Noemi Mendes Fernandes

Expanded phylogenetic analyses of the class Heterotrichea (Ciliophora, Postciliodesmatophora) using five molecular markers and morphological data.

Most studies of the molecular evolution of Heterotrichea have been based solely on the 18S-rDNA gene, which were inconsistent with morphological classification. Because of the limitations of single locus phylogenies and the recurring problem of lack of resolution of deeper nodes found in previous studies, we present hypotheses of the evolution of internal groups of the class Heterotrichea based on multi-loci analyses (18S-rDNA, 28S-rDNA, ITS1-5.8S-ITS2 region, COI and alpha-tubulin) and morphological data. Phylogenetic trees from protein coding gene data are presented for Heterotrichea for the first time. Phylogenetic analyses included Bayesian inference, maximum likelihood, maximum parsimony methods, and optimal trees were statistically compared to alternative topologies from the literature. Additionally, the Bayesian concordance approach (BCA algorithm) was used to assess the concordance factor between topologies obtained from isolated analyses. Because different loci may evolve at different rates, resulting in different gene topologies, we also estimated a species tree for Heterotrichea using the STAR coalescence-based method. The results show that: (1) single gene trees are inconsistent regarding the position of some heterotrichean families; (2) the concatenation of all data in a total-evidence tree improved the resolution of deep nodes among the heterotrichean families and genera; (3) the coalescent-based species tree is consistent with phylogenies based on the 18S-rDNA gene and shows Spirostomidae as the stem group of Heterotrichea; (4) however, the total-evidence tree suggests that the large Heterotrichea cluster is divided into nine lineages in which Peritromidae diverges at the base of the Heterotrichea tree.

Morphology and 18S rDNA gene sequence of Spirostomum minus and Spirostomum teres (Ciliophora: Heterotrichea) from Rio de Janeiro, Brazil

Species of Spirostomum Ehrenberg, 1838 are widely used as model organisms in ecological studies of environmental impacts and symbioses between ciliates and human pathogenic bacteria. However, the taxonomy of this genus is confused by the superficiality of the morphological descriptions of its included species, and the use of only a few characters for their differentiation. The present study provides details of total infraciliature, nuclear apparatus, morphometric data and 18S rDNA gene sequences of Spirostomum teres Claparede & Lachmann, 1858 and Spirostomum minus Roux, 1901, isolated from a sewage treatment plant and a freshwater lake in the city of Rio de Janeiro, Brazil, respectively. For the morphological descriptions of S. teres and S. minus, living cells were observed using bright-field and differential interference contrast (DIC) microscopy, the total infraciliature and nuclear apparatus were revealed by staining with protargol, and ciliary patterns were observed also with scanning electron microscopy (SEM). The complete sequences of the 18S rDNA of S. teres and S. minus were obtained using eukaryotic universal primers, and then compared with sequences of other species and populations of Spirostomum deposited in the GenBank database. Living S. minus measured 400-800 µm in length and 55-115 µm in width, with the following characteristics: adoral zone of membranelles approximately 112 µm long; inconspicuous paroral kinety; 30-40 kineties in somatic ciliature; moniliform macronucleus with 9-25 nodes, approximately 12 micronuclei; single and posterior contractile vacuole; and yellow-brown cytoplasm. Living and fully extended S. teres measured approximately 250 µm in length and 65 im in width, with the following characteristics: adoral zone of membranelles approximately 92 µm long; approximately 30 somatic kineties; compact macronucleus, approximately five micronuclei; macronuclear groove present; single and posterior contractile vacuole; and colorless cytoplasm. Evidence from 18S rDNA sequences confirms the identification of S. teres and suggests the existence of cryptic species closely related to S. minus. The use of silver impregnation technique (protargol) allowed the observation and description of a greater number of characters in S. minus and S. teres, thus assisting the research that require identification of these species.

