F. Thomas Ledig

GENE DIVERSITY AND GENETIC STRUCTURE IN A NARROW ENDEMIC, TORREY PINE (PINUS TORREYANA PARRY EX CARR.)

Recent reviews have suggested that tree species are the most variable of organisms, as measured by proportion of polymorphic loci or average heterozygosity (Hamrick, 1979; Hamrick et al., 1979). Average heterozygosity is greater than 0.30 for several conifers. By contrast, annual herbaceous species have a mean heterozygosity of 0.13 (Hamrick et al., 1979). Conifers have several mechanisms that promote outcrossing, and are expected to maintain high levels of genetic variation. Despite these mechanisms, it is uncertain whether the breeding system is capable of maintaining variability in small populations and in the absence of migration. While the genetic consequences of reduced population size have long been understood in theory, empirical evidence is scarce in plants, particularly in tree species. The potential to maintain high levels of genetic variation in reduced and scattered populations is important because of its implications for genetic resource conservation, and in fact, for the ability of species to respond to environmental change and avoid extinction. Because most conifers are commercially valued and exploited for lumber and paper products, much of the original forest in North America has been destroyed, and the tendency under management will be to reduce native populations to scattered relicts. One way of forecasting the fate of species reduced in numbers is to make use of natural experiments, by examining the genetic structure of species that occur in disjunct populations (Shaffer, 1981). We chose to investigate how much variation might be preserved in conifers by observing a narrow endemic, Torrey pine (Pinus torreyana Parry ex Carr.). The investigation also contributes to an understanding of the biogeography of the California Channel Islands. Torrey pine has the smallest population of any known pine. The 1973 count in the 445 ha (1,100 acre) Torrey Pines State Reserve on the Pacific Coast at San Diego, California was 3,401 mature trees (Calif. Dept. Parks Rec., 1975). Including seedlings, the Reserves naturalist estimated ca. 7,000 individuals in 1979 (H. Nicol, pers. comm.). The population includes only two other small stands, contiguous with the Reserve. Another population occurs on Santa Rosa Island, one of the Northern Channel Islands off the California coast near Santa Barbara. There may be 2,000 individuals on the northeast coast of the island. Climatic, edaphic, and floristic characteristics of the two sites were summarized by Haller (1967). The San Diego and Santa Rosa Island populations are separated by 280 km and the island is 40 km from the mainland. It is highly unlikely that there has been any significant opportunity for gene exchange within recent centuries. Nor is Torrey pine likely to exchange genes with its closest relatives, digger pine (Pinus sabiniana Dougl.) and Coulter pine (Pinus coulteri D. Don), both of which are allopatric. Controlled crosses with Digger pine succeed only with difficulty and there have been no hybrids with Coulter pine despite several attempts (Critchfield, 1966).

GENETIC DIVERSITY AND POPULATION STRUCTURE IN PITCH PINE (PINUS RIGIDA MILL.)

Electrophoretic studies of protein polymorphisms in plants have focused upon herbaceous species, primarily inbreeding annuals, in efforts to characterize the levels and patterns of genic variation within and between populations (Clegg and Allard, 1972; Gottlieb, 1973, 1975; Levin, 1975, 1978; Levy and Levin, 1975; Schaal, 1975; Roose and Gottlieb, 1976; Brown et al., 1978; and others). These studies have indicated that predominantly outbreeding species maintain higher levels of intrapopulation variation than predominantly inbreeding species, while inbreeders exhibit a greater degree of population differentiation than outbreeders (Brown, 1979; Hamrick et al., 1979). This relationship is by no means perfect as Levin (1978) points out, because of differences in ecological requirements, breeding systems, dispersal mechanisms, evolutionary history, and other factors which affect the genetic system (Grant, 1958, 1971; Brown, 1979; Hamrick et al., 1979). Whether longlived perennials such as forest trees conform to the general pattern is still an open question. Allozyme studies of forest tree species have suggested that levels of genic variation are exceptionally high in natural populations (Tigerstedt, 1973; Rudin et al., 1974; Lundkvist and Rudin, 1977; Yang et al., 1977; Hamrick, 1979; Hamrick et al., 1979; Lundkvist, 1979), that certain populations appear to be moderately inbred (Rudin et al., 1974; Mejnartowicz and Bergmann, 1975; Phillips and Brown, 1977), and that populations have become differentiated over relatively short distances (Sakai and Park, 1971; Mitton et al., 1977). However, many inferences have been drawn from only one or a few loci, or only from loci known to be highly polymorphic. Valid estimates of mating system parameters may be obtained by examining only a few loci, but for estimates of heterozygosity, genic diversity, and the extent of differentiation, a large number of loci is preferred (Lewontin, 1974; Nei, 1975). As part of a continuing study of the genetics and ecology of pitch pine (Pinus rigida Mill.), we have surveyed 21 enzymatic loci in 11 populations across the species range. Pitch pine occurs from coastal Maine and southern Quebec to northern Georgia and from the Atlantic Coast to central Ohio, but almost always on relatively infertile sites. In spite of a history of overexploitation, it appears to have retained appreciable variation (Ledig and Fryer, 1974). Pitch pine demonstrates clinal patterns of variation in cone serotiny (Ledig and Fryer, 1972), wood properties (Ledig et al., 1975), and seedling growth (Ledig et al., 1976), as well as seed and needle characters (Ledig, unpubl.). It is uncertain whether these clinal patterns are the result of gene flow among pockets of differential fitness or reflect a continuous gradient in selection pressures (Endler, 1977; Givnish, 1981). The objectives of the present study were to determine the levels of genic diversity characteristic of pitch pine, and to examine the organization of genic variability within the species and the patterning of genic differentiation between populations. Among other comparisons, we contrasted marginal vs. central populations and the

