grasses that were maintained in the greenhouse or a common garden. Live cultures were deposited in the ATCC or Centraalbureau voor Schimmelcultures (CBS; Utrecht, The Netherlands); endophyte herbarium specimens (Pollack 1967) were deposited in the Cornell University Plant Pathology Herbarium (CUP; Ithaca, New York); and endophyte-infected live plants and seeds were deposited in the Conservatoire Botanique National du Massif Central (CBNMC; Chavaniac-Lafayette, Haute-Loire, France).
Examination of endophyte morphology.— –
Endophyte colony morphology was examined and radial growth rates were measured from cultures grown on potato-dextrose agar (PDA) that were inoculated with small agar blocks (ca. 1 mm3) taken from the periphery of actively growing colonies. Cultures were incubated at 22 C in the dark. Radial measurements from at least eight colonies were made weekly until cultures were 6 wk old, except in the case of endophyte ATCC MYA-1228 from A. inebrians, which was grown 8 wk. Cultures were photographed and preserved as herbarium specimens (Pollack 1967).
Microscopic examination of fungal structures was achieved by inoculating 1.5% water agar or PDA plates with mycelium ground in sterile water as described by Moon et al (2002). From past observations Neotyphodium isolates tend to sporulate better on water agar than on nutrient agars. Plates were incubated at 22 C in the dark up to 10 d with regular monitoring for conidiophores. Agar blocks were mounted on slides and overlaid with a cover slip. Endophytes were examined with a Zeiss Axioskop light microscope (Carl Zeiss Microimaging Inc., Thornwood, New York) with 1000x magnification with oil immersion and Nomarski DIC optics. Images were captured with a digital AxioCam (Carl Zeiss Microimaging Inc.) operated by Openlab software (Improvision Inc., Lexington, Massachusetts). From these images hyphal widths and cell dimensions of mature conidia and conidiogenous cells were recorded for at least 20 structures per isolate.
DNA isolation.— –
Fungal DNA was isolated from single-spore purified cultures (if cultures sporulated) as described by Moon et al (2002). DNA was isolated from infected plant tissue by the DNeasy 96 Plant Kit (QIAGEN Inc., Valencia, California). Pseudostem sections from the crown to the ligule were harvested and the outer leaf sheathes were discarded. Each sample (50–100 mg wet weight) was placed with 400 µL homogenization buffer and a single 3 mm stainless steel bead into a well of a 96 deep-well plate and disrupted with a GenoGrinder (SPEX CertiPrep, Metuchen, New Jersey) with a 1.5 min pulse of 1500 strokes/min, which was repeated once after rotating the plate. DNA was isolated per the protocol provided with the DNeasy 96 Plant Kit.
DNA amplification and sequencing.— –
DNA fragments were amplified by polymerase chain reaction (PCR) from total genomic DNA of endophyte (1–10 ng) or infected plant (10–100 ng) as described by Craven et al (2001a, b) and Moon et al (2002). A ca. 1300 bp portion of the actin gene, actG, was amplified with PCR primers act1-exon1d-1 (5'-TAA TCA GTC ACA TGG AGG GT-3') and act1-exon6u-1 (5'-AAC CAC CGA TCC AGA CAG AGT-3'). Amplification of portions of tubB and tefA were performed as described by (Craven et al (2001a, b) and Moon et al (2002). PCR products were purified with a QIAquick PCR Purification Kit (QIAGEN Inc.) and sequenced with a BigDye kit version 1.1 or 3.1 Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, California) with fragment separation on an ABI Prism model 310 or 3730 genetic analyzer (Applied Biosystems) at the University of Kentucky Advanced Genetic Technologies Center. When the sequence traces indicated the presence of multiple alleles, individual alleles were analyzed by either cloning individual PCR products with a TOPO TA cloning kit (Invitrogen, Carlsbad, California) or with selective primers to amplify the individual alleles. Selective primers for tubB and tefA alleles were described by Craven et al (2001a, b) and Moon et al (2002). Primers act1-(706u)-selC (5'-CTG CCA AAC ATT GTC AGA ATC-3') and act1-(706u)-selG (5'-CTG CCA AAC ATT GTC AGA ATG-3'), which differ only at the 3'-most nucleotide, were paired with act1-exon1d-1 to amplify actG alleles from isolates MYA-1235 and MYA-1236. Gene sequences were deposited in GenBank and accession numbers are listed (SUPPLEMENTAL TABLE I).
