Geographic locality and host identity shape fungal endophyte communities in cupressaceous trees

Abstract

Understanding how fungal endophyte communities differ in abundance, diversity, taxonomic composition, and host affinity over the geographic ranges of their hosts is key to understanding the ecology and evolutionary context of endophyte–plant associations. We examined endophytes associated with healthy photosynthetic tissues of three closely related tree species in the Cupressaceae (Coniferales): two native species within their natural ranges [Juniperus virginiana in a mesic semideciduous forest, North Carolina (NC); Cupressus arizonica, under xeric conditions, Arizona (AZ)], and a non-native species planted in each site (Platycladus orientalis). Endophytes were recovered from 229 of 960 tissue segments and represented at least 35 species of Ascomycota. Isolation frequency was more than threefold greater for plants in NC than in AZ, and was 2.5 (AZ) to four (NC) times greater for non-native Platycladus than for Cupressus or Juniperus. Analyses of ITS rDNA for 109 representative isolates showed that endophyte diversity was more than twofold greater in NC than in AZ, and that endophytes recovered in AZ were more likely to be host-generalists relative to those in NC. Different endophyte genera dominated the assemblages of each host species/locality combination, but in both localities, Platycladus harboured less diverse and more cosmopolitan endophytes than did either native host. Parsimony and Bayesian analyses for four classes of Ascomycota (Dothideomycetes, Sordariomycetes, Pezizomycetes, Eurotiomycetes) based on LSU rDNA data (ca 1.2 kb) showed that well-supported clades of endophytes frequently contained representatives of a single locality or host species, underscoring the importance of both geography and host identity in shaping a given plant's endophyte community. Together, our data show that not only do the abundance, diversity, and taxonomic composition of endophyte communities differ as a function of host identity and locality, but that host affinities of those communities are variable as well.

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.