The relation of growth to heterozygosity in pitch pine

The connection between fitness and heterozygosity has eluded geneticists for decades. The classic form of the Neo-Darwinian argument hypothesizes that heterozygosity confers genetic homeostasis (Lerner, 1954); i.e., multiple, molecular forms of the same enzyme endow the organism with a broader range of tolerance to environmental variation because different forms may differ in their optima for temperature, pH, and other factors (Johnson, 1976). In maize (Zea mays L.), inbred lines and their hybrids are similar in growth rate when raised under constant conditions at optimal temperatures, but hybrid superiority becomes progressively more obvious as conditions deviate from the optimum (McWilliam and Griffing, 1965). Apparently, homozygotes in maize can only deal successfully with a narrow range of conditions as compared to heterozygotes. In a variety of organisms, including butterflies, fish, and oysters, variance for morphological traits decreased with increasing heterozygosity at enzyme loci (Eanes, 1978; Mitton, 1978; Zouros et al., 1980), and bilateral symmetry, a measure of developmental homeostasis, increased with heterozygosity in lizards and bivalves (Soule, 1979; Kat, 1982). Growth rate increased with heterozygosity in oysters and salamanders (Singh and Zouros, 1978; Zouros et al., 1980; Pierce and Mitton, 1982). The perennial herb, cylindric blazing star (Liatris cylindracea Michx.), can be roughly aged by counting rings in the corm, the subterranean perennating organ. In natural populations, heterozygosity was higher among older than among younger individuals, suggesting poor survival for homozygotes (Schaal and Levin, 1976). Grown in a uniform environment, biomass production and reproductive potential of the herb were weakly, but positively, related to the level of enzyme heterozygosity. However, Mitton and colleagues (Mitton and Grant, 1980; Knowles and Grant, 1981; Mitton et al., 1981) noted conflicting results in forest trees, which are among the most polymorphic of organisms (Hamrick, 1979; Hamrick et al., 1979). In only one of three tree species was there a positive association between mean width of the annual ring, taken as a measure of growth and fitness, and heterozygosity. In two of the species, variance in ring width increased with increasing heterozygosity and in the third, it decreased. The relationship between growth and heterozygosity in trees is confusing, and more data are needed to draw firm conclusions. Our objective was to relate growth to heterozygosity in pitch pine (Pinus rigida Mill.), for which we had information on 21 enzyme loci in several populations. We were also interested in whether heterozygotes had greater longevity and whether annual growth was more or less well-buffered in heterozygotes than in homozygotes. A positive association be-

RECENT EVOLUTION AND DIVERGENCE AMONG POPULATIONS OF A RARE MEXICAN ENDEMIC, CHIHUAHUA SPRUCE, FOLLOWING HOLOCENE CLIMATIC WARMING

