High Frequency of Sex and Equal Frequencies of Mating Types in Natural Populations of the Ciliate Tetrahymena Thermophila F

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8715-8718, September 1995 Population Biology

High frequency of sex and equal frequencies of mating types in natural populations of the ciliate Tetrahymena thermophila F. PAUL DOERDERt, MICHAEL A. GATES, FRANK P. EBERHARDT, AND MUHIrTIN ARSLANYOLU Department of Biology, Cleveland State University, Cleveland, OH 44115 Communicated by John R. Preer, Jr., Indiana University, Bloomington, IN, June 5, 1995

ABSTRACT In ciliate protists, sex involves the temporary To determine the incidence of sex and the frequencies of joining of two cells of compatible mating type, followed by multiple mating types in natural populations of Tetrahymena meiosis and exchange of gametic nuclei between conjugants. thermophila, we have extensively sampled ponds in which T. Reproduction is by asexual binary fission following conjuga- thermophila is resident. tion. For the many ciliates with fixed multiple mating types, frequency-dependent sex-ratio theory predicts equal frequen- MATERIALS AND METHODS cies of mating types, if sex is common in nature. Here, we report that in natural populations of Tetrahymena thermophila Study ponds are located in the Allegheny National Forest of sexually immature cells, indicative of recent conjugation, are northwestern Pennsylvania. Ponds are located 41.5° north found from spring through fall. In addition, the seven mating latitude, 78.9° longitude at -580 m above sea level and were types occur in approximately equal frequencies, and these constructed between 1950 and 1970. Ponds are spring fed, with frequencies appear to be maintained by interaction between the pH of the water ranging from 4.2 to 6.4. Samples of pond complex, multiple mat alleles and environmental conditions water (usually 150 ml but sometimes 30 ml) were collected and during conjugation. Such genotype-environment interaction placed in plastic bags in the field. Proteose peptone, penicillin determining mating type frequency is rare among ciliates. G, and streptomycin (0.1%, 3 mg/ml, and 3 mg/ml, respec- tively) were subsequently added to eliminate most nontetra- Sex is an extraordinarily successful eukaryotic invention usu- hymenine species. Two to three days later, bags were scored for ally associated with reproduction. However, in ciliates, repro- the presence of Tetrahymena-like cells, and from positive bags, duction is by binary fission, and sex is limited to the temporary 1-12 (usually 4) single cells were cloned into fresh medium conjugal union of two cells for purposes of recombination and [0.15% (wt/vol) cereal grass inoculated with Klebsiella pneu- rejuvenation-i.e., micronuclear exchange and macronuclear moniae]. Each clone was then tested for mating type (I-VII) and replacement. To recognize suitable partners, ciliates are dif- for the major cell surface immobilization serotype (Hi, H2, H3, ferentiated into mating types, a kind of self-not-self discrim- H4, J, K, or L) by standard procedures (11, 12). Mating-type ination system, as opposed to true sexes. Hurst and coworkers alleles were extracted in homozygous form by crossing isolates to (1, 2) have suggested that by exchanging only gametic nuclei at inbred genomic exclusion strain A*III (13, 14) to yield whole- conjugation, ciliates minimize conflict between cytoplasmic genome homozygotes (thus bypassing inbreeding). Genetically genomes and, therefore, are free to evolve multiple mating identical homozygotes were then crossed among themselves at types to maximize the choice of sexual partners. Simple 18°C, 28'C, and 37'C. After 3-10 fissions at the experimental extension of frequency-dependent sex-ratio theory suggests temperature (in which macronuclear development was complet- that multiple but fixed mating types should be equally frequent ed), cultures were returned to a constant 28'C, where a single cell in breeding populations, a suggestion supported by two theo- (karyonide) from each pair was serially transferred to sexual retical studies. Orias and Rohlf (3) constructed a deterministic maturity. Mating type was determined by mating each clone with model for three alleles at a single locus and found that there all seven mating type testers. is a stable equilibrium in which mating types are equally frequent. In a more general study, Iwasa and Sasaki (4) found RESULTS that evolutionary consequences greatly depend upon mating kinetics and sex-determining mechanisms. When opportuni- Ecological Distribution. T. thermophila is most commonly ties for finding suitable conjugal partners are temporally found in decaying vegetation at the mud-water interface and limited, the number of sexes-i.e., mating types-increases. in sympatry with other, unidentified Tetrahymena species (Fig. There is, however, limited information on ciliate sex and 1A). The frequency of positive samples varies directly with mating types in natural populations (5, 6) with which to test temperature, presumably as a consequence of bacterial food these models. supply. Ciliates typically have two types of nuclei: a germinal, Seasonal Distribution