Exploring the mycobiota of bromeliads phytotelmata in Brazilian Campos Rupestres.

The phytotelmata is a water-filled tank on a terrestrial plant, and it plays an important role in bromeliad growth and ecosystem functioning. Even though previous studies have contributed to elucidate the composition of the prokaryotic component of this aquatic ecosystem, its mycobiota (fungal community) is still poorly known. In the present work, ITS2 amplicon deep sequencing was used to examine the fungal communities inhabiting the phytotelmata of two bromeliads species that coexist in a sun-exposed rupestrian field of Southeastern Brazil, namely Aechmea nudicaulis (AN) and Vriesea minarum (VM). Ascomycota was the most abundant phylum in both bromeliads (57.1 and 89.1% in AN and VM respectively, on average), while the others were present in low abundance (< 2%). Mortierellomycota and Glomeromycota were exclusively observed in AN. Beta-diversity analysis showed that samples from each bromeliad significantly clustered together. In conclusion, despite the considerable within-group variation, the results suggested that each bromeliad harbor a distinct fungi community, what could be associated with the physicochemical characteristics of the phytotelmata (mainly total nitrogen, total organic carbon, and total carbon) and plant morphological features.

Different plant compartments, different yeasts: The example of the bromeliad phyllosphere.

The plant phyllosphere is one of the largest sources of microorganisms, including yeasts. In bromeliads, the knowledge of yeasts is dispersed and still incipient. To understand the extent of our knowledge of the subject, this review proposes to compile and synthesize existing knowledge, elucidating possible patterns, biotechnological and taxonomic potentials, bringing to light new knowledge, and identifying information gaps. For such, we systematically review scientific production on yeasts in bromeliads using various databases. The results indicated that the plant compartments flowers, fruits, leaves, and water tank (phytotelma) have been studied when focusing on the yeast community in the bromeliad phyllosphere. More than 180 species of yeasts and yeast-like fungi were recorded from the phyllosphere, 70% were exclusively found in one of these four compartments and only 2% were shared among all. In addition, most of the community had a low frequency of occurrence, and approximately half of the species had a single record. Variables such as bromeliad subfamilies and functional types, as well as plant compartments, were statistically significant, though inconclusive and with low explanatory power. At least 50 yeast species with some biotechnological potentials have been isolated from bromeliads. More than 90% of these species were able to produce extracellular enzymes. In addition, other biotechnological applications have also been recorded. Moreover, new species have been described, though yeasts were only exploited in approximately 1% of the existing bromeliads species, which highlights that there is still much to be explored. Nevertheless, it appears that we are still far from recovering the completeness of the diversity of yeasts in this host. Furthermore, bromeliads proved to be a good ecological model for prospecting new yeasts and for studies on the interaction between plants and yeasts. In addition, the yeast community diverged among plant compartments, establishing bromeliads as a microbiologically complex and heterogeneous mosaic.

Tremella ananatis sp. nov. and Tremella lamprococci sp. nov., two yeast species associated with bromeliads.

Eight yeast isolates with an affinity to the genus Tremella were obtained from bromeliads from different locations in Brazil. Although the formation of basidia and basidiocarp were not observed, on the basis of the results of sequence analysis of the D1/D2 domain of the large subunit (LSU) rRNA gene and internal transcribed spacer (ITS) region, we suggest that these isolates represent two novel species of the genus Tremella. These yeasts are phylogenetically related to Tremella saccharicola and Tremella globispora. Therefore, we propose Tremella ananatis sp. nov. and Tremella lamprococci sp. nov. as novel yeast species of the order Tremellales (Agaricomycotina, Basidiomycota). Sequence analysis revealed that Tremella ananatis sp. nov. differs by 11 and 28 nucleotide substitutions from Tremella saccharicola in the D1/D2 sequence and ITS region, respectively. Moreover, Tremella lamprococci sp. nov. differs by 15 and 29 nucleotide substitutions from Tremella globispora in the D1/D2 sequence and ITS region, respectively. The holotypes of Tremella ananatis sp. nov. and Tremella lamprococci sp. nov. are CBS 14568T and CBS 14567T, and the MycoBank numbers are MB840480 and MB840481, respectively.

