Original PaperSystematic Redefinition of the Hypotricha (Alveolata, Ciliophora) Based on Combined Analyses of Morphological and Molecular Characters
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.
Section snippets
Morphological Characters
The morphological matrix contained a total of 79 characters, of which 0–5 were continuous, and 6–78 discrete (Supplementary Material Data S1, S2).
Molecular Characters
After aligned and trimmed, the 18S matrix had 1,830 nucleotide positions (mean sequence length = 1,770.1 ± 6.0 ungapped positions), with pairwise identity of 91.3% and G + C content of 42.9%. Gaps and invariable sites comprised 3.3% and 46% of the positions, respectively. A total of 532 positions corresponded to parsimony-informative characters.
Phylogeny of the Hypotricha
The analyses
Phylogeny of the Hypotricha Inferred from Morphological and Molecular Characters
Separate analyses: Trees resulting from the 18S matrix under ML and parsimony (Supplementary Material Figs S2, S4) are in conformity with the array of phylogenetic patterns found in previous studies (e.g. Chen L. et al., 2017, Foissner et al., 2014, Gao et al., 2016, Heber et al., 2014; Park et al. 2017, 2020; Shao et al. 2014c; Singh and Kamra 2013, 2015a), thus, warrant no further discussion in the present context.
Interestingly enough, the ML and parsimony morphological trees (Supplementary
Conclusions
Since the early hypotrich molecular phylogenies were published, conflicts with traditional classification brought up the necessity of reconciling the new findings with morphology (Foissner et al. 2004); however, the molecular taxonomy of hypotrichs has made little progress in this subject so far (Foissner 2016). Analyzing morphological and molecular characters altogether truly reconciled the hypotrichs morphology with molecular data because both contributed to phylogenetic inference, enabling
Methods
Taxon sampling: The literature on ciliates contains more than 1,300 species of hypotrichs s. l. (i.e., including hypotrichs, euplotids and discocephalids) (Berger 2006a), of which a relatively small fraction has been object of thorough morphological studies employing nowadays standard microscopy techniques (e.g. protargol-impregnation and scanning electron microscopy), and had divisional morphogenesis investigated in detail (Berger, 1999, Berger, 2006b, Foissner et al., 2002). For the present
Conflict of interest
The author declares having no conflict of interest.
Acknowledgements
This study was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (Universal 437655/2018-0). I am thankful to Carlos Peña for the pbs.run TNT script, to my former scientific mentor Inácio Domingos da Silva-Neto for some insightful conversations about the ultrastructure of ciliates, and to my cherished Bárbara “Babita” do Nascimento Borges for patiently proofreading this rather long paper. I dedicate this study to my family.
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