Phylogenetic analysis.— –
Sequences were aligned with Clustal W (Chenna et al 2003). Sequences also included in these analyses were from representative Epichloë species and Neotyphodium species isolates from studies by Gentile et al (2005) and Moon et al (2004). Alignments were checked by eye for ambiguities and adjusted if necessary. Alignment gaps were treated as missing information. Alignments for actG and tubB are deposited in TreeBase under accession Nos. M3305 and M3306 respectively (study accession No. S1806).
Maximum likelihood (ML) analysis and estimation of posterior probabilities of nodes employed MrBayes version 3 (Ronquist and Huelsenbeck 2003), with a GTR+G model (lset nst = 6 rates = gamma). For both the tubB and actG alignment four chains (three heated at temp = 0.2) were run 450 000 generations, saving one out of every 100 trees (mcmc ngen = 450 000 printfreq = 5000 samplefreq = 100 nchains = 4). The first 2000 trees (200 000 generations) were discarded as burn-in, and the consensus tree and posterior support values were determined from the remaining 2500 trees.
To assess the robustness of the topology 1000 bootstrap replicates were run by maximum parsimony analyses, employing heuristic search in PAUP* 4.0b10 (Swofford 1998), in which character states were unordered and unweighted, and trees were built by 100 iterations of random taxon addition with a different number seed for each iteration.
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TAXONOMY |
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Colony 13–20 mm diam after 7 wk at 22 C on PDA (FIG. 1
) or 1% malt. Colonies raised from agar surface, slightly to strongly convoluted, white, felted with abundant aerial hyphae, colony margins distinct to superficial. Colony reverse tan centrally to cream at margin. Vegetative hyphae hyaline, septate, 2.3–3.4 µm wide in young cultures and up to 5 µm wide in older cultures. Conidiogenous cells hyaline, unbranched, generally thin and flexuous, 13–42 µm long, ca. 2–3 µm wide at base, tapering to ca. 0.8–1.0 µm at tip. Basal septum observed occasionally. Conidia hyaline, smooth, navicular or lunate, sometimes appearing ellipsoidal broadside, 5.5–8 x 2.0–3.5 µm (FIG. 2). Sporulation in culture moderate to abundant. Approx. 20% of isolates do not sporulate in culture on PDA. Species associated with grasses Melica ciliata L. and M. transsilvanica Schur. All French samples of M. ciliata observed harbored N. guerinii. Hyphae in planta extracellular, 2–3 µm wide, parallel to the axis of the cells, often twisted or sinusoidal. Hyphae strongly colored by acidic blue dyes, except the septa which remain hyaline. The mycelium is observed regularly: (i) in leaf sheaths, (ii) in the parenchymatous pith of the internodal region of the reproductive stem (surrounding the central lacuna), (iii) in the aleurone layer of the seed. Teleomorph not observed. Shows genetic similarity to Neotyphodium gansuense C.J. Li et Z.B. Nan and Epichloë typhina (Pers. : Fr.) Tul.
Etymology. – In honor of Pierre Guérin, a French mycologist who was one of the first to describe an endophyte in a grass (in the seeds of Lolium temulentum L.), the same year (1898) as the German researcher Vogl.
HOLOTYPE. – FRANCE, Isère, Mont-de-Lans, 1213 m, from culms of Melica ciliata B11, Jul 2001, leg. J.-J. Guillaumin, CBNMC BP 01-003 and BS 03-001.
