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Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Apartado Postal 70-275, Ciudad Universitaria, México, D.F. 04510, México
David S. Gernandt
Centro de Investigaciones Biológicas, Universidad Autónoma del Estado de Hidalgo, Plaza Juárez A.P. 1-69, Pachuca, Hidalgo 42001, México
Jeffrey K. Stone
Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon 97331-2902
Peter R. Johnston
Landcare Research, Private Bag 92170, Auckland, New Zealand
Ignacio H. Chapela
Division of Ecosystem Sciences, 334 Hilgard Hall, University of California, Berkeley, California 94720-3110
Rodolfo Salas-Lizana
Elena R. Alvarez-Buylla
Departamento de Ecología Evolutiva, Instituto de Ecología, Universidad Nacional Autónoma de México, Apartado Postal 70-275, Ciudad Universitaria, México, D.F. 04510, México
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Lophodermium comprises ascomycetous fungi that are both needle-cast pathogens and asymptomatic endophytes on a diversity of plant hosts. It is distinguished from other genera in the family Rhytismataceae by its filiform ascospores and ascocarps that open by a longitudinal slit. Nucleotide sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA were used to infer phylogenetic relationships within Lophodermium. Twenty-nine sequences from approximately 11 species of Lophodermium were analyzed together with eight sequences from isolates thought to represent six other genera of Rhytismataceae: Elytroderma, Lirula, Meloderma, Terriera, Tryblidiopsis and Colpoma. Two putative Meloderma desmazieresii isolates occurred within the Lophodermium clade but separate from one another, one grouped with L. indianum and the other with L. nitens. An isolate of Elytroderma deformans also occurred within the Lophodermium clade but on a solitary branch. The occurrence of these genera within the Lophodermium clade might be due to problems in generic concepts in Rhytismataceae, such as emphasis on spore morphology to delimit genera, to difficulty of isolating Rhytismataceae needle pathogens from material that also is colonized by Lophodermium or to a combination of both factors. We also evaluated the congruence of host distribution and several morphological characters on the ITS phylogeny. Lophodermium species from pine hosts formed a monophyletic sister group to Lophodermium species from more distant hosts from the southern hemisphere, but not to L. piceae from Picea. The ITS topology indicated that Lophodermium does not show strict cospeciation with pines at deeper branches, although several closely related isolates have closely related hosts. Pathogenic species occupy derived positions in the pine clade, suggesting that pathogenicity has evolved from endophytism. A new combination is proposed, Terriera minor (Tehon) P.R. Johnst.
Key words: endophyte, evolution, ITS region, pathogen, phylogeny, Rhytismataceae
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Saikkonen et al 1998) and have been found in all woody plants that have been examined (Carroll and Carroll 1978, Petrini et al 1982, Chapela and Boddy 1988, Espinosa-García and Langenheim 1990, Hata and Futai 1995, Stone et al 2000). Endophytic fungi of woody hosts are a highly diverse assemblage, primarily of ascomycetes, representing a range of symbiotic interactions with their host plants, from commensals and mutualists to latent pathogens. Most phylogenetic studies of above-ground endophytes have focused on grass symbionts (i.e., Schardl et al 1991, Glenn et al 1996, Reddy et al 1998, but see Gernandt et al 1997). Although both have been termed "endophytes", endophytes of grasses are limited to a relatively small group of genera in the Clavicipitaceae, while endophytic fungi of woody hosts are biologically and ecologically more diverse (Stone et al 2000). Phylogenetic studies of woody plant endophytes are needed to evaluate degrees of specialization to habitats and hosts, endophyte diversity and distribution patterns, and the evolutionary pathways leading to this widespread association.
