Investigating non-fungal eukaryotic diversity in snow in the Antarctic Peninsula region using DNA metabarcoding.

Snow is a unique microhabitat, despite being a harsh environment, multiple life forms have adapted to survive in it. While algae, bacteria and fungi are dominant microorganisms in Antarctic snow, little is known about other organisms that may be present in this habitat. We used metabarcoding to investigate DNA sequence diversity of non-fungal eukaryotes present in snow obtained from six different sites across the Maritime Antarctica. A total of 20 taxa were assigned to obtained sequences, representing five Kingdoms (Chromista, Protozoa, Viridiplantae and Metazoa) and four phyla (Ciliophora, Cercozoa, Chlorophyta and Cnidaria). The highest diversity indices were detected in Trinity Peninsula followed by Robert Island, Arctowski Peninsula, Deception Island, King George Island and Snow Island. The most abundant assignments were to Trebouxiophyceae, followed by Chlamydomonas nivalis and Chlamidomonadales. No taxa were detected at all sites. Three potentially new records for Antarctica were detected: two Ciliophora (Aspidisca magna and Stokesia sp.) and the green algae Trebouxia potteri. Our data suggested that similarities found between the sites may be more related with snow physicochemical properties rather than geographic proximity or latitude. This study provides new insights into the diversity and distribution of eukaryotic organisms in Antarctic snow.

A putatively extinct higher taxon of Spirotrichea (Ciliophora) from the Lower Cretaceous of Brazil.

Fossil microeukaryotes are key elements for understanding ancient ecosystems at microscopic level and improving the knowledge on the diversification of microbial life as a whole. We describe Palaeohypothrix bahiensis gen. et sp. nov., an exceptionally well-preserved Lower Cretaceous (Berriasian-Barremian; 145-125 Mya) amber-entrapped microeukaryote, identified as a spirotrich ciliate. The preservation of structures interpreted as the nuclear apparatus and remains of the ciliature revealed a novel ground plan, not found in modern Spirotrichea, thus representing a putatively extinct higher taxon lineage, viz. the Palaeohypotricha nov. tax. Based on cladistic analysis, the new taxon is hypothesized as phylogenetically related to the Protohypotrichia.

Incorporating mitogenome sequencing into integrative taxonomy: The multidisciplinary redescription of the ciliate Thuricola similis (Peritrichia, Vaginicolidae) provides new insights into the evolutionary relationships among Oligohymenophorea subclasses.

The evolutionary relationships among Oligohymenophorea subclasses are under debate as the phylogenomic analysis using a large dataset of nuclear coding genes is significantly different to the 18S rDNA phylogeny, and it is unfortunately not stable within and across different published studies. In addition to nuclear genes, the faster-evolving mitochondrial genes have also shown the ability to solve phylogenetic problems in many ciliated taxa. However, due to the paucity of mitochondrial data, the corresponding work is scarce, let alone the phylogenomic analysis based on mitochondrial gene dataset. In this work, we presented the characterization on Thuricola similis Bock, 1963, a loricate peritrich (Oligohymenophorea), incorporating mitogenome sequencing into integrative taxonomy. As the first mitogenome for the subclass Peritrichia, it is linear, 38,802 bp long, and contains two rRNAs, 12 tRNAs, and 43 open reading frames (ORFs). As a peculiarity, it includes a central repeated region composed of tandemly repeated A-T rich units working as a bi-transcriptional start. Moreover, taking this opportunity, the phylogenomic analyses based on a set of mitochondrial genes were also performed, revealing that T. similis, as a representative of Peritrichia subclass, branches basally to other three Oligohymenophorea subclasses, namely Hymenostomatia, Peniculia, and Scuticociliatia. Evolutionary relationships among those Oligohymenophorea subclasses were discussed, also in the light of recent phylogenomic reconstructions based on a set of nuclear genes. Besides, as a little-known species, T. similis was also redescribed and neotypified based on data from two populations collected from wastewater treatment plants (WWTPs) in Brazil and Italy, by means of integrative methods (i.e., living observation, silver staining methods, scanning and transmission electron microscopy, and 18S rDNA phylogeny). After emended diagnosis, it is characterized by: (1) the sewage habitat; (2) the lorica with a single valve and small undulations; (3) the 7-22 µm-long inner stalk; and (4) the presence of only a single postciliary microtubule on the left side of the aciliferous row in the haplokinety. Among Vaginicolidae family, our 18S rRNA gene-based phylogenetic analysis revealed that Thuricola and Cothurnia are monophyletic genera, and Vaginicola could be a polyphyletic genus.

