STEPHANOMERIA EXIGUA Ssp. CORONARIA THAT FLUCTUATES in POPULATION SIZE1

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GENETIC STABILITY IN A PERIPHERAL ISOLATE OF STEPHANOMERIA EXIGUA ssp. CORONARIA THAT FLUCTUATES IN POPULATION SIZE1

L. D. GOTTLIEB Department of Genetics, University of California, Davis 95616 Manuscript received September 5, 1973

ABSTRACT Allelic frequencies did not change at five polymorphic loci in seedlings grown from seeds collected in four consecutive years in a geographically peripheral population of the annual plant Stephanomeria ezigua ssp. coronaria (Compositae) , even though the population number fluctuated during this period by 50:i. Genetic stability is attributed to buffering effects provided by seed storage in the ground. Evidence described elsewhere suggests that this population was the recent progenitor of a new diploid species. The present re- sult indicates that fluctuations in number of individuals in the parental population were probably not involved in the origin of the new species.

ISOLATED populations on the geographical periphery of widespread species often undergo drastic and unpredictable expansions and contractions in numbers of individuals. Such fluctuations have been considered to set the stage for the establishment of novel genetic features and for speciation (CARSON1968; LEWIS1962, 1973). Evidence has been presented elsewhere (GOTTLIEB1973a) that a peripheral isolate of the annual plant Stephanomeria exigua ssp. coronaria (Compositae) in eastern Oregon was the recent progenitor of a new diploid species with which it is still sympatric. The derivative species, presently called “Malheurensis,” is self-pollinating and very similar morphologically to ssp. coronaria. Electrophoretic analysis demonstrated that its gene pool is a highly limited extraction of the alleles present in the progenitor population. The progenitor population (as well as the derivative) experiences considerable yearly fluctuations in number of individuals in response to certain climatic factors, particu- larly the amount and timing of precipitation. Consequently, it was worthwhile to determine whether these fluctuations affect its allelic frequencies and could have been implicated in the origin of “Malheurensis”. The environmental conditions required for seed germination and seedling survival are often not satisfied at the study site in many years because of insuffi- cient and irregular rainfall. However, since seeds remain viable for long periods of time (following storage in the laboratory, 10-20% of field-collected seeds are viable after five years), it is likely that the soil at the study site contains a pool of seeds contributed over many generations, with the mating group of any one

Supported by National Science Foundation Grant GB 29484X.

Genetics 76: 551-556 March, 1974. 552 L. D. GOTTLIEB year comprised of individuals produced in different generations. The' data presented in this paper document that the genetic input into this seed pool is stable from year to year regardless of the number of individuals in the mating population.

MATERIALS AND METHODS Stephanomeria exigua ssp. coronaria is encountered in a diverse series of habitats and plant communities from eastern Oregon and western Idaho south to the islands off the California coast near Santa Barbara (GOTTLIEB1971). The subspecies is diploid, has annual generations, and possesses a sporophytic self-incompatibility system making it an obligate outcrosser. The population described in this report is the most northern one known in the subspecies; it occupies a site approximately 50 acres in extent, located 25 miles south of Burns, Harney County, Oregon. The seeds of the study population must be exposed to cold temperatures before they can germinate (GOTTLIEB1973a). Consequently, seeds presumably germinate in early spring, and the seedlings grow initially as rosettes. After several months, the rosettes send up a central stem which develops side branches that bear the flowering heads. Flowering begins by early July and seeds are produced until the plants die in early September. In additiosn to exposure to low temperatures, an adequate precipitation in the spring is required for germination and seedling survival; but in eastern Oregon rainfall is highly unpredictable. Climatalogical data, available from the U.S. Weather Bureau Station at the nearby Malheur National Wildlife Refuge Headquarters, show that April, May and June, 1971, were wet with a total of 3.13 inches; the adult population in that year numbered approximately 25,000 individuals. The same months in 1972 were much drier, with a total of 1.06 inches, and the population numbered fewer than 500 individuals, a 50: 1 reduction. Average precipitation (35 years of data) for these three months is 2.64 inches. Census estimates were not made for earlier years except that following the dry spring (1.78 inches) of 1968, fewer than 100 individuals were observed. When precipitation is nearer to average values, my observations suggest that the mating group probably approximates 5000 individuals. Seeds collected from individuals growing in the field in 1969, 1970, 1971 and 1972 were available for study. The seeds were taken from plants sampled at ten-foot intervals along transects over the entire extent of the population. After germination in the laboratory, the seedlings were assayed by horizontal starch gel electrophoresis for three loci controlling glutamate oxaloacetate transaminases (GOT) and two controlling esterases (EST). Techniques are described in GOTTLIEB (1973a). These five loci were selected because they are highly polymorphic and their enzyme patterns can be unmistakenly determined. Formal genetic analysis o'f the electrophoretic patterns of the transminases has been reported (GOTTLIEB1973b), and the genetics of the esterases will be reported elsewhere. Twelve additional loci responsible for variation in electrophoretic mobility of enzymes, of which three are polymorphic by the five percent criterion, have been identified in this population (GOTTLIEB1973a and unpublished). The patterns governed by these three loci are not consistently readable and they are not included in this study. The data presented here for 1971 differs in some respects from that reported previously (GOTTLIEB1973a) which also was based on seeds of 1971. EST-I and EST-2 are now designated EST-2 and EST-3, respectively, since a third locus controlling more anodal esterases has been detected, and allele 43 is now called allele 44. The small differences in allelic frequencies between the two reports reflect the smaller sample size previously studied, differences in observed frequencies of rare alleles, and the improved resolu- tion obtained in the more recent gels.

