Description and Phylogeny of Tetrakeronopsis silvanetoi gen. nov., sp. nov. (Hypotricha, Pseudokeronopsidae), a New Benthic Marine Ciliate from Brazil

Pseudokeronopsidae Borror & Wicklow, 1983 are biotechnologically important ciliate protists which produce toxic defense substances; however, their diversity is still little known in Brazil. In the present study, Tetrakeronopsis silvanetoi, a new genus and species of marine pseudokeronopsid hypotrichs is described from samples of water with bottom sediment collected from the coast of São Paulo state. Its phylogenetic affinities to the “core urostyloids” are hypothesized based on analyses of the 18S-rDNA marker, and a new subfamily, the Nothoholostichinae subfam. nov., is erected to name the monophylum composed of pseudokeronopsids in which the anterior corona is usually formed by four frontal cirri. In addition, the new combination Monocoronella longissima comb. nov. is proposed for Nothoholosticha longissima (Dragesco & Dragesco-Kernéis, 1986) Li et al., 2009.


Introduction
Hypotrichs s. l. [1] are ciliates which generally exhibit a dorsoventrally flattened body with a polyhymenophore adoral zone and somatic cilia arranged in cirri along the ventral side and rows of dikinetids on the dorsal side of the body [2][3][4]. Their body architecture makes them pre-adapted to life in micropore habitats [5] and, like most ciliates, hypotrichs occur in edaphic, freshwater and marine sediments worldwide [6]. In marine environments, hypotrichs are present mostly as free-living microbenthic forms, which are omnivorous and compete for food with similar sized interstitial metazoa and other protists [7,8].
Among hypotrichs, representatives of the Pseudokeronopsidae are known to produce toxic substances, the keronopsins, which are used as chemical defense [9][10][11][12]. Hence, pseudokeronopsids offer biotechnological potential for drug discovery related studies, which makes expanding knowledge of their diversity highly desirable.
In Brazil, the most relevant studies on the diversity of benthic marine ciliates were those conducted by Kattar [13], who found 75 species along the coast of the states of São Paulo, Guanabara (nowdays part of Rio de Janeiro), Espírito Santo and Pernambuco; and by Wanick & Silva-Neto [14], who reported 32 species from Sepetiba bay, Rio de Janeiro. However, the diversity of benthic marine ciliates remains unknown for the majority of the Brazilian coast [15], and this includes the diversity of marine pseudokeronopsids.
The present study expands the knowledge on diversity of marine pseudokeronopsids from Brazil with the description of a new genus and species, namely Tetrakeronopsis silvanetoi gen. nov., sp. nov., found in environmental samples from the coast of São Paulo, Brazil. The systematics of the new organism is discussed based on comparative morphology and phylogenetic analyses of the 18S-rDNA marker, and a new subfamily, Nothoholostichinae subfam. nov., is proposed.

Morphology
Samples with water and bottom sediment were collected from Martim de Sá beach, in Caraguatatuba, a city on the coast of São Paulo state, Brazil, in May of 2006. The sampling location is a public beach, thus no specific permissions were required to collect the material necessary for the present study. No known endangered or protected species were involved in the present study.
The samples were brought to the laboratory, where aliquots were examined in Petri dishes under the stereomicroscope. For identification and description, the ciliates were further analyzed in vivo under bright field and DIC at 1006, 2006 and 1,0006 magnifications, and after protargol-impregnation and scanning electron microscopy, prepared according to Dieckmann [16] and Silva-Neto et al. [17], respectively. Measurements in Table 1 were taken from protargol-impregnated specimens at 1,0006 magnification, and descriptive statistics therein were calculated with the computer program GraphPad Prism 4 [18]. All measurements are in mm. Diagrams illustrating the ciliature pattern are schematic representations drawn with Adobe PhotoShop CS4, and were based on photographs of various protargol-impregnated specimens. The terminology adopted in the present study follows mostly Berger [2,19], and the classification shown in the phylogenetic tree is basically according to Chen et al. [20], except for Anteholosticha spp. (see discussion below).