Systematic Redefinition of the Hypotricha (Alveolata, Ciliophora) Based on Combined Analyses of Morphological and Molecular Characters.

The systematics of Hypotricha is one of the most puzzling problems in ciliate biology, having spanned numerous conflicting hypotheses with unstable relationships at various levels in molecular trees, for which the constant addition of newly discovered species has only increased the confusion. The hypotrichs comprise a remarkable morphologically diversified group of ciliates, and the phylogenetic potential of morphological traits is generally recognized. However, such characters were rarely used in phylogenetic reconstructions, and congruence with molecular data never assessed from simultaneous analyses. To properly reconciliate morphological and molecular information, maximum-likelihood and parsimony analyses of 79 morphological characters and 18S rDNA sequences were performed for 130 ingroup terminals, broadly sampled to represent the known hypotrich diversity. As result, well-supported and relatively stable clades were recovered, based on which the redefined Hypotricha comprises at least six higher taxa: The "arcuseriids", Holostichida, Parabirojimida, and the "amphisiellids", plus the two large clades Kentrurostylida nov. tax. (Hispidotergida nov. tax. and Simplicitergida nov. tax.) and Diatirostomata nov. tax. ("bistichellids", "kahliellids", Gonostomatida and Dorsomarginalia [Postoralida nov. tax. and Uroleptida]). Each taxon was circumscribed by synapomorphies, of which most were homoplastic, as the natural history of hypotrichs is portrayed by an outstanding quantity of convergences and reversions.

A new oligotrich (Ciliophora, Oligotrichia) from Argentina, with redefinition of Novistrombidium Song and Bradbury.

A new oligotrich similar to Novistrombidium was discovered in plankton samples from an artificial tributary of the Salado River, Buenos Aires province, Argentina, in summer 2010. Propecingulum fistoleramalliei sp. n. has an obovate and anteriorly truncated body, with a conspicuous ventral furrow, is flattened ventrally, and has a prominent right apical protrusion. It temporarily attaches to the substratum by a posterior mucous thread. Rod-shaped extrusomes arranged equidistantly and insert directly above the girdle kinety. The macronucleus is globular to ellipsoidal. The contractile vacuole is located in the left, anterior quarter of the cell and the adoral zone is composed of 30-35 collar, 9-14 buccal, and two thigmotactic membranelles. The girdle kinety is dextrally spiraled and ventrally open; the ventral kinety is posterior to anterior end of the girdle kinety. The oral primordium develops posterior to the right thigmotactic membranelle and anterior the stripe of extrusomes above left, lateral portion of the girdle kinety. The SSUrDNA phylogeny confirms one more time that Novistrombidium is not monophyletic; consequently, we elevate the subgenus Propecingulum up to genus rank and redefine the genus Novistrombidium.

Discomorphella pedroeneasi sp. nov. (Ciliophora, Odontostomatida): An anaerobic ciliate hosting multiple cytoplasmic and macronuclear endocytobionts.

Odontostomatids are among the less studied representatives of the Ciliophora. They are anaerobic microeukaryotes usually occurring as rare species in sapropelic environments. Here we describe a novel species of Discomorphella, named Discomorphella pedroeneasi sp. nov., using light and electron microscopy observations. Discomorphella pedroeneasi displays many complex morphological features, for which new terms are introduced, such as the auricules, epistomial fringe spacer, frontal awning, odontostomatid ciliary sockets, oral lips and ventral flap. Remarkably, D. pedroeneasi has at least six types of endocytobionts, including intramacronuclear ones, and organelles ultrastructurally similar to the M/H bodies of the odontostomatid Saprodinium dentatum. Based on the new data and revision of the literature, we propose two new diagnostic characters for species separation within Discomorphella: the fringe spacer ratio and the posterior fringe ratio. The taxonomy of Discomorphella is revised and an identification key is provided.

Fine Structure and Molecular Phylogeny of Parametopidium circumlabens (Ciliophora: Armophorea), Endocommensal of Sea Urchins.