RESULTS AND DISCUSSION The data inTable 1 demonstrate that even though the population of S. ezigua ssp. coronaria numbered approximately 5000 individuals in 1969 and 1970, flushed to 25,000 in 1971, and crashed to fewer than 500 in 1972, allelic frequencies at all loci remained remarkably stable. The chi-square test of independence GENETIC STABILITY IN STEPHANOMERIA 553

TABLE 3

Allelic frequencies obserued for five loci in seedlings grown from seed dlected in each of four years in a peripheral populution of Stephanomeria exigua ssp. coronaria

Year Locus and allele 1969 (12,16) 1970 (14,25) 1971 (43,137) 1972 (52,125) GOT-1-5 1 0.31 0.24 0.18 0.20 -54 0.69 0.76 0.82 0.80 GOT-2-31 - 0.02 0.01 0.02 -38 1.00 0.98 0.99 0.98 GOT-3- 8 0.03 - 0.01 0.01 -1 1 0.13 0.19 0.16 0.19 -1 9 0.84 0.81 0.83 0.80 EST-- - - -

The numbers in the parentheses are the average number of plant families sampled and the total number of individuals in these families. The alleles are identified according to the migration in mm of their enzyme products. * Frequency of homozygous individuals only. calculated on the basis of observed numbers of alleles showed no significant changes over years: for GOT-I, x;= 3.88, P < 0.25; for GOT-2, x:= 1.93, P < 0.50; for GOT-3, xi= 5.01, P < 0.50. For the more highly polymorphic esterase loci, the calculations were made using the two most frequent alleles and pooling the additional alleles in low frequency into a single class: for EST-2, xi = 10.97, P < 0.05; and for EST-3, x: = 7.59, P < 0.25. The small fluctuations which occurred may reflect sampling errors attributable to the much smaller number of seeds available for the 1969 and 1970 samples, or possible differences in seed viability (not seedling survival since following germination seedling survival was uniformly high for all four age classes). The important observation is that regardless of the size of the mating group in the different years the allelic frequencies of their progenies fluctuated with a very narrow amplitude and that even rare alleles were not lost. Comparison of observed and expected numbers of heterozygous individuals suggests that the population is mating at random (Table 2). Only EST-2 showed a significant heterozygote deficiency but, since genetic analysis has identified 5 54 L. D. GOTTLIEB TABLE 2

Proportion and number of observed and expected individuals heterozygous for five loci in seedlings grown from seed collected in four consecutive years in a population of Stephanomeria exigua ssp. coronaria

Year Locus 1969 1970 1971 1972 GOT-I ob) 0.25(4) 0.41(11) 0.26 (35) 0.23 (29) * ex) 0.43 (6.9) 0.36(9.7) 0.30( 41 .I ) 0.32(40.6) GOT-2 ob) 0.00 0.04(1) 0.02(3) 0.05 (6) ex) 0.00 om(1.1) 0.012(2.7) 0.04 (5.0) GOT-3 ob) 0.31(5) 0.23 (6) 0.25 (37) 0.27 (34) ex) 0.28(4.5) 0.31(8.1) 0.29 (42.3) 0.32(40.3) EST-2 ob) 0.40(6) 0.4Q(9) 0.32(42)*** 0.30(38)*** ex) 0.57(9.1) 0.57( 11.9) 0.61 (81.1) 0.61 (77.5) EST-3 ob) 0.53(8) 0.48(11) 0.48(63)* 0.50(59) ex) 0.58(8.7) 0.65(15.0) 0.63 (82.5) 0.60(70.2)