Lophodermium Chevall. is a large and complex genus in the family Rhytismataceae (Rhytismatales, Ascomycota). This genus offers a great opportunity to study the evolutionary history of the important and widespread symbiotic relationships among endophytes and woody plants. Approximately 103 species have been described on different plant groups (Kirk et al 2001). Lophodermium is conspicuous on senescent and abscised conifer needles and recognizable in culture by its anamorphic states; it is among the most common endophytes isolated from Pinus, Abies and Picea (Stone et al 2000). Although more than 20 species of Lophodermium are known to colonize needles of coniferous trees and shrubs, only one of these, L. seditiosum, is a major pathogen, causing needle cast of pines in forest nurseries and plantations (Minter and Millar 1980, Sinclair et al 1987). In most Lophodermium species from pine, ascocarps usually are found on recently abscised needles. These fungi colonize healthy needles, where they reside as endophytes within the asymptomatic host tissue; ascocarps typically mature after needle abscission (Deckert et al 2001). Lophodermium species have been described from several other plant families and are especially diverse on Poaceae and Ericales (Cannon and Minter 1983, Johnston 1989, 1992, 2001, Ellis and Ellis 1997).
Lophodermium is characterized by apothecia covered with a clypeus, which opens in a single longitudinal slit, and hyaline, filiform, nonseptate ascospores. Delimitation of Lophodermium species in pines is based on characters observed in mid-point cross sections of mature ascocarps. These characters include the position of the ascocarp in relation to the host's epidermis and hypodermis, the number and position of displaced epidermal cells, the presence and pigmentation of distinctive "lip" cells along the slit zone and the degree of pigmentation of the subhymenial lower wall associated with the presence of a perimeter line. Characters related to the external appearance include the presence of zone lines or perimeter lines, the size and shape of the ascocarp and its position in relation to the stomatal rows (Darker 1967, Minter 1981).
Several authors have emphasized the artificial nature of the separation of many genera in Rhytismataceae, including Lophodermium (Darker 1967, Johnston 1989, 1990, Spooner 1991). Early classifications based generic delimitation primarily on ascospore shape. More recent classifications include characteristics of the ascocarp, asci and ascospores, as well as the anamorph. For example, Johnston (1988, 1989) delineated six different groups with 21 Lophodermium species from New Zealand based on distinctive developmental features of the ascocarps. The same developmental features that defined some of these groups were shared by species in different genera within the family, including Hypoderma, Meloderma and Coccomyces.
A comprehensive monograph of Lophodermium is a difficult task, considering its putative artificial constitution, its complexity and the number of species ascribed to the genus. In this study we used internal transcribed spacer (ITS) region sequences from nuclear ribosomal (nr) DNA to infer phylogenetic relationships for Lophodermium species with different host preferences and geographic distributions. The ITS region has been used widely to resolve intraspecific and interspecific phylogenetic relationships, providing insights into the utility of conventional morphological characters and species delimitation in fungi (Lee and Taylor 1992, Carbone and Kohn 1993, Harrington and Potter 1997, Johnston and Jones 1997, Jacobs and Rehner 1998). Monophyly of the genus is tested by the inclusion of six other genera in the family Rhytismataceae. The range of interactions between Lophodermium and its hosts also provides an opportunity to study levels of specificity and to trace the transition between pathogenicity and endophytism. Finally, by mapping morphological and developmental characters used for species delimitation onto the molecular phylogeny, we investigate levels of convergent evolution in this group and whether certain morphological characters can be attributed to common descent or rather to anatomical similarities in their hosts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) obtained from GenBank were included, although they were missing the first 40 bp of ITS1. Isolates of Elytroderma deformans, Lirula macrospora, Meloderma desmazieresii, Colpoma quercinum and Tryblidiopsis pinastri were obtained from culture collections. All five of these genera occurred in a Rhytismataceae clade with Lophodermium in a phylogenetic analysis of ascomycete small-subunit nrDNA sequences (Gernandt et al 2001). Elytroderma deformans, L. macrospora and M. desmazieresii share characteristics with some species in Lophodermium, including host substrate, ascocarp structure and development, and anamorph morphology (Darker 1967, Cannon and Minter 1983, Johnston 1988, 1989). Terriera minor differs from Lophodermium in structure of the ascomatal primordium and in the ascospores lacking a gelatinous sheath (Johnston 2001, as Lophodermium minus). We included these four species to test the monophyly of Lophodermium, while Tryblidiopsis pinastri and Colpoma quercinum, two morphologically more distinct members of Rhytismataceae, were used to root the phylogenetic trees (Nixon and Carpenter 1993). Tryblidiopsis pinastri and C. quercinum occur on dead stems and branches and have distinctive erumpent ascomata.
