Assessing the utility of SoilGrids250 for biogeographic inference of plant populations.

Inclusion of edaphic conditions in biogeographical studies typically provides a better fit and deeper understanding of plant distributions. Increased reliance on soil data calls for easily accessible data layers providing continuous soil predictions worldwide. Although SoilGrids provides a potentially useful source of predicted soil data for biogeographic applications, its accuracy for estimating the soil characteristics experienced by individuals in small-scale populations is unclear. We used a biogeographic sampling approach to obtain soil samples from 212 sites across the midwestern and eastern United States, sampling only at sites where there was a population of one of the 22 species in Lobelia sect. Lobelia. We analyzed six physical and chemical characteristics in our samples and compared them with predicted values from SoilGrids. Across all sites and species, soil texture variables (clay, silt, sand) were better predicted by SoilGrids (R 2: .25-.46) than were soil chemistry variables (carbon and nitrogen, R 2 ≤ .01; pH, R 2: .19). While SoilGrids predictions rarely matched actual field values for any variable, we were able to recover qualitative patterns relating species means and population-level plant characteristics to soil texture and pH. Rank order of species mean values from SoilGrids and direct measures were much more consistent for soil texture (Spearman r S = .74-.84; all p < .0001) and pH (r S = .61, p = .002) than for carbon and nitrogen (p > .35). Within the species L. siphilitica, a significant association, known from field measurements, between soil texture and population sex ratios could be detected using SoilGrids data, but only with large numbers of sites. Our results suggest that modeled soil texture values can be used with caution in biogeographic applications, such as species distribution modeling, but that soil carbon and nitrogen contents are currently unreliable, at least in the region studied here.

From individual leaves to forest stands: importance of niche, distance decay, and stochasticity vary by ecosystem type and functional group for fungal community composition.

Community assembly is influenced by environmental niche processes as well as stochastic processes that can be spatially dependent (e.g. dispersal limitation) or independent (e.g. priority effects). Here, we sampled senesced tree leaves as unit habitats to investigate fungal community assembly at two spatial scales: (i) small neighborhoods of overlapping leaves from differing tree species and (ii) forest stands of differing ecosystem types. Among forest stands, ecosystem type explained the most variation in community composition. Among adjacent leaves within stands, variability in fungal composition was surprisingly high. Leaf type was more important in stands with high soil fertility and dominated by differing tree mycorrhizal types (sugar maple vs. basswood or red oak), whereas distance decay was more important in oak-dominated forest stands with low soil fertility. Abundance of functional groups was explained by environmental factors, but predictors of taxonomic composition within differing functional groups were highly variable. These results suggest that fungal community assembly processes are clearest for functional group abundances and large spatial scales. Understanding fungal community assembly at smaller spatial scales will benefit from further study focusing on differences in drivers for different ecosystems and functional groups, as well as the importance of spatially independent factors such as priority effects.

Characterizing natural variability of lignin abundance and composition in fine roots across temperate trees: a comparison of analytical methods.

Lignin is an important root chemical component that is widely used in biogeochemical models to predict root decomposition. Across ecological studies, lignin abundance has been characterized using both proximate and lignin-specific methods, without much understanding of their comparability. This uncertainty in estimating lignin limits our ability to comprehend the mechanisms regulating root decomposition and to integrate lignin data for large-scale syntheses. We compared five methods of estimating lignin abundance and composition in fine roots across 34 phylogenetically diverse tree species. We also assessed the feasibility of high-throughput techniques for fast-screening of root lignin. Although acid-insoluble fraction (AIF) has been used to infer root lignin and decomposition, AIF-defined lignin content was disconnected from the lignin abundance estimated by techniques that specifically measure lignin-derived monomers. While lignin-specific techniques indicated lignin contents of 2-10% (w/w) in roots, AIF-defined lignin contents were c. 5-10-fold higher, and their interspecific variation was found to be largely unrelated to that determined using lignin-specific techniques. High-throughput pyrolysis-gas chromatography-mass spectrometry, when combined with quantitative modeling, accurately predicted lignin abundance and composition, highlighting its feasibility for quicker assessment of lignin in roots. We demonstrate that AIF should be interpreted separately from lignin in fine roots as its abundance is unrelated to that of lignin polymers. This study provides the basis for informed decision-making with respect to lignin methodology in ecology.