Specimens examined. – CBS 112035 from the HOLOTYPE. CBS 112034 from M. ciliata A4, FRANCE, Puy de Marmant, near Clermont-Ferrand, Puy-de-Dôme, Auvergne, Jun 2001, leg. J.-J. Guillaumin and C. Ravel, PARATYPE CBNMC BP 01-002 and BS 01-003; CBS 112036 from M. ciliata B18, FRANCE, Les Ougiers, commune de Saint-Christophe en Oisans, Isère, Jul 2001, leg. J.-J. Guillaumin, PARATYPE CBNMC BP 01-004 and BS 03-002; CBS 113029 from M. ciliata A15, FRANCE, Auvergne, sous Grandeyrolles, Jun. 2001, leg. J.-J. Guillaumin and C. Ravel CBNMC BP 01-001 and BS 01-001; A28 from M. ciliata, FRANCE, Auvergne, Puy Long, Jun 2001, leg. J.-J. Guillaumin and C. Ravel CBNMC BS 01-002; PARATYPE ATCC MYA-1236 from M. ciliata, GREECE, Voiotia, near Delphi, Sep 1989, leg. M. Baltisberger, CUP 65638; ATCC MYA-1235 from M. transsilvanica, SWITZERLAND, Canton Grisons, Schuls, Jul 1990, leg. A. Leuchtmann, CUP 65637. In addition 30 M. ciliata plants from the Auvergne region (Département Puy-de-Dôme, around Clermont-Ferrand) and 30 M. ciliata plants from the French Alps, (Départements Isère and Hautes-Alpes) were grown at INRA Clermont-Ferrand in 2001–2003. The endophyte was present in all 60 living plants and could be isolated from 55 of them.
N. funkii K.D. Craven et C.L. Schardl, sp. nov. FIGS. 3, 4
Coloniae 21–26 mm diam aetate 6 hebdomadum ad 22 C in PDA, elevatae, leviter convolutae in centro, complanatae peripheriam versus, modice coactae, albae; coloniae reversae eburnae in centro, albidae peripheriam versus. Hyphae septatae, 1.9–3.2 µm latae et fortiter contortae ad emersum ex planta. Conidia in cultura moderate abunda; cellulae conidiogenae 18–65 µm, ca. 2.5 µm latae ad basim, ca. 1.5 µm angustatae ad apicem, plerumque septatae ad basim; conidia hyalina, ellipsoidea vel lunata, 6.5–10 x 3–4.5 µm, aliquando nonnulla in apice phialidis fasciculata. Affinis genetice Epichloës elymi C.L. Schardl et A. Leuchtmann et E. festucae A. Leuchtmann C.L. Schardl et M.R. Siegel.
Colony 21–26 mm diam after 6 wk at 22 C on PDA (FIG. 3); colonies raised, slightly convoluted in center, and flattening toward perimeter; lightly felted, white, colony margins superficial. Colony reverse tan centrally to cream at margin. Hyphae septate, 1.9–3.2 µm wide, and highly convoluted on emergence from plant tissue. Sporulation in culture moderately abundant; conidiogenous cells 18–65 µm long, ca. 2.5 µm wide at base, tapering to ca. 1.5 µm at tip, basal septum observed fairly often; conidia hyaline, ellipsoidal to lunate, 6.5–10 x 3–4.5 µm, often associated in clusters of 2–4 conidia per conidiogenous cell (FIG. 4). Shows genetic similarity to Epichloë elymi Schardl et Leuchtm. and E. festucae Leuchtm. Schardl et Siegel.
Etymology. – In honor of American grass breeder, C. Reed Funk, whose studies have highlighted the importance of the symbiosis between grasses and epichloë endophytes.
HOLOTYPE. – USA, South Fork, Colorado, infecting Achnatherum robustum, 2000, leg. T.A. Jones, CUP 65764.
Specimens examined. – ATCC MYA-2583 from the HOLOTYPE, CUP 65764, isolates E4097-E4100 from Achnatherum robustum, USA, South Fork, Colorado, 2000, leg. T.A. Jones.
Neotyphodium gansuense var. inebrians C.D. Moon et C.L. Schardl, var. nov. FIG. 5
Coloniae 5–8 mm diam aetate 8 hebdomadum ad 22 C in PDA, elevatae, maxime convolutae, albidae vel eburneae, ceraceae, reverso eburneae. Hyphae septate, 2.8–4.2 µm latae. Conidia in cultura non observata. Affinis genetice Neotyphodii gansuensis C.J. Li et Z.B. Nan.
