Journal of Eukaryotic Microbiology ISSN 1066-5234 ORIGINAL ARTICLE A Proposed Timescale for the Evolution of Armophorean Ciliates: Clevelandellids Diversify More Rapidly Than Metopids Peter Vd’acn ya , L’ubomır Rajtera, Thorsten Stoeckb & Wilhelm Foissnerc a Department of Zoology, Comenius University in Bratislava, Bratislava, Slovakia b Department of Ecology, University of Kaiserslautern, Kaiserslautern, Germany c FB Ecology and Evolution, University of Salzburg, Salzburg, Austria Keywords ABSTRACT 18S rRNA gene; Clevelandella; endosym- bionts; Metopus; Nyctotherus; paraphyly; Members of the class Armophorea occur in microaerophilic and anaerobic habi- perizonal stripe. tats, including the digestive tract of invertebrates and vertebrates. Phyloge- netic kinships of metopid and clevelandellid armophoreans conflict with Correspondence traditional morphology-based classifications. To reconcile their relationships P. Vd’acn y, Department of Zoology, Faculty and understand their morphological evolution and diversification, we utilized of Natural Sciences, Comenius University in the molecular clock theory as well as information contained in the estimated Bratislava, Ilkovicova 6, SK-842 15 Bratislava, time trees and morphology of extant taxa. The radiation of the last common Slovakia ancestor of metopids and clevelandellids very likely occurred during the Paleo- Telephone number: +421 2 60296170; zoic and crown diversification of the endosymbiotic clevelandellids dates back FAX number: +421 2 60296333; to the Mesozoic. According to diversification analyses, endosymbiotic cleve- e-mail: [email protected] landellids have higher net diversification rates than predominantly free-living metopids. Their cladogenic success was very likely associated with sharply Received: 10 April 2018; revised 11 May isolated ecological niches constituted by their hosts. Conflicts between tradi- 2018; accepted June 1, 2018. tional classifications and molecular phylogenies of metopids and clevelandellids Early View publication July 1, 2018 very likely come from processes, leading to further diversification without extinction of ancestral lineages as well as from morphological plesiomorphies doi:10.1111/jeu.12641 incorrectly classified as apomorphies. Our study thus suggests that diversifica- tion processes and reconstruction of ancestral morphologies improve the understanding of paraphyly which occurs in groups of organisms with an apparently long evolutionary history and when speciation prevails over extinction. THE class Armophorea unites microaerophilic or anaerobic, the digestive tract of invertebrates and vertebrates (for a bacterivorous ciliates carrying an adoral zone of mem- review, see Corliss 1979 and Earl 1972). branelles that is involved in motion and food acquisition. Lynn Molecular relationships among and within armophorids (2004) established this group solely on the gene coding for and clevelandellids conflict with traditional morphology- the small subunit ribosomal RNA and ranked it as a riboclass, based classifications (Jankowski 2007; Lynn 2008). Specif- because no morphological synapomorphies were detected. ically, the species rich genus Metopus is nonmonophyletic According to Lynn’s (2008) textbook, the Armophorea are and the family Metopidae is consistently depicted as para- subdivided into the orders Armophorida and Clevelandellida. phyletic encompassing the monophyletic order Cleve- Armophorids are common in anoxic water habitats, but live landellida (Bourland et al. 2014, 2017a,b; Li et al. 2016, also in oxygen-depleted soils (Esteban et al. 1995; Foissner 2017a,b; Lynn and Wright 2013; da Silva-Neto et al. 2016). 1998; Foissner et al. 1992, 2002; Hu 2014; Sacca 2012) and Ontogenetic data excluded metopids from the order Armo- some are even endocommensals of sea urchins (Biggar and phorida and are now considered as a distinct order, Wenrich 1932; Kattar 1982; da Silva-Neto et al. 2016). On Metopida (Foissner and Agatha 1999; Vd’acn y and Foiss- the other hand, clevelandellids are exclusively inhabitants of ner 2017a). The classification of the armophorid family © 2018 International Society of Protistologists Journal of Eukaryotic Microbiology 2019, 66, 167–181 167 Phylogeny of Metopids and Clevelandellids Vd’acn y et al. Caenomorphidae is unstable in phylogenetic analyses. It PCR products were purified using the NucleoSpin Gel and could be a sister group of metopids and clevelandellids or PCR clean-up Kit (Macherey-Nagel, Duren,€ Germany) and could represent a distinct lineage at the base of the class cloned into a plasmid vector using the pGEMâ-T and the Spirotrichea or the infraphylum Intramacronucleata (da pGEMâ-T Easy Vector Systems (Promega, Fitchburg, Wis- Silva Paiva et al. 2013). consin, United States). Recombinant