Correlated evolution in an ectomycorrhizal host-symbiont system.

Mechanisms of diversification in fungi are relatively poorly known. Many ectomycorrhizal symbionts show preference for particular host genera or families, so host-symbiont selection may be an important driver of fungal diversification in ectomycorrhizal systems. However, whether ectomycorrhizal hosts and symbionts show correlated evolutionary patterns remains untested, and it is unknown whether fungal specialisation also occurs in systems dominated by hosts from the same genus. We use metabarcoding of ectomycorrhizal fungi collected with hyphal ingrowth bags from Nothofagus forests across southern New Zealand to investigate host-symbiont specialisation and correlated evolution. We examine how ectomycorrhizal communities differ between host species and look for patterns of host-symbiont cophylogeny. We found substantial differences in ectomycorrhizal communities associated with different host taxa, particularly between hosts from different subgenera (Lophozonia and Fuscospora), but also between more closely related hosts. Twenty-four per cent of fungal taxa tested showed affiliations to particular hosts, and tests for cophylogeny revealed significant correlations between host relatedness and the fungal phylogeny that extended to substantial evolutionary depth. These results provide new evidence of correlated evolution in ectomycorrhizal systems, indicating that preferences among closely related host species may represent an important evolutionary driver for local lineage diversification in ectomycorrhizal fungi.

Zeta diversity differentiates factors driving community assembly of rare and common ectomycorrhizal fungi.

A fundamental goal in community ecology is to understand what factors drive community assembly processes. The factors affecting ectomycorrhizal fungal communities are unknown in many regions, particularly in the southern hemisphere. We investigate community assembly using ITS2 metabarcoding of ectomycorrhizal fungi sampled from 3943 hyphal ingrowth bags buried in 81 Nothofagus forests across New Zealand's South Island. By applying zeta diversity analysis and multisite generalized dissimilarity modelling (MS-GDM) we quantify the effects of 43 biotic and environmental variables on community turnover. Unlike traditional beta diversity analyses that are heavily influenced by rare species, the zeta diversity framework differentiates between factors driving turnover of rare and common species, providing a more complete picture of community dynamics. We found that community assembly was dominated by deterministic rather than stochastic processes and identified ecological factors affecting all taxa, as well as others that were specifically important to rare or common taxa. Soil variables were important drivers of turnover for all species, whereas ground cover variables, forest patch size, precipitation and host tree identity had greater effects on rarer species, and tree size and temperature effects were specific to more common species. Interestingly, the effect of temperature on common species is in line with recent evidence from other Kingdoms, pointing to possible generality, and highlighting the importance of considering common species. Applying these methods to fungi has allowed us to identify the distinct ecological processes that structure rare and common taxa during community assembly. This has important implications for understanding the functional effects of community responses to environmental change.

Purple haze: Cryptic purple sequestrate Cortinarius in New Zealand.

CORTINARIUS: is a species-rich ectomycorrhizal genus containing taxa that exhibit agaricoid or sequestrate basidiome morphologies. In New Zealand, one of the most recognizable and common Cortinarius species is the purple sequestrate fungus, C. porphyroideus. We used genome skimming of the almost 100-y-old type specimen from C. porphyroideus to obtain the nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS barcode) and partial nuc rDNA 28S (28S) sequences. The phylogenetic position of C. porphyroideus was established, and we found that it represents a rarely collected species. Purple sequestrate Cortinarius comprise multiple cryptic species in several lineages. We describe four new species of Cortinarius with strong morphological similarity to C. porphyroideus: Cortinarius diaphorus, C. minorisporus, C. purpureocapitatus, and C. violaceocystidiatus. Based on molecular evidence, Thaxterogaster viola is recognized as Cortinarius violaceovolvatus var. viola. These species are associated with Nothofagus (southern beech) and have very similar morphology to C. porphyroideus but are all phylogenetically distinct based on molecular data.

Fungal diversity in canopy soil of silver beech, Nothofagus menziesii (Nothofagaceae).