Phylogenetic Analyses
To assure correct identification of the ciliates used in the molecular analyses, clonal cultures were made from single specimens picked from ordinary cultures and transferred to Petri dishes with boiled sea water and crushed rice grains. Specimens were then isolated for DNA extraction and amplification of the 18S marker following Paiva et al. [21]. The obtained sequence was added to a data matrix containing 54 other sequences representing the ''core urostyloids'' [22][23][24], which is the largest monophylum of urostyloids recovered in molecular phylogenies, but also the group in which pseudokeronopsids are included, e.g. [20][21][22][23][24]26]. Seven additional sequences of representatives of Discocephalida Wicklow, 1982, sensu [27] were included as an outgroup [25][26][27].  The sequences were aligned based on their eukaryotic 18S-rRNA secondary structure, using the SINA web aligner (http://www. arb-silva.de/aligner) [28] with its default settings. Next, the nucleotide matrix was inspected in the computer program BioEdit v7.0.6 [29] and their alignment refined by eye, considering the structural similarity among sequences. Genetic distances ( Table 2) were calculated with the program MEGA 5 [30], using pairwise deletion as treatment for gaps and missing data. The nucleotide sequence of Tetrakeronopsis silvanetoi obtained in the present study was deposited in NCBI/GenBank (access code: KF730314). Bayesian inference (BI) and maximum likelihood (ML) analyses were performed to hypothesize the phylogenetic affinities of T. silvanetoi within the studied taxa sample. Both analyses employed the TrN+I ( = 0.4687)+C ( = 0.4828) nucleotide substitution model, selected via the Akaike information criterion (AIC) [31,32] in MODELTEST 3.7 [33]. The BI was performed with the program MrBayes 3.2.1 implemented in the CIPRES Science Gateway (http://www.phylo.org) [34]. It was based on two independent Markov Chain Monte Carlo (MCMC) simulations run with four chains of 1,000,000 generations, and trees sampled each 200 (temperature of heat chains = 0.2). The first 100,000 generations were discarded as burn-in. For ML, the sequences were analyzed with the program PhyML 3.0 [35] using an initial BioNJ topology, and improving its likelihood via subtree pruning and regrafting (SPR) branch-swapping moves to achieve the ML tree. Node stability in BI was assessed via posterior probabilities calculated from a 50% majority-rule consensus of the trees kept after burn-in, and in ML via 1,000 bootstrap pseudoreplicates. The trees were rooted a posteriori according to outgroup position [36].
In the present study, we did not assess the phylogeny of the Urostyloidea Bütschli, 1889 as a whole, which are generally hypothesized as non-monophyletic, e.g. [21,24,26,[37][38][39], but rather focused our analyses on the core urostyloids.

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix ''http://zoobank.org/''. The LSID for this publication is: urn:lsid:zoobank.org:pub: E5464A1D-5EA0-45A3-8D1E-26230E691A8C. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Diagnosis
Pseudokeronopsidae sensu Chen et al. [20] with an atypical bicorona in which the anterior corona is usually formed by four frontal cirri. Table 2. Distance matrix of 18S-rDNA sequences of representatives of the Pseudokeronopsidae.  (Table 3).

Etymology
Greek, composite of prefix tetra-(four) and Keronopsis Penard, 1922. Named after the number of cirri composing the anterior corona; feminine.

Diagnosis
Nothoholostichinae with two frontoterminal cirri; buccal, pretransverse and transverse cirri present; midventral complex formed by cirral pairs only; one left and one right marginal cirral row; caudal cirri absent.

Species Included
Up to the present time, only the type species T. silvanetoi is assigned to Tetrakeronopsis.

Etymology
T. da S. Paiva proposed the epithet ''silvanetoi'' in dedication to his former doctoral advisor Prof. Dr. Inácio Domingos da Silva Neto, who collected the environmental samples in which this new species was found.

Diagnosis
Tetrakeronopsis measuring , 280640 mm in vivo (N = 15); body slender, flexible and acontractile, of pale yellow coloration under the stereomicroscope, exhibiting a conspicuous longitudinal ventral groove; rusty colored cortical granules present but scarce; cytoplasm with many globular and ring-shaped inclusions. Adoral zone with crown and lapel membranelles separated by a gap; right portion of lapel membranelles separated by a gap; on average 14 membranelles in the crown and 43 in the lapel; two parabuccal and one (rarely two) buccal cirri; midventral complex formed by 37 cirral pairs; 70 left and 73 right marginal cirri; two pretransverse and 5 transverse cirri; three dorsal kineties. Nuclear apparatus with, on average, 38 macronuclear nodules and five micronuclei.