of Immature Isolates Indicating Re- diploid micronucleus capable of meiosis and mitosis and a cent Conjugation. The life cycle of T. thermophila includes an somatic, compound macronucleus controlling the phenotype immature phase after conjugation, during which cells are of the cell. At conjugation, haploid gametic nuclei meiotically unable to mate and therefore do not react with mating-type derived from the micronuclei are reciprocally exchanged, and, testers; the immaturity period typically is 60-100 fissions (15). following fertilization, new macronuclei are derived from Using T. thermophila-specific antisera (16), we were able to mitotic products of zygotic micronuclei, while the old macro- identify T. thermophila independent of the state of maturity nuclei are destroyed (7). Replacement of the macronucleus not (Fig. 1B). Immature cells, indicative of recent conjugation, only initiates a new life cycle that may include change in mating were found throughout the collecting season, with two appar- type but also is associated with rejuvenating (8) and heterotic ent peaks: one in late spring/early summer and one in late effects (9, 10). summer/early autumn. Because - 15-20 fissions elapsed between collection and testing isolated clones for maturity, the shown in Fig. 1B are underestimates of the true The publication costs of this article were defrayed in part by page charge frequencies payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8715 Downloaded by guest on September 26, 2021 8716 Population Biology: Doerder et al. Proc. Natl. Acad. Sci. USA 92 (1995) A B

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FIG. 1. (A) Fraction of samples positive for the presence of Tetrahymena-like species (Tetrahymena) and fraction of positive samples containing T. thermophila (th) collected at the surface (ws) or at the bottom (wm) at depths of 15-30 cm. The number of samples is indicated in parentheses. Pond (i) and average daily high/low air (E) temperatures (for Warren, PA) are for 1987-1993. (B) Seasonal distribution of immature T thermophila isolates in 1987-1992 and 1993. All clones reacting with T thermophila-specific antisera were tested for mating type by using all seven mating types. Mature clones reacted with one or (usually) more mating-type testers. Immature clones failed to react with any of the testers. Randomly selected clones eventually became mature (10-100 fissions), confirming the validity of the method. The number of samples is indicated in parentheses. (C) Frequencies of mating types I-VII in three study ponds (bars, sample sizes in parentheses) and throughout the region (line, n = 5822). (D) Seasonal distribution of mating types in pond SG29. Sample sizes are shown in parentheses. (E and F) Effect of temperature on mating-type frequencies for mat-6 (E) and mat-7 (F) homozygotes. Temperatures were 18°C, 28°C, and 37°C. Sample size of karyonides is shown in parentheses. Line shows distribution of mating types in pond SG29. frequency of immaturity. Immature isolates required an ad- Samples also frequently yielded more than one mating type. ditional 10-50 fissions to become mature. The seasonal fre- For example, among 495 samples from which four or more quencies of immature isolates-e.g., >25%-indicate fre- clones were isolated, 211 (42.6%) contained two or more quent conjugation. mating types. As expected, samples with multiple mating types Equal Frequencies of Multiple Mating Types. Because were more common in July, when the density of T. thermophila mating-type frequencies determine the probability that two appears to be the highest. Among 87 bottom samples, from mature cells are compatible, we also have examined mating- which 12 clones were isolated, 63 (72.4%) contained four or type distribution among 5822 clones of T. thermophila isolated more mating types and 2 (2.3%) contained all seven mating from these ponds (Fig. 1C). All seven previously known mating types. Among 42 control (surface) samples, 23 (54.8%) con- types (15) were present in each pond sampled (including ponds tained four or more mating types. This difference is significant not shown in Fig. 1). The seven mating types occurred in (G = 16.129, df = 6, P < 0.05) and is consistent with increased approximately equal frequency in ponds throughout the study localized abundance around food sources. area, thus tending to maximize the probability that two mature Multiple Mating Type Alleles. Studies on inbred strains cells are of complementary mating type. show that each mat allele specifies a frequency distribution of Downloaded by guest on September 26, 2021 Population Biology: Doerder et aL Proc. Natl. Acad. Sci. USA 92 (1995) 8717 Table 1. Frequency distributions for T. thermophila mating-type alleles at 28°C Frequency of mating type Allele Pond Sample size, n I II III IV V VI VII mat-4 CRWP 96 0.47 0.31 0.06 0 0.09 0.06 0 mat-S CRWP 63 0 0.18 0 0.38 0.06 0.32 0.06 mat-6 SG29 185 0 0.30 0 0.26 0.26 0.09 0.08 mat-7 SG29 78 0 0.36 0.01 0.26 0.18 0.077 0.12 Frequencies were obtained from homozygotes as described in Materials and Methods. Zero (0) values for I, IV, and VII identify type A and B alleles (see text), whereas III is found