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327 Abstract Our understanding of the evolutionary diversiﬁ cation of ciliates in the past two decades particularly has depended upon the interaction between conceptual views and technological advanc- es. Transmission electron microscopy precipitated a revolution in our views of what characters might be signiﬁ cant in inferring deep phylogenetic relation- ships. The ﬁ brillar patterns of somatic kinetids were considered crucial, based on the notion of the struc- tural conservation of these cortical components. Molecular phylogenetic analyses have been used to test the conclusions based on electron microscopy. In the main, phylogenetic relationships inferred from sequences of the small subunit and large subunit rRNA genes have conﬁ rmed the major class- es, and suggested several new ones (i.e., Classes ARMOPHOREA and PLAGIOPYLEA). In addi- tion, the rRNA genes demonstrated a fundamental subphyletic division – now named the Subphyla Postciliodesmatophora and Intramacronucleata. Protein gene sequences (e.g., elongation factor 1α, α-tubulin, and histone H3 and H4) provide conﬁ r- mation for some clades. Using the rRNA phylogeny, the evolution of some major character states, particularly nuclear ones, can be assessed. Keywords Phosphoglycerate kinase, intramem- branous particles, ciliary necklace The progress in our understanding of the evolution- ary diversification of ciliates has depended upon an interaction between conceptual views and techno- logical advances . On the conceptual side, our views of which characters or features of ciliates were most important in revealing common ancestry have changed ( see Chapter 1 ). Briefly, in the 18th and 19th centuries, overall ciliation patterns and the dominance of the “ spirotrich ” oral region divided the ciliates into “holotrichs” and “ spirotrichs ”. In the first half of the 20th century, ontogenetic patterns , particularly revealed by silver-staining organisms at cell division, received greater weight and aligned taxa that had previously been distantly separated (e.g., chonotrichs and suctoria were related to the cyrtophorines ). In the latter half of the 20th century, transmission electron microscopy revealed a whole new set of cytoskeletal charac- ters, particularly the somatic kinetid patterns. The diversity of these somatic kinetid patterns initially suggested eight major clades or classes (Small & Lynn, 1981, 1985). In the 1970s, microbiologists studying prokaryo- tes had been successfully using small subunit (SSU) rRNA genes to resolve relationships among this group whose members were not rich in mor- phological features (Stackebrandt & Woese, 1981). By the mid-1980s, several research groups began sequencing SSUrRNA genes of ciliates (Elwood, Olsen, & Sogin, 1985; Sogin & Elwood, 1986; Sogin, Swanton, Gunderson, & Elwood, 1986a), demonstrating that ciliates, even with this small sampling of species, appeared to be monophyletic and yet showed very deep divergences, equivalent to the genetic distances between the classical plant and animal “kingdoms”. The first denser samplings of species, using both the SSUrRNA (Lynn & Sogin, 1988; Sogin & Elwood) and the large subunit (LSU) rRNA (Baroin et al., 1988), provided enough taxon density to demonstrate Chapter 16 Deep Phylogeny, Gene Sequences, and Character State Evolution – Mapping the Course of Ciliate Evolution 328 16. Deep Phylogeny, Gene Sequences, and Character State Evolution utility in testing the deeper relationships predicted by ultrastructural research. The molecular phylogenetic approach is now a recognized method for testing and establishing phylogenetic relationships among organisms, and has been particularly fruitful in revealing the broad lines of evolutionary descent among eukaryotes. However, it rests on the basic assumption that phylogenetic trees based on genes truly represent the phylogeny of the organisms. Ultimately, our confidence in so-called “gene trees” increases when multiple and unlinked genes show patterns congruent with each other and with organismal phylogenies constructed on other features, such as morphology. It is the purpose of this chapter to briefly review the deep phylogeny of ciliates as inferred from features of cortical ultrastructure , primarily, and then to examine how this topology is congruent with gene tree topologies derived from rRNA genes and several protein coding genes. This will provide a consensus phylogenetic tree of the currently recognized classes of ciliates, which will provide the basis for a final discussion of the evolution of character states in the phylum. It is this distribution of character states that, in part, forms the rationale for the higher classification presented in Chapter 17 . 16.1 Deep Phylogeny and Ultrastructure The transmission electron microscope provided a technical approach that opened up literally a vast array of detailed character information with which to investigate the cellular morphology of protists. Initially, there was a preoccupation with cortical fibrillar systems, an approach pioneered by Pitelka (1969). Later, comparative analyses of these cortical patterns, especially of somatic kinetids , suggested eight major clades or classes of ciliates: (1) Class KARYORELICTEA ; (2) Class SPIROTRICHEA ; (3) Class LITOSTOM- ATEA ; (4) Class PHYLLOPHARYNGEA ; (5) Class COLPODEA ; (6) Class NASSOPHOREA ; (7) Class PROSTOMATEA ; and (8) Class OLIGOHYMENOPHOREA (Lynn, 1981; Small & Lynn, 1981, 1985). As discussed in Chapter 1 , arrangement of these classes into subphyla based on morphology has not been supported by molec- ular analyses (see below). While divided into subphyla by Small and Lynn (1985), the classes emerged “bush-like” from the common ancestor (Fig. 16.1). Bardele (1981) analyzed the arrays of intramem- branous particles of cilia in 68 genera, representing a broad diversity of ciliates. These particle array patterns were classified into a ciliary necklace that ringed the base of the cilium, ciliary plaques , ciliary rosettes , single- and double-stranded longitudinal rows, and orthogonal arrays covering most of the cilium. His analysis suggested six major assem- blages: (1) SPIROTRICHA , corresponding to the Class SPIROTRICHEA ; (2) GYMNOSTOMATA , which included representatives of the Classes LITOSTOMATEA and PROSTOMATEA ; (3) TRICHOSTOMATA , which included representatives from the Classes LITOSTOMATEA and COLPODEA ; (4) ENTO- DINIOMORPHA , which included repre- sentatives from the Class LITOSTOMATEA ; (5) HYPOSTOMATA + SUCTORIA , corresponding to the ClassPHYLLOPHARYNGEA ; and (6) HYMEN- OSTOMATA + PERITRICHA + ASTOMATA , corre- sponding to the Class OLIGOHYMENOPHOREA . Bardele’s “ciliate bush” was anchored in a gymnos- tome -like form and radiated out from there. While there was some broad agreement with the clades based on cortical ultrastructure, the particle array character set was not rich enough to tease out the details of this diversification (Fig. 16.2). Bardele (1987, 1989) turned his “ciliate bush” upside down as he reviewed the data arising from his laboratory on the ultrastructure of ontogeny , and particularly stomatogenesis , in ciliates. These observations, coupled with the conception that the ciliate ciliature arose by proliferation from the paroral (Eisler, 1989, 1992), suggested that gym- nostomy – a simple, anterior oral region – may have arisen repeatedly as a derived and secondary feature of oral apparatus evolution and not as a pri- mary feature. Bardele (1989) concluded by doubt- ing that many of the major groups suggested by Small and Lynn (1981, 1985) would be confirmed to be monophyletic, and he strongly argued that a research program in ontogeny would reveal this view to be true. By the early 1990s, there was general agree- ment among morphologiststhat 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 classin our macrosystem. The deeper relationships among