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DNA extraction and amplification –
A modification of the procedure of Doyle and Doyle (1987) was used to extract DNA from fungal cultures. Samples were ground in 1.5 mL tubes with sand and 1 mL of 60 C 2x CTAB supplemented with 2% sodium bisulfite, then incubated at 37 C for 1–2 h. Two chloroform : isoamyl alcohol (24:1) extractions were performed, DNA was precipitated in cold isopropanol, and the pellet was washed with 70% ethanol and dissolved in TE (10 mM Tris-HCL pH 7–8, 0.1 mM EDTA). PCR reactions were performed in 100 µL volumes with 1.5 U ReplithermTM DNA polymerase (Epicentre Technologies, Madison, Wisconsin), 1x ReplithermTM buffer, 1.5 mM MgCl2, 4% DMSO, 2% BSA, 200 µmol each dNTP, 1.5 µmol each primer and approximately 50 ng sample DNA. The primers used to amplify the ITS region were ITS1F: 5' CTT GGT CAT TTA GAG GAA GTA A 3' (White et al 1990) and, NL6A: 5' CAA GTG CTT CCC TTT CAA CA 3' (Egger 1995). These primers anneal to the 18S small- and 28S large-subunit nuclear ribosomal RNA genes, respectively. PCR conditions were 40 cycles at 94 C, denaturation for 45 s, 55 C annealing for 45 s and 72 C extension for 90 s. Reactions were terminated after a final extension at 72 C for 9 min. Products were purified with QIAquick gel extraction kits (Qiagen, Chatsworth, California). Cycle sequencing with dye terminator chemistry was performed using an ABI model 373A fluorescent sequencer (Applied Biosystems, Foster City, California). Products were sequenced in both 5' to 3' and 3' to 5' directions.
Sequence analyses –
Sequence alignments were performed using CLUSTALW (Thompson et al 1994) with a gap weight = 40, gap length weight = 5, and adjusted manually by visual inspection in Genetic Data Environment (Smith et al 1994). Phylogenetic analyses were conducted with PAUP* version 4.0b10 (Swofford 2002) using the parsimony criterion. Heuristic searches were performed with these options: 500 replicates of random-addition sequence, tree bisection reconnection (TBR) branch swapping and no maxtree limit. Gaps were treated as missing data to minimize homology assumptions. All characters were equally weighted and unordered. Support for branching topologies was evaluated with 500 replicates of bootstrapping (Felsenstein 1985). A maxtree limit of 10 000, random-addition sequence with 10 replicates and TBR branch swapping were used in the bootstrap analysis. To examine the number of additional steps required to collapse each clade, Bremer support/decay indices (Bremer 1988, 1994) were calculated by using the command "keep trees" for trees 1–4 steps longer and by the constraint consensus analysis for each clade with longer steps (Morgan 1997). In both methods, the above heuristic search parameters were used.
