Foliar mycobiome remains unaltered under urban air-pollution but differentially express stress-related genes.

Air pollution caused by tropospheric ozone contributes to the decline of forest ecosystems; for instance, sacred fir, Abies religiosa (Kunth) Schltdl. & Cham. forests in the peri-urban region of Mexico City. Individual trees within these forests exhibit variation in their response to ozone exposure, including the severity of visible symptoms in needles. Using RNA-Seq metatranscriptomic data and ITS2 metabarcoding, we investigated whether symptom variation correlates with the taxonomic and functional composition of fungal mycobiomes from needles collected in this highly polluted area in the surroundings of Mexico City. Our findings indicate that ozone-related symptoms do not significantly correlate with changes in the taxonomic composition of fungal mycobiomes. However, genes coding for 30 putative proteins were differentially expressed in the mycobiome of asymptomatic needles, including eight genes previously associated with resistance to oxidative stress. These results suggest that fungal communities likely play a role in mitigating the oxidative burst caused by tropospheric ozone in sacred fir. Our study illustrates the feasibility of using RNA-Seq data, accessible from global sequence repositories, for the characterization of fungal communities associated with plant tissues, including their gene expression.

Deep sequencing across multiple host species tests pine-endophyte specificity.

PREMISE: Foliar fungal endophytes vary in their distributions across landscapes or plant host taxa, indicative of specialized ecologies and host specific adaptations. Accounts of specialization, however, depend on the taxonomic breadth and geographic range of the host plants included in each study. A broad region-scale study or deep sampling of diverse potential host species still remains relatively rare but is becoming increasingly possible with high-throughput sequencing. METHODS: Amplicon sequencing was used to rapidly identify the fungal endophytic community among six pine (Pinus, Pinaceae) species co-occurring across northeastern United States and to test for site and host specialization. We focused on the endophytic genus Lophodermium (Rhytismataceae), whose species members are thought to specialize on different pine species, to test if amplicon sequencing could rapidly verify previously implied or discover new patterns of host specificity. RESULTS: While amplicon sequencing could analyze more samples at greater depths and recover greater numbers of unique Lophodermium taxa than when endophyte communities were surveyed with traditional culturing methods, patterns of specialization were not better supported. This may be because amplicon sequencing can indiscriminately capture non-host specific organisms found incidentally from plant tissues or because we have overestimated host-specificity in the past with biased culturing techniques. CONCLUSIONS: Amplicon sequencing can quickly identify patterns of host specificity by allowing large-scale surveys but has limitations in quantifying the level of intimacy of these relationships.

Thelephora versatilis and Thelephora pseudoversatilis: two new cryptic species with polymorphic basidiomes inhabiting tropical deciduous and sub-perennial forests of the Mexican Pacific coast.

Thelephora is a genus of ectomycorrhizal basidiomycetes with basidiomes of varied shape which has been poorly studied in tropical ecosystems. In this paper, we present Thelephora versatilis and Thelephora pseudoversatilis, two new species collected in the same localities of deciduous and sub-perennial tropical forests of Jalisco, Mexico. Basidiomes of both species are brownish gray to violet brown with clavarioid-mesopodal, sub-resupinate or completely resupinate growth forms. In turn, phylogenetic analyses using nrDNA ITS sequences showed that these species are not closed related, nevertheless they are part of a well-supported clade conformed by several species of Thelephora, Tomentella and some undescribed Thelephorales. Morphological segregation of these species was attained by analyzing spore and hyphae characters using a wide sample. Significant statistical differences between the new species were observed regarding spore size, spine size and context hyphae width. This work exemplifies the relevance of integrating both morphological and molecular data, as well of the use of an appropriate sample size in order to discriminate among morphological cryptic species.

Neotropical Clavulina: Two new species from Mexico and a re-evaluation of Clavulina floridana.