these clades have not been strongly resolved. Cameron et al. (2001) performed statistical analyses and concluded that there was good statistical support for the Classes KARYORELICTEA , HETEROTRICHEA , SPIROTRICHEA , LITOSTOMATEA , PHYLL- OPHARYNGEA , PROSTOMATEA , and PLAGI OPYLEA . The Classes COLPODEA and NAS- SOPHOREA were often associated in their analyses, while the Class OLIGOHYMENOPHOREA often did not form a well supported clade. Review of the deeper topology demonstrated in the studies cited above provides no doubt of a deep bifurcation in the phylum, providing confir- mation for the Subphylum Postciliodesmatophora to include the Classes KARYORELICTEA and HETEROTRICHEA , and providing support for the Subphylum Intramacronucleata (Lynn, 1996a, 2004). There is no consistent deep topol- ogy within the intramacronucleates, although the following assemblages receive some support: SPIROTRICHEA + ARMOPHOREA ; NASSO- PHOREA + COLPODEA ; PROSTOMA-TEA + PLAGIOPYLEA ; and PHYLLOPHARNYGEA + ( NASSOPHOREA + COLPODEA ) + ( PROSTOMA- TEA + PLAGIOPYLEA ) + OLIGOHYMENOPHO- REA . Based on an analysis of our SSU rRNA database, a summary tree provides support for some of these groupings (Fig. 16.3). 16.2.2 Protein Gene Sequences There is a handful of studies that examine protein sequences, both as nucleotides and as amino acids, to provide further tests of the robustness of our understanding of relationships among ciliates. An underlying problem with using protein genes to 16.2 Deep Phylogeny and Gene Sequences 331 Fig. 16.3. A phylogenetic tree based on sequences of the small subunit rRNA gene and using the profile- neighbor-joining method implemented in Profdist ver. 0.9.6.1 (Friedrich et al., 2005). Note that the two subphyla – Postciliodesmatophora and Intramacronucleata - are strongly supported at >90%. Some classes are strongly supported (e.g., KARYORELICTEA , HETEROTRICHEA , ARMOPHOREA , LITOSTOMATEA , PHYLLOPHARYNGEA , PLAGIOPYLEA ). Six “terminal” clades consistently cluster: the Classes PHYLLOPHARYNGEA , COLPODEA , NASSOPHOREA , PLAGIOPYLEA , PROSTOMATEA , and OLIGOHYMENOPHOREA ) (cf. Fig. 16.5). We still have no rationalization outside of sequence data for this grouping. *Indicates support <20% reconstruct the phylogeny of ciliates is the relatively high rate of protein diversification in the phylum, and especially in ciliate clades whose macronu- clear genomes are extensively fragmented (Zufall, McGrath, Muse, & Katz, 2006). Nevertheless, protein phylogenetic studies can be divided into two groups – those that have sequenced a small number of representative genera from across the phylum and those that have provided a larger sam- pling of species. Initial studies of the actin genes of ciliates indicated that the phylum was not recovered as a monophyletic group due to the high rela- tive evolutionary rate of this gene in ciliates (Philippe & Adoutte, 1998). Kim, Yura, Go, and Harumoto (2004) have extended the sampling to about 20 genera of ciliates from five classes. Again, the ciliates are not recovered as a mono- phyletic group, although several classes appear to be: the Class LITOSTOMATEA and Class OLIGOHYMENOPHOREA . Elongation factor 1α (EF-1α) is a protein that, in addition to its role in protein synthesis, probably interacts with actin in the cytoskeleton of ciliates. It also shows unusually high rates of evolution, and again ciliates are not recovered as a monophyletic assemblage (Moreira, Le Guyader, & Philippe, 1999). In an update of this research, Moreira, Kervestin, Jean-Jean, and Philippe (2002) provided sequences of eukaryotic release factor 1 (eRF1) and factor 3 (eRF3) in addition to sequences of EF-1α and elongation factor 2 (EF-2). The genus sampling of eRF3 was too low to draw any definitive conclusions, but ciliates again were not recovered as monophyletic using either EF-1α or eRF1. With seven genera representing five classes, the ciliates were recovered as monophyletic with EF-2 (Moreira et al., 