Morphological character analysis –
Nineteen primarily qualitative morphological characters were scored for most of the isolates (Table II and Table III). Characters were chosen based on availability and on traditional use for taxonomic assignment of Lophodermium species in pines and were scored based on comparison of isolates with published descriptions (Darker 1967, Johnston 1989, 1992, 2001, Minter 1981, Minter and Hettige 1983). All morphological characters were coded as unordered and were optimized onto the trees obtained from the ITS region analysis by using unweighted parsimony with multistate taxa treated as polymorphic. The molecular data matrix is available in TreeBASE (M1360).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Morales et al 1993, Jasalavich et al 1995). The length of the ITS alignment was 484 bp, including 201 variable positions, 162 of which were phylogenetically informative. The 34 complete ITS region sequences ranged in length from 424 to 455 bp. Most length variation was in ITS1 (range 122–150 bp, mean = 129.5 ± 8.4), due mainly to the presence of an 18 bp insertion present in both isolates of T. minor and partially shared with outgroup and L. piceae in 13 positions. The 5.8S rDNA gene was 158 bp in all species. The ITS2 varied between 143 and 158 bp (mean = 149.1 ± 4.9) and had a higher G + C content (59%) than ITS1 and 5.8S (46–47%), a pattern that also is characteristic of angiosperms, some genera in the family Pinaceae and other groups of fungi (Baldwin et al 1995, Kuhls et al 1997, O'Donnell et al 1998, Liston et al 1999). The uncorrected intraspecific percentage of nucleotide differences was 5.6% between L. pinastri isolates, 2.9% between L. agathidis isolates, 1.3% between T. minor isolates, 0.9% between L. indianum isolates, 0.4% between L. australe isolates, 0% between L. conigenum isolates, and ranged from 0% to 7.6% between L. baculiferum isolates and from 0.2% to 3.8% between L. nitens isolates. Intrageneric nucleotide differences were as high as 19.5% between L. agathidis and L. conigenum.
Phylogenetic analysis – The heuristic search recovered eight equally most-parsimonious trees (Fig. 1). Twenty-one clades had bootstrap support greater than 50%, 14 of which were greater than 90%. Decay indices ranged from 0 in the two branches uniting closely related species at the tips of the tree that collapsed in the strict-consensus tree (not shown) to 19 steps on the branch uniting two isolates of L. agathidis. A Lophodermium clade was resolved in the strict-consensus tree but received low bootstrap and decay support: (61% bootstrap, decay index = 2).
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), and the M. desmazieresii sequence from P. strobus had only a single nucleotide difference from an undescribed Lophodermium species from P. ayacahuite. Furthermore, Elytroderma deformans CBS181.68 occupied a solitary branch within the Lophodermium clade. The E. deformans strain did not match any Lophodermium isolate sequenced (8.0–13.7% nucleotide divergence from other members of the pine clade). The occurrence of these three sequences in the Lophodermium clade suggests either human error or the need to synonymize one or both of these genera under Lophodermium (see below). Regardless, these three sequences are considered representatives of Lophodermium in the following description of the results. At the base of the pine clade, L. molitoris and Lophodermium sp. 5 were in a tenuous sister relationship to three L. baculiferum isolates paraphyletic to two Leptostroma decipiens isolates (Fig. 1). A fourth L. baculiferum isolate was in an intermediate position between this group and the remaining species of Lophodermium from pines. The remainder of the pine clade included L. nitens, L. pinastri, L. indianum, L. conigenum, L. australe, several undescribed species from Mexican pines, and the Meloderma and Elytroderma sequences. The highly similar L. conigenum and L. australe sequences formed a clade, with the L. australe sequences paraphyletic to the two L. conigenum sequences. Two undescribed Lophodermium isolates from P. ayacahuite and P. douglasiana, a Meloderma sequence from P. strobus, and two (paraphyletic) L. indianum isolates together formed a well-supported clade.
Other Lophodermium species were from a phylogenetically diverse assemblage of hosts and were paraphyletic with respect to the pine clade. Two L. agathidis isolates from Agathis (Araucariaceae) and Metrosideros (Myrtaceae), respectively, in New Zealand were monophyletic and in a sister position to L. actinothyrium isolated from Poaceae in Argentina. Lophodermium piceae was in a sister position to all other Lophodermium sequences. The branch at the base of the Lophodermium clade had low bootstrap support (61%). Of the remaining genera, two monophyletic Terriera sequences formed a weakly supported sister relationship to Lirula macrospora and Tryblidiopsis pinastri formed a weakly supported sister relationship to Colpoma quercinum.