Plant growth-promoting traits of yeasts isolated from the tank bromeliad Vriesea minarum L.B. Smith and the effectiveness of Carlosrosaea vrieseae for promoting bromeliad growth.

Yeasts can play important roles in promoting plant growth; however, little information is available in this regard for yeasts in water of bromeliad tanks. Here, we characterize the ability of 79 yeast isolates from tank bromeliad Vriesea minarum, an endangered species, to solubilize phosphate, secrete siderophores, and synthesize indole-3-acetic acid (IAA). The results showed that 67.8% of all assayed yeast isolates mobilized inorganic phosphate; 40.0% secreted siderophores; and 89.9% synthetized IAA and IAA-like compounds. Among the species studied, Carlosrosaea vrieseae UFMG-CM-Y6724 is highlighted for producing IAA (76.1 µg mL-1) and siderophores, and solubilizing phosphate. In addition, evaluation of the effects of filtrate containing IAA-like compounds produced by the C. vrieseae on the development and photosynthetic performance of V. minarum seedlings found it to improve seedling growth equal to that of commercial IAA. These results demonstrate that C. vrieseae can produce compounds with great potential for future use as biofertilizer agents.

Remarkably Complex Microbial Community Composition in Bromeliad Tank Waters Revealed by eDNA Metabarcoding.

To investigate patterns of biotic community composition at different spatial scales and biological contexts, we used environmental DNA metabarcoding to characterize eukaryotic and prokaryotic assemblages present in the phytotelmata of three bromeliad species (Aechmea gamosepala, Vriesea friburgensis, and Vriesea platynema) at a single Atlantic Forest site in southern Brazil. We sampled multiple individuals per species and multiple tanks from each individual, totalizing 30 samples. We observed very high levels of diversity in these communities, and remarkable variation across individuals and even among tanks from the same individual. The alpha diversity was higher for prokaryotes than eukaryotes, especially for A. gamosepala and V. platynema samples. Some biotic components appeared to be species-specific, while most of the biota was shared among species, but varied substantially in frequency among samples. Interestingly, V. friburgensis communities (which were sampled at nearby locations) tended to be more heterogeneous across samples, for both eukaryotes and prokaryotes. The opposite was true for V. platynema, whose samples were more broadly spaced but whose communities were more similar to each other. Our results indicate that additional attention should be devoted to within-individual heterogeneity when assessing bromeliad phytotelmata biodiversity, and highlight the complexity of the biotic assemblages gathered in these unique habitats.

Vishniacozyma alagoana sp. nov. a tremellomycetes yeast associated with plants from dry and rainfall tropical forests.

Plants are important reservoirs of described and undescribed species of yeast. During a study of yeasts associated with bromeliads from the Northeast region of Brazil (collected in 2013-2017), analysis of the D1/D2 domain of the LSU rRNA and internal transcribed spacer (ITS) region identified eleven strains of yeasts as representing an unknown species of the genus Vishniacozyma. The species may have a diverse habitat in Brazil as a strain was collected from a flowering plant (Acanthaceae) in 1994. As a consequence, we propose Vishniacozyma alagoana sp. nov. as a member of the tremellomycetes yeasts (Agaricomycotina, Basidiomycota). Vishniacozyma alagoana sp. nov. was found in Atlantic Forest (a tropical rainforest) and the Caatinga (a seasonally dry tropical forest) associated with bromeliads in northeast and southeastern Brazil. The proposed novel species is related to Vishniacozyma taibaiensis and distinguished by eight nucleotide substitutions in the D1/D2 domain and seventeen in the ITS region. In addition, Vishniacozyma alagoana sp. nov. differs from V. taibaiensis by the ability to assimilate ribitol. The holotype is CBS 15966T.

Yeast in plant phytotelmata: Is there a "core" community in different localities of rupestrian savannas of Brazil?

