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(Gates & Berger, 1974; Gates, 
Powelson, & Berger, 1975; Powelson, Gates, & 
Berger, 1975). Others have applied these approaches 
to resolving morphological species within genera, 
such as Colpoda (Foissner & Schubert, 1983; 
Lynn & Malcolm, 1983) and Ancistrum (Berger & 
Hatzidimitriou, 1978). Gates (1977, 1978b, 1979) 
assessed the pattern of variation on the ventral 
surface of the hypotrich Euplotes by measuring 
all possible distances between cirri. By converting 
these intercirral distances to a relative frequency 
distribution of scaled intercirral distances, he was 
able to show that these distributions corresponded 
with relationships determined by mating tests. 
 3.1.3 Genetics 
 Interbreeding, in which two populations are considered
to be members of different species if mating tests 
fail to produce fertile offspring, is the essential 
criterion for recognition of a biological species .
Ciliates are usually stimulated to conjugate in 
the laboratory by starvation, the analogue of the 
natural stimulus, which is depletion of the food 
resource. Once starved, known species and mating 
types can be used as testers to identify unknowns. 
Studies recognizing new species of Tetrahymena
(Nyberg, 1981a; Simon, Meyer, & Preparata, 1985) 
and Paramecium (Aufderheide, Daggett, & Nerad, 
Fig. 3.3. A Argyromes of six types, demonstrating the diversity of patterns that can provide significant taxonomic 
character information, particularly at the species level. Top row: the hymenostome Colpidium, the peniculine 
Frontonia, the prostome Bursellopsis; Bottom row: the prostome Pelagothrix, the colpodean Pseudoplatyophrya, and 
the prostome Urotricha. Note that the three prostomes have quite different patterns (redrawn from various sources). 
B Examples of an anterior suture or secant system (top) and two posterior suture or secant systems (bottom)
3.1 At the Genus-Species Level 79
80 3. Characters and the Rationale Behind the New Classification
1983) have used this approach. Genetic techniques 
have been used to explore the biology of the sibling 
species complexes of Euplotes (e.g., Dini & Gianni,
1985; Génermont, Machelon, & Demar, 1985; 
Luporini & Dini, 1977) and the biogeography of 
Paramecium (Komala & Przybos, 1990; Przybos 
& Fokin, 1997). 
 Many morphological species may, in fact, be 
sibling or cryptic species groups (Curds, 1985). 
However, the genetic approach, while most rigor-
ous, is difficult in practice since both a complete 
set of viable reference strains must be main-
tained and the taxonomist must have competence 
with genetic techniques. Fortunately, biochemi-
cal and genetic correlates have now been found 
for several sibling species complexes (see 3.1.4 
GENE SEQUENCES), and these studies provide 
metrics to discover how common cryptic species 
of ciliates are. 
 3.1.4 Isoenzymes and Biochemistry 
 Isoenzymes are enzymatic proteins that share the 
same biochemical function, but they are coded by 
structurally different alleles. This structural differ-
ence is revealed by their differential movement in an 
electrophoretic gel. Based on earlier work on isoen-
zymes (e.g., Allen, Byrne, & Cronkite, 1971; Tait, 
1970), Sonneborn (1975) established Linnean names 
for the sibling species or syngens of the Paramecium 
aurelia sibling species complex . Allen et al. (1983a, 
1983b) have applied this approach to other species 
of Paramecium . Nanney and McCoy (1976) likewise 
established Linnean names for the 12 syngens of the 
Tetrahymena pyriformis sibling species complex, fol-
lowing earlier isoenzyme studies (e.g., see Allen & 
Weremuik, 1971; Borden, Whitt, & Nanney, 1973a, 
1973b). Species of the hypotrich Euplotes (Machelon 
& Demar, 1984; Schlegel, Kramer, & Hahn, 1988; 
Valbonesi, Ortenzi, & Luporini, 1985) and the sti-
chotrich Stylonychia (Ammermann, Schlegel, & 
Hellmer, 1989) have also demonstrated different 
isoenzyme patterns. 
