Laura R. P. Utz

Molecular Phylogenetics of Subclass Peritrichia (Ciliophora: Oligohymenophorea) Based on Expanded Analyses of 18S rRNA Sequences

ABSTRACT. Phylogenetic relationships among peritrich ciliates remain unclear in spite of recent progress. To expand the analyses performed in previous studies, and to statistically test hypotheses of monophyly, we analyzed a broad sample of 18s rRNA sequences (including 15 peritrich genera), applying a conservative alignment strategy and several phylogenetic approaches. The main results are that: (i) the monophyly of Peritrichia cannot be rejected; (ii) the two main clades of Sessilida do not correspond to formally recognized taxa; (iii) the monophyly of genera Vorticella and Epistylis is significantly rejected; and (iv) morphological structures commonly used in peritrich taxonomy may be evolutionarily labile.

Expanded Phylogenetic Representation of Genera Opercularia and Epistylis Sheds Light on the Evolution and Higher-Level Taxonomy of Peritrich Ciliates (Ciliophora: Peritrichia)

ABSTRACT. 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.

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.

Characterization of plant growth-promoting bacteria inhabiting Vriesea gigantea Gaud. and Tillandsia aeranthos (Loiseleur) L.B. Smith (Bromeliaceae)

Microorganisms that live inside and around a plant can supply it with essential substances, such as phytohormones and essential nutrients. The present investigation aimed to isolate and characterize the phyllosphere, the endophytic, and the water tank bacteria associated with Vriesea gigantea and Tillandsia aeranthos. The bacteria were tested for siderophore and indole-3-acetic acid (IAA) production, phosphate solubilization, and presence of the nif H gene. Genetic diversity of the bacterial isolates was evaluated by rep-PCR. Sixty-eight bacterial strains were isolated from 3 different microhabitats of V. gigantea and from 2 microhabitats of T. aeranthos bromeliad plants. Gram-positive, spore-forming bacilli comprised most bacterial isolates. All isolates produced IAA in vitro in presence of very low amounts of tryptophan. More than 70% of the evaluated bacteria presented the ability of siderophore production and phosphate solubilization, and possessed the nif H gene. It was not possible to distinguish well-defined groups of isolates based on the bromeliad species and microhabitat they inhabit using genetic characterization by rep-PCR. Water tanks presented the most abundant diversity compared with phyllosphere and endophytes, probably due to the high nutrient concentration, which promotes an ideal environment for complex microbial communities.

Evolution of Variations in the Common Pattern of Stomatogenesis in Peritrich Ciliates: Evidence from a Comparative Study Including a New Description of Stomatogenesis in Pseudepistylis songi Peng et al.,

The stomatogenesis of peritrich ciliates is an important developmental process but has been studied relatively little for such a large, diverse taxon. Complex oral structures and an inability of staining techniques to reveal them clearly have been the major factors hindering investigation of this process. In the present study, an improved method of staining with protargol was used to investigate the entire process of stomatogenesis in a large, colonial species of peritrich, Pseudepistylis songi, and to compare it to descriptions of stomatogenesis in several other species. We found that P. songi and other peritrichs have the same general type of ophryobuccokinetal stomatogenesis, with the parental oral complex being inherited by one daughter and the new oral complex by the other daughter; however, some differences between individual taxa appear to have arisen in the course of evolution. Reorganization of the entire germinal kinety (Gk) to form the germinal anlage may be plesiomorphic, with restriction of reorganization to its abstomal part being apomorphic. Development of the entire new haplokinety of one daughter cell (2Hk) from the germinal band or its homologue also appears to be plesiomorphic, with development of peristomial and infundibular parts of 2Hk from separate rudiments being apomorphic. Furthermore, development of the new Gk of the parental oral complex (1Gk) from the entire infundibular part of the parental haplokinety (1Hk) may be plesiomorphic, and development from just the abstomal part may be apomorphic. Finally, development of the Gk of the new oral complex (2Gk) from residual kinetosomes of the germinal band appears to be plesiomorphic.

Morphology and Morphogenesis of a Freshwater Ciliate, Epistylis chlorelligerum Shen, 1980 (Ciliophora, Peritrichia)

ABSTRACT. An oligohalobic peritrichous ciliate, Epistylis chlorelligerum Shen, 1980, was collected from a ditch in Hangzhou, China. The morphology, oral infraciliature, and morphogenesis of the species were studied using living and protargol‐impregnated specimens. Zooids of E. chlorelligerum are 160–230 × 50–60 μm in vivo, and characterized by green‐colored endoplasm containing symbiotic algae. The oral infraciliature presents a well‐developed filamentous reticulum linked to the circular fiber of the cytostome; the outer two rows of P3 extend adstomally over P1 and usually enfold it. During binary fission, one daughter cell inherits most part of the old buccal apparatus and the reorganized haplokinety and germinal kinety (Hk′ and G′), and new buccal apparatus of the other daughter cell is mostly developed from the original germinal kinety (G) and haplokinety (Hk): new peniculi 2, 3 (2P2, 2P3), new haplokinety (2Hk), and new germinal kinety (2G) are formed from G, while the new peniculus 1 (2P1) and its peristomial extention (2Pk) originate from Hk. The epistomial membrane can be observed until the two sets of buccal apparatus begin to separate from each other.

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.

Effects of Seasonality and Dispersal on the Ciliate Community Inhabiting Bromeliad Phytotelmata in Riparian Vegetation of a Large Tropical River

This study evaluated the influence of rainfall amount on the abundance, species richness, and species occurrence and abundance distribution of the ciliate community associated with the bromeliad Aechmea distichantha. The plants were collected from a rock wall of about 10‐km long at the left bank of Paraná River. We assessed the effects of both spatial and temporal variables on the community attributes, as well as whether plants geographically closer have a similar abundance distribution and species composition. The ciliate community was substantially distinct between both hydrological periods, with greater values of species richness and abundance in the rainy period. No spatial structuring (differences in the species occurrence and abundance distribution among strata) or geographical similarity (similarity in ciliate species composition among the plants) was found. Multiple regression analysis showed a positive relationship only between the ciliate abundances and water volumes for both periods. Although few of the formulated predictions were confirmed, our study provides valuable information on the ecological aspects of the ciliate community inhabiting bromeliad phytotelmata.

Description of Epistylis riograndensis n.sp. (Ciliophora: Peritrichia) found in an artificial lake in Southern Brazil.

Epistylis riograndensis n. sp., a freshwater peritrich hosting symbiotic algae in its cytoplasm, was collected from an artificial lake, in a Botanical garden in Southern Brazil. Its detailed morphology was investigated using live and silver-stained specimens. The colonial sessile E. riograndensis has elongate zooids measuring, on average, 162 μm in length and 45 μm in width. A single contractile vacuole located near the infundibulum and a C-shaped macronucleus located transversely in the adoral half of the cell were also observed. The oral infraciliature revealed in silver-stained specimens was typical of peritrich ciliates. Three infundibular polykineties consisting of 3 rows of kinetosomes were observed. Molecular analyses of 18s rDNA placed E. riograndensis among other Epistylis species in the Order Vorticellida.

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.