Ephemeral microbial communities usually undergo priority effect and result in higher diversity with a few representatives of each species. Community structure of yeasts in bromeliad tanks was compared between two rupestrian savanna (Cerrado) areas in Brazil and to yeasts isolated from water holes in the same areas. Water samples were collected from 60 tanks of bromeliads Bromelia karatas and Encholirium sp. and rock holes at the Karstic Area of Aurora, Tocantins State and 60 tanks of Vriesea minarum (Bromeliaceae) and Paepalanthus bromelioides (Eriocaulaceae) at Serra do Cipó National Park, Minas Gerais State in Brazil. The yeast diversity comprised 90 species from which 60% are basidiomycetous yeasts usually associated with phylloplane, soils, and aquatic habitats. The species Papiliotrema laurentii, Rhodotorula mucilaginosa, Pa. nemorosus, and Pseudozyma hubeiensis were the most frequent species associated with bromeliads. Eighteen yeast species, two ascomycetous and 16 basidiomycetous, were consistently isolated from the substrates in both areas and may represent a core community in bromeliads in rupestrian fields. Singlets occurred in 38 to 69% of samples, and 32 species were isolated only once. Our findings reinforce the ephemeral nature of the yeast communities associated with tank-forming plants in which individual phytotelmata act as patches or aquatic islands prone to rapid colonization-extinction rates receiving inocula from plant and soil debris. Ephemeral rock holes also represent a transitory habitat for yeast species associated with plants and soil.

Cosmarium bromelicola sp. nov. (Desmidiaceae, Zygnematophyceae), a new desmid species from Northeast Brazil / Cosmarium bromelicola sp. nov. (Desmidiaceae, Zygnematophyceae), uma nova espécie de desmídia do nordeste do Brazil

Abstract Cosmarium bromelicola sp. nov. is a new desmid species described from samplings carried out in bromeliad tanks (phytotelmata) from an area of rocky outcrops at Serra da Jiboia, Bahia State, Northeast Brazil. Presence of subtrapeziform cells with a deep depression at the apical region and twisted X-shaped cell in side view are the diagnostic features for the species. Relationships with the morphologically closest taxa are discussed.

Resumo Cosmarium bromelicola sp. nov. é uma nova espécie de desmídia descrita a partir de amostragens realizadas em tanques de bromélias (fitotelmata) de uma área de afloramentos rochosos na Serra da Jiboia, Bahia, nordeste do Brasil. A presença de células trapeziformes com uma depressão profunda na região apical e célula em forma de X em vista lateral são as características diagnósticas da espécie. Relações com os táxons morfologicamente próximos são discutidas.

Cosmarium bromelicola sp. nov. (Desmidiaceae, Zygnematophyceae), a new desmid species from Northeast Brazil.

Cosmarium bromelicola sp. nov. is a new desmid species described from samplings carried out in bromeliad tanks (phytotelmata) from an area of rocky outcrops at Serra da Jiboia, Bahia State, Northeast Brazil. Presence of subtrapeziform cells with a deep depression at the apical region and twisted X-shaped cell in side view are the diagnostic features for the species. Relationships with the morphologically closest taxa are discussed.

Hydrogenotrophic methanogenesis is the dominant methanogenic pathway in neotropical tank bromeliad wetlands.

Several thousands of tank bromeliads per hectare of neotropical forest create a unique wetland ecosystem that emits substantial amounts of CH4 . Tank bromeliads growing in the forest canopy (functional type-II tank bromeliads) were found to emit more CH4 than tank bromeliads growing on the forest floor (functional type-I tank bromeliads) but the reasons for this difference and the underlying microbial CH4 -cycling processes have not been studied. Therefore, we characterized archaeal communities in bromeliad tanks of the two different functional types in a neotropical montane forest of southern Ecuador using terminal-restriction fragment length polymorphism (T-RFLP) and performed tank-slurry incubations to measure CH4 production potential, stable carbon isotope fractionation and pathway of CH4 formation. The archaeal community composition was dominated by methanogens and differed between bromeliad functional types. Hydrogenotrophic Methanomicrobiales were the dominant methanogens and hydrogenotrophic methanogenesis was the dominant methanogenic pathway among all bromeliads. The relative abundance of aceticlastic Methanosaetaceae and the relative contribution of aceticlastic methanogenesis increased in type-I tank bromeliads probably due to more oxic conditions in type-I than in type-II bromeliads leading to the previously observed lower in situ CH4 emissions from type-I tank bromeliads but to higher CH4 production potentials in type-I tank bromeliad slurries.