Morphological and combined analyses – Preliminary parsimony analyses indicated that inclusion of three taxa with a moderate to high amount of missing morphological data (Leptostroma sp., Meloderma, and Elytroderma) resulted in unresolved trees when analyzed apart from ITS (not shown). Deleting these three taxa reduced the number of informative characters from 19 to 18 (character 10, ascospore shape, was no longer informative) but resulted in a more resolved tree (Fig. 2). Nevertheless, only four branches received bootstrap values greater than 50%, of which only two agreed in topology with the ITS tree, one uniting Lophodermium baculiferum and its anamorph, Leptostroma decipiens (95% bootstrap) and the other uniting Tryblidiopsis and Colpoma (61% bootstrap).
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(0.50) were: ascomata opening, ascomata shape, lip cell color, ascospore septa, paraphyses shape and conidium shape (Table III). Two ecological characters had CI values 0.50: host and substrate of fructification. Ascomata position had relatively low consistency (CI = 0.40; see distribution in Fig. 1).
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) are indicated in Fig. 3. Based on our incomplete sampling, species or clades on closely related hosts (within the same pine subsection) included L. conigenum (P. radiata), the clade including Lophodermium sp. 4, Leptostroma sp., Meloderma from CBS, and three L. nitens isolates (P. ayacahuite, P. chiapensis and P. strobus), and the clade of two L. decipiens isolates (P. ponderosa) and three L. baculiferum isolates (P. ponderosa and P. montezumae). Lophodermium species on intermediately related hosts (different subsection, same section) included L. indianum (P. greggii and P. hartwegii) and L. australe (P. pseudostrobus and P. palustris). Lophodermium species from more distantly related pine hosts include L. pinastri (P. ponderosa and P. sylvestris) and L. baculiferum 2 (P. contorta), which did not group with the other three L. baculiferum isolates from P. ponderosa and P. montezumae. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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erected the genus Meloderma, he pointed out similarities in ascus shape and in the position of the developing ascocarp in relation to the host tissue between Meloderma desmazieresii and Lophodermium species from pines. More recent studies have described similarities in ascocarp development related to the internal differentiation of the upper wall of unopened ascocarps between M. desmazieresii and Lophodermium from pines (Johnston 1988), anamorphs (Minter 1980) and ascospore discharge mechanisms (Minter and Cannon 1984). Both Meloderma and Elytroderma have been segregated from Lophodermium species mainly based on ascospore morphology. Species of Meloderma have short, cylindrical ascospores versus the filiform ascospores in Lophodermium. Elytroderma is distinguished by its large, clavate, 1-septate ascospores.
In the ITS results reported here, Elytroderma deformans from P. ponderosa and two strains of Meloderma desmazieresii from P. ayacahuite and P. strobus occurred in the Lophodermium pine clade but in distinct parts of the tree. If this placement is not a result of human error (see below), then it suggests that ascus shape and position of the developing ascocarp should be weighted more heavily than ascospore shape in the delineation of Lophodermium because Elytroderma and Meloderma have similar ascocarp development patterns. Characters related to the patterns of development of the sterile tissues of the ascocarps have proven to be more useful than spore shape in defining other groups within the Rhytismataceae (Johnston 1988, 1989). The taxonomic significance of these characters remains uncertain because ascocarp development is poorly understood in most species. Furthermore, different Lophodermium species that share closely related hosts may have similar ascocarp developmental patterns, particularly if the anatomy and disposition of the available tissues in the hosts constrain ascocarp development. However, the possibility that ascocarp development is a reliable criterion for the delimitation of genera comprising natural groups within the Rhytismataceae should stimulate further ontogenetic studies.