Clavulina comprises ca. 90 described species distributed worldwide in both tropical and temperate regions. However, only one species (C. floridana) has been described so far from tropical North America. We used morphological and molecular data from three DNA loci (nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 [ITS], a portion of nuc 28S rDNA [28S], and a fragment of DNA-directed RNA polymerase II second largest subunit [RPB2]) from basidiomata and ectomycorrhizas collected in tropical ecosystems from three biogeographic provinces of Mexico and one tropical province in the USA to investigate the phylogenetic and taxonomic diversity of Clavulina in the region. Nine new species-level clades were discovered, two of which are proposed as new species (C. arboreiparva and C. tuxtlasana). Specimens of C. floridana recently collected in Florida were included in our analyses, for which a modern description is provided. In addition, C. floridana is a new record for Mexico. The diversity of Clavulina in tropical North America is comparable to that found in lowland tropical South America. However, some of the species found in tropical deciduous forests produce small, rare, and inconspicuous basidiomata, which easily go unnoticed, and therefore are poorly represented in collections. Many species remain undescribed in tropical regions of North America.

Fungal catalases: function, phylogenetic origin and structure.

Most fungi have several monofunctional heme-catalases. Filamentous ascomycetes (Pezizomycotina) have two types of large-size subunit catalases (L1 and L2). L2-type are usually induced by different stressors and are extracellular enzymes; those from the L1-type are not inducible and accumulate in asexual spores. L2 catalases are important for growth and the start of cell differentiation, while L1 are required for spore germination. In addition, pezizomycetes have one to four small-size subunit catalases. Yeasts (Saccharomycotina) do not have large-subunit catalases and generally have one peroxisomal and one cytosolic small-subunit catalase. Small-subunit catalases are inhibited by substrate while large-subunit catalases are activated by H(2)O(2). Some small-subunit catalases bind NADPH preventing inhibition by substrate. We present a phylogenetic analysis revealing one or two events of horizontal gene transfers from Actinobacteria to a fungal ancestor before fungal diversification, as the origin of large-size subunit catalases. Other possible horizontal transfers of small- and large-subunit catalases genes were detected and one from bacteria to the fungus Malassezia globosa was analyzed in detail. All L2-type catalases analyzed presented a secretion signal peptide. Mucorales preserved only L2-type catalases, with one containing a secretion signal if two or more are present. Basidiomycetes have only L1-type catalases, all lacking signal peptide. Fungal small-size catalases are related to animal catalases and probably evolved from a common ancestor. However, there are several groups of small-size catalases. In particular, a conserved group of fungal sequences resemble plant catalases, whose phylogenetic origin was traced to a group of bacteria. This group probably has the heme orientation of plant catalases and could in principle bind NADPH. From almost a hundred small-subunit catalases only one fourth has a peroxisomal localization signal and in fact many fungi lack a peroxisomal catalase. Catalases have a deep buried active site and H(2)O(2) has to go through a long passage to reach it. In all known structures of catalases, the major channel has common features, particularly in the straight and narrow final section that is positioned perpendicular to the heme. Besides, other conserved channels are present in catalases whose function remains to be elucidated. One of these channels intercommunicates the major channels from the two R-related subunits. In three of the four known large-subunits catalase structures, the heme b is partially transformed into heme d. In Neurospora crassa, this occurs in vivo and is related to oxidative stress conditions in which singlet oxygen is produced. A pure source of singlet oxygen oxidizes catalases purified from different sources and singlet oxygen quenchers prevent oxidation. A second modification is observed in N. crassa catalase-1, in which the tyrosine that forms the fifth coordination bound to the heme iron makes a covalent bond with a vicinal cysteine, similarly to the tyrosine-histidine bonding found in Escherichia coli hydroperoxidase II. Molecular dynamics has been used to determine how H(2)O(2) reaches the enzyme active site and how products exit the protein. We found that the bottleneck of the major channel seems to disappear in water and is wide open in the presence of substrate. Amino acid residues exhibiting an increased residence time for H(2)O(2) are abundant at the protein surface and at the entrances to the major channel. The net effect of this is an increased H(2)O(2)/H(2)O ratio in the major channel. Once in the final section of this channel, H(2)O(2) is retained and tends to occupy specific sites while water molecules have a higher turnover rate and occupy different sites. Despite the intense study of catalases our knowledge of this enzyme is still limited and in need of new studies and different approaches.

A comparative analysis of Lophodermium fissuratum, sp. nov., found in haploxylon pine needles in the Pacific Northwest, and other Lophodermium endophytes.

