Cap 16

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