Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees
Introduction
Recent studies have begun to elucidate the diversity and ubiquity of fungal endophytes associated with terrestrial plants, providing a framework for understanding the evolutionary context of these plant–fungus associations (reviewed by Stone et al., 2000, Schulz and Boyle, 2005, Arnold and Lutzoni, 2007). Despite a strong foundation laid by a growing number of surveys, the degree to which endophytes differ in abundance, diversity, taxonomic composition, and host affinity over the geographic ranges of particular host lineages remains unclear. Resolving the importance of locality and host identity in shaping these aspects of endophyte communities is important for understanding fundamental aspects of endophyte symbioses.
Previous studies have indicated that the abundance, diversity, and species composition of endophytes can be strongly influenced by the locality in which a given plant occurs (e.g. Carroll and Carroll, 1978, Petrini et al., 1982, Bills and Polishook, 1992, Fisher et al., 1994, Hata and Futai, 1996, Bayman et al., 1998, Arnold et al., 2001, Higgins et al., 2007). For example, different microhabitats vary in inoculum potential, with higher infection frequencies observed beneath forest canopies versus in forest clearings (Arnold & Herre 2003) or in more mesic microhabitats relative to proximate sites that are more xeric (Petrini et al., 1982, Petrini, 1996). At larger geographic scales, endophytes are frequently more abundant in tropical regions than in boreal, arctic, or some temperate regions (Arnold & Lutzoni 2007), although local climate, short-term weather patterns, and some disturbance regimes can override latitudinal gradients (see Suryanarayanan et al., 2000, Arnold et al., 2007, Higgins et al., 2007). Similarly, diversity of endophytes differs at small scales as a function of land use history, vegetation cover, and other factors. For example, Gamboa & Bayman (2001) found higher endophyte diversity in leaves of Guarea guidonia in a forest preserve relative to a disturbed forest area in Puerto Rico. At larger scales, endophyte diversity varies as a function of latitude and annual rainfall (Arnold & Lutzoni 2007), although the contributions of co-varying factors, such as plant diversity, remain to be explored. Finally, the species composition of endophyte assemblages also differs as a function of localities. For example, Arnold et al. (2003) found distinctive endophyte communities associated with Theobroma cacao at multiple sites in Panama. In a study of endophytes and saprotrophs associated with closely related palms in Australia and Brunei Darussalam, Fröhlich & Hyde (1999) found that only 27 of 189 fungal species were shared between localities. Similarly, Fisher et al. (1995) found that only two of 25 species of endophytes recovered from Dryas octopetala (Rosaceae) in Switzerland also occurred in the same host in Norway.
By comparing endophyte assemblages in a single host species or genus in multiple sites, these studies have shown that plants interact with distinctive endophyte communities across the geographic areas in which they grow. However, it is unclear whether plants growing in different localities experience not only a distinctive abundance, diversity, and species assemblage of endophytes, but also a different degree of relative host specificity. Do sympatric plant species differ in terms of the relative host specificity or host-generalism of the endophytes they harbour? Do different regions or environmental conditions select for more or less host-specific endophytes? To our knowledge, these questions have not been addressed previously.
Most studies examining host affinities of endophytes have focused on distantly related host species that co-occur within particular geographic areas, and have yielded conflicting results. For example, a study involving endophytes of mistletoe and fir established that despite close physical proximity of these two plant taxa, endophyte communities remained exclusively associated with their particular host (Petrini 1996). Similarly, Suryanarayanan et al. (2000) found distinctive endophytes in Cuscuta reflexa relative to its host plants, and Arnold et al. (2000) found in lowland Panama that distantly related hosts, growing within metres of one another, bore distinctive endophyte communities. Yet Cannon & Simmons (2002) recovered similar endophyte communities from phylogenetically diverse angiosperm trees in a tropical forest reserve. Suryanarayanan et al. (2004) recovered the same endophyte species from diverse plant species in mangroves, dry deciduous forest, and other tropical forest types (Phyllosticta capitalensis), and Mohali et al. (2005) found that Lasiodiplodia theobromae occurs in multiple hosts and sites at a global scale. Suryanarayanan et al. (2005) found no evidence for host specificity among endophytes of cacti in the southwestern USA, yet Higgins et al. (2007) found that most genotypes of endophytes recovered from arctic and boreal plants were associated with only one host species. Thus, research to date remains in conflict with regard to the prevalence of host specificity among endophytes, and the potential for host specificity of endophyte communities to differ over the range of a plant taxon, or among species within a particular site, is not clear.
To our knowledge, no study to date has concurrently assessed the importance of host identity and locality in shaping endophyte abundance, diversity, taxon composition, and relative host specificity by simultaneously sampling pairs of closely related plants in geographically distinct sites. Molecular tools are especially useful in this regard, providing a framework for explicitly comparing sterile endophytes and providing a tool to disentangle potentially cryptic species for which morphological characters are of limited use (Lacap et al., 2003, Arnold et al., 2007). In turn, phylogenetic analyses provide insight into the evolutionary associations of fungal lineages with hosts and localities, and can provide much-needed insight into the degree to which particular clades of fungi are more or less likely to form host-specific associations with plants. Unfortunately, endophyte surveys are often restricted in terms of molecular phylogenetics due to the cost associated with sequencing informative loci for numerous and often phylogenetically diverse isolates (see Arnold et al., 2007, Higgins et al., 2007). In particular, there is a need to examine the ability of particular loci to infer topologies that are consistent with reconstructions provided by multi-locus analyses (e.g., Lutzoni et al., 2004, James et al., 2006) and to maximize the phylogenetic power provided by loci that can be sequenced rapidly and at reasonable cost.