Lophodermium is a large fungal genus consisting of over 100 named species, with ca. 38 of these commonly found as endophytes of pine needles. In this study, we use both morphological and sequencing data to describe a new Lophodermium species associated with haploxylon pines from the Pacific Northwest. This new species resembled the morphology of L. nitens, another commonly occurring species from the same geographic regions and host species. They both present dark subcuticular ascocarps without lips. However, the upper walls of their ascocarps are different, as the new species forms an inward V-shaped folding, not present in L. nitens. Phylogenies using nuc rDNA internal transcribed spacer barcodes (ITS1-5.8S-ITS2 = ITS), partial D1-D2 domains of nuc rDNA 28S, and partial sequences of the nuc actin gene confirmed that this species represents a unique lineage not closely related to L. nitens. We discuss the current state of the phylogeny in light of all currently available sequences from pine-associated Lophodermium species.

Double-digest RADseq loci using standard Illumina indexes improve deep and shallow phylogenetic resolution of Lophodermium, a widespread fungal endophyte of pine needles.

The phylogenetic and population genetic structure of symbiotic microorganisms may correlate with important ecological traits that can be difficult to directly measure, such as host preferences or dispersal rates. This study develops and tests a low-cost double-digest restriction site-associated DNA sequencing (ddRADseq) protocol to reveal among- and within-species genetic structure for Lophodermium, a genus of fungal endophytes whose evolutionary analyses have been limited by the scarcity of informative markers. The protocol avoids expensive barcoded adapters and incorporates universal indexes for multiplexing. We tested for reproducibility and functionality by comparing shared loci from sample replicates and assessed the effects of numbers of ambiguous sites and clustering thresholds on coverage depths, number of shared loci among samples, and phylogenetic reconstruction. Errors between technical replicates were minimal. Relaxing the quality-filtering criteria increased the mean coverage depth per locus and the number of loci recovered within a sample, but had little effect on the number of shared loci across samples. Increasing clustering threshold decreased the mean coverage depth per cluster and increased the number of loci recovered within a sample but also decreased the number of shared loci across samples, especially among distantly related species. The combination of low similarity clustering (70%) and relaxed quality-filtering (allowing up to 30 ambiguous sites per read) performed the best in phylogenetic analyses at both recent and deep genetic divergences. Hence, this method generated sufficient number of shared homologous loci to investigate the evolutionary relationships among divergent fungal lineages with small haploid genomes. The greater genetic resolution also revealed new structure within species that correlated with ecological traits, providing valuable insights into their cryptic life histories.

Phylogenetics of Lophodermium from pine.

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.

Genetic diversity of Histoplasma capsulatum isolated from infected bats randomly captured in Mexico, Brazil, and Argentina, using the polymorphism of (GA)(n) microsatellite and its flanking regions.

The genetic diversity of 47 Histoplasma capsulatum isolates from infected bats captured in Mexico, Brazil, and Argentina was studied, using sequence polymorphism of a 240-nucleotides (nt) fragment, which includes the (GA)(n) length microsatellite and its flanking regions within the HSP60 gene. Three human clinical strains were used as geographic references. Based on phylogenetic analyses of 240-nt fragments achieved, the relationships among H. capsulatum isolates were resolved using neighbour-joining and maximum parsimony methods. The tree topologies obtained by both methods were identical and highlighted two major clusters of isolates. Cluster I had three sub-clusters (Ia, Ib, and Ic), all of which contained Mexican H. capsulatum samples, while cluster II consisted of samples from Brazil and Argentina. Sub-cluster Ia included only fungal isolates from the migratory bat Tadarida brasiliensis. An average DNA mutation rate of 2.39 × 10(-9) substitutions per site per year was estimated for the 240-nt fragment for all H. capsulatum isolates. Nucleotide diversity analysis of the (GA)(n) and flanking regions from fungal isolates of each cluster and sub-cluster underscored the high similarity of cluster II (Brazil and Argentina), sub-clusters Ib, and Ic (Mexico). According to the genetic distances among isolates, a network of the 240-nt fragment was graphically represented by (GA)(n) length haplotype. This network showed an association between genetic variation and both the geographic distribution and the ecotype dispersion of H. capsulatum, which are related to the migratory behaviour of the infected bats studied.