Drivers of fungal diversity and community biogeography differ between green roofs and adjacent ground-level green space.

Green roof soils are usually engineered for purposes other than urban biodiversity, which may impact their fungal communities, and in turn impact the health of plants in the urban ecosystem. We examined the drivers of fungal diversity and community composition in soil of green roofs and adjacent ground-level green spaces in three Midwestern USA cities-Chicago, Cleveland, and Minneapolis. Overall, fungal communities on green roofs were more diverse than ground-level green spaces and were correlated with plant cover (positively) and roof age (negatively) rather than abiotic soil properties. Fungal community composition was distinct between roof and ground environments, among cities, and between sampling sites, but green roofs and their immediately surrounding ground-level green space showed some similarity. This suggests dispersal limitation may result in geographic structuring at large spatial scales, but dispersal between roofs and their neighbouring sites may be occurring. Different fungal taxonomic and functional groups were better explained when roofs were classified either by depth (extensive or intensive) or functional intent of the roof design (i.e. stormwater/energy, biodiversity, or aesthetics/recreation). Our results demonstrate that green roofs are an important reservoir of fungal diversity in the urban landscape, which should be considered in future green roof design.

Arbuscular Mycorrhizal Tree Communities Have Greater Soil Fungal Diversity and Relative Abundances of Saprotrophs and Pathogens than Ectomycorrhizal Tree Communities.

Trees associating with different mycorrhizas often differ in their effects on litter decomposition, nutrient cycling, soil organic matter (SOM) dynamics, and plant-soil interactions. For example, due to differences between arbuscular mycorrhizal (AM) and ectomycorrhizal (ECM) tree leaf and root traits, ECM-associated soil has lower rates of C and N cycling and lower N availability than AM-associated soil. These observations suggest that many groups of nonmycorrhizal fungi should be affected by the mycorrhizal associations of dominant trees through controls on nutrient availability. To test this overarching hypothesis, we explored the influence of predominant forest mycorrhizal type and mineral N availability on soil fungal communities using next-generation amplicon sequencing. Soils from four temperate hardwood forests in southern Indiana, United States, were studied; three forests formed a natural gradient of mycorrhizal dominance (100% AM tree basal area to 100% ECM basal area), while the fourth forest contained a factorial experiment testing long-term N addition in both dominant mycorrhizal types. We found that overall fungal diversity, as well as the diversity and relative abundance of plant pathogenic and saprotrophic fungi, increased with greater AM tree dominance. Additionally, tree community mycorrhizal associations explained more variation in fungal community composition than abiotic variables, including soil depth, SOM content, nitrification rate, and mineral N availability. Our findings suggest that tree mycorrhizal associations may be good predictors of the diversity, composition, and functional potential of soil fungal communities in temperate hardwood forests. These observations help explain differing biogeochemistry and community dynamics found in forest stands dominated by differing mycorrhizal types. IMPORTANCE Our work explores how differing mycorrhizal associations of temperate hardwood trees (i.e., arbuscular [AM] versus ectomycorrhizal [ECM] associations) affect soil fungal communities by altering the diversity and relative abundance of saprotrophic and plant-pathogenic fungi along natural gradients of mycorrhizal dominance. Because temperate hardwood forests are predicted to become more AM dominant with climate change, studies examining soil communities along mycorrhizal gradients are necessary to understand how these global changes may alter future soil fungal communities and their functional potential. Ours, along with other recent studies, identify possible global trends in the frequency of specific fungal functional groups responsible for nutrient cycling and plant-soil interactions as they relate to mycorrhizal associations.

Coordination between compound-specific chemistry and morphology in plant roots aligns with ancestral mycorrhizal association in woody angiosperms.