Using pairs of related species of the conifer family Cupressaceae from a mesic forest [central North Carolina (NC)] and from a xeric desert environment [southern Arizona (AZ)], we used a culture-based approach to examine the foliar endophyte communities of two native species (Cupressus arizonica, AZ and Juniperus virginiana, NC) and a co-occurring, non-native species (Platycladus orientalis in both AZ and NC). Our objectives were to: (1) examine the abundance, diversity, taxonomic composition, and host preferences of endophytic fungi of these cupressaceous hosts; (2) infer the relationships of endophytes associated with these trees in a broad phylogenetic context; (3) evaluate an accelerated phylogenetic search strategy for rapidly and robustly diagnosing these phylogenetic relationships using the LROR-LR7 region of the LSU rDNA; and (4) generate hypotheses regarding the relative importance of geographic locality and host identity in structuring the endophyte communities associated with these ecologically and economically important trees.
Section snippets
Host species
The cypress family, Cupressaceae (Coniferales), is widely distributed in warm-temperate regions and contains 110–130 species of evergreen trees and shrubs in 25–30 genera (Fralish & Franklin 2002). Within the Cupressoideae, the genera Cupressus and Juniperus comprise 70–86 species, whereas Platycladus is monotypic (Platycladus orientalis). Cupressus arizonica (AZ cypress) is indigenous in the western United States and northern Mexico (Little, 1950, USDA, NRCS, 2004). Juniperus virginiana
Results
From 960 tissue samples, 229 isolates of endophytic fungi were recovered in culture. Isolation frequency, defined as the percent of tissue segments bearing cultivable endophytes, was more than threefold greater for hosts from NC (56.3 %) than AZ (15.8 %), and was 2.5 to four times greater in Platycladus (AZ, 25.5 %; NC, 80.2 %) than in Cupressus (AZ; 6 %) or Juniperus (NC; 32.4 %; Table 1).
Among 109 isolates sequenced for ITS rDNA, we recovered 35 unique ITS rDNA genotypes (90 % ITS rDNA sequence
Discussion
We used a culture-based approach, paired with sequencing of both a fast-evolving locus (ITS rDNA) and a phylogenetically informative locus (LSU rDNA), to assess the diversity and taxon composition of fungal endophytes among native and non-native conifers in two geographically distinct localities. We found that the abundance, diversity, species composition, and relative host affinity of endophyte communities differed as a function of locality and host species. Cultivable endophytes were present
Acknowledgements
We thank the University of Arizona's Division of Plant Pathology and Microbiology and College of Agriculture and Life Sciences for supporting this work. Additional support from the National Science Foundation [DEB-0343953 to A.E.A. (and James W. Dalling); DEB-0200413 to A.E.A.] is gratefully acknowledged. We thank David Maddison for sharing pre-release versions of MacClade and Mesquite, and for advice with regard to phylogenetic analyses. We are grateful to Rebecca Porter, Mary Shimabukuro,
References (50)
- et al.
Fungal endophytes in dicotyledonous neotropical trees: patterns of abundance and diversity
Mycological Research
(2001) - et al.
Distribution and dispersal of Xylaria endophytes in two tree species in Puerto Rico
Mycological Research
(1998) - et al.
Host affinity and geographic structure among boreal and arctic endophytes from three major plant lineages
Molecular Phylogenetics and Evolution
(2007) The parsimony ratchet, a new method for rapid parsimony analysis
Cladistics
(1999)- et al.
The endophytic continuum
Mycological Research
(2005) - et al.
Characterization of the melanin pigment of a cosmopolitan fungal endophyte
Mycological Research
(2004) - et al.
Endophytic fungi associated with cacti in Arizona
Mycological Research
(2005) - et al.
Phylogenetic species recognition and species concepts in fungi
Fungal Genetics and Biology
(2000) - et al.
Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics
- et al.
Diversity and host range of foliar fungal endophytes: are tropical leaves biodiversity hotspots?
Ecology
(2007)
Diversity and phylogenic affinities of foliar fungal endophytes in loblolly pine inferred by culturing and environmental PCR
Mycologia
Canopy cover and leaf age affect colonization by tropical fungal endophytes: ecological pattern and process in Theobroma cacao (Malvaceae)
Mycologia
Fungal endophytes limit pathogen damage in a tropical tree
Proceedings of the National Academy of Sciences, USA
Are tropical fungal endophytes hyperdiverse?
Ecology Letters
Recovery of endophytic fungi from Chamaecyparis thyoides
Sydowia
Studies on the incidence of coniferous needle endophytes in the Pacific Northwest
Canadian Journal of Botany
Diversity and host preference of leaf endophytic fungi in the Iwokrama Forest Reserve, Guyana
Mycologia
The Comparative RNA Web (CRW) Site: an online database of comparative sequences and structure information for ribosomal, intron, and other RNAs
BMC Bioinformatics
Base-calling of automated sequencer traces using Phred. II. Error probabilities
Genome Research
Base–calling of automated sequencer traces using Phred. I. Accuracy assessment
Genome Research
Mutualistic asexual endophytes in a native grass are usually parasitic
American Naturalist
A comparative study of fungal endophytes in xylem and bark of Eucalyptus nitens in Australia and England
Sydowia
Fungal endophytes from the leaves and twigs of Quercus ilex L. from England, Majorca, and Switzerland
New Phytologist
Fungal endophytes of Dryas octopetala from a high arctic polar semidesert and from the Swiss Alps
Mycologia
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