* Significant, P < 0 025, *** P < 0.001. a null allele at this locus ( GOTTLIEBunpublished), it is likely that the deficit can be ascribed to the inability to recognize the heterozygous null individuals. The number of seeds that germinate and produce individuals that survive to maturity in an annual plant population is generally considered to depend largely on the number of available “safe sites” (HARPERet al. 1961) which, in turn, depends on moisture availability in the spring, adequate temperature, and other micro- and macro-environmental factors. From this ecological point of view, a population builds up a seed pool because the appropriate environmental factors permitting germination and seedling survival are not present every year. In this population of ssp. coronaria, an average plant produces, on a conservative esti- mate, about 1000 seeds. Since the number of plants per year may average 5000, the seed pool in the soil may contain at least lo6 seeds, many times more than the number of safe sites. If the degree of genetic variation among seeds is small relative to the chance environmental factors that permit individual seeds to germinate and produce reproductive individuals, then the large seed pool func- tions as a brake that retards or perhaps even prevents genetic responses, at least for the short term. This inertial effect would operate for a number of generations even when the genetic input to the seed pool differs from its own genetic consti- tution. For example, HARPER(1957) has pointed out that seed storage in the ground has provided stability for weeds of agricultural fields even though cul- tural practices have changed profoundly. The observation that genetic stability characterizes seed input suggests that regardless of any differential effects of natural selection acting on individuals in the population, there is little net change in the population genotype. Analysis of allelic frequencies in seedlings and individuals that mature in the field can test GENETIC STABILITY IN STEPHANOMERIA 555 this directly. In sum, the data indicate that over the present four-year period the pace of evolution is very slow in this peripheral population of ssp. coronaria. Similar results were obtained for blue and white flowered plants of the desert annual Linanthus parryae, which maintained a stable population phenotype for flower color over a 15-year period that included severe fluctuations in population size ( EPLING,LEWIS and BALL1960). Relative to the central populations in California, the study population has had to adapt to more rigorous environmental conditions. The cold temperature requirement for breaking seed dormancy is a key adaptation in this regard and one which is absent from Californian populations. In California, seeds of ssp. coronaria remain viable for a number of years, but they germinate readily under the cool moist conditions regularly occurring in the fall months. Seed dormancy in the Oregon population was presumably selected as a means of assuring that when germination occurs, plants survive to maturity. The acquisition of this adaptation not only shifted germination from the fall to the spring but, since the amount of rainfall in the spring is notably irregular, seeds probably also were selected that had some means of “measuring” if the available moisture in the soil was sufficient for seedling survival. Seed storage permits the population to maintain whatever genetical, physiological and developmental devices it uses to adapt to its harsh habitat without disturbance brought about by genetic drift. Thus, even though the population inhabits an unpredictable environment which directly affects the size of its annual mating group, it is protected from having to respond genetically. Seed storage is a common adaptation in plants. Its prevalence appears to limit the utility of the population flush and crash for the release of genetic variability or the origin of new species (CARSON1968). It is noteworthy that species of Clarkia are among the few annual plants in which severe reductions in population number may be associated with certain factors that permit speciation and, in these species, very little or no seed storage occurs (LEWIS1962). Since expan- sion and contraction of the Oregon population of S. ezigua ssp. coronaria have little short-term genetic effect, there is no need to postulate that fluctuations in population size were required for the origin of the derivative species. Para- doxically, the new species may have arisen in a population which itself was not evolving.

LITERATURE CITED CARSON,H. L., 1968 The population flush and its genetic consequences. In: Population Biology and Evolution. Edited by R. C. LEWONTIN.Syracuse Univ. Press, Syracuse, N. Y. EPLING,C., H. LEWIS and F. BALL, 1960 The breeding group and seed storage: a study in population dynamics. Evolution 14: 238-255. GOTTLIEB, L. D., 1971 Evolutionary relationships in the outcrossing diploid annual species of Steephanomeria (Compositae). Evolution 25: 312329. __, 1973a Genetic differentia- tion, sympatric speciation, and the origin of a diploid species of Stephanomeria. Amer. J. Bot. 60: 545-553. -, 1973b Genetic control of glutamate oxaloacetate transaminase iso- zymes in the diploid plant Stephanomeria ezigua and its allotetraploid derivative. Biachem. Genet. 9: 97-107. 556 L. D. GOTTLIEB HARPER,J. L., 1957 The ecological significance of dormancy and its importance in weed control. Proc. IVth Inter’l. Congress of Crop Protection, Hamburg. Vol. 1 : 415-420. HARPER,J. L., J. N. CLATWORTHY,I. H. MCNAUGHTONand G. R. SAGAR,1961 The evolution and ecology of closely related species living in the same area. Evolution 15: 209-227. LEWIS,H., 1962 Catastrophic selection as a factor in speciation. Evolution 16: 257-271. -, 1973 The origin of diploid neospecies in CZarkia. Am. Naturalist 107: 161-170. Corresponding editor: R. ALLARD