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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 ISOENZYMES AND BIOCHEMISTRY and 3.1.5 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 \u201chomologues\u201d 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 choice. 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,