under other circumstances (see Fig. 1 E and F). five or six possible mating types (15, 17, 18). These alleles fall immature T thermophila (16), supporting the notion that into two categories distinguished by the critical mating types I, conjugation occurs with some seasonal periodicity. IV, and VII. In the A category, IV and VII are never All seven mating types of T. thermophila were found in all expressed; in the B category, I is never expressed. Relative of the ponds sampled. No eighth mating type was found, nor frequencies of mating types further distinguish among mat has an eighth type been found in other collections (18) (F.P.D., alleles. We have identified what appear to be at least six mat unpublished data), implying a closed set of seven mating types. alleles, each falling into either the A or B category and thus These seven mating types are specified by mat alleles which suggesting that these' two' general patterns may be fixed. resemble previously identified mat alleles. We have identified Mating-type frequencies associated with four of the alleles in at least six putative mat alleles and have preliminary evidence homozygous form are shown in Table 1. Since these frequen- for additional alleles. It is possible that each pond has a unique cies were obtained in each instance by crossing identical s'et of mat alleles (or modifying genes). Although these alleles whole-genome homozygotes, possible effects of modifying fall into the previously identified type A (no IV or VII) and genes were eliminated. These frequencies differ substantially type B (no I) families, they differ substantially in the frequen- from those previously described, particularly with respect to III cies of specified mating types. This is important because and V (see ref. 19). similarity between alleles isolated from Illinois and Massachu- The available data suggest that additional mat alleles exist. setts had led to speculation that, with few exceptions, alleles For example, the frequency of VII associated with mat-6 and within each category differed little in mating-type frequency mat-7 is in both instances lower than the observed frequency (18). of VII in pond SG29 (Fig. 1 E and F). Since any type A alleles The seven mating types occur in approximately equal fre- that must be present would reduce the frequency of VII, there quency throughout the study area. This agrees with Kosaka's must be an allele with a higher frequency of VII. Indeed, we observations for Paramecium bursaria (23) and also with an have preliminary evidence for such an allele (data not shown). extension of frequency-dependent sex-ratio theory, which There also must be an allele with a higher frequency of III and predicts equal frequencies of fixed multiple mating types. A lower frequency of II. Similar arguments apply to ponds rare mating type, for example, should increase in frequency CRWP and 343S. The observation that eight of nine isolates because any cell with that type would have an increased (from which the mat alleles were extracted) were heterozygous probability of finding a compatible conjugal partner. Theoret- for mat alleles (as determined by appropriate crosses) also ical models based on simple multiple alleles (each specifying supports the notion of additional mat alleles. one mating type) support this idea (3, 4). What makes' the Temperature Effects on Mating-Type Frequency. Frequency multiple mating types of T. thermophila unique is that each distributions specified by mat' alleles are affected by environ- mating type (mat) allele specifies a frequency distribution of mental conditions during macronuclear development at con- mating types rather than a single fixed type. Mating type of a jugation, specifically temperature (19, 20) and nutritional state conjugant is erased with the destruction of its old macronu- (21, 22). Such effects generally are allele-specific and do not cleus, and each new macronucleus (karyonide) is indepen- affect every mating type. One such environmental effect is dently fixed for the mating type subsequently inherited through evident with the reciprocal seasonal variation ip frequencies of each macronuclear division during binary fission (15, 17). IV and VII in pond SG29 (Fig. 1D); similar variation was seen We hypothesize that mating-type frequencies in a given in pond CRWP but not in pond 343S (not shown). The effect pond are due to three interacting variables: (i) the frequency of temperature on two of the mat alleles (type B from SG29) of multiple mat alleles, (ii) the array of mating types specified is shown in Fig. 1 E and F. Allelic differences are clearly by each allele, and (iii) the effect ofenvironmental variables on evident (compare III, IV, V, and VI) and give rise to the the determination of mating type during macronuclear devel- hypothesis that the mating-type-frequency distribution in opment. Testing this hypothesis requires characterizing addi- ponds is due to complex interaction between multiple 'alleles tional mat alleles and, because mating-type frequencies are not and environmental conditions' during conjugation. It is also necessarily intermediate in heterozygotes (15, 24), determin- possible that there are