Recent studies on fine root functional traits proposed a root economics hypothesis where adaptations associated with mycorrhizal dependency strongly influence the organization of root traits, forming a dominant axis of trait covariation unique to roots. This conclusion, however, is based on tradeoffs of a few widely studied root traits. It is unknown how other functional traits fit into this mycorrhizal-collaboration gradient. Here, we provide a significant extension to the field of root ecology by examining how fine root secondary compounds coordinate with other root traits. We analyzed a dataset integrating compound-specific chemistry, morphology and anatomy of fine roots and leaves from 34 temperate tree species spanning major angiosperm lineages. Our data uncovered previously undocumented coordination where root chemistry, morphology and anatomy covary with each other. This coordination, aligned with mycorrhizal colonization, reflects tradeoffs between chemical protection and mycorrhizal dependency, and provides mechanistic support for the mycorrhizal-collaboration gradient. We also found remarkable phylogenetic structuring in root chemistry. These patterns were not mirrored by leaves. Furthermore, chemical protection was largely decoupled from the leaf economics spectrum. Our results unveil broad organization of root chemistry, demonstrate unique belowground adaptions, and suggest that root strategies and phylogeny could impact biogeochemical cycles through their links with root chemistry.

Independent evolutionary changes in fine-root traits among main clades during the diversification of seed plants.

Changes in fine-root morphology are typically associated with transitions from the ancestral arbuscular mycorrhizal (AM) to the alternative ectomycorrhizal (ECM) or nonmycorrhizal (NM) associations. However, the modifications in root morphology may also coincide with new modifications in leaf hydraulics and growth habit during angiosperm diversification. These hypotheses have not been evaluated concurrently, and this limits our understanding of the causes of fine-root evolution. To explore the evolution of fine-root systems, we assembled a 600+ species database to reconstruct historical changes in seed plants over time. We utilise ancestral reconstruction approaches together with phylogenetically informed comparative analyses to test whether changes in fine-root traits were most strongly associated with mycorrhizal affiliation, leaf hydraulics or growth form. Our findings showed significant shifts in root diameter, specific root length and root tissue density as angiosperms diversified, largely independent from leaf changes or mycorrhizal affiliation. Growth form was the only factor associated with fine-root traits in statistical models including mycorrhizal association and leaf venation, suggesting substantial modifications in fine-root morphology during transitions from woody to nonwoody habits. Divergences in fine-root systems were crucial in the evolution of seed plant lineages, with important implications for ecological processes in terrestrial ecosystems.

Patterns in spatial distribution and root trait syndromes for ecto and arbuscular mycorrhizal temperate trees in a mixed broadleaf forest.

Functional differences between trees with arbuscular (AM) or ectomycorrhizal (ECM) partnerships influence important ecological processes including nutrient cycling, community assembly, and biomass allocation patterns. Although most broadleaf temperate forests show both mycorrhizal types, relatively few studies have addressed functional difference among coexisting mycorrhizal tree species. The maintenance of ECM associations usually requires higher C investment than AM, leading to (A) lower root biomass and (B) more conservative root trait syndromes in ECM tree species compared to AM species. Here we quantified the representation and trait syndromes of 14 canopy tree species associated with either AM or ECM fungi in a natural forest community. Our results showed that, whereas species root abundance was proportional to basal area, some ECM tree roots were largely under-represented (up to ~ 33%). Most of the under-representation was due to lower than expected root abundance of Quercus rubra and Fagus grandifolia. Functional root traits in tree species were similar, with the exception of higher tissue density in ECM species. Moreover, closely related AM and ECM exhibited similar traits, suggesting inherited trait syndrome from a common ancestor. Thus, we found little evidence of divergent functional root trait syndromes between mycorrhizal types. Cores dominated by ECM species influenced trait distribution at the community level, but not total biomass, suggesting that mycorrhizal affiliation may have a stronger effect on the spatial distribution of traits but not on biomass stocks. Our results present an important step toward relating belowground carbon dynamics to species traits, including mycorrhizal type, in broadleaf temperate forests.