The occurrence of both Elytroderma and Meloderma in the Lophodermium pine clade further suggests that ascospore shape, such as filiform versus clavate or septate, is a reliable character for species, but not generic, delimitation. Studies of other ascomycetes have reported convergence in ascospore and conidium shape (e.g., Gaudet et al 1989, Wingfield et al 1994). The inconsistent placement of the two M. desmazieresii sequences might be the result of parallel modifications in ascospore shape (from filiform to cylindrical). If so, M. desmazieresii might be polytypic and hitherto undetected differences might separate these two strains into separate species. This is supported by the geographical separation of the strains; the CBS isolate of M. desmazieresii was obtained from P. ayachahuite at a pinetum in Texcoco, central Mexico, and the Deckert et al isolate was obtained from P. strobus in eastern Canada. If, as the ITS results suggest, ascospore shape is not a reliable character for genus-level differences, M. desmazieresii might not be monotypic and actually represent Lophodermium species with reduced ascospores.
An alternate explanation is that the Elytroderma and Meloderma sequences used in this study are misidentified Lophodermium. If so, the presumed Elytroderma deformans CBS181.68 sequence occurs on a solitary branch within the Lophodermium clade and does not match any Lophodermium species yet sequenced. The CBS Meloderma isolate from P. ayacahuite in central Mexico has a high sequence similarity with L. nitens strains throughout North America (Deckert et al 2002; this study, Figs. 1 and 3). Although the Meloderma strain from Deckert et al was different from our sequence, it still occurred in the Lophodermium pine clade, in a sister relationship to Lophodermium sp. 1. Given the disagreement between the two Meloderma sequences and the limitation of having only a single Elytroderma sequence, the possibility that the Elytroderma and at least one of the two Meloderma isolates might have been contaminated by Lophodermium, either during isolation or subsequent handling, must be considered. We have no details regarding the care used in the isolation of the CBS cultures, but the Meloderma from P. strobus was isolated after positive identification of the ascocarp and under careful observation of the ascospores (Deckert et al 2002). Small-subunit sequences based on the two CBS isolates in question have been included in a previous study as Elytroderma and Meloderma (Gernandt et al 2001). The uncertain identities of these cultures suggest caution when relying on cultures and sequences from third parties, particularly when these cultures are sterile and their identity cannot be rechecked. Despite these uncertainties, we hypothesize that Meloderma and Elytroderma are members of Lophodermium, although this interpretation deserves further study.
Species delineation –
Morphological characters traditionally used for species delimitation showed different degrees of phylogenetic value (Table III). Most characters were homoplastic with respect to the molecular phylogenetic reconstruction. The depth of embedding of the ascocarp, a key character for distinguishing pine inhabiting Lophodermium species (Minter 1981), showed much plasticity on the ITS tree (Fig. 1). There might be multiple sources of incongruence between the phylogenetic hypotheses and the morphological characters used for species delimitation. One possibility is that closely related species or even the same species can colonize hosts with different needle anatomies, resulting in differences in the location of the ascocarp in relation to the number of pine cell layers in the needle. This could be the case for the clade that comprises L. indianum and two undescribed species of Lophodermium (sp. 1 and sp. 2). Pinus hartwegii and P. greggii have 2–3 layers of hypodermal cells, while P. ayacahuite has only one (Farjon and Styles 1997). This anatomical difference limits the sites available for ascocarp insertion in P. ayacahuite and similar soft pines and thus constrains the potential morphological characters related to the embedding of the ascocarp.
Most Lophodermium taxa with unique combinations of morphological characters also have unique molecular sequences. The five putative novel species from Mexico have unique ITS sequences. Only Leptostroma sp. from P. ayacahuite has an identical nucleotide sequence to that of Lophodermium sp. 4 from P. chiapensis, suggesting that they are conspecific. Nevertheless, some morphologically different species have identical or almost-identical sequences, while molecular variations are comparable to interspecific differences for L. pinastri (5.6%) and L. baculiferum (as high as 7.6%). These exceptions can be explained in part by dependence of morphological characters used for species delimitation, such as depth of embedding of the ascocarp, on the anatomy of the hosts and partly by reproductive isolation preceding morphological divergence. In the former instance, different species of Lophodermium in anatomically similar hosts may be morphologically similar and the same species in anatomically different hosts may be morphologically different. In the latter instance, ecological differences independent from host anatomy can accompany morphological differences. Minter and Millar (1980) studied the biology and ecology of a group of Lophodermium species inhabiting Pinus sylvestris: L. seditiosum, L. pinastri and L. conigenum. They reported marked differences in their habitat use, fructification and sporulation periods, as well as in their pathogenicity. The phylogenetic separation of these species in the ITS tree corroborates their species status and suggests that such ecological differences might be used to supplement morphological evidence in delineating species.