Colony 5–8 mm diam after 8 wk at 22 C on PDA (FIG. 5); colonies raised from agar surface, highly convoluted, cream to light tan, waxy, colony margins distinct. Colony reverse tan. Hyphae septate at branching junctions, 2.8–4.2 µm wide, although occasionally bulging to 6.5 µm wide. Sporulation not observed in culture. Shows genetic similarity to Neotyphodium gansuense C.J. Li et Z.B. Nan.
Etymology. – Varietal form of Neotyphodium gansuense referring to the host species and psychoactive effects of the mycotoxins.
HOLOTYPE. – CHINA, Xinjiang Province infecting Achnatherum inebrians, autumn 1993, leg. K.F. Min, I. Fletcher, and P.S. Harris, CUP 65636.
Specimens examined. – ATCC MYA-1228 from the HOLOTYPE, CUP 65636; ATCC MYA-1187 from A. inebrians, CHINA, Xinjiang Province, autumn 1993, leg. K.F. Min, I. Fletcher, and P.S. Harris, CUP 65626.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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; SUPPLEMENTAL TABLE II). Colonies of the new species, N. guerinii, from M. ciliata and M. transsilvanica, were white and cottony with abundant aerial hyphae (FIG. 1).
The morphology of the N. funkii isolate MYA-2583 (FIG. 3) was consistent with observations by Kaiser et al (1996), who extensively sampled endophyte isolates from 10 A. robustum populations in New Mexico and Colorado. Colonies grew at a moderate rate and were white and cottony. Conidia of MYA-2583 had a much narrower size range than the extraordinary range (2.5–16 µm long) reported by Kaiser et al (1996). A distinctive feature of MYA-2583 was the frequent occurrence of multiple spores associated with individual conidiogenous cells (FIG. 4), where often 2–4 conidia were clustered adjacent to, or in the immediate vicinity of, the apex of a conidiogenous cell. This feature has been documented for other isolates from the same host species, and in scanning electron micrographs the clusters of conidia appear to be encased in mucilage (Kaiser et al 1996). Similar clusters can be observed on young stromata of Epichloë species and are characteristic of E. glyceriae cultures (Schardl and Leuchtmann 1999) but otherwise are uncommon in cultured epichloë endophytes.
Consistent with the N. gansuense description, isolates from A. inebrians in Gansu Province grew to 30 mm diam after only 4 wk on PDA at 22 C and had a white cottony appearance (FIG. 7) (Li et al 2004). These characteristics differed considerably from those of isolates from A. inebrians in Xinjiang Province. The latter, described herein as N. gansuense var. inebrians, emerged from the host tissue slowly, taking 6–8 wk to be visible by eye (FIG. 5). Colonies of N. gansuense var. inebrians were slow growing, raised, waxy and highly convoluted, consistent with the reports of Bruehl et al (1994) and Miles et al (1996). Within colonies hyphae were too densely packed to examine directly, so colony material was finely ground in sterile water, inoculated on water agar and grown at 22 C. From these preparations growing hyphae were observed, but spores were not apparent, in contrast to N. gansuense isolates that produced spores under stress (Li et al 2004).
The colony morphology of N. chisosum (FIG. 6) and its growth rate were consistent with its taxonomic description (White and Morgan-Jones 1987), although we did not observe conidia or conidiogenous cells in cultures. It is possible that the ability to conidiate has been lost in subcultures of the ex type isolate.
Phylogenetic relationships.— –
Isolates of the three newly proposed taxa previously were subjected to phylogenetic analysis based on tubB and tefA haplotypes, indicating their unique evolutionary origins within the Epichloë and Neotyphodium group (Moon et al 2004). In the present study more extensive phylogenetic analyses have been undertaken with additional representative isolates of N. guerinii and N. funkii and also involving sequence from an additional gene, actG, that has been demonstrated to be unlinked to tubB and tefA in Mendelian analysis of E. typhina (Craven et al 2001a). Furthermore sequence data have been obtained from N. gansuense and a tubB sequence available in GenBank (accession No. DQ675589
[GenBank]
) is attributed to a Neotyphodium sp. from Achnatherum sibiricum in Inner Mongolia, China.