A worldview of root traits: the influence of ancestry, growth form, climate and mycorrhizal association on the functional trait variation of fine-root tissues in seed plants.

Fine-root traits play key roles in ecosystem processes, but the drivers of fine-root trait diversity remain poorly understood. The plant economic spectrum (PES) hypothesis predicts that leaf and root traits evolved in coordination. Mycorrhizal association type, plant growth form and climate may also affect root traits. However, the extent to which these controls are confounded with phylogenetic structuring remains unclear. Here we compiled information about root and leaf traits for > 600 species. Using phylogenetic relatedness, climatic ranges, growth form and mycorrhizal associations, we quantified the importance of these factors in the global distribution of fine-root traits. Phylogenetic structuring accounts for most of the variation for all traits excepting root tissue density, with root diameter and nitrogen concentration showing the strongest phylogenetic signal and specific root length showing intermediate values. Climate was the second most important factor, whereas mycorrhizal type had little effect. Substantial trait coordination occurred between leaves and roots, but the strength varied between growth forms and clades. Our analyses provide evidence that the integration of roots and leaves in the PES requires better accounting of the variation in traits across phylogenetic clades. Inclusion of phylogenetic information provides a powerful framework for predictions of belowground functional traits at global scales.

Initial nitrogen enrichment conditions determines variations in nitrogen substrate utilization by heterotrophic bacterial isolates.

BACKGROUND: The nitrogen (N) cycle consists of complex microbe-mediated transformations driven by a variety of factors, including diversity and concentrations of N compounds. In this study, we examined taxonomic diversity and N substrate utilization by heterotrophic bacteria isolated from streams under complex and simple N-enrichment conditions. RESULTS: Diversity estimates differed among isolates from the enrichments, but no significant composition were detected. Substrate utilization and substrate range of bacterial assemblages differed within and among enrichments types, and not simply between simple and complex N-enrichments. CONCLUSIONS: N substrate use patterns differed between isolates from some complex and simple N-enrichments while others were unexpectedly similar. Taxonomic composition of isolates did not differ among enrichments and was unrelated to N use suggesting strong functional redundancy. Ultimately, our results imply that the available N pool influences physiology and selects for bacteria with various abilities that are unrelated to their taxonomic affiliation.

A global Fine-Root Ecology Database to address below-ground challenges in plant ecology.

Variation and tradeoffs within and among plant traits are increasingly being harnessed by empiricists and modelers to understand and predict ecosystem processes under changing environmental conditions. While fine roots play an important role in ecosystem functioning, fine-root traits are underrepresented in global trait databases. This has hindered efforts to analyze fine-root trait variation and link it with plant function and environmental conditions at a global scale. This Viewpoint addresses the need for a centralized fine-root trait database, and introduces the Fine-Root Ecology Database (FRED, http://roots.ornl.gov) which so far includes > 70 000 observations encompassing a broad range of root traits and also includes associated environmental data. FRED represents a critical step toward improving our understanding of below-ground plant ecology. For example, FRED facilitates the quantification of variation in fine-root traits across root orders, species, biomes, and environmental gradients while also providing a platform for assessments of covariation among root, leaf, and wood traits, the role of fine roots in ecosystem functioning, and the representation of fine roots in terrestrial biosphere models. Continued input of observations into FRED to fill gaps in trait coverage will improve our understanding of changes in fine-root traits across space and time.

Comparison of pectin-degrading fungal communities in temperate forests using glycosyl hydrolase family 28 pectinase primers targeting Ascomycete fungi.