Fragmentation and reduction in population size are expected to reduce genetic diversity. However, examples from natural populations of forest trees are scarce. The range of Chihuahua spruce retreated northward and fragmented coincident with the warming climate that marked the early Holocene. The isolated populations vary from 15 to 2441 trees, which provided an opportunity to test whether census number is a good predictor of genetic diversity. Mean expected heterozygosity, He, based on 24 loci in 16 enzyme systems, was 0.093 for 10 sampled populations, which is within the range reported for conifers. However, estimates varied more than twofold among populations and He was closely related to the logarithm of the number of mature trees in the population (rHe,N = 0.93). Diversity among populations, FST, was 24.8% of the total diversity, which is higher than that observed in almost all conifer species studied. Neis genetic distance, D, was not related to geographic distance between populations, and D̄ was 0.033, which is higher than estimates for most wide‐ranging species. Most populations had excess homozygosity and the fixation index, FIS, was higher than that reported for all but one species of conifer. Nm, the number of migrants per generation, was 0.43 to 0.76, depending on estimation procedure, and is the smallest observed in conifers. The data suggest that populations of Chihuahua spruce have differentiated by drift and that they are effectively isolated. The results illustrate how a combination of paleontological observation and molecular markers can be used to illuminate recent evolutionary events. Multilocus estimates of outcrossing for two small populations were zero (complete selfing) and 0.153, respectively, which are in striking contrast to the near complete outcrossing observed in most conifers. The high fixation index and a high proportion of empty seeds (45%) suggest that inbreeding may be a serious problem for conservation of Chihuahua spruce.

The Fitness Consequences Of Multiple‐Locus Heterozygosity: The Relationship Between Heterozygosity And Growth Rate In Pitch Pine (Pinus Rigida Mill.)

Positive correlations between measures of “fitness” and the number of electrophoretic loci for which an individual is heterozygous have been observed in many species. Two major hypotheses have been proposed to explain this phenomenon: inbreeding depression and overdominance. Until recently, there has been no way to distinguish between these hypotheses. The overdominance model devised by Smouse (1986) is used here in a reanalysis of Ledig et al.‘s (1983) study of heterozygosity and growth rate in eight populations of pitch pine and is contrasted with an inbreeding‐depression analysis. Ledig et al. (1983) regressed mean growth rate per heterozygosity class on the number of heterozygous loci, a method of analysis which, although it points to general trends in the data, does not differentiate between hypotheses. The correlations they obtained in four populations were significant only because regressing on the means eliminates most of the sum of squares for error and does not weight the unequally sized heterozygosity classes. Reanalysis of Ledig et al.‘s data using individuals, not means, showed no significant correlations between heterozygosity and fitness.

Decoupled mitochondrial and chloroplast DNA population structure reveals Holocene collapse and population isolation in a threatened Mexican-endemic conifer.

Chihuahua spruce (Picea chihuahuana Martínez) is a montane subtropical conifer endemic to the Sierra Madre Occidental in northwestern México. Range‐wide variation was investigated using maternally inherited mitochondrial (mtDNA) and paternally inherited chloroplast (cpDNA) DNA markers. Among the 16 mtDNA regions analysed, only two mitotypes were detected, while the study of six cpDNA microsatellite markers revealed eight different chlorotypes. The average cpDNA diversity (H = 0.415) was low but much higher than that for mtDNA (H = 0). The distribution of mitotypes revealed two clear nonoverlapping areas (GST = NST = 1), one including northern populations and the second one including the southern and central stands, suggesting that these two regions may represent different ancestral populations. The cpDNA markers showed lower population differentiation (GST = 0.362; RST = 0.230), implying that the two ancestral populations continued to exchange pollen after their initial geographic separation. A lack of a phylogeographic structure was revealed by different spatial analyses of cpDNA (GST > RST; and samova), and reduced cpDNA gene flow was noted among populations (Nm = 0.873). Some stands deviated significantly from the mutation–drift equilibrium, suggesting recent bottlenecks. Altogether, these various trends are consistent with the hypothesis of a population collapse during the Holocene warming and suggest that most of the modern P. chihuahuana populations are now effectively isolated with their genetic diversity essentially modelled by genetic drift. The conservation efforts should focus on most southern populations and on the northern and central stands exhibiting high levels of genetic diversity. Additional mtDNA sequence analysis confirmed that P. martinezii (Patterson) is not conspecific with P. chihuahuana, and thus deserves separate conservation efforts.

EVIDENCE FOR AN EXTREME BOTTLENECK IN A RARE MEXICAN PINYON: GENETIC DIVERSITY, DISEQUILIBRIUM, AND THE MATING SYSTEM IN PINUS MAXIMARTINEZII

Maxipiñon (Pinus maximartinezii Rzedowski), which is confined to a single population of approximately 2000 to 2500 mature trees, covers about 400 ha in southern Zacatecas, Mexico. Genetic diversity measured by expected heterozygosity was 0.122, which is moderate for pines. However, percentage polymorphic loci was low, 30.3%. The fixation index (F) of 0.081 indicated only slight heterozygote deficiency. Mating system analysis indicated a significant but low level of selling; the multilocus outcrossing rate, tm, was 0.816. The mean of single locus estimates, ts, was smaller (0.761), perhaps suggesting mating among relatives, although the difference between tm and ts was not statistically significant.