2002). Moreira et al. specu- lated that these accelerated rates of evolution in the ciliates may be due to loss of interaction of these proteins with cytoskeletal elements or may be a co-evolutionary phenomenon linked with the extremely fast-evolving actins of ciliates. The 70 kDa heat shock proteins (Hsp70) comprise a multigene family that has been divided into three major subfamilies: (1) prokaryotic, mitochondrial, and chloroplast proteins; (2) eukaryotic cytosolic and nuclear proteins; and (3) eukaryotic proteins localized in the endoplasmic reticulum (Budin & Philippe, 1998). Budin and Philippe (1998) dem- onstrated that Hsp70 subfamily sequences from Euplotes and Paramecium confirmed the ciliates as a monophyletic group. Baroin et al. (1998) provided sequences of phosphoglycerate kinase (PGK) for seven species representing three classes – Classes HETEROTRICHEA , SPIROTRICHEA , and OLIGOHYMENOPHOREA – and showed that the phylum was monophyletic, although these data could be compared to only a limited sam- pling of other eukaryotes. Thus far, only three protein genes – EF-2, Hsp70, and PGK – have confirmed the monophyly of the ciliates. The last two proteins that have been studied – the tubulins and histones – also comprise multigene families, but they have been much more exten- sively sampled across the phylum. Baroin et al. (1998) provided nucleotide and amino acid sequences for α-tubulins from representatives of seven classes – Classes KARYORELICTEA , HETEROTRICHEA , SPIROTRICHEA , LITOST- OMATEA , COLPODEA , NASSOPHOREA , and OLIGOHYMENOPHOREA . Israel, Pond, Muse, and Katz (2002) have added sequence data for the Classes ARMOPHOREA and PHYLLOPHARYNGEA . Although both studies only compared the ciliate sequences to alveolate sister taxa, the ciliates were monophyletic. Overall, although taxon sampling was low, most classes appeared to be monophyletic, excepting the Classes HETEROTRICHEA and SPIROTRICHEA . While the classes were generally supported, there was no consistently recoverable deep topology (Fig. 16.4) (Israel et al., 2002). The ciliates were also recov- ered as a monophyletic group based on β-tubulin sequences (Philippe & Adoutte, 1998). Bernhard and Schlegel (1998) provided the first analyses of variation among the histone genes H3 and H4 in six classes – Classes HETERO- TRICHEA , SPIROTRICHEA , COLPODEA , NASSOPHOREA , PROSTOMATEA , and OLI-GOHYMENOPHOREA . Katz, Bornstein, Lasek-Nesselquist, and Muse (2004) have expanded the database, adding sequences from representatives of the Classes ARMOPHOREA , COLPODEA , PHYLLOPHARYNGEA , and OLIGOHYMENOPHOREA . Thus, only repre- sentatives of the Class PLAGIOPYLEA are mis- sing. In unconstrained analyses of H4 nucleotides, the ciliates were not monophyletic, but they were monophyletic based on amino acid sequences (Katz et al., 2004). Based on amino acids, classes were generally monophyletic (Fig. 16.5). The deep 16.2 Deep Phylogeny and Gene Sequences 333 334 16. Deep Phylogeny, Gene Sequences, and Character State Evolution Fig. 16.4. A phylogenetic tree derived from a neighbor-joining analysis of the amino acid sequences of the α-tubulin gene. The numbers on the branches represent bootstrap percentages for neighbor-joining (NJ) and maximum parsi- mony (MP) while support estimates are provided for puzzle quartet analysis (PZ). The dots indicate branches with very low support values or inconsistent topology; P1 and P2 refer to paralogs of the α-tubulin gene. (Redrawn from Israel et al., 2002.) topology was generally unresolved, although four classes were often associated – Classes COLPO- DEA , NASSOPHOREA , PROSTOMATEA , and OLIGOHYMENOPHOREA (CONP, Fig. 16.5) (Bernhard & Schlegel, 1998;Katz et al., 2004). The unusual ciliate Protocruzia , which we place in the Class SPIROTRICHEA ( see Chapter 17 ), is associated with karyorelicteans (Bernhard & Schlegel, 1998) or the four-class assemblage (Katz et al., 2004), based on H4 nucleotide sequences. However, this genus