Deposition of Type-specimens
Type slides (protargol-impregnation) of T. silvanetoi were deposited in the collection of Laboratório de Protistologia, Dept. de Zoologia, Inst. de Biologia, Universidade Federal do Rio de Janeiro, under the accession codes IBZ-UFRJ0014-3-holotype (marked with ink on the slide) and various paratypes; and IBZ-UFRJ0014-4-paratypes.
Frontal ciliature arranged in an atypical bicorona invariably formed by four frontal and two parabuccal cirri, each , 13 mm  Midventral pairs slightly oblique above body equator, displaying a typical urostyloid zig-zag pattern; below the equatorial region, midventral pairs become less oblique, almost aligned as a straight row. Pairs located close to transverse cirri are more spaced than in the rest of the complex. Cirri of midventral complex , 12 mm long, with the right cirrus of each pair slightly thicker than the left one ( Figure 2C). Two thin pretransverse and an almost longitudinal set of 4-6 about 17 mm long transverse cirri in the posterior region of the body.
Marginal ciliature composed of one left and one right cirral rows bearing , 11-12 mm long cilia. Left marginal row with 55-98 cirri, beginning dorsally, at 20-30 mm from anterior end of body, shifting to ventral side and running along left margin, terminating close to posterior end of body; right marginal row with 54-90 cirri, beginning at , 16-23 mm away from anterior end of Dorsal ciliature composed of three rows of , 3 mm long bristles; middle row always beginning slightly anteriorly in relation to the other two; all three rows terminate at about the same level, on average 8.3 mm (N = 6) from posterior end of body; caudal cirri lacking (Figures 1D; 2E, G).
Morphogenesis. Only one very early divider, two late dividers, and some middle-stage reorganizers were present in the studied slides, from which some aspects of morphogenesis were unveiled (Figures 3C-G). The adoral zone of the proter is fully renewed and develops within the parental one. Gaps in adoral membranelles of lapel are likely formed in very late dividers or after cytokinesis. Ventral primordia of the proter develop without participation from cirri of rear corona; however, buccal cirrus very likely participates in the process. Midventral complex is formed as usual for pseudokeronopsid urostyloids, that is, from many short ventral primordia that each produces a cirral pair; the rightmost and second rightmost primordia each produces a pretransverse cirrus; transverse cirri are produced by the rightmost four to six primordia. The two frontoterminal cirri originate, as usual, from the rightmost ventral primordium. New marginal cirral rows and dorsal kineties originate from within parental structures. The parental ciliature is completely reabsorbed after cytokinesis. Macronuclear nodules become dumbbell shaped in very early dividers, each exhibiting a replication band. Unfortunately, it was not possible to determine if nodules fuse in a single mass prior to division or divide individually. Those four terminals consisted of a strongly supported cluster which was an adelphotaxon of the Pseudokeronopsis spp.+Uroleptopsis citrina Kahl, 1932 group; thus, the Pseudokeronopsidae were always dichotomized, with one branch containing the species in which the anterior portion of the bicorona is formed by four frontal cirri, and the other branch containing those with a typical bicorona, namely the Nothoholostichinae and the Pseudokeronopsinae, respectively (Figure 4). Inspection of the nucleotide matrix revealed positions with putative molecular synapomorphies of the Nothoholostichinae that were unique within the analyzed sequences according to the 18S rRNA secondary structure: 529 G; 531 A; 767 C; 816 T; 1537 A, shown in Figure 5.

Molecular Phylogeny of
The p-distance between the 18S fragment of T. silvanetoi and that of H. pulchra was 0.026, while between T. silvanetoi and N. fasciola, but also Apoholosticha sinica, it was 0.025. Within-group mean pdistances were 0.022 and 0.014 for the Nothoholostichinae and Pseudokeronopsinae, respectively, and the mean p-distance between both clusters was 0.046 (Table 2).
In comparison with Nothoholosticha fasciola, T. silvanetoi differs in the presence of two frontoterminal cirri (vs. lacking in N. fasciola); however, unlike in H. pulchra, the cirri that are homologous to the frontoterminal ones are not generated in N. fasciola [41]. In their paper, Li et al. [41] transferred Anteholosticha longissima (Dragesco & Dragesco-Kernéis, 1986) Berger, 2006 to Nothoholosticha due to its supposed lack of frontoterminal cirri. According to Berger [2], the lack of such cirri in the original description by Dragesco & Dragesco-Kernéis [42] is possibly due to confusion with the right marginal row, which is indicated by the far anteriorly extending row [2]. Additionally, the frontal cirri are five in number and organized in a single corona, resembling Monocoronella Chen et al., 2011b. Because of such, we transfer Nothoholosticha longissima to that genus as M. longissima comb. nov. This new combination is likely provisory since M. longissima apparently lacks buccal cirri, while all congeners display at least one [43]. It is worthy of note that a redescription of this organism, as suggested by Berger [2], is necessary to verify the absence of the buccal cirrus and elucidate other features, such as the dorsal ciliature pattern.