Fungi have developed a wide assortment of enzymes to break down pectin, a prevalent polymer in plant cell walls that is important in plant defense and structure. One enzyme family used to degrade pectin is the glycosyl hydrolase family 28 (GH28). In this study we developed primers for the amplification of GH28 coding genes from a database of 293 GH28 sequences from 40 fungal genomes. The primers were used to successfully amplify GH28 pectinases from all Ascomycota cultures tested, but only three out of seven Basidiomycota cultures. In addition, we further tested the primers in PCRs on metagenomic DNA extracted from senesced tree leaves from different forest ecosystems, followed by cloning and sequencing. Taxonomic specificity for Ascomycota GH28 genes was tested by comparing GH28 composition in leaves to internal transcribed spacer (ITS) amplicon composition using pyrosequencing. All sequences obtained from GH28 primers were classified as Ascomycota; in contrast, ITS sequences indicated that fungal communities were up to 39% Basidiomycetes. Analysis of leaf samples indicated that both forest stand and ecosystem type were important in structuring fungal communities. However, site played the prominent role in explaining GH28 composition, whereas ecosystem type was more important for ITS composition, indicating possible genetic drift between populations of fungi. Overall, these primers will have utility in understanding relationships between fungal community composition and ecosystem processes, as well as detection of potentially pathogenic Ascomycetes.

A toolkit for ARB to integrate custom databases and externally built phylogenies.

UNLABELLED: Researchers are perpetually amassing biological sequence data. The computational approaches employed by ecologists for organizing this data (e.g. alignment, phylogeny, etc.) typically scale nonlinearly in execution time with the size of the dataset. This often serves as a bottleneck for processing experimental data since many molecular studies are characterized by massive datasets. To keep up with experimental data demands, ecologists are forced to choose between continually upgrading expensive in-house computer hardware or outsourcing the most demanding computations to the cloud. Outsourcing is attractive since it is the least expensive option, but does not necessarily allow direct user interaction with the data for exploratory analysis. Desktop analytical tools such as ARB are indispensable for this purpose, but they do not necessarily offer a convenient solution for the coordination and integration of datasets between local and outsourced destinations. Therefore, researchers are currently left with an undesirable tradeoff between computational throughput and analytical capability. To mitigate this tradeoff we introduce a software package to leverage the utility of the interactive exploratory tools offered by ARB with the computational throughput of cloud-based resources. Our pipeline serves as middleware between the desktop and the cloud allowing researchers to form local custom databases containing sequences and metadata from multiple resources and a method for linking data outsourced for computation back to the local database. A tutorial implementation of the toolkit is provided in the supporting information, S1 Tutorial. AVAILABILITY: http://www.ece.drexel.edu/gailr/EESI/tutorial.php.

Aggregated and complementary: symmetric proliferation, overyielding, and mass effects explain fine-root biomass in soil patches in a diverse temperate deciduous forest landscape.

Few studies describe root distributions at the species level in diverse forests, although belowground species interactions and traits are often assumed to affect fine-root biomass (FRB). We used molecular barcoding to study how FRB of trees relates to soil characteristics, species identity, root diversity, and root traits, and how these relationships are affected by proximity to ecotones in a temperate forest landscape. We found that soil patch root biomass increased in response to soil resources across all species, and there was little belowground vertical or horizontal spatial segregation among species. Root traits and species relative abundance did not explain significant variation in FRB after correcting for soil fertility. A positive relationship between phylogenetic diversity and FRB indicated significant belowground overyielding attributable to local root diversity. Finally, variation in FRB explained by soil fertility and diversity was reduced near ecotones, but only because of a reduction in biomass in periodically anoxic areas. These results suggest that symmetric responses to soil properties are coupled with complementary species traits and interactions to explain variation in FRB among soil patches. In addition, landscape-level dispersal among habitats and across ecotones helps explain variation in the strength of these relationships in complex landscapes.

Conspecific plant-soil feedback scales with population size in Lobelia siphilitica (Lobeliaceae).