Host niche may determine disease-driven extinction risk.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) drives declines and extinctions in amphibian communities. However, not all regions and species are equally affected. Here, we show that association with amphibian aquatic habitat types (bromeliad phytotelmata versus stream) across Central America results in the odds of being threatened by Bd being five times higher in stream microhabitats. This differential threat of Bd was supported in our study by a significantly lower prevalence of Bd in bromeliad-associated amphibian species compared to riparian species in Honduran cloud forests. Evidence that the bromeliad environment is less favorable for Bd transmission is exemplified by significantly less suitable physicochemical conditions and higher abundance of Bd-ingesting micro-eukaryotes present in bromeliad water. These factors may inhibit aquatic Bd zoospore survival and the development of an environmental reservoir of the pathogen. Bromeliad phytotelmata thus may act as environmental refuges from Bd, which contribute to protecting associated amphibian communities against chytridiomycosis-driven amphibian declines that threaten the nearby riparian communities.

Carlosrosaea hohenbergiae sp. nov. and Carlosrosaea aechmeae sp. nov., two tremellaceous yeasts isolated from bromeliads in north-eastern Brazil.

Yeast surveys associated with different bromeliads in north-eastern Brazil led to the proposal of two novel yeast species, Carlosrosaea hohenbergiae sp. nov. and Carlosrosaea aechmeae sp. nov., belonging to the Tremellales clade (Agaricomycotina, Basidiomycota). Analysis of the sequences of the internal transcribed spacer (ITS) region and D1/D2 domain of the LSU rRNA gene suggested an affinity with a phylogenetic lineage that includes recently reclassified Carlosrosaeavrieseae. Six isolates of the novel species were obtained from different bromeliad species collected in three Atlantic Forest fragments in Alagoas state, Brazil. Ca. hohenbergiae sp. nov. differs by 69 and 12 nucleotide substitutions in the ITS and D1/D2 domain, respectively, from Ca. vrieseae. The type strain is UFMG-CM-Y405T (=BSB 34T=CBS 14563T), Mycobank 819227. Ca. aechmeae sp. nov. is represented by one strain isolated from Aechmea constantinii leaves. Ca. aechmeae sp. nov. differs from the related species Ca. hohenbergiae and Ca. vrieseae by 36 and 65 nucleotide substitutions, respectively, in the ITS region and by 12 and 15 nucleotide substitutions in the D1/D2 domain, respectively. The type strain of Ca. aechmeae sp. nov. is UFMG-CM-Y6095T (=BM 94T=CBS 14578), Mycobank 819228.

Functional structure of the bromeliad tank microbiome is strongly shaped by local geochemical conditions.

Phytotelmata in tank-forming Bromeliaceae plants are regarded as potential miniature models for aquatic ecology, but detailed investigations of their microbial communities are rare. Hence, the biogeochemistry in bromeliad tanks remains poorly understood. Here we investigate the structure of bacterial and archaeal communities inhabiting the detritus within the tanks of two bromeliad species, Aechmea nudicaulis and Neoregelia cruenta, from a Brazilian sand dune forest. We used metagenomic sequencing for functional community profiling and 16S sequencing for taxonomic profiling. We estimated the correlation between functional groups and various environmental variables, and compared communities between bromeliad species. In all bromeliads, microbial communities spanned a metabolic network adapted to oxygen-limited conditions, including all denitrification steps, ammonification, sulfate respiration, methanogenesis, reductive acetogenesis and anoxygenic phototrophy. Overall, CO2 reducers dominated in abundance over sulfate reducers, and anoxygenic phototrophs largely outnumbered oxygenic photoautotrophs. Functional community structure correlated strongly with environmental variables, between and within a single bromeliad species. Methanogens and reductive acetogens correlated with detrital volume and canopy coverage, and exhibited higher relative abundances in N. cruenta. A comparison of bromeliads to freshwater lake sediments and soil from around the world, revealed stark differences in terms of taxonomic as well as functional microbial community structure.

Kockovaella libkindii sp. nov., a yeast species isolated from water tanks of bromeliad.

