Cytonuclear disequilibrium and genetic drift in a natural population of ponderosa pine.

We measured the cytonuclear disequilibrium between 11 nuclear allozyme loci and both mitochondrial and chloroplast DNA haplotypes in a natural population of ponderosa pine (Pinus ponderosa, Laws). Three allozyme loci showed significant associations with mtDNA variation, while two other loci showed significant association with cpDNA. However, the absolute number of individuals involved in any of the associations was small, such that in none of the nuclear-organellar combinations was the difference between observed and expected numbers >11 individuals. Patterns of association were not consistent across loci or organellar genomes, suggesting that they are not the result of mating patterns, which would act uniformly on all loci. This pattern of disequilibria is consistent with the action of genetic drift and with existing knowledge of the structure of this population and thus does not imply the action of other evolutionary processes. The overall magnitude (normalized disequilibrium) of associations was greater for maternally inherited mtDNA than for paternally inherited cpDNA, though this difference was neither large nor significant. Such significant disequilibria involving the paternally inherited organelle indicate that not only are there a limited number of seed parents, but the effective number of pollen parents is also limited.

Phylogeography of the plains killifish, Fundulus zebrinus.

Drainage systems of the Great Plains and western Gulf Slope underwent substantial changes through diversions and stream captures during the Pleistocene, either as the result of the glacial advances or through independent geologic processes. The distributions of a variety of fishes that range across west-central North America, such as the plains killifish (Fundulus zebrinus), are thought to be the product of this Pleistocene influence. We examined the geographic pattern of genetic variation in F. zebrinus using three allozyme loci (n = 793), mitochondrial DNA restriction fragment length polymorphisms (RFLPs, n = 352), and sequencing of the mitochondrial cytochrome oxidase I (COI, n = 23) in an attempt to understand the roles of dispersal and vicariance. The phylogeographic patterns were concordant between the allozyme and mitochondrial data with the exception of the population in the North Canadian River. The populations fell into three geographic assemblages, which we designated as northern, central, and southern. A large phylogenetic break (average Roger's D = 0.702; average sequence divergence in RFLPs = 4.6%; average sequence divergence in COI = 5.5%) separated the northern/central and southern assemblages. The northern region was likely colonized sometime during the mid-Pleistocene. Fish in the Brazos and Pecos Rivers probably reached these drainages through stream captures of the Red River. The large phylogenetic break between the northern/central and southern clades supports previous attempts to recognize two species of plains killifish: F. zebrinus and F. kansae.

Population and subspecific genetic differentiation in the foxtail pine (Pinus balfouriana).

We performed an allozyme survey of genetic differentiation in Pinus balfouriana, a subalpine conifer endemic to California that is comprised of two allopatric subspecies, one in the Klamath Mountains and the other in the southern Sierra Nevada. Although the two subspecies are morphologically distinct and gene flow between them is virtually nonexistent, we observed much higher levels of differentiation among populations within a subspecies than between the two subspecies. Differentiation is particularly strong in the Klamath populations (multilocus FST = 0.242), which are small, isolated, and ecologically marginal. We attribute this strong differentiation to the mountain island effect, in which populations restricted to high elevations become isolated from each other on different mountains separated by unsuitable intervening habitat, with consequent reduced gene flow allowing populations to evolve independently. Populations of P. balfouriana in the Klamath region only exist scattered on the few highest ridges and peaks that rise above 2,000 m, which defines the lower limit of the species elevational distribution. This pattern of distribution has allowed genetic drift and allelic sorting through historical events to produce strong population-level differentiation, which was likely in place before the two subspecies were geographically separated. Because P. balfouriana occurs on both serpentine soils and nonserpentine soils in the Klamath Mountains, we tested for genetic differentiation between populations growing on serpentine versus nonserpentine soils and our results were equivocal. Our data, combined with several other studies of conifers, show that the mountain island effect can produce significant genetic differentiation in conifers whose life-history traits of widely dispersed pollen, long generation times, and high outcrossing rates would lead us to predict a more homogenous population genetic structure.

Effects of specialization on genetic differentiation in sister species of bark beetles.