plasmids were intro- To reconstruct the evolutionary history of armophorean duced into the host organism Escherichia coli (strain ciliates and to improve understanding of their phylogenetic JM109). After cultivation of transformed bacteria, plasmids relationships, diversification dynamics, and morphological were isolated using the PureYieldTMPlasmid Miniprep Sys- evolution, we utilized the molecular clock theory as well tem (Promega, Fitchburg, Wisconsin, United States) and as information contained in the estimated time trees and sequenced on an ABI 3730 automatic sequencer (Macro- morphology of extant taxa. Diversification dynamics is only gen, Amsterdam, The Netherlands) with the M13 forward rarely examined in ciliates due to the lack of fossil records and reverse primers. (Rajter and Vd’acn y 2016; Vd’acn y 2015; Vd’acn y et al. 2017; Wright and Lynn 1997). However, this problem Alignment and tree-building methods could be overcome by the fossil appearance of hosts of the exclusively endosymbiotic clevelandellids, whose ori- New sequences were checked and trimmed at the 50 and gin cannot precede that of their host groups. Similar 30 ends in Chromas ver. 2.33 (Technelysium Pty Ltd.) and assumptions were already proposed by Williams and Cole- assembled into contigs using BioEdit ver. 7.2.5 (Hall man (1992) and Wright and Lynn (1997), pioneers of diver- 1999). An alignment of new and all available 18S rRNA gence time estimates using molecular clock in ciliates. gene sequences of free-living metopids and endosymbi- In this study, we attempt to address outstanding ques- otic clevelandellids was constructed on the GUIDANCE2 tions about (i) the molecular clock rate of the 18S rRNA server (available at http://guidance.tau.ac.il/ver2/), using gene in ciliates; (ii) the diversification patterns of free-living the MAFFT algorithm and 100 bootstrap repeats (Sela and endosymbiotic ciliates; and (iii) the paraphyly problems et al. 2015). Since the score of the resulting alignment in ciliates in the light of time scale and adaptive change in was very high (>0.95), no special masking strategy was a new ecological situation. employed. Caeonomorphid taxa were selected as out- group, because they are morphologically most similar to metopids and clevelandellids within the SAL super-cluster, MATERIALS AND METHODS and a sister group relationship of caenomorphids and metopids + clevelandellids cannot be excluded by statisti- Sample collecting and processing cal tree topology tests (da Silva Paiva et al. 2013). All newly sequenced metopids were collected from the GTR + I + Γ was selected as the best evolutionary sub- upper 5 cm litter and soil layer of the floodplain of the stitution model for maximum likelihood and Bayesian anal- Murray River at the Landside of Ryans road near to the yses using jModelTest ver. 0.1.1 under the Akaike town of Albury, Southeast Australia (S36°060 E146°540). Information Criterion (AIC, Guindon and Gascuel 2003; The material was sampled in February 2006, air-dried for 3 Posada 2008). Maximum likelihood analyses were per- weeks, and stored in a plastic bag. Metopids were reacti- formed in PHYML ver. 3.0 with SPR tree-rearrangement vated from resting cysts in summer 2006, using the non- and 1,000 nonparametric bootstrap replicates on the South flooded Petri dish method, as described in Vd’acn y and of France bioinformatics platform (available at http:// Foissner (2012). A more detailed description of the sample www.atgc-montpellier.fr/phyml/) (Guindon et al. 2010). is available in Vd’acn y and Foissner (2017a,b). Bayesian analyses were run on the CIPRES portal ver. 3.1 Isolated metopids were studied using a combination of (available at http://www.phylo.org/), using the program detailed in vivo observation, silver impregnation, and scan- MrBayes (Ronquist et al. 2012) on XSEDE ver. 3.2.6 ning electron microscopy, as described by Foissner (Miller et al. 2010). Bayesian inference was performed (2014). Briefly, living cells were observed at low (50– with four chains running simultaneously for 5,000,000 gen- 4009) and high (10009, oil immersion) magnifications with erations. Prior parameters for stationary base frequencies, bright field and differential interference contrast. The cil- rate matrix for substitutions, gamma distribution shape iary pattern of fixed cells was revealed with protargol and and proportion of invariable sites, as estimated in jModelT- silver carbonate impregnation. Identification followed Kahl est, were implemented into Bayesian analyses using the (1932), Foissner and Agatha (1999), Foissner et al. (2002), ‘prset’ command. Every 1000th tree was sampled and the Bourland and Wendell (2014), and Vd’acn y and Foissner first 25% of the sampled trees were considered as burn- (2017b). in and discarded prior