Adventitious roots in canopy soils associated with silver beech (Nothofagus menziesii Hook.f. (Nothofagaceae)) form ectomycorrhizal associations. We investigated the extent to which canopy ectomycorrhizal communities contribute to overall diversity of ectomycorrhizal fungi associated with silver beech. Hyphal ingrowth bags were buried for 12 months in canopy and terrestrial soils of five trees at one site. We used amplicon sequencing of the nuclear ribosomal internal transcribed spacer 2 region (ITS2) to assess diversity of both ectomycorrhizal and non-ectomycorrhizal OTUs in hyphal ingrowth bags. There was a significant difference in ectomycorrhizal fungal community diversity between the terrestrial and canopy hyphal ingrowth bag communities. Ectomycorrhizal community composition of the terrestrial and canopy environments was also significantly different. Some ectomycorrhizal taxa were significantly differentially represented in either the terrestrial or canopy environment. The hyphal ingrowth bags also accumulated non-ectomycorrhizal species. The non-ectomycorrhizal fungi also had significantly different diversity and community composition between the canopy and terrestrial environments. Like the ectomycorrhizal community, some non-ectomycorrhizal taxa were significantly differentially represented in either the terrestrial or canopy environment. The canopy soil microhabitat provides a novel environment for growth of ectomycorrhizal adventitious roots and enables the spatial partitioning of ectomycorrhizal and non-ectomycorrhizal fungal diversity in the forest.

Contrasting bacterial communities in two indigenous Chionochloa (Poaceae) grassland soils in New Zealand.

The cultivation of grasslands can modify both bacterial community structure and impact on nutrient cycling as well as the productivity and diversity of plant communities. In this study, two pristine New Zealand grassland sites dominated by indigenous tall tussocks (Chionochloa pallens or C. teretifolia) were examined to investigate the extent and predictability of variation of the bacterial community. The contribution of free-living bacteria to biological nitrogen fixation is predicted to be ecologically significant in these soils; therefore, the diazotrophic community was also examined. The C. teretifolia site had N-poor and poorly-drained peaty soils, and the C. pallens had N-rich and well-drained fertile soils. These soils also differ in the proportion of organic carbon (C), Olsen phosphorus (P) and soil pH. The nutrient-rich soils showed increased relative abundances of some copiotrophic bacterial taxa (including members of the Proteobacteria, Bacteroidetes and Firmicutes phyla). Other copiotrophs, Actinobacteria and the oliogotrophic Acidobacteria showed increased relative abundance in nutrient-poor soils. Greater diversity based on 16S rRNA gene sequences and the Tax4Fun prediction of enhanced spore formation associated with nutrient-rich soils could indicate increased resilience of the bacterial community. The two sites had distinct diazotrophic communities with higher diversity in C. teretifolia soils that had less available nitrate and ammonium, potentially indicating increased resilience of the diazotroph community at this site. The C. teretifolia soils had more 16S rRNA gene and nifH copies per g soil than the nutrient rich site. However, the proportion of the bacterial community that was diazotrophic was similar in the two soils. We suggest that edaphic and vegetation factors are contributing to major differences in the composition and diversity of total bacterial and diazotrophic communities at these sites. We predict the differences in the communities at the two sites will result in different responses to environmental change.

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand.

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non-heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%-42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.

Morphological and molecular data for Australian Hebeloma species do not support the generic status of Anamika.

The first collection of a macrofungal agaric species, with morphological features similar to already described Anamika species, has been found in association with animal bones in north Queensland, Australia. This species also shares features with several, commonly occurring and previously described Australian Hebeloma species. An integrated morphological and molecular study has resulted in the conclusion that all Anamika species belong in Hebeloma. As a result, already described species of Anamika are recombined as H. indicum (K.A. Thomas, Peintner, M.M. Moser and Manim.) B.J. Rees & Orlovich, H. angustilamellatum (Zhu L. Yang and Z.W. Ge) B.J. Rees & Orlovich and H. lactariolens (Clémençon and Hongo) B.J. Rees & Orlovich. A. phylogenetic tree based on ribosomal ITS sequences examines the relationship of these species with other Hebeloma species from both hemispheres. Four new species, Hebeloma youngii B.J. Rees, H. nothofagetorum B.J. Rees, H. subvictoriense B.J. Rees, H. lacteocoffeatum B.J. Rees, and one form, H. aminophilum f. hygrosarx B.J. Rees, are described as new from Australia.

Piracy in the high trees: ectomycorrhizal fungi from an aerial 'canopy soil' microhabitat.