We investigated the relative importance of resource use and geography on genetic differentiation in the sister-species pair of generalist and specialist bark beetles: Dendroctonus ponderosae and D. jeffreyi (Coleoptera: Scolytidae). In two regions, where the distributions of these species overlap, we collected specimens of the generalist from multiple host species and specimens of the specialist from its single host species. Using allozyme techniques, we uncovered genetic differentiation between generalist populations on different host species in the same region (one locus in each region). However, a much stronger pattern of differentiation was found between specialist populations in the two distantly separated regions (three loci). With mtDNA, we found no significant differentiation between regions in the specialist, or among host species in the generalist, although there was some differentiation between regions in the generalist (AMOVA, P < 0.05). Overall, the generalist populations maintained approximately 10 times the genetic variation in mtDNA as the specialist populations, which suggests that the specialist either has generally smaller population sizes than the generalist, or has experienced a historical population bottleneck.

Paternity and gene dispersal in limber pine (Pinus flexilis James).

This study provides empirical information on intrapopulation gene dispersal via pollen, the size of genetic 'neighbourhoods', and interpopulation gene flow in a long-lived conifer, limber pine (Pinus flexilis). We used allozyme loci for a paternity analysis of 518 seeds produced in an isolated population of limber pine located in north-eastern Colorado, U.S.A., separated by 2 km from the nearest conspecific trees and nearly 100 km from populations in the Rocky Mountains. We also used indirect techniques (FST analyses) to estimate gene flow rates among subdivisions of the study population and among five widely separated populations. Within the main study population limber pine exhibited a polymorphism level of 50%, observed heterozygosity of 0.159, and 2.36 alleles per polymorphic locus. Mountain populations were slightly more variable. The main study population showed significant differentiation in allozymes among neighbouring subpopulations. The mean FST was 0.031 and the gene flow rate among subpopulations was estimated as 7.8 migrants per generation. Among widely separated populations the mean FST was 0.035 and the gene flow rate was estimated as 6.9 migrants per generation. The paternity analysis indicated a best estimate of 6.5% pollen immigration (minimum 1.1%) from populations 2 km to 100+ km away. For 4% of the seeds examined, paternity could be ascribed to a single tree in the study population. Fractional paternity and likelihood methods were used to estimate pollen dispersal distances for the remainder of the seeds. Mean pollen dispersal distance was estimated at 140 m using the fractional method, similar to results from the other techniques. This compares with a mean distance of 172 m between potential mates. These results suggest near-panmictic pollen dispersal over this population, which covers about 15 ha. The observed allozyme differences and surprisingly low estimates of among-subpopulation gene flow are ascribed to a probable restriction of gene dispersal by seeds.

Glacial refugia of limber pine (Pinus flexilis James) inferred from the population structure of mitochondrial DNA.

To make inferences about the glacial refugia that harboured the limber pine, Pinus flexilis James, we examined the range-wide population structure of mitochondrial DNA (mtDNA) with eight size variants in the second intron of nad1. The data consisted of haplotypes from 704 trees collected from 40 localities. The value of FST for these populations was 0.80, which is a much larger value than has been reported for allozymes and chloroplast DNA (cpDNA) in limber pine, and it suggests that the number of seeds moving among localities per generation is approximately 0.12. Gene flow of this magnitude would allow mutation and subsequent genetic drift to have a substantial impact on the population structure of mtDNA. The majority of the mtDNA haplotypes are restricted to minor portions of the geographical range. The data are consistent with mtDNA differentiation in seven glacial refugia, followed by dispersal out of those refugia.

Variation in allozymes and stomatal size in pinyon (Pinus edulis, Pinaceae), associated with soil moisture.

Microgeographic allozyme variation was examined in pinyon pine, Pinus edulis, among five collection sites in Owl Canyon, Colorado. Relatively dry and moist sites were identified by associated plant communities and the sizes and densities of trees. Three moist sites and two dry sites were compared, and because all sites were within 600 m of one another, isolation by distance was not considered as a viable explanation of genetic differentiation between sites. Allelic frequencies at glycerate dehydrogenase (Gly) differed by 14% between moist and dry areas, and the pattern of microgeographic variation found here-allele 3 higher in frequency on dry sites-was consistent with previous studies of microgeographic variation in contrasting moist and dry sites. Trees within one of the dry sites were examined to test the hypothesis that stomata sizes and densities are heterogeneous among Gly genotypes. Heterozygotes had the longest and widest stomata; the stomatal area of heterozygotes was 28% greater than the stomatal area of homozygotes. Whereas the stomatal areas of the two homozygotes were similar, their shapes did not overlap when projected on a bivariate plot of length and width. These results suggest that stomatal shape may play a role in adapting pinyon to heterogeneity in soil moisture.