Projections of suitable habitat for rare species under global warming scenarios

UNLABELLED PREMISE OF THE STUDY Modeling the contemporary and future climate niche for rare plants is a major hurdle in conservation, yet such projections are necessary to prevent extinctions that may result from climate change. • METHODS We used recently developed spline climatic models and modified Random Forests statistical procedures to predict suitable habitats of three rare, endangered spruces of Mexico and a spruce of the southwestern USA. We used three general circulation models and two sets of carbon emission scenarios (optimistic and pessimistic) for future climates. • KEY RESULTS Our procedures predicted present occurrence perfectly. For the decades 2030, 2060, and 2090, the ranges of all taxa progressively decreased, to the point of transient disappearance for one species in the decade 2060 but reappearance in 2090. Contrary to intuition, habitat did not develop to the north for any of the Mexican taxa; rather, climate niches for two taxa re-materialized several hundred kilometers southward in the Trans-Mexican Volcanic Belt. The climate niche for a third Mexican taxon shrank drastically, and its two mitotypes responded differently, one of the first demonstrations of the importance of intraspecific genetic variation in climate niches. The climate niche of the U.S. species shrank northward and upward in elevation. • CONCLUSION The results are important for conservation of these species and are of general significance for conservation by assisted colonization. We conclude that our procedures for producing models and projecting the climate niches of Mexican spruces provide a way for handling other rare plants, which constitute the great bulk of the worlds endangered and most vulnerable flora.

A POCKET OF VARIABILITY IN PINUS RIGIDA

Steady state gene frequencies around a pocket of differential fitness have been formulated by Hanson (1966) in a generalization of the work of Haldane (1948). A pocket of differential fitness would result in a pocket-of-variability, assuming that the radius of the area of contrasting fitness was large in relation to the vagility of the organism. Conversely, the absence of a pocket-of-variability is predicted whenever dispersal is broad relative to the size of the pocket of differential fitness. Around the pocket, a cline would be established through the interaction of migration and selection. The slope of the cline would depend on the width of the band of intermediate fitness, being steepest where the band was absent. The change in gene frequency with distance from the pocket would be rapid in the band of intermediate fitness values and approach limiting values asymptotically in the areas of contrasting fitness. The serotinous cone habit in pitch pine (Pinus rigida Mill.) provided us an example of a pocket-of-variability. A serotinous cone is one that remains closed for at least one year after maturity. The cone scales are held together with a resin seal and heat is required for prompt opening. Normally, temperatures in the tree crown do not reach levels sufficient to open serotinous cones. Serotinous cones do open and shed seed following a fire, so that serotiny seems to be an adaptation to fire, ensuring reproduction at the most opportune time in relation to site availability . To properly relate patterns of variability to Hansons model, characteristics controlled by a single gene should be considered. For Jack pine (Pinus banksiana Lamb.) there is evidence that the closedcone characteristic may be under simple genetic control (Rudolph et aI., 1959). As-

Genic diversity, genetic structure, and mating system of Brewer spruce (Pinaceae), a relict of the Arcto-Tertiary forest

Brewer spruce (Picea breweriana), a relict of the widespread Arcto-Tertiary forests, is now restricted to a highly fragmented range in the Klamath Region of California and Oregon. Expected heterozygosity for 26 isozyme loci, averaged over 10 populations, was 0.121. More notable than the relatively high level of diversity when compared to other woody endemics was the strong decrease in expected heterozygosity with latitude. Differentiation (F(ST)) was 0.152, higher than values for many north temperate conifers with larger distributions. The number of migrants per generation (Nm) was 1.34 or 2.70, depending on the method of estimation. Inbreeding appeared low; F(IS) was only 0.003, in agreement with multilocus population outcrossing rates (t(m)), which were generally well above 0.90. No difference in t(m) was found between isolated vs. clustered trees. However, the number of seeds per cone was greatest in the densest populations; t(m) is a measure of effective outcrossing after mortality in the embryonic stage, whereas a reduced number of seeds per cone indicates self pollination. Selfing increased after logging; outcrossing rate before logging was 0.961 and after logging, 0.756. Despite Brewer spruces narrow, fragmented distribution, the outlook for its conservation was good, with the exception of possible negative effects of logging.