The tubB phylogeny (FIG. 8) provided further support for the unique phylogenetic origins of the new Neotyphodium taxa described in this study. A key finding was the close relationship between the ex A. inebrians isolates ATCC MYA-1187 and ATCC MYA-1228. These isolates had been informally designated ‘N. inebrians’ (Moon et al 2004) but here are described as N. gansuense var. inebrians because of their close phylogenetic relationship to N. gansuense from the same host species.
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The presence of two tubB haplotypes in N. guerinii isolates indicated a hybrid origin of this species as well. One of its tubB haplotypes grouped in a well supported clade with N. gansuense and N. gansuense var. inebrians and was related particularly closely to the tubB sequence from the ex A. sibiricum endophyte. The other tubB haplotype in N. guerinii grouped with E. typhina sequences. Included in this analysis were isolates from other Melica species, which previously were characterized by molecular phylogenetics. One of these endophytes has been described as Neotyphodium melicicola (Moon et al 2002), and the other had phylogenetic and morphological characteristics consistent with Neotyphodium tembladerae Cabral et J.F. White (Gentile et al 2005). Sequences from N. guerinii did not group with those of these other two species. Although N. guerinii and N tembladerae both had tubB haplotypes related to those of E. typhina, these haplotypes were distant from each other in the E. typhina clade. Furthermore, there was no indication of any contribution from Epichloë glyceriae to any of the endophytes from Melica species, even though E. glyceriae is associated with another genus (Glyceria) within grass tribe Meliceae.
Additional phylogenetic analysis was conducted based on actG haplotype sequences for representatives of the three new taxa and N. chisosum, along with sequences from other Neotyphodium and Epichloë species (FIG. 9). The phylogenetic origins of N. funkii, N. guerinii and N. gansuense var. inebrians from actG analyses were consistent with those determined by tubB analysis. From each N. gansuense var. inebrians isolate only a single actG haplotype was detected, and this grouped with sequences from N. gansuense that, together with one of the actG haplotypes from N. guerinii, formed a distinct and well supported clade. Consistent with the tubB results N. guerinii had a second actG haplotype, which was placed in the E. typhina clade. Two haplotypes also were identified in N. funkii, and these grouped in the E. elymi and E. festucae clades.
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) (Moon et al 2004) and might indicate aneuploidy resulting from the loss of redundant genetic material after its hybrid origin. However, the apparent lack of multiple actG haplotypes also might be due to false-negative amplifications, if priming sites had diverged between gene copies. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSTAXONOMYRESULTSDISCUSSIONLITERATURE CITED |
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). From Achnatherum inebrians of northwestern China, N. gansuense var. inebrians was described. This is morphologically and geographically distinct from N. gansuense, to which it is closely related.
Host and geographic associations.— –
The nonhybrid Neotyphodium endophytes that are not known to be closely related to extant Epichlöe spp. tend to exhibit considerable phylogeographical structure and some association of host and phylogenetic relationships, suggestive of host and endophyte co-evolution. As demonstrated, each sexual (Epichloë) species is associated with a particular host tribe in Europe or North America (Craven et al 2001a), and N. aotearoae is a nonhybrid associated with Echinopogon ovatus in Australia and New Zealand (Moon et al 2002). In keeping with this pattern our phylogenetic analyses grouped nonhybrid isolates from A. inebrians collected in different provinces of China, and their clade included an isolate (which we presume is also nonhybrid) from a congeneric host, A. sibiricum.