is at the base of the intrama- cronucleate clade (Bernhard & Schlegel, 1998) or associated with the spirotrichs (Katz et al., 2004), based on amino acid sequences (Fig. 16.5). Fig. 16.5. A phylogenetic tree derived from a neighbor-joining analysis of the amino acid sequences of the histone H4 gene. The dots indicate bootstrap percentages >70%. Clades indicated by capital letters correspond to the respective classes. Note that only the Classes COLPODEA and PROSTOMATEA are supported >70%, but species sampling in these is very low. P1, P2, etc. indicate paralogs. (Redrawn from Katz et al., 2004.) Overall, the protein sequence database provides us with little confidence in the deep phylogeny of the ciliates. Proteins refute or confirm the monophyly of the phylum. Since there is no doubt from a morphological perspective that the ciliates are monophyletic, reinforced strongly by the rRNA sequence databases, we must consider those protein molecules refuting this monophyly to be aberrant in some way, perhaps due to very high relative rates of evolution (Katz et al., 2004; Moreira et al., 2002; Zufall et al., 2006). The major assemblages suggested by the SSUrRNA database, including the Classes COLPODEA , NASSOPHOREA , PROSTOMATEA , and OLIGOHYMENOPHOREA , are supported at least by H4 amino acid sequences (cf. Figs. 16.3, 16.5). 16.3 Character State Evolution The review of gene sequence data for rRNA and protein genes, excluding those proteins with unu- sually high relative rates of evolution (i.e., actins, elongation factors), leaves us to conclude that the Phylum Ciliophora is monophyletic, supporting the classical view based on morphology. The sampling 16.3 Character State Evolution 335 336 16. Deep Phylogeny, Gene Sequences, and Character State Evolution density of sequence information across the phylum is really only significant for the SSUrRNA gene, for which we now have representatives sequenced for all major classes and most major subclasses or orders. Based on this gene, a simplified topology has been constructed to use in our evaluation of the evolution of character states within the phylum (Figs. 16.6, 16.7). This analysis will provide some of the evidential basis for the higher classification presented in Chapter 17 . The ciliate tree is deeply divided into two major lineages. Mapping the presence of post- ciliodesmata on the tree demonstrates that this character is restricted to one of these two major lin- eages, which is now recognized as the Subphylum Postciliodesmatophora (Fig. 16.6A) (Lynn, 1996a). The next five characters are all related to nuclear features. The other major lineage of ciliates has the major unifying feature of dividing the macronucleus primarily by using intramacronuclear microtubules . Distribution of this character on the tree supports recognition of the Subphylum Intramacronucleata (Fig. 16.6B) (Lynn, 1996a). The other major lineage with dividing macronuclei uses extramacronuclear microtubules in the division process. Distribution of this character on the tree supports recognition of the Class HETEROTRICHEA , which is also char- acterized by postciliodesmata whose ribbons are separated by a single microtubule (Fig. 16.6C) ( see Chapter 6 ). The third nuclear character is the pres- ence of non-dividing macronuclei. Distribution of this character on the tree supports recognition of the Class KARYORELICTEA , which is also character- ized by postciliodesmata whose ribbons are sepa- rated by the 2+ribbon+1 microtubular arrangement (Fig. 16.6D) ( see Chapter 5 ). As noted earlier, the topology of the tree does not permit us to unambigu- ously conclude how dividing macronuclei evolved within the phylum. One view is that macronuclei gained the ability to divide using both intra- and extramacronuclear microtubules. This was followed by a loss of division in the karyorelicteans , an emphasis on extramacronuclear microtubules in heterotrichs , and an emphasis on intramacronuclear microtubules in all