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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!