A comparison of population differentiation across four classes of gene marker in limber pine (Pinus flexilis James).

We examined genetic differentiation among seven populations of limber pine using four classes of gene marker. Among-population differentiation was much higher for maternally inherited mitochondrial DNA polymorphisms than for paternally inherited chloroplast DNA, indicating that wind-dispersed pollen is the main agent of gene flow. Chloroplast DNA differentiation is consistent with gene flow estimated in a prior paternity analysis. Using the estimates of seed and pollen flow derived from mtDNA and cpDNA differentiation, we predicted the value of Fst expected at nuclear loci. Allelic frequency differentiation at seven allozyme loci was relatively homogeneous across loci and consistent with the level of differentiation predicted from the organellar haplotypes. By contrast four of the nine randomly applied polymorphic DNA (RAPD) markers we examined were more strongly differentiated than this prediction, suggesting the action of diversifying selection. However, the differentiated RAPDs and mtDNA were concordant in dividing the populations into two groups, suggesting some historical division. Simulations show that such historical division can increase the interlocus variance in Fst, but neither a historical nor an equilibrium model could account for the joint distribution of Fst estimates across both allozyme and RAPD loci. Thus at least one group of loci appears to be experiencing natural selection.

Zoogeographic Distributions of the Sibling Species Mytilus galloprovincialis and M. trossulus (Bivalvia: Mytilidae) and Their Hybrids in the North Pacific.

Diagnostic length differences in a PCR amplified fragment of the gene for byssal adhesive protein were used to study the zoogeographic distribution of Mytilus galloprovincialis and M. trossulus along the west coast of North America and in Japan. The distributions of M. galloprovincialis and M. trossulus are patchy, although an overall geographic pattern emerges. M. galloprovincialis was the only species found on either Kyushu or Honshu, and it was the most abundant mussel from Tomales Bay to San Diego, California. M. trossulus was the only bay mussel found on Hokkaido and in Alaska, and it was by far the most abundant mussel along the coasts of Washington and Oregon. Mytilus galloprovincialis and M. trossulus are sympatric and hybridize near Whidbey Island, Washington, in San Francisco Bay, and in San Diego Bay. A second diagnostic anonymous nuclear PCR marker was used to examine the extent of hybridization at Palo Alto, California. At this site, genotypes appeared to be a mixture of M. galloprovincialis, F1 hybrids between M. galloprovincialis and M. trossulus, and backcrosses between the F1's and M. galloprovincialis. The discontinuity between the zoogeographic distributions of these two species at about 40{deg}-41{deg}N latitude in both the eastern and western Pacific suggests that temperature is a factor in determining their present distribution and limiting their dispersal to other regions.

Selection of breeding stock in pigs favours 6PGD heterozygotes.

Associations between genetic variation at four enzyme polymorphisms and growth rate, feed conversion and economic index were studied in commercial lines of pigs. These lines were also tested for the fit of observed genotypic frequencies to expected genotypic frequencies at the four polymorphic loci. Genotypic distribution fit Hardy-Weinberg expectations in four pure-breeding lines but crosses among lines produced excesses in heterozygosity at the locus coding for 6-phosphogluconate dehydrogenase (6PGD) due to differences in allelic frequencies among lines. Selection of breeding individuals further enhanced the heterozygosity at 6PGD to 73-93 per cent but it had no impact on a linked locus that was sometimes in linkage disequilibrium with 6PGD. The relationship between feed conversion and individual heterozygosity was examined in one population sample; feed conversion increased with individual heterozygosity.

Correlation between the individual heterozygosity of parents and their offspring.

Specific formulations are derived for the correlation between the heterozygosity of a randomly mating parent and its offspring for a diallelic locus, and for the correlation when multiple loci are considered. The expected correlation is maximal, approaching r = 0.50, when allelic frequencies are highly asymmetric, and it is zero when the allelic frequencies are equal to 0.50. Parent-offspring correlations, up to a maximum of 0.50 for diallelic loci, indicate that levels of heterozygosity can respond to selection. Multilocus allozyme data from limber pine, Pinus flexilis, and from horses of standardbred and thoroughbred breeds are used to demonstrate correlations between a parent and its offspring. The Spearman rank correlation between the heterozygosity of a limber pine and the mean heterozygosity of her offspring is r = 0.45. Correlations in the horses range from r = 0.16 to 0.32.