genes which modify mating-frequency ing mating-type frequencies in heterozygotes. Additionally, distributions, but evidence for such genes is inconclusive. The genes which modify the frequency distributions specified by effect of nutritional state on the new alleles has not been various mat alleles must be identified. studied. The present results also suggest a unifying model concerning the life history of T thermophila. We postulate that cells are attracted to food sources, where they feed and reproduce. DISCUSSION Among these cells are those of different mating type, as well The frequency and tempo of sex in natural populations of as immature cells that may reach maturity upon further fission. ciliates is little studied. In this large field study of ciliate sex, Upon exhaustion of the food supply, mating occurs, and after we find evidence that conjugal sex occurs frequently in natural sex, with its rejuvenative effects (for discussion see ref. 8), cells populations of T thermophila. Since conjugation is stimulated disperse to new food sources. This model is consistent with the by mild starvation, such frequent conjugation is consistent with chemosensory nature of T thermophila (25) and accounts for local and seasonal variation in bacterial food supply. The peak the higher frequency of multiple mating types in bottom of immaturity in late spring coincides with a conjugation- samples, where food sources are more prevalent. It also is dependent switch in the surface proteins used to identify consistent with the density-dependent nature of costimulation Downloaded by guest on September 26, 2021 8718 Population Biology: Doerder et al. Proc. Natl. Acad. Sci. USA 92 (1995) leading to conjugation (26, 27). Dispersal after conjugation 2. Hurst, L. D. & Hamilton, W. D. (1992) Proc. R Soc. London B also means that cells are less likely to encounter relatives at 247, 189-194. new food sources, therefore facilitating outbreeding (5). We 3. Orias, E. & Rohlf, F. J. (1964) Evolution 18, 620-629. 4. Iwasa, Y. & Sasaki, A. (1987) Evolution 41, 49-65. note that the model fulfills the requirement of Iwasa and 5. Sonneborn, T. M. (1957) in The Species Problem, ed. Mayr, E. Sasaki (4) that high frequencies of multiple mating types are (Am. Assoc. Adv. Sci., Washington, DC), pp. 155-324. possible when there is limited time in which to find a mating 6. Dini, F. & Nyberg, D. (1993) Adv. Microb. Ecol. 13, 85-154. partner, in this case, after starvation and before dispersal. 7. Ray, C., Jr. (1956) J. Protozool. 3, 88-96. However, the mating-type determination mechanism of T. 8. Bell, G. (1988) Sex and Death in Protozoa (Cambridge Univ. thermophila is more complex than considered by previous Press, Cambridge, U.K.). theoretical studies, and no extant theoretical model explains 9. Nanney, D. L. & Doerder, F. P. (1972) Genetics 72, 227-238. at seven in T 10. Doerder, F. P. & DeBault, L. E. (1978) Chromosoma 69, 1-19. the fixity of mating types thermophila. 11. Orias, E. & Bruns, P. J. (1976) Methods Cell Biol. 12, 247-284. In conclusion, it appears that wild T. thermophila mates 12. Smith, D. L., Berkowitz, M. S., Potoczak, D., Krause, M., Raab, often and that interaction between complex mating-type al- C., Quinn, F. & Doerder, F. P. (1992) J. Protozool. 39, 420-428. leles and the environment results in distributions of multiple 13. Allen, S. L., File, S. K. & Koch, S. L. (1967) Genetics 55,823-837. mating types that tend to maximize the probability that any two 14. Weindruch, R. H. & Doerder, F. P. (1975) Mech. Age. Dev. 4, mature cells are sexually compatible. The postulated rejuve- 263-279. nating (8) and heterotic (9, 10) consequences of conjugation 15. Nanney, D. L., Caughey, P. A. & Tefankjian, A. (1955) Genetics observed in the laboratory therefore presumably apply to wild 40, 668-680. 16. Saad, Y. & Doerder, F. P. (1995) Eur. J. Protistol. 31, 45-53. populations as well. 17. Nanney, D. L. (1956) Am. Nat. 90, 291-307. 18. Phillips, R. B. (1968) Genet. Res. 11, 211-214. We thank Dennis Burian, Chris Caprette, Nathan Doerder, Nora 19. Nanney, D. L. (1960) Physiol. Zool. 33, 146-151. Doerder, Greg Everett, Bryan Graham, Tom Jones, Anton Kozelj, 20. Nanney, D. L., Meyer, E. B. & Chen, S. S. (1977) Differentiation Sandy Kulp, Greg LaCrosse, James Little, Manuel Mendoza, Barry 9, 119-130. Mita, Jack Thatcher, and Fred Wolfffor assistance in the field. We also 21. Orias, E. & Baum, M. P. (1984) Dev. Genet. 4, 145-158. thank Ferdinand Apolonio for expert assistance in the laboratory and 22. Nanney, D. L., Meyer, E. B. & Portnoy, S. (1980) Differentiation an anonymous reviewer for suggestions concerning theoretical models. 16, 49-60. This work was supported by personal funds, Research Challenge 23. Kosaka, T. (1991) J. Protozool. 38, 140-148. funding, and by National Science Foundation Grant DEB9220784 to 24. Nanney, D. L. (1959) Genetics 44, 1173-1184. F.P.D. and M.A.G. 25. Leick, V. & Hellung-Larsen, P. (1992) BioEssays 14, 61-66. 26. McCoy, J. W. (1972) J. Exp. Zool. 180, 271-278. 1. Hurst, L. D. (1992) Proc. R. Soc. London B 248, 135-140. 27. Finley, M. J. & Bruns, P. J. (1980) Dev. Biol. 79, 81-94. Downloaded by guest on September 26, 2021