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.

Characterization of ciliate diversity in bromeliad tank waters from the Brazilian Atlantic Forest.

Bromeliads are a diverse group of plants that includes many species whose individuals are capable of retaining water, forming habitats called phytotelmata. These habitats harbor a diversity of organisms including prokaryotes, unicellular eukaryotes, metazoans, and fungi. Among single-celled eukaryotic organisms, ciliates are generally the most abundant. In the present study, we used Illumina DNA sequencing to survey the eukaryotic communities, especially ciliates, inhabiting the tanks of the bromeliads Aechmea gamosepala and Vriesea platynema in the Atlantic Forest of southern Brazil. Filtered sequences were clustered into distinct OTUs using a 99% identity threshold, and then assigned to phylum and genus using a BLAST-based approach (implemented in QIIME) and the SILVA reference database. Both bromeliad species harbored very diverse eukaryotic communities, with Arthropoda and Ciliophora showing the highest abundance (as estimated by the number of sequence reads). The ciliate genus Tetrahymena was the most abundant among single-celled organisms, followed by apicomplexan gregarines and the ciliate genus Glaucoma. Another interesting finding was the presence and high abundance of Trypanosoma in these bromeliad tanks, demonstrating their occurrence in this type of environment. The results presented here demonstrate a hidden diversity of eukaryotes in bromeliad tank waters, opening up new avenues for their in-depth characterization.

Epistylis portoalegrensis n. sp. (Ciliophora, Peritrichia): A New Freshwater Ciliate Species from Southern Brazil.

The peritrich ciliate Epistylis portoalegrensis n. sp. was found in two bodies of freshwater located in Porto Alegre, Southern Brazil. Morphological features were investigated using live and protargol-stained specimens. The zooids presented a vase to cylindrical shape narrowed at the scopula, and a mean size of 131 × 37 µm in vivo. A C-shaped macronucleus lay in the middle of the cell close to a single contractile vacuole. The oral infraciliature was typical for the genus, with all infundibular polykineties composed by three distinct rows of kinetosomes. Colonies are often nonbranched with no lateral stalk, carrying several zooids stemming from a single point. Specimens from the two sampling sites showed identical arrangement of the infraciliature, similar morphometry, identical 18S rDNA sequences, and a single nucleotide difference across the more variable ITS regions. Molecular phylogenetic analyses placed E. portoalegrensis in a well-supported clade containing other Epistylis species, within the order Vorticellida.

Multiple lines of evidence shed light on the occurrence of paramecium (ciliophora, oligohymenophorea) in bromeliad tank water.

Phytotelmata are vegetal structures that hold water from the rain, and organic matter from the forest and the soil, resulting in small, compartmentalized bodies of water, which provide an essential environment for the establishment and development of many organisms. These microenvironments generally harbor endemic species, but many organisms that are found in lakes and rivers, are also present. Here, we report, for the first time, the occurrence of the ciliate genus Paramecium in the tank of the bromeliad species Aechmaea distichantha. The identification of the Paramecium species was performed based on live observations, protargol impregnation, scanning electronic microscopy, and sequencing of the 18s rRNA. The absence of Paramecium from bromeliad tank water was highlighted in several earlier investigations, and may be due to the fact that this species is unable to make cysts. The occurrence of Paramecium multimicronucleatum in our samples may be explained by the proximity between the bromeliads and the river, a potential source of the species. Further, we also believe that the counting methodology used in our study provides a more accurate analysis of the species diversity, since we investigated all samples within a maximum period of 6 h after sampling, allowing minimum loss of specimens.

Impacts of the Cretaceous Terrestrial Revolution and KPg extinction on mammal diversification.

Previous analyses of relations, divergence times, and diversification patterns among extant mammalian families have relied on supertree methods and local molecular clocks. We constructed a molecular supermatrix for mammalian families and analyzed these data with likelihood-based methods and relaxed molecular clocks. Phylogenetic analyses resulted in a robust phylogeny with better resolution than phylogenies from supertree methods. Relaxed clock analyses support the long-fuse model of diversification and highlight the importance of including multiple fossil calibrations that are spread across the tree. Molecular time trees and diversification analyses suggest important roles for the Cretaceous Terrestrial Revolution and Cretaceous-Paleogene (KPg) mass extinction in opening up ecospace that promoted interordinal and intraordinal diversification, respectively. By contrast, diversification analyses provide no support for the hypothesis concerning the delayed rise of present-day mammals during the Eocene Period.

Expanded phylogenetic representation of genera Opercularia and Epistylis sheds light on the evolution and higher-level taxonomy of peritrich ciliates (Ciliophora: Peritrichia).

We have generated 18S rRNA sequences for peritrichs collected in Brazil, including four Opercularia species, two different populations of Epistylis plicatilis (one epibiont and another free-living), and one additional Epistylis species. Our Opercularia species clustered with the previously available Opercularia microdiscum, corroborating the monophyly of this genus. The Epistylis sampled here clustered with previously sequenced species of this genus. The two populations of E. plicatilis collected in Brazil clustered closely together despite their different ecological contexts, whereas both were very divergent from the sample assigned to the same species previously sampled in China. If affirmed by additional morphological corroboration of species assignment, this observation would indicate that samples from different continents morphologically allocated in the same species may in fact belong to distant evolutionary lineages. More broadly, our results support the recognition of two major clades within Peritrichia. Given the robustness of their support, we suggest that these two clades should be formally recognized as orders, and propose the names Vorticellida and Operculariida to designate them. Furthermore, Epistylis species occurred in both orders, tending to occupy basal positions. This suggests that characters used to define this genus may be plesiomorphic for Peritrichia, so that Epistylis may in fact represent an assemblage of basal species retaining ancestral features.