The mantle of dead organic material ("canopy soil") associated with the mats of vascular and nonvascular epiphytes found on the branches of trees in the temperate rainforests along the southwestern coast of the South Island of New Zealand were examined for evidence of ectomycorrhizal fungi. DNA sequencing and cluster analysis were used to identify the taxa of fungi present in 74 root tips collected from the canopy soil microhabitat of three old growth Nothofagus menziesii trees in the South West New Zealand World Heritage Area. A diverse assemblage of ectomycorrhizal fungi was found to infect an extensive network of adventitious canopy roots of Nothofagus menziesii in this forest, including 14 phylotypes from nine genera of putative ectomycorrhizal fungi. Seven of the genera identified previously were known to form ectomycorrhizas with terrestrial roots of Nothofagus: Cortinarius, Russula, Cenococcum, Thelephora/Tomentella, Lactarius and Laccaria; two, Clavulina and Leotia, previously have not been reported forming ectomycorrhizas with Nothofagus. Canopy ectomycorrhizas provide an unexpected means for increased host nutrition that may have functional significance in some forest ecosystems. Presumably, canopy ectomycorrhizas on host adventitious roots circumvent the tree-ground-soil nutrient cycle by accessing a wider range of nutrients directly in the canopy than would be possible for non-mycorrhizal or arbuscular mycorrhizal canopy roots. In this system, both host and epiphytes would seem to be in competition for the same pool of nutrients in canopy soil.

The influence of probiotic treatment on sulfasalazine metabolism in rat.

Probiotics are live microorganisms claimed to exert beneficial effects on the host. This study investigated their effect on the metabolism and pharmacokinetics of sulfasalazine (SSZ), a drug whose efficacy depends on metabolism by azoreductase (AR) in the gut microbiota to sulfapyridine (SP) and 5-acetylsalicylic acid (5-ASA). The probiotic strains Lactobacillus acidophilus L10, Bifidobacterium lactis B94 and Streptococcus salivarius K12 possessed AR activity and a corresponding ability to metabolize SSZ. Treatment of male Wistar rats (n = 5) with oral 2 g doses of a mixture of the three probiotics (total dose 1.8 × 109 cfu) every 12 h for 3 days resulted in a significant increase (p < 0.05) in AR activity in ex vivo colon contents with a corresponding increase in SSZ metabolism. Similar probiotic treatment of male Wistar rats (n = 8) followed by an oral 100 mg/kg dose of SSZ produced high plasma levels of SP, but pharmacokinetic parameters of SSZ and SP were not significantly different from control rats given SSZ. These results indicate that probiotic strains possess AR activity and can metabolize SSZ. Treatment with probiotics increases AR activity in the gut microbiota but has no effect on plasma levels of SSZ and SP following a subsequent oral dose of SSZ.

Detection and analysis of endogenous badnaviruses in the New Zealand flora.

BACKGROUND AND AIMS: Badnaviruses and their host-integrated DNA occur in tropical crops and a few northern temperate species. Following the discovery of a badnavirus on a subantarctic island with floristic links to New Zealand, we postulated that badnaviruses exist in the New Zealand flora. Badnavirus reverse transcriptase (RT) sequences consist of variable regions flanked by highly conserved regions. This study used RT sequences to detect and characterize badnavirus sequences in the New Zealand flora and to investigate their utility for the study of broader aspects of plant biology. METHODOLOGY: Molecular diversity of RT sequences was analysed using polymerase chain reaction and denaturing gradient gel electrophoresis (DGGE). In a study of the genus Melicytus, internal transcribed spacer (ITS) sequences were compared with the RT data. PRINCIPAL RESULTS: No freely replicating badnaviruses were detected but more than half of the species (37/60) contained RT sequences. Phylogenetic analysis of 21 RT sequences formed monophyletic groups distinct from other species and from badnaviruses. No frameshift mutations occurred in any of the sequences translated in silico. More detailed study of the genus Melicytus indicated broader applications for our approach. Analysis of RT sequences revealed the presence of a previously unrecognized species (confirmed using ITS). Inheritance of DGGE profiles by Melicytus ramiflorus seedlings suggested that this species may undergo apomixis. CONCLUSIONS: The presence of integrated badnavirus sequences in a wide range of taxa from this Southern Hemisphere flora indicates that these sequences may be common in many temperate regions. Potential to activate viruses from these sequences should be considered when placing these species in tissue culture or under other forms of abiotic or genomic stress. Analysis of endogenous RT sequences shows potential for the study of systematics, phylogenetics and plant reproductive biology.