Host specificity –
Some Lophodermium species represented by more than one isolate had noticeably divergent ITS sequences (Fig. 1), with higher divergence found in isolates from different host species. This was the case for the Lophodermium baculiferum species complex, including the anamorph, Leptostroma decipiens. ITS intraspecific nucleotide divergence in this complex correlated with the phylogenetic relationships of the hosts. All individuals isolated from Pinus ponderosa and originating from a small geographic range in Oregon had identical sequences. Isolates of the same morphospecies taken from P. montezumae across a relatively extensive geographic range in Mexico also had little nucleotide variation (0.24%). Lophodermium baculiferum isolates from different pine species had higher nucleotide divergence. Isolates from hosts in the same subsection had a mean nucleotide divergence of 4.2%, whereas isolates from different subsections had a mean nucleotide divergence of 6.1%. Pinus ponderosa and P. montezumae are closely related members of subsection Ponderosae (Price et al 1998), and their L. baculiferum isolates have intermediate levels of divergence, despite the geographical isolation of their hosts. In contrast, L. baculiferum isolated from P. contortae in subsection Contortae (Price et al 1998) displayed higher nucleotide divergence, despite the overlapping geographic distributions of P. ponderosa and P. contorta along the West Coast. The successive increase in nucleotide divergence for L. baculiferum isolates in relation to phylogenetic divergence in their hosts is consistent with host-mediated speciation.
Levels of intraspecific variation among L. baculiferum isolates were comparatively high (up to 6.1%). As in previous studies (Carbone and Kohn 1993, Morales et al 1993, Ko et al 1997), divergence among isolates from different hosts may be high enough to justify splitting them into separate species (or subspecies). This is consistent with the idea that speciation has occurred in the fungi in parallel with host speciation. Mating studies and reciprocal inoculations might be appropriate to investigate whether molecular divergence and host specificity justify the segregation of additional species in this group. Molecular and morphological studies of additional L. baculiferum isolates would be useful to decide whether the current taxonomy is appropriate.
Although some Lophodermium species, such as L. baculiferum, may be undergoing host-mediated speciation, others show evidence of host switching. The L. indianum, Lophodermium sp. 1, Meloderma MD3 and Lophodermium sp. 2 clade has low sequence divergence, despite having a host distribution that includes three subsections and both subgenera of Pinus (Figs. 1 and 3). Species of L. pinastri occur on two different sections of hard pines, although high sequence divergence among the strains could be explained by inadequate taxonomic sampling or by ancient host tracking followed by extinction on more closely related hosts. The L. agathidis isolates collected exclusively from New Zealand but from different hosts also offer an example of a generalist species capable of host switching. Lophodermium agathidis has a wide host and geographic range through tropical and subtropical regions (Johnston 2001). Despite the phylogenetic distance of the hosts (a conifer and an angiosperm), ITS nucleotide divergence between the two individuals of L. agathidis (2.9%) were within the range of other conspecifics in this study.
Cryptic speciation may be occurring in similar taxa on different hosts, and therefore differences in habitat and behavior may indicate genetic discontinuities. Chemical and morphological characters of the pines hosts, such as needle anatomy and secondary metabolites, may influence fungal colonization. There is evidence that specific host-fungus relationships occur between Leptostroma anamorphs and their hosts (Sieber-Canavesi et al 1991), and this is reflected by the fact that host preference and substrate often provide a guide for identifying Lophodermium species. Similar needle anatomy and secondary compounds might permit host switching and colonization, especially between closely related pines in sympatry (Espinosa-García et al 1993). Horizontal transfer in Lophodermium by broadcasting of ascospores may increase gene flow and preclude specialization in some species, but host tracking seems to be occurring in others.
