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2001; Kusch, Welter, Stremmel, & Schmidt, 2000; Mollenbeck, 1999). Since RAPD fingerprinting depends upon PCR , large numbers of cells are, in principle, not required. However, the technique does have significant problems, including variation introduced due to inefficiencies in the PCR and due to variations in band intensity. For these reasons, more predictable approaches are to be preferred. The techniques discussed so far have all assessed variation based on nuclear genetic variation, which may be more constrained both within and between species. A promising new approach is the \u201c bar- code \u201d gene, mitochondrial cytochrome c oxidase 1 ( cox 1 ), which has been successfully applied to a variety of animal groups (Hajibabaei, Janzen, Burns, Hallwachs, & Hebert, 2006; Hebert, Cywinska, Ball, & DeWaard, 2003; Hebert, Stoeckle, Zemlak, & Francis, 2004). Barth, Krenek, Fokin, and Berendonk (2006) demonstrated that cox 1 could be effectively used to separate out several Paramecium species, with interspecific divergences ranging from 12\u201327%, while Lynn and Strüder-Kypke (2006) and Chantangsi, Lynn, and Brandl (2007) have demonstrated simi- lar levels of divergence in cox 1 between species of Tetrahymena that are identical based on the SSrRNA gene sequence. Barth et al. (2006) showed signifi- cant intrahaplogroup variation within Paramecium caudatum and Paramecium multimicronucleatum , suggesting that these species may, in fact, be sibling species complexes , while Chantangsi et al. (2007) have demonstrated that isolates of Tetrahymena iden- tified to species on the basis of isozyme patterns have apparently been misclassified. 3.1.6 Summary The approaches presented above provide different methods of assessing variation within species and between species within genera. We cannot recom- mend one of these approaches over another. Rather, a modern description of a new species of ciliate should, where possible, include data provided by observa- tion of living organisms, stained organisms, and gene sequence data (e.g. see Agatha, Strüder-Kypke, Beran, & Lynn, 2005; Modeo, Petroni, Rosati, & Montagnes, 2003; Rosati, Modeo, Melai, Petroni, & Verni, 2004). Comparison of these datasets with previous descriptions should then enable one to conclude whether an isolate is indeed new. As our databases of gene sequences increase, it has been demonstrated that fluorescence in situ hybridiza- tion can be used to identify species (Fried, Ludwig, Psenner, & Schleifer, 2002), and environmental gene sequences can be linked to morphology using both light and scanning electron microscopy (Stoeck, Fowle, & Epstein, 2003). While body size is important, body size on its own is seldom sufficient to distinguish a species. Indeed, there are many other quantitative traits not correlated with size that may ultimately be dis- criminatory. Just as there are no hard and fast rules for determining whether an isolate is a new spe- cies, it is also difficult to provide any for the genus level. In general, one can say that genera should be differentiated on the basis of significant qualitative characters. And one may reasonably ask \u2013 what is a significant qualitative character? Again, there are no hard and fast rules, and what characters are con- sidered important may depend upon whether the taxonomist is a \u201c lumper \u201d or a \u201c splitter \u201d \u2013 what is a significant qualitative character for a \u201csplitter\u201d may 3.1 At the Genus-Species Level 81 82 3. Characters and the Rationale Behind the New Classification not be so for a \u201clumper\u201d (Corliss, 1976). In general, it is our view that \u201csignificant\u201d at the generic level should at least included qualitative differences in body shape, pattern of the somatic kineties, and organization of the oral structures . As noted in Chapter 1, oral variations are likely to directly affect growth and reproductive rates, enhancing the relative fitness and fixation of new oral variants (Lynn, 1979b). Thus, it is often the case that new genera are distinguished on the basis of variations in oral features, as well as qualitative variations in somatic features. 3.2 Above the Genus-Species Level Above the level of genus and species, it is even more difficult to provide guidance on what fea- tures can be used to generally distinguish a family, an order, a class, or a subphylum. Corliss (1976, 1979) discussed the \u201c gap size of distinctness \u201d as a conceptual way to identify the discontinuities that separate these higher taxa. As he noted, \u201cone should be able to recognize a gap of \u2018sufficient\u2019 (how defined?!?) magnitude between any two groups of species before proposing their formal separation into different higher taxa\u201d (p. 59, Corliss, 1979). Indeed, it is often the case that higher taxa show these discontinuities with respect to each other, and they often exhibit what Corliss (1979) termed a shared \u201c constellation of characters \u201d, which fur- ther supports their separation. While a \u2018sufficient\u2019 gap size of distinctness and a shared constellation of characters often characterize higher taxa, there must be at least one synapomorphic or shared derived character that can be used to establish the monophyly of the group. Thus, to identify major monophyletic clades, we must ultimately search for characters that are highly conserved over time. As Lynn (1976a, 1981) has argued, conservation of biological structure, espe- cially in regard to the ciliate cortex, becomes more conserved as we investigate lower levels of biological organization (i.e., organellar complexes , organelles ), which we discuss in more detail below (see 3.2.1 ULTRASTRUCTURE, ESPECIALLY OF THE CORTEX). These highly conserved \u2018characters\u2019 may also be morphogenetic sequences or developmental patterns, which appear as structural similarities, especially in the division ontogeny of ciliates, unit- ing different major taxa into higher assemblages (see 3.2.2 MORPHOGENETIC PATTERNS). In the present day, the ultimate signals of common descent are the primary and secondary structures of gene and amino acid sequences (see 3.2.3 GENE AND PROTEIN SEQUENCES). 3.2.1 Ultrastructure, Especially of the Cortex Since the late 1960s and early 1970s, electron microscopic investigations of ciliates have provided a substantial increase in the number of characters available to determine relationships. As argued in Chapter 1 and elsewhere (Lynn, 1976a, 1981), there are good reasons to believe that similarities at this level of biological organization reveal much more ancient common ancestry. The diversity of somatic and oral kinetids of ciliates has been described (Grain, 1969, 1984; Lynn, 1981, 1991; de Puytorac & Grain, 1976). Lynn (1976a, 1979a, 1981) has argued that somatic kinetid features are more strongly conserved than oral features (Fig. 3.4). Application of these criteria \u2013 lower levels of bio- logical organization more conserved and \u201csomatic over oral\u201d \u2013 has enabled us to establish a number of the major classes of ciliates (Lynn & Small, 1997, 2002; Small & Lynn, 1981, 1985). While cortical characters have been of primary importance, the fine structure of other features has also been helpful: variations in the particle distribu- tions on ciliary membranes (Bardele, 1981) and in the substructure of extrusomes , like toxicysts and tri- chocysts (Hausmann, 1978; Rosati & Modeo, 2003). The multitude of ultrastructural characters has meant that several studies have used both phenetic and cladistic approaches assisted by computer to assess relationships among ciliates. These stud- ies have ranged from a broad assessment at the phylum level (Lynn, 1979a; de Puytorac, Grain, & Legendre, 1994; de Puytorac, Grain, Legendre, & Devaux, 1984) to focussed treatments of classes and orders (Lipscomb & Riordan, 1990, 1992). Nevertheless, there are clear signs that mor- phostatic