Proc. Nati. Acad. Sci. USA Vol. 91, pp. 2542-2546, March 1994 Evolution Evolutionary diversification of fungal endophytes of tall fescue grass by hybridization with EpichloO species (Festhwa annaca/Acrnuonium conop um/C lavptee/mut symblots/jtubu gns) HUEI-FUNG TSAI*, JIH-SHIOU LIu*, CHUCK STABENt, MICHAEL J. CHRISTENSENt, GARRICK C. M. LATCH*, MALCOLM R. SIEGEL*, AND CHRISTOPHER L. SCHARDL*§ *Department of Plant Pathology, S-305 Agricultural Science Building, University of Kentucky, Lexington, KY 40546-0091; thomas Hunt Morgan School of Biological Science, University of Kentucky, Lexington, KY 40506-0225; and tAgResearch, Grasslands Research Centre, Private Bag 11008, Palmerston North, New Zealand Communicated by Peter H. Raven, November 29, 1993 ABSTRACT The mutulisc aoatin of tall fescue (Festuca arundinacca) with seed-borne Hnal symbionts (en- dophytes) are Important for fitness of the grass host and Its surival under biotic and ablotic stress. The tall fescue endo- phytes are asexual relatives of biological species (mating pop- ulations) of genus EpichWoe (Clavlcipitaceae), sexual fungi that cause grass choke disease. Isozyme studies have s d considerable genetic disit among endophytes of tall fescue. Phylogenetic relationships among seven Isolates from tall fes- cue, three from meadow fescue (a probable ancestor of tall fescue), and nine EpkhkA Isolates from other host species were Investigated by comparing sequences of noncodin segments of the P-tubulin (tub2) and rRNA (-rn) genes. Whereas each EpichloE isolate and meadow fescue endophyte had only a single tub2 gene, most tall fescue endophytes had two or three distinct tub2 coples. Phylogenetic analysis of tub2 sequences indicated that the presence Ofmultip copies in the tall fescue endophytes was a consequence of hybridization with Ephklol species. At least three hybridization events account for the dbution and relationships of tWb2 genes. These results suggest that inter- speific hybridization is the major cause of genetic diversifi- cation of the tail fescue endophytes. Mutualistic fungal endophytes are important for the fitness of many grass hosts, such as hexaploid tall fescue (Festuca arundinacea), on which the endophytes can confer enhanced growth and fecundity, protection from herbivores and para- sites, and a profound increase in drought tolerance (1). These endophytes spend their entire life cycles within host tissues, are maternally transmitted in seed, are completely nonpath- ogenic, and are asexual. They are evolutionarily derived from the sexual (teleomorphic) fungi of genus Epichloe (Ascomy- cotina, Clavicipitaceae), which cause grass choke disease (2). During the pathogenic stage of Epichloe (3, 4) the affected inflorescence of the host is sterilized and the fungus, if crossed with an opposite mating type of the same biological species (i.e., mating population), produces and disseminates ascospores (Fig. 1). Epichloe species vary widely in the relative importance of sporulation (choke disease) versus seed transmission during their life cycles. For example, the symbiont of F. rubra depicted in Fig. 1 typically exhibits a high level of seed transmission and provides biological pro- tection to its host (5). More antagonistic Epichloe species completely sterilize infected plants (6). Thus, symbioses with Epichloe and endophytes span the spectrum from antagonism to mutualism, making them an ideal system for studying the evolution of fungal mutualists. The endophytes contribute important and heritable genetic diversity to their symbiotic systems (7). Because they are FiG. 1. Life cycles of Epichloe sp. MP-ll in Festuca rubra (refs. 3 and 4; C.L.S. and M.R.S., unpublished data). Fungal structures are shown in circles. In the asexual cycle, highly convoluted hyphae grow imtercellularly in leaf sheaths (a), floral meristems (b), and in the ovules of the florets (c) such that the fungus is transmitted in the next generation of seed (d). In the sexual cycle, the fungus also grows intercellularly in vegetative leaf sheaths without causing symptoms (e), but then emerges from the leaf sheath surrounding the immature host inflorescence, produces spermatia, and arrests inflorescence maturation (f). Fertilization occurs by transfer of spermatia of opposite mating type (g). If the parents are conspecific [same mating population (MP)], perithecia containing asci develop (h) and fila- mentous ascospores are ejected (i). Germinating ascospores initiate cycles of asexual sporulation (conidiation) and are postulated to cause infection of host florets (j) and ultimately of seed (k). Endophytes of tall fescue and meadow fescue are only known to undergo the asexual cycle. maternally transmitted (8), produce several classes of anti- herbivore alkaloids (9), and confer other fitness enhance- ments to their hosts (1), the diversity of the endophytes probably plays a major role in the long-term fitness and adaptability of the grass-fungus symbiotic entities. A recent survey assessing allozyme variation, morphological charac- teristics, and profiles of protective alkaloids indicated con- siderable diversity among tall fescue endophytes (10). Three taxa were described as F. arundinacea endophyte taxonomic grouping one (FaTG-1 = Acremonium coenophialum), FaTG-2, and FaTG-3. Endophytes of meadow fescue (Fes- tuca pratensis) were also surveyed. This grass is the likely Abbreviations: FaTG, Festuca arundinacea endophyte taxonomic grouping; ITS, internal transcribed spacer; MP, mating population; IVS, intervening sequence. §To whom reprint requests should be addressed. 2542 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.