Plant-soil interactions directly affect plant success in terms of establishment, survival, growth and reproduction. Negative plant-soil feedback on such traits may therefore reduce the density and abundance of plants of a given species at a given site. Furthermore, if conspecific feedback varies among population sites, it could help explain geographic variation in plant population size. We tested for among-site variation in conspecific plant-soil feedback in a greenhouse experiment using seeds and soils from 8 natural populations of Lobelia siphilitica hosting 30-330 plants. The first cohort of seeds was grown on soil collected from each native site, while the second cohort was grown on the soil conditioned by the first. Our goal was to distinguish site-specific effects mediated by biotic and/or abiotic soil properties from those inherent in seed sources. Cohort 1 plants grown from seeds produced in small populations performed better in terms of germination, growth, and survival compared to plants produced in large populations. Plant performance decreased substantially between cohorts, indicating strong negative feedback. Most importantly, the strength of negative feedback scaled linearly (i.e., was less negative) with increasing size of the native plant population, particularly for germination and survival, and was better explained by soil- rather than seed-source effects. Even with a small number of sites, our results suggest that the potential for negative plant-soil feedback varies among populations of L. siphilitica, and that small populations were more susceptible to negative feedback. Conspecific plant-soil feedback may contribute to plant population size variation within a species' native range.

The spatial scaling of saprotrophic fungal beta diversity in decomposing leaves.

Assembly of fungal communities remains poorly understood in part because of the daunting range of spatial scales that may be involved in this process. Here, we use individual leaves as a natural sampling unit, comprising spatially distinct habitat and/or resource patches with unique histories and suites of resources. Spatial patterns in fungal beta diversity were tested for consistency with the metacommunity paradigms of species sorting and neutral dynamics. Thirty senesced leaves were collected from the forest floor (O horizon) in replicate upland forest, riparian forest and vernal pool habitats. We quantified spatial distance between leaves, and fungal community composition was assayed by terminal restriction fragment length polymorphism. Significant distance-decay relationships were detected at all but one upland site. This is the first study where changes in fungal community composition were quantified across discrete adjacent habitat patches, providing evidence that fungal distance decay is operational at a scale of centimetres. Although leaves of differing lignin contents were sampled from each site, leaf type was not consistently important in explaining variation in fungal community composition. However, depth of a leaf within the forest floor significantly influenced community composition at five of six sites. Environmental heterogeneity associated with depth could include moisture gradients, relative influence of soil or spore colonization, and impact of forest floor biotic community (i.e. collembola and earthworms). Because the influence of spatial distance and depth on fungal community composition could not be disentangled, both species-sorting and neutral processes may be embedded within the distance-decay relationships that we found.

Taxa-area relationship and neutral dynamics influence the diversity of fungal communities on senesced tree leaves.

This study utilized individual senesced sugar maple and beech leaves as natural sampling units within which to quantify saprotrophic fungal diversity. Quantifying communities in individual leaves allowed us to determine if fungi display a classic taxa-area relationship (species richness increasing with area). We found a significant taxa-area relationship for sugar maple leaves, but not beech leaves, consistent with Wright's species-energy theory. This suggests that energy availability as affected plant biochemistry is a key factor regulating the scaling relationships of fungal diversity. We also compared taxa rank abundance distributions to models associated with niche or neutral theories of community assembly, and tested the influence of leaf type as an environmental niche factor controlling fungal community composition. Among rank abundance distribution models, the zero-sum model derived from neutral theory showed the best fit to our data. Leaf type explained only 5% of the variability in community composition. Habitat (vernal pool, upland or riparian forest floor) and site of collection explained > 40%, but could be attributed to either niche or neutral processes. Hence, although niche dynamics may regulate fungal communities at the habitat scale, our evidence points towards neutral assembly of saprotrophic fungi on individual leaves, with energy availability constraining the taxa-area relationship.

Cultivable bacteria from ancient algal mats from the McMurdo Dry Valleys, Antarctica.