Evolution of pathogenicity –
The phylogenetic results provide evidence of at least one instance of pathogenicity evolving from endophytism within the studied pine-inhabiting fungi. Lophodermium seditiosum, which occurs in a derived part of the pine clade, attacks two- and three-needle pines, causing needle death during their first growing season and sometimes killing seedlings (Hansen and Lewis 1997). This species has the widest host range within the genus Pinus, reported from more than 40 different host species. Evidence for endophytism in several Lophodermium species included in this study has been reported (Minter et al 1978, Minter and Millar 1980, Suske and Acker 1989, Wilson et al 1994).
Elytroderma deformans and Meloderma desmazieresii are considered serious pathogens of several pine species. Elytroderma deformans causes "witches broom" and severe defoliation (Weir 1916, Childs et al 1971), and Meloderma desmazieresii causes a needle blight (Hansen and Lewis 1997). If any of the three sequences representing these two genera are confirmed to be correct, then their position nested within endophytic species of Lophodermium further would support the idea that pathogenicity has evolved from endophytism in this group. Furthermore, because of their separation in the ITS tree from L. seditiosum, confirmation of either Meloderma sequence would reveal a second derivation of pathogenism from endophytism. Other studies also have shown evidence of multiple origins of pathogenic fungi from nonpathogenic relatives (Bowman and Taylor 1993, Bowman et al 1996).
Indirect evidence supports the view that most Lophodermium species are endophytes; their fruiting bodies frequently are associated with recently senescent or dead tissue, and viable infections commonly are found in living tissues of asymptomatic hosts (Carroll and Carroll 1978, Hata and Futai 1993, 1996, Stone et al 2000). Other representatives of Rhytismataceae, including Colpoma quercinum and Tryblidiopsis pinastri, also are considered endophytes. They colonize living branches and play an important role in natural decay and shedding of dead branches (Livsey 1993, Livsey and Minter 1994, Kowalski and Kehr 1996). Delaying reproduction until needle senescence might represent a less adaptive strategy compared to pathogenic fungi that aggressively colonize host tissue after infection, allowing them to reproduce within a year after infection, but severe needle pathogens deplete substrate availability for subsequent infections because only current-season needles are available commonly for colonization (Sinclair et al 1987). In contrast, endophytes can colonize a wider range of needle age classes and therefore their populations should not be as vulnerable as pathogen populations in years when environmental conditions are unfavorable for ascospore dispersal and colonization. The ability to occupy different, often longer-lived host substrates may permit pathogenic fungi to ameliorate such environmental variability; Lophodermium seditiosum can be found fruiting on pine cones, and E. deformans also invades shoots and twigs.
The molecular approach used in this study helped to interpret relationships between the genus Lophodermium and selected species in the family Rhytismataceae by providing phylogenetic resolution that we were unable to obtain with morphological characters traditionally used in taxonomic studies, but many questions remain. A better understanding of the biological and evolutionary relationships among Lophodermium and related genera might be guided by considering host associations, which are useful in studying cospeciation, host switching and the evolution of endophytism and pathogenism. Ideally, future studies of such patterns will involve formal comparison of robust, fully resolved phylogenies for plant hosts and Rhytismataceae parasites, backed by improved species delineation and clearer understanding of the ecological role of each species. Inclusion of other members of Lophodermium and species of allied genera also will be required before a decision can be made on the most appropriate way to classify this diverse genus.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Instituto Nacional de Ecología, SEMARNAT, Periférico Sur 5000 5° Piso, Col. Cuicuilco, Insurgentes, México, D.F. 04530, Mexico
Accepted for publication February 23, 2003.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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