other ciliates (Hammerschmidt et al., 1996). The other view is that dividing macro- nuclei evolved twice independently from non-divid- ing macronuclei (Katz, 2001; Orias, 1991a). The next two nuclear characters are related to the molecular processing of macronuclear DNA. Following conjugation , the formation of poly- tene chromosomes and extensive chromosomal fragmentation can occur as the new macronu- cleus differentiates (Jahn & Klobutcher, 2002; Prescott, 1994; Raikov, 1996). The distribution of this combined feature is restricted to three classes – SPIROTRICHEA , ARMOPHOREA , and Fig. 16.6. Character evolution in the ciliates using a phylogenetic tree whose deep topology is based on the consensus of gene sequences, primarily from the small subunit rRNA and histone H4 genes (cf. Figs. 16.3, 16.5). A Presence of postciliodesmata . B Presence of intramacronuclear microtubules to divide macronu- cleus. C Presence of extramacronuclear microtubules to divide macronucleus. D Presence of non-dividing macronuclei. KA , Class KARYORELICTEA ; HE , Class HETEROTRICHEA ; SP , Class SPIROTRICHEA ; AR , Class ARMOPHOREA ; LI , Class LITOSTOMATEA ; PH , Class PHYLLOPHARYNGEA ; CO , Class COLPODEA ; NA , Class NASSOPHOREA ; PL , Class PLAGIOPYLEA ; PR , Class PROSTOMATEA ; OL , Class OLIGOHYMENOPHOREA PHYLLOPHARYNGEA (Fig. 16.7A). Riley and Katz (2001) argued that chromosomal fragmen- tation may have had multiple origins. However, these three lineages often find their place at the “base” of the intramacronucleate radiation in gene sequence trees, sometimes separated by the Class LITOSTOMATEA (Fig. 16.7A). Thus, a common molecular mechanism of polytenization and genome fragmentation possibly underlies the explosive diversification of intramacronucleates . This mechanism has been refined or lost secondar- ily, at least twice, as this radiation diverged: it may have been lost in the common ancestor to the Class LITOSTOMATEA and in the common ancestor of the NASSOPHOREA - OLIGOHYMENOPHOREA clade (Fig. 16.7A). The final nuclear feature is the presence of replication bands , which pass through the macro- nuclear karyoplasm during the S phase of DNA synthesis. Distribution of this character is restricted to lineages in the Class SPIROTRICHEA , and with the exception of Protocruzia , provides a rationale for the monophyly of this group (Fig. 16.7B) ( see Chapter 7 ). Finally, two features that have been considered important in systematic discussions are the pres- ence of somatic monokinetids or somatic dikinetids and the kinds of stomatogenesis . Lynn and Small (1981) argued that the dikinetid state was likely the ancestral state for the ciliates, considering that the majority of flagellate taxa believed to be sister taxa to the ciliates had dikinetids. Distribution of the monokinetid character state on the ciliate tree is consistent with this view as four of the “early” emerging classes – KARYORELICTEA , HETEROTRICHEA , SPIROTRICHEA , and AR- MOPHOREA – are characterized by somatic diki- netids (Fig. 16.7C). In fact, the character state distribution of monokinetids suggests a “gain” of this character as the common ancestor of the litostomes, phyllopharyngeans, and their sister taxa arose, with an independent second- ary evolution of the somatic dikinetid character in the Class COLPODEA and within the Class OLIGOHYMENOPHOREA (Fig. 16.7C). Ontogenetic features have assumed a cen- tral place in ciliate systematics since the early researches of Fauré-Fremietand his group (Fauré- Fremiet, 1948a, 1950a, 1950b). Corliss (1968) Fig. 16.7. Character evolution in the ciliates using a phylogenetic tree whose deep topology is based on the consensus of gene sequences, primarily from the small subunit rRNA and histone H4 genes (cf. Figs. 16.3, 16.5). A Presence of polytene chromo- somes and chromosal fragmentation during macronu- clear development . B Presence of replication bands during S phase of macronuclear DNA synthesis. Note that the genus