Description and Phylogeny of Urostyla grandis wiackowskii subsp. nov. (Ciliophora, Hypotricha) from an Estuarine Mangrove in Brazil

Interphase specimens, aspects of physiological reorganization and divisional morphogenesis were investigated in a strain of a hypotrichous ciliate highly similar to Urostyla grandis Ehrenberg, (type species of Urostyla), collected from a mangrove area in the estuary of the Paraíba do Sul river (Rio de Janeiro, Brazil). The results revealed that albeit interphase specimens match with the known morphologic variability in U. grandis, morphogenetic processes have conspicuous differences. Parental adoral zone is entirely renewed during morphogenesis, and marginal cirri exhibit a unique combination of developmental modes, in which left marginal rows originate from multiple anlagen arising from innermost left marginal cirral row, whereas right marginal ciliature originates from individual within‐row anlagen. Based on such characteristics, a new subspecies, namely U. grandis wiackowskii subsp. nov. is proposed, and consequently, U. grandis grandis Ehrenberg, stat. nov. is established. Bayesian and maximum‐likelihood analyses of the 18S rDNA unambiguously placed U. grandis wiackowskii as adelphotaxon of a cluster formed by other U. grandis sequences. The implications of such findings to the systematics of Urostyla are discussed.

Morphology and 18S rDNA gene sequence of Blepharisma sinuosum Sawaya, 1940 (Ciliophora: Heterotrichea) from Brazil

The morphology and morphometric data of seven populations of Blepharisma sinuosum from southeastern Brazil were investigated. The description is based on live observations, protargol impregnation, and scanning electron microscopy. Blepharisma sinuosum measures 75-255μm in length and 25-93μm in width and has a spindle-shaped body, pink color, a single contractile vacuole located at the posterior end, 50 adoral membranelles, a conspicuous paroral, 17-35 somatic kineties, a moniliform macronucleus with 2-7 connected nodules, and 3-20 micronuclei. Morphological comparisons with similar species were performed and suggest that B. americanum is the junior synonym of B. sinuosum. The 18S rDNA gene sequence of B. sinuosum was obtained and compared with that of other Blepharisma species. The length and GC content of the obtained sequence is 1652bp and 47.03%, respectively, and has a very high structural similarity (99.9%) with the B. undulans sequence. The validity of the classification of Blepharisma species in morphonuclear subgenera is also discussed.

Quantitative study of Trichodina heterodentata (Ciliophora: Mobilia) infrapopulations infesting tadpoles of a Brazilian endemic toad Rhinella pombali (Anura: Bufonidae)

In this work we investigated the quantitative aspects of the relationship between Rhinella pombali (Baldissera, Caramaschi & Haddad, 2004) tadpoles and their trichodinid ectoparasites. Specimens were collected from a small stream located at an agricultural area between rainforest fragments in southeastern Brazil. A total of 80 tadpoles were investigated. Trichodina heterodentata Duncan, 1977 was the only trichodinid ciliate species recovered. All tadpoles were naturally infested by T. heterodentata with a mean intensity of 695.14 ± 33.33 trichodinid ciliates per tadpole. A positive correlation was observed between the intensity of the infestation and tadpole weight. Intensity of infestation did not significantly influence mean trichodinid diameter in the populations studied. This is the first study investigating density-dependent effects in interspecific interactions between trichodinids and anurans.

Redescription and Phylogenetic Position of Condylostoma arenarium Spiegel, 1926 (Ciliophora, Heterotrichea) from Guanabara Bay, Brazil

Details on Condylostoma arenarium infraciliature have not been described; therefore, it is considered a poorly known species. The lack of detailed description on C. arenarium morphology caused several misidentifications that have accumulated in the literature. In this study, we present the first complete description of C. arenarium infraciliature based on protargol‐impregnated organisms and scanning electron microscopy. We also have inferred the phylogenetic position of this species based on 18S rRNA sequences. The main characteristics of C. arenarium population from Guanabara Bay are as follows: in vivo elongated body shape with 350–600 μm length × 70–220 μm width, they are highly contractile when subjected to disturbances, green‐yellowish cortical granules are present, contractile vacuoles absent, V‐shaped peristome comprises approximately 1/5 of the total length, adoral zone with 83–145 membranelles, 1–2 small frontal cirrus observed only in impregnated specimens, 10–15 fiber‐like stripes arranged transversely on the inner wall of the oral cavity, 30–45 somatic kineties, moniliform macronucleus with 15–20 nodules. Some observations on morphogenesis of C. arenarium were also included. In phylogenetic analyses, C. arenarium clustered with Condylostoma sp. within a clade composed of three C. curva sequences with high support values.