The McMurdo Dry Valleys in Antarctica are a favorable location for preservation of dormant microbes due to their persistent cold and dry climate. In this study, we examined cultivable bacteria in a series of algal mat samples ranging from 8 to 26539 years old. Cultivable bacteria were found in all samples except one (12303 years old), but abundance and diversity of cultivable bacteria decreased with increasing sample age. Only members of the Actinobacteria, Bacteroidetes, and Firmicutes were found in the ancient samples, whereas bacteria in the 8-year-old sample also included Cyanobacteria, Proteobacteria, and Deinococcus-Thermus. Isolates of the Gram-positive spore-forming bacterium Sporosarcina were found in 5 of 8 samples. The growth of these isolates at different temperatures was related to the phylogenetic distance among genotypes measured by BOX-PCR. These findings suggest that adaptation to growth at different temperatures had occurred among Sporosarcina genotypes in the Dry Valleys, causing the existence of physiologically distinct but closely related genotypes. Additionally, fully psychrophilic isolates (that grew at 15°C, but not 25°C) were found in ancient samples, but not in the modern sample. The preservation of viable bacteria in the Dry Valleys could potentially represent a legacy of bacteria that impacts on current microbial communities of this environment.

Metacommunity organization of soil microorganisms depends on habitat defined by presence of Lobelia siphilitica plants.

We tested regional-scale spatial patterns in soil microbial community composition for agreement with species sorting and dispersal limitation, two alternative mechanisms behind different models of metacommunity organization. Furthermore, we tested whether regional metacommunity organization depends on local habitat type. We sampled from sites across Ohio and West Virginia hosting populations of Lobelia siphilitica, and compared the metacommunity organization of soil microbial communities under L. siphilitica to those in adjacent areas at each site. In the absence of L. siphilitica, bacterial community composition across the region was consistent with species sorting. However, under L. siphilitica, bacterial community composition was consistent with dispersal limitation. Fungal community composition remained largely unexplained, although fungal communities under L. siphilitica were both significantly different in composition and less variable in composition than in adjacent areas. Our results show that communities in different local habitat types (e.g., in the presence or absence of a particular plant) may be structured on a regional scale by different processes, despite being separated by only centimeters at the local scale.

Evolutionary analysis of glycosyl hydrolase family 28 (GH28) suggests lineage-specific expansions in necrotrophic fungal pathogens.

Glycosyl hydrolase family 28 (GH28) is a set of structurally related enzymes that hydrolyze glycosidic bonds in pectin, and are important extracellular enzymes for both pathogenic and saprotrophic fungi. Yet, very little is understood about the evolutionary forces driving the diversification of GH28s in fungal genomes. We reconstructed the evolutionary history of family GH28 in fungi by examining the distribution of GH28 copy number across the phylogeny of fungi, and by reconstructing the phylogeny of GH28 genes. We also examined the relationship between lineage-specific GH28 expansions and fungal ecological strategy, testing the hypothesis that GH28 evolution in fungi is driven by ecological strategy (pathogenic vs. non-pathogenic) and pathogenic niche (necrotrophic vs. biotrophic). Our results showed that GH28 phylogeny of Ascomycota and Basidiomycota sequences was structured by specific biochemical function, with endo-polygalacturonases and endo-rhamnogalacturonases forming distinct, apparently ancient clades, while exo-polygalacturonases are more widely distributed. In contrast, Mucoromycotina and Stramenopile sequences formed taxonomically-distinct clades. Large, lineage-specific variation in GH28 copy number indicates that the evolution of this gene family is consistent with the birth-and-death model of gene family evolution, where diversity of GH28 loci within genomes was generated through multiple rounds of gene duplication followed by functional diversification and loss of some gene family members. Although GH28 copy number was correlated with genome size, our findings suggest that ecological strategy also plays an important role in determining the GH28 repertoire of fungi. Both necrotrophic and biotrophic fungi have larger genomes than non-pathogens, yet only necrotrophs possess more GH28 enzymes than non-pathogens. Hence, lineage-specific GH28 expansion is the result of both variation in genome size across fungal species and diversifying selection within the necrotrophic plant pathogen ecological niche. GH28 evolution among necrotrophs has likely been driven by a co-evolutionary arms race with plants, whereas the need to avoid plant immune responses has resulted in purifying selection within biotrophic fungi.