In contrast to the nonhybrids, taxonomic relationships of the hosts were not necessarily reflected in the evolutionary relationships of the hybrid endophytes. Notably, one of the ancestors of N. guerinii apparently derived from the N. gansuense clade. Host tribes Stipeae (including Achnatherum species) and Meliceae (including Melica and Glyceria species) are not closely related, having diverged from each other and most other tribes early in evolution of the grass subfamily Pooideae (Soreng and Davis 1998). Therefore the association of N. gansuense with one of the ancestors of N. guerinii was not attributable to host relationships but relates instead to geography because both species have Eurasian hosts.
The endophyte taxa from the two North American Achnatherum species, N. chisosum from A. eminens and N. funkii from A. robustum, had different hybrid origins, and there was no indication of genomic contribution from the N. gansuense clade. There also was little indication of phylogeographic structure in their relationships; the inferred ancestral species of each of these hybrids are associated with different continents. Specifically, ancestors of N. chisosum are related to North American E. amarillans and Eurasian E. bromicola and E. typhina whereas, N. funkii appears to derive from North American E. elymi and Eurasian E. festucae. Despite the fact that E. typhina and E. festucae are found only in European grasses (including many that humans have spread to other continents), hybrids with ancestral relationships to E. typhina, E. festucae or both are common worldwide (Gentile et al 2005, Moon et al 2002, 2004). This pattern suggests either that such hybrids have spread widely or that E. typhina and E. festucae exist or recently existed in Eurasia, southern Africa, Australia, and South and North America, where they regularly have hybridized with each other or other endophyte species.
Of interest, N. chisosum represents the first endophyte of a native North American grass with a demonstrable relationship to E. bromicola, which has been identified only in Eurasia. The corresponding tubB haplotype from N. chisosum seems to have a basal relationship to the E. bromicola clade, which might indicate an early colonization of A. eminens or ancestral grass species or alternatively might indicate an undiscovered relative of E. bromicola in North America.
Possible origins of N. gansuense and N. guerinii.— –
The hosts of N. guerinii and N. gansuense are in distantly related tribes (Kellogg 1998), Meliceae and Stipeae respectively, so the relationship between N. gansuense and one of the N. guerinii ancestors cannot be explained by a history of vertical transmission over the course of host speciation. Instead, this relationship suggests an Epichloë species that recently has become extinct or might yet be discovered. For the sake of this discussion we will refer to that hypothetical species as ‘Epichloë gansuensis’. Like most extant Epichloë species, ‘E. gansuensis’ probably had a strong or exclusive association with grasses related at the tribe or even genus level (Schardl and Leuchtmann 2005). (Epichloë typhina is the only exception, but even so related genotypes of E. typhina tend to have the same host.) Nevertheless, a key event in evolution of N. guerinii or N. gansuense was probably a cross-tribe jump of ‘E. gansuensis’.
An alternative scenario involving transmission of asexual N. gansuense or N. guerinii between hosts seems unlikely given the importance of ascospores in horizontal transmission (Brem and Leuchtmann 1999, Chung and Schardl 1997). Therefore we currently favor the simplest hypothesis, that a sexual member of the N. gansuense clade originally was associated with an Achnatherum species but gave rise by ascospore-mediated host jump to a symbiont in a common ancestor of M. ciliata and M. transsilvatica. The new host plant already might have had a resident endophyte derived from E. typhina or a descendant plant might have become infected by E. typhina, and the co-occurring endophytes then would have fused hyphae and nuclei to generate N. guerinii. The close relationship between the ‘E. gansuensis’ ancestor of N. guerinii and the recently deposited sequence from an endophyte of A. sibiricum suggests that populations of A. sibiricum or a close relative still might harbor a sexual representative of the N. gansuense clade.
Concluding remarks.— – The multidisciplined approach of using both morphological and phylogenetic analyses in the identification and description of new Neotyphodium species provides a comprehensive picture of the species as well as their evolutionary origins. Based on such criteria we have proposed three new Neotyphodium taxa: N. gansuense var. inebrians, N. funkii and N. guerinii. This work forms part of an ongoing project that seeks to better understand the origins and processes that drive the evolution of this unique group of fungal symbionts.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Present address: AgResearch Limited, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand. 
2 Present address: Plant Biology Division, Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401.
3 Corresponding author. E-mail: schardl{at}uky.edu
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