Morphology and Phylogenetic Position of an Unusual Stentor polymorphus (Ciliophora: Heterotrichea) Without Symbiotic Algae

We investigated the live morphology, infraciliature, and small subunit rRNA gene sequences of an unusual population of Stentor polymorphus without symbiotic algae that was isolated from the southeastern region of Brazil. The morphological and molecular data confirmed the identity of this strain as S. polymorphus. The Brazilian S. polymorphus organism is 850–2,000 μm in length in vivo and has colorless cortical granules, a moniliform macronucleus with 6–12 nodules, somatic ciliature composed of 50–60 kineties, a single contractile vacuole located to the left of the cytostome, and a conspicuous oral pouch, and it does not build a lorica. Based on the phylogenetic analyses, the Brazilian S. polymorphus was located within a cluster consisting of four other S. polymorphus sequences, with high support values using both the Bayesian inference and maximum likelihood algorithms. Our study presents the first report of a S. polymorphus population without its symbionts under natural conditions. On the basis of our findings, we propose that the presence or absence of symbiotic algae should not be used as a taxonomic character for the identification of Stentor species.

Resting cysts of the pigmented ciliate blepharisma sinuosum sawaya, 1940 (ciliophora: heterotrichea)

The morphology of Blepharisma sinuosum resting cysts and the dynamics of pigmentation at different stages of encystment are presented for the first time. Cyst morphometrics are similar to other Blepharisma species, with three‐wall layers, bacteria surrounding the ectocyst, a conical plug, and wrinkly surface toward the plug in mature stages. The vegetative moniliform macronucleus changes to a horseshoe shape, and at early stages, the cystic cytoplasm is homogeneously pigmented, comprising a contractile vacuole; later, pigments polarize toward the plug, decorate the cortical layer, and become brownish. This work reinforces the potential role of pigment dynamics on cyst biology.

Description and phylogenetic position of Corlissina maricaensis gen. nov., sp. nov. (Karyorelictea, Geleiidae), a novel interstitial ciliate from Brazil, with redefinition of the family Geleiidae

Corlissina maricaensis gen. nov., sp. nov. was obtained from samples of sediment collected in a brackish lagoon of Maricá city, Rio de Janeiro state, Brazil. The morphological description was based on live observations, after protargol staining, and scanning electron microscopy. The novel species has a cylindrical body shape that is slightly contractile, 230-550 × 35-65 μm, a cytoplasm with many globular inclusions, one row of irregular cortical granules between each somatic kinety, approximately 40-62 somatic kineties, two globular macronuclei measuring 9-24 μm and one micronucleus of approximately 4-9 μm. A subapical oral cavity was approximately 20-80 × 9-25 μm, with an adoral zone on the left side of the buccal field, which was composed of 32-60 polykineties and a paroral at the right side that was composed of 40-57 short polykineties. The new genus is distinguished from other geleiids by a loop-shaped posterior end of the paroral ciliature, made up of two rows of short polykineties, and the oralization of the central superior kinety (K0i), forming a row of dikinetids that borders the adoral zone internally, followed by several rows of monokinetids. In the phylogenetic analyses, the novel species was recovered as the sister group of Parduczia orbis with full support values based on 18S rRNA gene sequences. This work also indicates some problems in the definitions of the Geleiidae and proposes a new diagnosis for this karyorelictid family.

Ontogeny and Molecular Phylogeny of Apoamphisiella vernalis Reveal Unclear Separation between Genera Apoamphisiella and Paraurostyla (Protozoa, Ciliophora, Hypotricha).

Morphology and divisional morphogenesis of the hypotrich ciliate Apoamphisiella vernalis are investigated based on two populations from Brazil. Typical specimens of A. vernalis replicates its ventral ciliature from six fronto-ventral-transverse (FVT) anlagen independently formed for proter and opisthe, plus one or more short anlagen located between IV and V, which form surplus transverse cirri. Dorsal morphogenesis occurs as in typical oxytrichid dorsomarginalians, viz., with formation of three anlagen and fragmentation of the rightmost one. Dorsomarginal kineties are formed near anterior end of right marginal cirral row anlagen. Various anomalous specimens exhibiting more than two long ventral rows were found, which are explained by increasing the number of FVT anlagen and/or the number of cirri produced by anlagen. Comparative ontogeny and phylogenetic analyses based on the 18S rDNA reveal that Apoamphisiella vernalis is closely affine to North American and European strains of the Paraurostyla weissei complex. Their reduced genetic distances and conspicuous morphological variability show that both genera can overlap, which implies the necessity of re-evaluating the contextual relevance of some morphological characters commonly used for genus-level separation within hypotrich taxa.