Associations between enzyme genotypes and dark respiration in perennial ryegrass, Lolium perenne L.

In this study, we determined whether relationships existed between dark respiration and genotype at five enzyme polymorphisms in perennial ryegrass, Lolium perenne L. Positive correlations were found between Q 10 of dark respiration and genotype at the phosphoglucomutase (PGM) and 6-phosphogluconate dehydrogenase (6PGD) loci. Plants doubly homozygous for the common allele at these loci were found to have Q 10 values 20% higher than those for double heterozygotes. In plants that were heat stressed for five consecutive days, Q 10 was found to be negatively correlated with apparent vigor after stressing. Individuals homozygous for PGM and 6PGD (with higher Q 10 values) exhibited more apparent damage following the stress than heterozygous individuals. Both PGM and 6PGD occupy positions in metabolism with regulatory potential. Although caution must be used in assigning causal relationships, the results suggest that specific forms of these enzymes are directly related to, or are correlated with, the determinants of respiratory efficiency in L. perenne.

Allozyme heterozygosity and growth in the tiger salamander, Ambystoma tigrinum.

Statistically significant, positive correlations were found between individual heterozygosity at seven protein polymorphisms and size in five natural populations of tiger salamanders. When these same populations were sampled later in the same year, the positive associations between heterozygosity and size were no longer evident. A pair mating was conducted in the laboratory, and offspring were distributed randomly to four replicate populations maintained in the laboratory. In two to these four replicates, a significant, positive association between protein heterozygosity and size was observed. The laboratory results suggest that the associations seen in natural populations are due to associations between protein heterozygosity and growth rate early in the larval period.

The distribution of individual heterozygosity in natural populations.

Estimation of the distribution of the level of individual heterozygosity within natural populations is explored with both Monte-Carlo simulation studies and data from natural populations. Simulations indicate that heterozygosities estimated from as few as a dozen randomly chosen loci may, to some degree, reflect (r = 0.35) heterozygosity determined by 100 independent loci. The shape of the expected distribution of heterozygosity is heavily dependent upon levels of heterozygosity at the loci. Complete genetic data for 12 loci from 997 Fundulus heteroclitus are used to describe the distributions of heterozygosity for different localities, for age classes and for sexes. The distributions deviate from normality. Distributions from different localities are not different, but the distributions are heterogeneous among age classes at one of two localities and are heterogeneous between the sexes.

Observations on the genetic structure and mating system of ponderosa pine in the Colorado front range.

Variation of peroxidase enzymes is analyzed both in mature needle tissue and in open-pollinated seedling families of ponderosa pine, Pinus ponderosa, and is identified as being controlled by a single Mendelian locus. Variation at this locus, analyzed in 1, 386 individuals, is used in the analysis of population differentiation and the mating system. Significant variation of gene frequencies is detected over distances of several hundred meters, and is found to be associated with slopes of different aspects. Ponderosa pine is wind-pollinated, and an analysis of the mating system indicates that the level of outcrossing is greater than 90 %. Selection specific for different environments is evidently strong enough to overcome the homogenizing force of migration and produce population fissuring in ponderosa pine.

Morphological adaptation to thermal stress in a marine fish, Fundulus heteroclitus.

Populations of Fundulus heteroclitus (Cyprinodontidae), a coastal marine fish, were studied in control and artificially heated environments on the north shore of Long Island to determine patterns of variation in morphology and the extent to which this variation reflected adaptation to environmental characteristics. Principal components and discriminant function analyses were used to analyze variation in and among seventeen morphological characters. Fishes living in water artificially heated by a power plant exhibited marked divergence from control populations in head morphology, and convergence with a population sampled at more southern latitudes. Hence, these differences were interpreted as adaptations to warm environments. Greater morphological variation is detected at the heated locality than at control localities, and this may be partially due to a breakdown in developmental homeostasis, and partially due to selection favoring phenotypes that are rare in this environment.