that the ciliates could by arranged into from 8 to 11 major clades or classes, although there was some disagreement on how these might be related at deeper levels (Lynn & Corliss, 1991; de Puytorac, 1994a; de Puytorac et al., 1993). The early researches into rRNA gene sequences suggested that molecular phylogenetics would be a productive approach to test the robust- ness of these morphology-based phylogenies and classifications. 16.2 Deep Phylogeny and Gene Sequences It is not our intention in this section to present an exhaustive review of molecular phylogenetic stud- ies on ciliates. Instead, studies will be cited that have tested the monophyly of the major classes, as suggested by morphological analysis, and that also provide some evidence of the deeper structure to the relationships among classes. Often, these deeper relationships have not been strongly sup- ported by “statistical” approaches, like bootstrap analysis or likelihood probabilities. However, if a consensus emerges based on different genes, both rRNA and proteins, we will use this to construct a tree with which to examine the broad evolution of character states within the phylum ( 16.3 Character State Evolution ). The basic approach for gene sequencing remains the same, but has developed to be much more efficient since the days of cloning genes into vectors in the 1980s. In brief, conserved regions of genes are used to design polymerase chain reaction (PCR) primers, which enable ampli- fication of the gene of interest (e.g., Bernhard Fig. 16.1. Phylogeny of the Phylum Ciliophora as presented by Small and Lynn (1985). Eight major monophyletic lineages (= classes) are thought to have diversified from a karyorelictean ancestor, one that exhibited the ancestral state of nuclear dimorphism . The thickness of each clade represents generic diversity. Each clade is characterized by a schematic of its kinetid, which is diagrammed as if viewed from the inside of the cell. The key to the kinetid structures is as follows: ( a ) kinetosome; ( b ) overlapping postciliary microtubular ribbons forming postciliodesma ; ( c ) convergent postciliary microtubular ribbon; ( d ) divergent postciliary microtubular ribbon; ( e ) striated kinetodesmal fibril ; ( f ) radial transverse microtubular ribbon; ( g ) tangential transverse microtubular ribbon; ( h ) overlapping trans- verse microtubular ribbons, the so-called transversodesma . (Redrawn from Small & Lynn, 1985.) 16.2 Deep Phylogeny and Gene Sequences 329 330 16. Deep Phylogeny, Gene Sequences, and Character State Evolution & Schlegel, 1998; Medlin, Elwood, Stickel, & Sogin, 1988). The PCR-amplified genes may then be cloned into a plasmid vector, amplified in bacteria, purified, and then sequenced (e.g., Baroin-Tourancheau, Villalobo, Tsao, Torres, & Pearlman, 1998; Greenwood, Schlegel, Sogin, & Lynn, 1991b; Hirt et al., 1995). As is often the case now, the PCR-amplified genes are directly sequenced (e.g., Lynn & Strüder-Kypke, 2005). In either case, both strands of the DNA should be sequenced to corroborate the sequence reads. 16.2.1 Ribosomal RNA Sequences The initial studies on rRNA gene sequences , using both SSUrRNA (Lynn & Sogin, 1988) and LSUrRNA (Baroin et al., 1988), confirmed the cili- ates as a monophyletic group. Later studies have served to solidify this confirmation and provide substantial support for the ciliates as the sister taxon to the dinoflagellates and apicomplexans in the alveolate clade (Leander & Keeling, 2003; Van de Peer, Van der Auwera, & De Wachter, 1996). Thus, the classical view of ciliates long being regarded as monophyletic is strongly supported by rRNA gene sequences. In the intervening years, species sampling has increased with the aim of determining how robust the monophyly of the major classes has been. Based on partial LSUrRNA gene sequences, Baroin- Tourancheau, Delgado, Perasso, and Adoutte (1992) provided evidence of the deep genetic divergences among five of the major classes (i.e. Classes KARYO- RELICTEA , SPIROTRICHEA , LITOSTOMATEA , COLPODEA , and NASSOPHOREA ), and their results united the Classes PROSTOMATEA and Fig. 16.2. Schematic view of the phylogeny of ciliates based on characterization of the particle arrays in ciliary mem- branes, revealed by the freeze fracture technique . The particle array patterns can be classified into a ciliary necklace that ringed the base of the cilium (virtually all groups), ciliary plaques (see Hymenostomatida ), ciliary rosettes (see Frontonia ), single- (see Hypotrichida , “ Karyorelictina ”, and SUCTORIA ) and double-stranded (see SPIROTRICHA , PERITRICHA , and HYPOSTOMATA ) longitudinal rows, and orthogonal arrays (see Tracheloraphis and Spirostomum ). (Redrawn from Bardele, 1981.) OLIGOHYMENOPHOREA . They did not sample the Class PHYLLOPHARYNGEA . Numerous studies on the SSUrRNA have now confirmed the major classes, but also suggested the recognition of new ones. Greenwood et al. (1991b) demonstrated the basal branching of the heterot- richs , separating them from the other spirotrichs , a result confirmed by subsequent studies (Hirt et al., 1995; Rosati, Modeo, Melai, Petroni, & Verni, 2004), and justifying their elevation to class rank (de Puytorac, 1994a). This added a ninth class to the Small and Lynn (1981, 1985) system. Greenwood, Sogin, and Lynn (1991a) added sequences of oli- gohymenophoreans to demonstrate the integrity of this group, which has been confirmed by later studies (Strüder-Kypke, Wright, Fokin, & Lynn, 2000b). Phyllopharyngeans were shown to be genetically distinct by Leipe, Bernhard, Schlegel, and Sogin (1994), and this has been subsequently confirmed (Riley & Katz, 2001; Snoeyenbos-West, Cole, Campbell, Coats, & Katz, 2004). Leipe et al. (1994) first demonstrated the genetic distinct- ness of the Class LITOSTOMATEA , and this has been subsequently confirmed (Cameron, Adlard, & O’Donoghue, 2001; Wright & Lynn, 1997b). Hirt et al. (1995) added members of the Classes KARYORELICTEA and HETEROTRICHEA to confirm the sister group relationship of these two taxa, and also demonstrated their genetic distinctness. In their study of the evolution of ciliate hydrogenosomes , Embley et al. (1995) demonstrated the genetic distinctness of the pla- giopyleans , intriguingly including Plagiopyla and Trimyema , two genera not suspected to be closely related on the basis of morphology – a so-called “riboclass” (Lynn, 2004). This has been subse- quently confirmed (Lynn & Strüder-Kypke, 2002), supporting the elevation of plagiopylids as the tenth class (de Puytorac, 1994a). Bernhard, Leipe, Sogin, and Schlegel (1995) provided evidence of the genetic distinctness of nassulid ciliates, now placed in the Class NASSOPHOREA . Throughout these intervening years, the Class SPIROTRICHEA with the heterotrichs removed, was confirmed as a monophyletic group to which Protocruzia was attached (Hammerschmidt et al., 1996) as well as the morphologically distinct genera – Phacodinium (Shin et al., 2000) and Licnophora (Lynn & Strüder-Kypke, 2002). Stechmann, Schlegel, and Lynn (1998) provided evidence of the distinctness of the Classes PROSTOMATEA and COLPODEA , while Lynn, Wright, Schlegel, and Foissner (1999) added species density to solidify the genetic dis- tinctness of the COLPODEA . Embley et al. (1995) had demonstrated that the armophorid Metopus spp. were not closely related to the heterotrichs , disproving this classical rela- tionship. The independence of this lineage was clinched by the addition of a substantial number of additional armophorid sequences, demonstrating them to form a sister taxon with several species of the clevelandellid nyctotherids (van Hoek et al., 2000b). Lynn (2004) elevated this group to class rank as the Class ARMOPHOREA , establishing the eleventh class