Interspecies Relationships in the Paramecium aurelia Complex: Acid Phosphatase Variation

Up to five zones of acid phosphatase activity appear in gels after electrophoresis of detergent-treated extracts from 13 of the 14 species of the Paramecium aurelia complex. The overall pattern is somewhat similar for all species; differences in intensity and mobility of individual zones permit the grouping of these sibling species into eight groups. All 14 species can be identified using the procedure of enzyme electrophoresis, although two of them are more similar than is usually the case. Problems of misclassification are discussed in terms of the nature and frequency of variants. With the judicious choice of enzymes used to screen new stocks, these problems can be circumvented. Species relationships are updated using 11 enzymes. A dendrogram constructed from the matrix of genetic distances shows four clusters of species: (i) P. biaurelia, P. triaurelia; (ii) P. primaurelia, P. pentaurelia, P. sexaurelia, P. novaurelia; (iii) P. septaurelia, P. undecaurelia, P. tredecaurelia, P. quadecaurelia; and (iv) P. tetraurelia. P. octaurelia. P. decaurelia, P. dodecaurelia. Distances between the species are large, on the order of the differences between Drosophilu species. The species are characterized by an extraordinary lack of geographical differentiation and great morphological similarity, which contrasts strongly with the molecular differentiation. SSESSING biological relationships- among organisms that A show few morphological differences-by use of morpho- logical criteria alone may lead to gross underestimation of their true evolutionary distance. This problem is particularly severe in microorganisms including the ciliated protozoa, which, in addition, lack a fossil record. Originally, the taxon "Paramecium aurelia" was used to lump together a number of organisms that were morphologically sim- ilar but between which gene flow did not always occur. As more information became available, particularly through application of biochemical techniques, classification of this group of organ- isms achieved finer resolution. At first, the 14 subgroups were called "varieties" on the basis of specificity of the mating types, that is, the specific selectivity of the breeding relations (see 30, and references therein). Subsequently the 14 varieties were des- ignated "biological species," or "syngens," but not given species status, on the grounds that mating type identification was dif- ficult to learn and apply to new strains and that it was undesir- able to base identification on living material that had to be maintained in the laboratory and could easily be lost or mixed up (30). When it became possible to freeze and store paramecia in liquid nitrogen efficiently, and the syngens could be identified by simple biochemical procedures (such as starch gel electro- phoresis of enzymes), species status was at last given to the 14 syngens (3 1, 32), now considered sibling species of an "aurelia complex." Previous work on intra- and interspecies enzyme variation in this complex has enabled all 14 species to be distinguished on the basis of phenotypic differences observed for nine enzymes (4-6, 34, 36). Seven out of nine of these enzymes show intra- specific variation that is controlled by alleles at seven loci, EstA, EstB, EstC,, HBD, IDH,, IDH,, GPI (7, 8, 19, 33, 35, 36). Intraspecific variation has also been observed for three other enzymes, anodal esterase C (EstC,), acid phosphatase (Acp), and malic dehydrogenase (MDH), but genetic studies have been carried out only for MDH (14, 37). EstC, is not found in all species, and MDH has been examined only in P. tetraureha. Acp is found in all species examined and is the subject of this paper. For some enzymes the variant forms are unique and do not coincide in mobility with those observed in interspecies comparisons (34). For the esterases the majority of the variants in P. primaurelia, P. tetraurelia, and P. octaurelia possess an electrophoretic mobility characteristic of a subtype observed in another species, whereas the mobilities of most of the variants in P. biaurelia are unique, as are the common forms in this species (8). About half of the variant forms for GPI coincide with forms found in other species (36). The frequency of intra- specific variation differs among enzymes (see Table VIII in Ref. 14). Less than 2% of the stocks were variant for seven enzymes, and 3-7% were variant for four other enzymes in the species complex as a whole, with P. biaurelia showing hypervariability for EstA and EstC,. The frequency of variation was higher for GPI, 12.4% (36), and similar to that observed for MDH, mtDNA (mitochondria1 DNA), and rDNA (ribosomal DNA) in P. tet- raurelia (24, 2 5, 3 7). Interspecies comparisons at nine enzyme loci led to estima- tion of genetic distances between the species of the aurelia com- plex (1). Not only were the differences between species large, but dendrograms constructed from the matrix of genetic dis- tances indicated a more complex set of relationships between the species than that originally proposed by Sonneborn. Com- parison of the genetic distances for this complex with similar values obtained for the Tetrahymena PYrlformis complex and Drosophila sibling and nonsibling species led us to strongly urge species status for members of the P. aurelia complex. Electrophoretic resolution of the acid phosphatases in the P. I Supported by a research grant, GM-15879, from the National In- stitute of General Medical Sciences, U.S. Public Health Service. We thank and acknowledge cont~butions of Robert Preston and Robert Elkus who explored and developed methods for the extraction and electrophoresis of the acid phosphatases of Paramecium. We also thank Almuth H. Tschunko for drawing Fig. 1. This is the third in a series of papers dedicated to the memory of Tracy M. Sonneborn.