Protocruzia does not have this feature although it clusters with the Class SPIROTRICHEA (cf. Figs. 16.3, 16.5). C Presence of somatic monoki- netids . D Presence of buccokinetal (black), parakinetal (dark grey), telokinetal (grey), apokinetal (white), and mixokinetal (half black: half grey) modes of stoma- togenesis . KA , Class KARYORELICTEA ; HE , Class HETEROTRICHEA ; SP , Class SPIROTRICHEA ; AR , Class ARMOPHOREA ; LI , Class LITOSTOMATEA ; PH , Class PHYLLOPHARYNGEA ; CO , Class COL- PODEA ; NA , Class NASSOPHOREA ; PL , Class PLAGIOPYLEA ; PR , Class PROSTOMATEA ; OL , Class OLIGOHYMENOPHOREA 16.3 Character State Evolution 337 338 16. Deep Phylogeny, Gene Sequences, and Character State Evolution affirmed this view, and presented the basis of the current classification of stomatogenetic types (Corliss, 1979). Foissner (1996b) has updated and refined the classification of types, and provided a phylogenetic scenario for the evolution of these sto- matogenetic types , assuming that the buccokinetal mode was ancestral or plesiomorphous. Foissner (1996b) noted that evidence for this assumption is weak, but he used as support the model proposed by Eisler (1992) for the evolution of the ciliate cortex. Distribution of all buccokinetal modes on the tree is not consistent with this view (Fig. 16.7D). Instead, the most broadly distributed mode is the telokinetal mode (Fig. 16.7D). Thus, Eisler’s model (Eisler, 1992; Schlegel & Eisler, 1996) may be incorrect. Alternatively, soon after the ancestral cortex evolved by this “paroral model” of evolution (Eisler, 1992), a telokinetal mode of stomatogen- esis may have evolved as the cell division process. As we have argued elsewhere, and is confirmed by this analysis, modes of stomatogenesis should be used only as descriptive features at this deep level. The usefulness of stomatogenetic characters is highest when characterizing and comparing genera and species. It is also useful in broadly associating ciliates into different clades based on the details of the stomatogenetic process rather than the mode itself (e.g. phyllopharyngean merotelokinetal vs. colpodean merotelokinetal ; see Foissner, 1996b). A final feature that we have not mapped on the tree, but which has been discussed by several research groups, is the evolution of hydrogeno- somes from mitochondria (Embley et al., 1995; van Hoek et al., 2000b). Hydrogenosomes have been found in all species so far examined of the Classes ARMOPHOREA and PLAGIOPYLEA , which are not closely related (Figs. 16.6, 16.7), and in select members of the Classes LITOSTOMATEA and OLIGOHYMENOPHOREA . The latter evidence – origin within a class – demonstrates unambigu- ously the adaptive nature of the hydrogenosome (Fenchel & Finlay, 1990b, 1991a). 16.4 Summary We have provided this discussion as an approach to demonstrating how to rationalize morphologi- cal and molecular features of the ciliates. This approach can also serve as the basis for provid- ing evidence of the robustness of a classification or suggesting deeper subdivisions, which may not be inspired immediately by morphology (e.g., Subphylum Intramacronucleata ; see Lynn, 1996a). As the species sampling for our gene sequence database expands, this approach may be productively extended “higher” in the tree, testing relationships among subclasses within classes and orders within subclasses. For exam- ple, the increased species sampling of SSUrRNA genes of suctorians provided very preliminary genetic evidence that the Orders Exogenida , Endogenida , and Evaginogenida may capture the evolutionary diversification of the suctorians (Snoeyenbos et al., 2004). Extensive sampling within the Class OLIGOHYMEN-OPHOREA has confirmed the monophyly of the major sub- classes classically based on morphology (Affa’a, Hickey, Strüder-Kypke, & Lynn, 2004; J.C. Clamp et al., 2008; Greenwood et al., 1991a; Lynn & Strüder-Kypke, 2005; Strüder-Kypke et al., 2000b). Yet, clearly, much work remains to be done!