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.

Introduction

Within the class Spirotrichea Bütschli, 1889, the Hypotricha Stein, 1859 (=Stichotrichia Small & Lynn, 1985), are a morphologically diverse group of ciliates with a dorsoventrally flat body; at least one left and one right marginal cirral row; and a highly diversified fronto-ventral-transverse (FVT) cirral field, originated by numerous ontogenetic patterns (Berger 1999, 2006b, 2008, 2011; Lynn 2008). The hypotrichs are typically substrate-oriented organisms, adapted to life in microporous sediments, occurring in freshwater, brackish and saltwater, as well as terrestrial habitats worldwide, where they behave as omnivorous consumers, feeding mostly on other protists and bacteria, with the largest species capable of capturing microscopic metazoans, such as rotifers (de Castro et al., 2016, Foissner et al., 2002, Lynn, 2008).

The systematics of Hypotricha is one of the most confuse subjects in ciliate biology, particularly because of inconsistencies between early molecular phylogenies and morphology-based classifications (Bernhard et al., 2001, Foissner et al., 2004; Foissner and Stoeck 2006, 2008; Paiva et al., 2009, Schmidt et al., 2007, Strüder-Kypke and Lynn, 2003; Yi et al. 2008a,b). Ever since, manifold studies pursued the improvement of knowledge on the phylogenetic affinities of Hypotricha using mostly the 18S rDNA (e.g. de Castro et al., 2016, Foissner et al., 2014, Li et al., 2010, Paiva et al., 2014), while others also made use of further molecular markers, such as the ITS1-5.8S-ITS2 and 28S rDNA, alpha-tubulin and actin, in either separate or concatenated approaches (Gao et al., 2016, Hu et al., 2011; Huang et al. 2014, 2016; Lyu et al. 2018; Yi et al. 2018a; Yi and Song 2011). Regardless of the effort, consistent, resolute patterns were not yet found for the deep diverging lineages along the hypotrichs tree. As consequence, the traditional higher taxa intermix, and trees constantly have many unstable branches at various levels (e.g. Bharti et al. 2014; Chen et al. 2013a, b; Foissner et al., 2014, Heber et al., 2014, Huang et al., 2014, Park et al., 2017; Shao et al. 2014a, b; Singh and Kamra 2015a, b).

The confusion intrinsic to hypotrich systematics is generally ascribed to numerous ciliature pattern convergences and to the weak phylogenetic signal of 18S rDNA (the most frequently analyzed gene) at assorted levels, which results in weakly supported wildcard branches (e.g., Berger, 2008, Foissner et al., 2002, Foissner and Stoeck, 2008, Paiva et al., 2009, Shao et al., 2014b). However, neither the monophyly of the many nominal taxa from diverging, but yet co-existing classifications (Berger 2006b, 2008; Lynn 2008), nor the inconsistencies with molecular studies were ever assessed from actual test of character congruence in combined analyses (Assis, 2009, De Pinna, 1991, Kluge, 1989, Sober, 1988). Phylogenetic analyses of hypotrichs based on morphology are remarkably scarce in the literature (e.g. Berger, 2006b, Berger and Foissner, 1997; Eigner 1997, 1999; Eigner and Foissner 1992; Shao et al., 2006, 2008; Wiackowski 1988), and although important steps towards the building of our understanding on hypotrichs natural history, they often focused on particular subgroups and used relatively narrow taxonomic sampling, thus not testing the monophyly of the commonly recognized higher taxa within the Hypotricha as a whole.

As consequence of the aforementioned problems, the systematics of hypotrichs has become a conundrum, for which the constant discovery of new species and genera every year, without robust taxonomic allocation, rendered it “hopelessly complicated” (Foissner, 2016, Foissner et al., 2002).

It is well-accepted nowadays that integrating morphological characters with molecular ones may improve previously unresolved relationships and expose phylogenetic patterns previously unseen, as both kinds of data, when analyzed simultaneously, can complement each other (e.g. Andreasen and Bremer, 2000, Cordie and Budd, 2016, Heethoff et al., 2011, Heikkilä et al., 2015, Miller et al., 1997, Silva-Júnior and Paiva, 2018, Wahlberg et al., 2005). As for ciliates, this kind of approach has been used to infer the position of the synhymeniid Zosterodasys (Kivimaki et al. 2009), to study the phylogeny of Litostomatea (Vďačný and Foissner, 2013, Vďačný et al., 2015, Vďačný and Rajter, 2015), and of Heterotrichea (Fernandes et al. 2016).

Thus, aiming to untangle the confusion pervading the systematics of Hypotricha, the present study intends to (i) hypothesize their internal phylogeny from maximum-likelihood and cladistic analyses of combined morphological and molecular (18S rDNA) matrices of a broad taxonomic sample; (ii) compare the results with the literature, focusing on the two mostly used hypotrich classification systems – those of Berger (2008) and Lynn (2008); and (iii) to outline the herein inferred Hypotricha higher taxa based on synapomorphies.