Metopid armophoreans are ciliates commonly found in anaerobic environments worldwide; however, very little is known of their fine structure. In this study, the metopid Parametopidium circumlabens (Biggar and Wenrich 1932) Aescht, 1980, a common endocommensal of sea urchins, is investigated for the first time with emphasis on transmission electron microscopy, revealing several previously unknown elements of its morphology. Somatic dikinetids of P. circumlabens have a typical ribbon of transverse microtubules, an isolated microtubule near triplets 4 and 5 of the anterior kinetosome, plus two other microtubules between anterior and posterior kinetosomes, a short kinetodesmal striated fiber and long postciliary microtubules. In the dikinetids of the perizonal stripe, the kinetodesmal fiber is very pronounced, and there is a conspicuous microfibrillar network system associated with the kinetosomes. A new structure, shaped as a dense, roughly cylindrical mass surrounded by microtubules, is found associated with the posterior kinetosome of perizonal dikinetids. The paroral membrane is diplostichomonad and the adoral membranelles are of the "paramembranelle" type. Bayesian inference and maximum-likelihood analysis of the 18S-rDNA gene unambiguously placed P. circumlabens as sister group of the cluster formed by ((Atopospira galeata, Atopospira violacea) Metopus laminarius) + Clevelandellida, corroborating its classification within the Metopida.

Phylogenetic study of Class Armophorea (Alveolata, Ciliophora) based on 18S-rDNA data.

The 18S rDNA phylogeny of Class Armophorea, a group of anaerobic ciliates, is proposed based on an analysis of 44 sequences (out of 195) retrieved from the NCBI/GenBank database. Emphasis was placed on the use of two nucleotide alignment criteria that involved variation in the gap-opening and gap-extension parameters and the use of rRNA secondary structure to orientate multiple-alignment. A sensitivity analysis of 76 data sets was run to assess the effect of variations in indel parameters on tree topologies. Bayesian inference, maximum likelihood and maximum parsimony phylogenetic analyses were used to explore how different analytic frameworks influenced the resulting hypotheses. A sensitivity analysis revealed that the relationships among higher taxa of the Intramacronucleata were dependent upon how indels were determined during multiple-alignment of nucleotides. The phylogenetic analyses rejected the monophyly of the Armophorea most of the time and consistently indicated that the Metopidae and Nyctotheridae were related to the Litostomatea. There was no consensus on the placement of the Caenomorphidae, which could be a sister group of the Metopidae + Nyctorheridae, or could have diverged at the base of the Spirotrichea branch or the Intramacronucleata tree.

Redescription of Licnophora chattoni Villeneuve-Brachon, 1939 (Ciliophora, Spirotrichea), associated with Zyzzyzus warreni Calder, 1988 (Cnidaria, Hydrozoa).

Licnophora chattoni, found in association with Zyzzyzus warreni, a tubulariid hydroid epizoic in sponges from São Sebastião (SP, Brazil), is redescribed and illustrated using light and electron microscopy. The ciliate has a flexible, transparent body formed by an oval anterior region linked to the posterior basal disc via a flexible neck region. Numerous cortical granules are observed scattered throughout the body and densely packed along the neck. The adoral zone is formed by about 81 external and 24 infundibular paramembranelles. The paroral membrane, formed by a row of long cilia arranged in monokinetids, extends through a groove in the body to the adhesive disc. Two dorsal kinetids are present along the right body margin and around the neck. The adhesive disc (18µm in diameter) lacks cilia in the area above the velum. The velum covers a row of dikinetids bearing long cilia and four dikineties, two or three of which are interrupted on the ventral surface. Nine to twelve macronuclear nodules connected by isthmuses are distributed in the cytoplasm, plus two nodules located in the adhesive disc and between those there is an ovate micronucleus.

Description of a mirror-image doublet from a Brazilian strain of Gastrostyla setifera (Engelmann, 1862) Kent, 1882 (Ciliophora: Stichotrichia).

The present work describes a mirror-image doublet morph found in a Brazilian strain of the stichotrich ciliate Gastrostyla setifera (Engelmann, 1862) Kent, 1882, obtained from a sample of water and sediment from an urban freshwater stream in the city of Juiz de Fora (Minas Gerais, Brazil). The doublet measured 63 x 49 microm, and had two components where the right one has upside-down arranged adoral membranelles with a set of small oblique membranelles at its proximal (posterior) end. The fronto-ventral-transverse ciliature comprised 34 cirri, mostly related to the left component, as well as two marginal rows. On the dorsal surface there were eight dorsal kineties and six caudal cirri. Dorsomarginal kineties were lacking. The macronucleus comprised one irregular-shaped transverse nodule and two smaller roughly ellipsoid longitudinal nodules.