Phylogeny
The phylogenetic trees obtained in the present study are generally in agreement with the literature concerning the internal kinships of the core urostyloids [20,23,24,44], including their families and the paraphyly of pseudourostylids, which is recurrent in the literature [22,23,40]. In addition, the spreading of Anteholosticha spp. among various clusters also corroborates previous phylogenetic studies on urostyloids [23,24,26,27,45]. The heterogeneity of the genus Anteholosticha was already pointed out by Berger [2,46]. Huang et al. [24] [24]), which consistently groups outside the core urostyloids in molecular trees [24,26]. Hence, Anteholosticha will likely be split into more genera in the future.
Moreover, the data supporting the phylogenetic patterns herein recovered, as expressed by posterior probabilities and bootstrap values, is also consistent with the literature, since the 18S core urostyloid clusters are hypothesized mostly with strong data support among the hypotrichs phylogeny [21,23].
The dichotomy separating the pseudokeronopsids in which the anterior corona is formed by four frontal cirri and those with a typical bicorona has been progressively shown in the literature, as further representatives of the former were discovered and subjected to phylogenetic analyses [20,23,40,41]. The inclusion of T. silvanetoi further corroborated this emerging pattern, consequently leading to the naming of a natural group, the Nothoholostichinae. Foundation for the Erection of Nothoholostichinae Subfam. nov. and Tetrakeronopsis gen. nov In their paper, Borror & Wicklow [47] included the Thigmokeronopsinae Wicklow, 1981 in the Pseudokeronopsidae, thus subdividing it into two subfamilies. Molecular data has suggested the former to be a monophyletic taxon that belongs to the Urostylidae instead of Pseudokeronopsidae, as recently proposed by Huang et al. [24]. Based on molecular phylogenetic analyses [20,23,24,40], the Pseudokeronopsidae were consistently split into two natural groups -the Pseudokeronopsinae Borror & Wicklow, 1983 and the newly erected Nothoholostichinae. The phylogenetic pattern recovered in the present paper suggests the typical bicorona of the Pseudokeronopsinae to be a plesiomorphic feature, inherited from a pseudourostylid-like last common ancestor. Hence, the peculiar composition of the anterior corona in Apoholosticha, Heterokeronopsis, Nothoholosticha and Tetrakeronopsis is herein regarded as a putative synapomorphy of the Nothoholostichinae within the Pseudokeronopsidae, and a feature that likely evolved by a reduction in the number of frontal primordia. Curiously, the morphometric data from Heterokeronopsis pulchra, shown in [40], indicate that occasional specimens may have five frontal cirri. Hence, some slight variations in the four-cirri pattern are expected to occur in the atypical bicorona of Nothoholostichinae.
Additional features which may be of taxonomic relevance for diagnosing the Nothoholostichinae are the presence of a longitudinal groove left of midventral complex and the split of adoral zone by a small gap (which also occurs in Uroleptopsis Kahl, 1932). Contractile vacuoles, when present, are generally located behind the equatorial level of body, and cytoplasm contains numerous inclusion bodies, which are also present in the Pseudokeronopsinae [2,14,23,40,41]. Moreover, as mentioned in the above section, specific conserved positions that may provide a molecular identity for the Nothoholostichinae among the core urostyloids were found in our alignment ( Figure 5), thus strengthening the establishment of this new subfamily.
Lastly, the new genus Tetrakeronopsis is established based on a unique combination of morphologic features for a nothoholostichine pseudokeronopsid (Table 3), namely, the presence of two frontoterminal cirri; presence of buccal, transverse and distinct pretransverse cirri; and lack of a posterior midventral cirral row.