 Genetic diversity between ciliate species is exceed-
ingly great, indicating a considerable evolutionary 
age of species or extremely rapid molecular 
evolution at these isoenzyme loci. Thus, these 
techniques are generally robust and reliable for 
distinguishing and identifying species (but see 3.1.5
GENE SEQUENCES). However, there are two 
major disadvantages to using isoenzymes. First, 
there is the need to have an efficient cultivation 
technique for the species of interest, one that 
yields significant protein biomass to enable resolu-
tion of these molecules. Second, since isoenzyme 
patterns are often complex, there is a strong pos-
sibility that “homologues” are not being identified 
unambiguously. Because of this, and because DNA 
techniques can now be carried out on much smaller 
numbers of cells, even single cells, isoenzymes 
have been displaced as systematic molecules of 
 3.1.5 Gene Sequences 
 Allen and Li (1974) began sequence diversity 
studies on ciliates with their analysis of DNA-
DNA hybridization of Tetrahymena species against 
syngen 1 (i.e., Tetrahymena thermophila ) as the 
reference standard. They showed considerable 
genetic diversity within the genus. However, as 
with isoenzyme techniques, this approach required 
substantial amounts of DNA and therefore was best 
used for ciliates that could be easily cultivated. 
Moreover, the invention of DNA sequencing tech-
nologies (e.g., Sanger, Nicklen, & Coulson, 1977) 
enabled a direct comparison of primary DNA 
sequence similarity between species. This pro-
vides greater resolution than the single percentage 
that DNA hybridization could provide, and also 
obviates the need to maintain cultures to continu-
ally obtain the DNA of the reference standard(s). 
Ultimately the invention of the polymerase chain 
reaction ( PCR ) (Mullis & Faloona, 1987) has 
enabled us to amplify DNA from small numbers 
of cells, even single cells, so that cultivation is no 
longer an absolute necessity for the application of 
molecular genetic techniques. 
 Genetic diversity among Tetrahymena species 
was first compared using small subunit rRNA 
( SSUrRNA ) gene sequences by Sogin, Ingold, 
Karlok, Nielsen, & Engberg (1986a), who cloned 
the SSUrRNA genes and demonstrated sequence 
identity in some species pairs and up to 33 
differences between others. The histone H3II/
H4II regions of the Tetrahymena genome were 
amplified by PCR and sequence analyses demon-
strated relationships among species similar to those 
derived from SSUrRNA comparisons, and further-
more differentiated all species uniquely (Brunk, 
Kahn, & Sadler, 1990; Sadler & Brunk, 1992). 
A similar approach using a portion of the large 
subunit rRNA ( LSUrRNA ) gene showed consider-
able genetic diversity among Tetrahymena species, 
and generally corroborated groupings based on 
other molecular methods (Nanney, Meyer, Simon, 
& Preparata, 1989; Nanney, Park, Preparata, & 
Simon, 1998; Preparata et al., 1989). Paramecium
species can be distinguished using SSrRNA gene 
sequences (Strüder-Kypke, Wright, Fokin, & Lynn, 
2000a) and heat shock protein 70 (Hori, Tomikawa, 
Przybos, & Fujishima, 2006). 
 In addition to direct sequence comparisons, PCR 
has also been used to generate randomly ampli-
fied polymorphic DNA ( RAPD ) and has been used 
in conjunction with restriction enzymes to digest 
SSUrRNA. Both approaches generate fragments of 
varying length that provide patterns diagnostic for 
species. Jerome and Lynn (1996) showed that different 
Tetrahymena species could be identified by discrete 
 restriction fragment length patterns or riboprints . The 
application of RAPD fingerprinting has been used to 
assess differences among Paramecium (Fokin, Stoeck, 
& Schmidt, 1999; Skotarczak, Przybos, Wodecka, & 
Maciejewska, 2004; Stoeck & Schmidt, 1998; Stoeck, 
Przybos, Kusch, & Schmidt, 2000a, Stoeck, Welter, 
Seitz-Bender, Kusch, & Schmidt, 2000b) and Euplotes
species (Chen, Song, & Warren,