During a study of yeast community associated with water tanks (phytotelmata) of the bromeliad Vriesea minarum, two strains of a novel stalk-forming yeast species were found. The sequences of the region spanning the ITS and D1/D2 domains of the large subunit rRNA gene showed that this species belongs to the genus Kockovaella. The novel species differs by 14 or more nucleotide substitutions in the D1/D2 domains and by 26 or more substitutions in the ITS-5.8S region from all other Kockovaella species. We describe this species as Kockovaella libkindii sp. nov. The type strain of Kockovaella libkindii sp. nov. is UFMG-CM-Y6053T (=UFMG-BRO-488T=CBS 12685T). The MycoBank number is MB 817710.

Decomposition of plant-sourced carbon compounds by heterotrophic betaproteobacteria isolated from a tropical Costa Rican bromeliad.

Betaproteobacteria were the most common isolates from the water-filled tank of a Costa Rican bromeliad. Isolates included eight species from the orders Neisseriales and Burkholderiales, with close relatives recovered previously from tropical soils, wetlands, freshwater, or in association with plants. Compared to close relatives, the isolates displayed high temperature and comparatively low pH optima, reflecting the tropical, acidic nature of the bromeliad tank. Bromeliad-associated bacteria most closely related to Chromobacterium, Herbaspirillum, and Aquitalea were all isolated exclusively at pH 6, while Ralstonia, Cupriavidus, and three species of Burkholderia were isolated mostly at pH 4. Activity profiles for the isolates suggest pervasive capabilities for the breakdown of plant-sourced organics, including d-galacturonic acid, mannitol, d-xylose, and l-phenylalanine, also reflecting a niche dominated by decomposition of leaves from the overlying canopy, which become entrained in the tanks. Metabolic activity profiles were overlapping between the Burkholderiales, isolated at pH 4, and the Neisseriales, isolated at pH 6, suggesting that plant material decomposition, which is presumably the underlying process sustaining the tank community and possibly the plant itself, occurs in the tanks at both pH extremes. These results suggest that bromeliad-associated betaproteobacteria may play an important role in the cycling of carbon in this unusual aquatic habitat.

Bullera vrieseae sp. nov., a tremellaceous yeast species isolated from bromeliads.

Two independent surveys of yeasts associated with different bromeliads in different Brazilian regions led to the proposal of a novel yeast species, Bullera vrieseae sp. nov., belonging to the Tremellales clade (Agaricomycotina, Basidiomycota). Analysis of the sequences in the internal transcribed spacer (ITS) region and D1/D2 domain of the LSU rRNA gene suggested affinity to a phylogenetic lineage that includes Bullera miyagiana and Bullera sakaeratica. Six isolates of the novel species were obtained from different bromeliads and regions in Brazil. Sequence analysis of the D1/D2 domains of the large subunit of the rRNA gene showed that the novel species differs from B. miyagiana and B. sakaeratica by 85 and 64 nt substitutions, respectively and by more than 75 nt substitutions in the ITS region. Phenotypically, Bullera vrieseae sp. nov. can be distinguished from both species based on the assimilation of meso-erythritol, which was negative for B. vrieseae sp. nov. but positive for the others, assimilation of d-glucosamine, which was positive for B. vrieseae sp. nov. but negative for B. miyagiana and of l-sorbose, which was negative for B. vrieseae sp. nov. but positive for B. sakaeratica. The novel species Bullera vrieseae sp. nov. is proposed to accommodate these isolates. The type strain of Bullera vrieseae sp. nov. is UFMG-CM-Y379T (BRO443T; ex-type CBS 13870T).

The diversity and extracellular enzymatic activities of yeasts isolated from water tanks of Vriesea minarum, an endangered bromeliad species in Brazil, and the description of Occultifur brasiliensis f.a., sp. nov.