The effects of an orally administered probiotic on sulfasalazine metabolism in individuals with rheumatoid arthritis: a preliminary study.

AIM: To carry out a pilot study to investigate the effect of short-term oral probiotic administration on the metabolism of sulfasalazine (SSZ) in patients with rheumatoid arthritis (RA) stabilized on SSZ. METHODS: Twelve subjects with RA taking stable doses of SSZ for a minimum of 3 months prior to the study, received a probiotic preparation contained three strains of bacteria (1.8 x 10(9) CFU/day) twice daily for 1 week. Single point blood and 12-h urine samples were taken before and after probiotic treatment and 3 weeks following discontinuation of probiotics, for determination of SSZ and its metabolites. The presence of the probiotic bacteria in the feces of patients was investigated by denaturing gradient gel electrophoresis (DGGE). RESULTS: Adverse events recorded were three instances of gastrointestinal disturbance and one flare of RA. Plasma and urinary levels of SSZ and its metabolites showed no statistically significant changes after probiotic administration and the incidence of gastrointestinal disturbance did not appear to be ascribed to higher sulfapyridine plasma levels. Probiotic-specific DGGE bands were detected in the feces of some patients after the treatment period. CONCLUSIONS: Short-term treatment of RA patients with a multi-strain probiotic did not significantly influence SSZ metabolism as has been demonstrated in animal models.

Detection and Specific Enumeration of Multi-Strain Probiotics in the Lumen Contents and Mucus Layers of the Rat Intestine After Oral Administration.

Although the detection of viable probiotic bacteria following their ingestion and passage through the gastrointestinal tract (GIT) has been well documented, their mucosal attachment in vivo is more difficult to assess. In this study, we investigated the survival and mucosal attachment of multi-strain probiotics transiting the rat GIT. Rats were administered a commercial mixture of the intestinal probiotics Lactobacillus acidophilus LA742, Lactobacillus rhamnosus L2H and Bifidobacterium lactis HN019 and the oral probiotic Streptococcus salivarius K12 every 12 h for 3 days. Intestinal contents, mucus and faeces were tested 6 h, 3 days and 7 days after the last dose by strain-specific enumeration on selective media and by denaturing gradient gel electrophoresis. At 6 h, viable cells and DNA corresponding to all four probiotics were detected in the faeces and in both the lumen contents and mucus layers of the ileum and colon. Viable probiotic cells of B. lactis and L. rhamnosus were detected for 7 days and L. acidophilus for 3 days after the last dose. B. lactis and L. rhamnosus persisted in the ileal mucus and colon contents, whereas the retention of L. acidophilus appeared to be relatively higher in colonic mucus. No viable cells of S. salivarius K12 were detected in any of the samples at either day 3 or 7. The study demonstrates that probiotic strains of intestinal origin but not of oral origin exhibit temporary colonisation of the rat GIT and that these strains may have differing relative affinities for colonic and ileal mucosa.

A novel supermatrix approach improves resolution of phylogenetic relationships in a comprehensive sample of danthonioid grasses.

Phylogeny reconstruction is challenging when branch lengths vary and when different genetic loci show conflicting signals. The number of DNA sequence characters required to obtain robust support for all the nodes in a phylogeny becomes greater with denser taxon sampling. We test the usefulness of an approach mixing densely sampled, variable non-coding sequences (trnL-F; rpl16; atpB-rbcL; ITS) with sparsely sampled, more conservative protein coding and ribosomal sequences (matK; ndhF; rbcL; 26S), for the grass subfamily Danthonioideae. Previous phylogenetic studies of Danthonioideae revealed extensive generic paraphyly, but were often impeded by insufficient character and taxon sampling and apparent inter-gene conflict. Our variably-sampled supermatrix approach allowed us to represent 79% of the species with up to c. 9900 base pairs for taxa representing the major clades. A 'taxon duplication' approach for taxa with conflicting phylogenetic signals allowed us to combine the data whilst representing the differences between chloroplast and nuclear encoded gene trees. This approach efficiently improves resolution and support whilst maximising representation of taxa and their sometimes composite evolutionary histories, resulting in a phylogeny of the Danthonioideae that will be useful both for a wide range of evolutionary studies and to inform forthcoming realignment of generic delimitations in the subfamily.