The diversity of yeast species collected from the bromeliad tanks of Vriesea minarum, an endangered bromeliad species, and their ability to produce extracellular enzymes were studied. Water samples were collected from 30 tanks of bromeliads living in a rupestrian field site located at Serrada Piedade, Minas Gerais state, Brazil, during both the dry and rainy seasons. Thirty-six species were isolated, representing 22 basidiomycetous and 14 ascomycetous species. Occultifur sp., Cryptococcus podzolicus and Cryptococcus sp. 1 were the prevalent basidiomycetous species. The yeast-like fungus from the order Myriangiales, Candida silvae and Aureobasidium pullulans were the most frequent ascomycetous species. The diversity of the yeast communities obtained between seasons was not significantly different, but the yeast composition per bromeliad was different between seasons. These results suggest that there is significant spatial heterogeneity in the composition of populations of the yeast communities within bromeliad tanks, independent of the season. Among the 352 yeast isolates tested, 282 showed at least one enzymatic activity. Protease activity was the most widely expressed extracellular enzymatic activity, followed by xylanase, amylase, pectinase and cellulase activities. These enzymes may increase the carbon and nitrogen availability for the microbial food web in the bromeliad tank of V. minarum. Sequence analyses revealed the existence of 10 new species, indicating that bromeliad tanks are important sources of new yeasts. The novel species Occultifur brasiliensis, f.a., sp. nov., is proposed to accommodate the most frequently isolated yeast associated with V. minarum. The type strain of O. brasiliensis, f.a., sp. nov. is UFMG-CM-Y375(T) (= CBS 12687(T)). The Mycobank number is MB 809816.

Hagleromyces gen. nov., a yeast genus in the Saccharomycetaceae, and description of Hagleromyces aurorensis sp. nov., isolated from water tanks of bromeliads.

Three strains of a novel yeast species were isolated from water tanks (phytotelmata) of a bromeliad species collected in the state of Tocantins, Brazil. Analysis of sequences for the region spanning the SSU rRNA gene, the internal transcribed spacer, the 5.8S rRNA gene and the D1/D2 domains of the LSU rRNA gene and RNA polymerase II gene showed that these novel yeasts belong to a species that is distinct from all recognized ascomycetous yeast species. Based on the results of gene sequence analyses, a novel species representing a new genus in the Saccharomycetaceae is proposed. The novel species is assigned to the genus Hagleromyces gen. nov. The three isolates of the novel yeast species failed to form sexual spores alone or in mixtures. The name Hagleromyces aurorensis sp. nov. is proposed to accommodate these isolates. The type strain of H. aurorensis sp. nov. is UFMG-CM-Y311(T) ( = CBS 13264(T)).

Tank bromeliad water: similar or distinct environments for research of bacterial bioactives?

The Atlantic Rainforest does not have a uniform physiognomy, its relief determines different environmental conditions that define the composition of its flora and fauna. Within this ecosystem, bromeliads that form tanks with their leaves hold water reservoirs throughout the year, maintaining complex food chains, based mainly on autotrophic and heterotrophic bacteria. Some works concluded that the water held by tank bromeliads concentrate the microbial diversity of their ecosystem. To investigate the bacterial diversity and the potential biotechnology of these ecosystems, tank bromeliads of the Neoregelia cruenta species from the Atlantic Rainforest in Brazil were used as models for this research. Bacteria isolated from these models were tested for production of bioactive compounds. DGGE of the water held by tank bromeliads was performed in different seasons, locations and sun exposure to verify whether these environmental factors affect bacterial communities. The DGGE bands profile showed no grouping of bacterial community by the environmental factors tested. Most of the isolates demonstrated promising activities in the tests performed. Collectively, these results suggest that tank bromeliads of the N. cruenta species provide important habitats for a diverse microbial community, suggesting that each tank forms a distinct micro-habitat. These tanks can be considered excellent sources for the search for new enzymes and/or new bioactive composites of microbial origin.

Kazachstania rupicola sp. nov., a yeast species isolated from water tanks of a bromeliad in Brazil.

Two isolates of a novel yeast species were obtained from water tanks (phytotelmata) of the bromeliad Vriesea minarum collected in a tableland ('campo rupestre') ecosystem in Brazil. The sequences of the D1/D2 domains of the large-subunit rRNA gene showed that this species is related to Kazachstania exigua and others, from which it differs by 8-10 nucleotide substitutions. The novel species Kazachstania rupicola sp. nov. is proposed to accommodate these isolates. The type strain is UFMG-BRO-80(T) ( = CBS 12684(T)  = CBMAI 1466(T)).