Genic Heterozygosity and Variation in Permanent Translocation Heterozygotes of the OENOTHERA BIENNIS Complex

Genic heterozygosity and variation were studied in the permanent translocation heterozygotes Oenothera biennis I, Oe. biennis II, Oe. biennis III, Oe. strigosa, Oe. parviflora I, Oe. parviflora II, and in the related bivalent formers Oe. argillicola and Oe. hookeri. From variation at 20 enzyme loci, we find that translocation heterozygosity for the entire chromosome complex is accompanied by only moderate levels of genic heterozygosity: 2.8% in Oe. strigosa, 9.5% in Oe. biennis and 14.9% in Oe. parviflora. Inbred garden strains of Oe. argillicola exhibited 8% heterozygosity; neither garden nor wild strains of Oe. hookeri displayed heterozygosity and only a single allozyme genotype was found. The mean number of alleles per locus is only 1.30 in Oe. strigosa, 1.40 in Oe. biennis, and 1.55 in Oe. parviflora, compared to 1.40 in Oe. argillicola. Clearly, the ability to accumulate and/or retain heterozygosity and variability has not been accompanied by extraordinary levels of either. Clinal variation is evident at some loci in each ring-former. A given translocation complex may vary geographically in its allozymic constitution. From gene frequencies, Oe. biennis I, II, and III, Oe. strigosa and Oe. hookeri are judged to be very closely related, whereas Oe. argillicola seems quite remote; Oe. parviflora is intermediate to the two phylads. Gene frequencies also suggest that Oe. argillicola diverged from the Euoenothera progenitor about 1,000,000 years ago, whereas most of the remaining evolution in the complex has occurred within the last 150,000 years.     

Examination of Allelic Variation at the Hexokinase Loci of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS by Different Methods

Recently a number of electrophoretic techniques have been applied to reveal the presence of additional genetic variation among the electrophoretic mobility classes of the highly polymorphic xanthine dehydrogenase (XDH ) and esterase-5 (est-5) loci. We examined the hexokinase loci of Drosophila pseudoobscura and D. persimilis using a variety of techniques to determine whether further allelic variation could be revealed for these much less polymorphic loci and to analyze the nature of the known variation at the hexokinase-1 (hex-1) locus. The following studies were conducted: 135 strains of the two species from six localities were examined with buffer pH ranging from 5.5 to 10.0; 40 strains of D. pseudoobscura and 9 strains of D. persimilis from Mather were studied using starch gel concentrations ranging from 8.5 to 15.5% and were examined for differences in heat stability and reactivity to the thiol reagent pCMSA; strains were also tested for susceptibility to urea denaturation and differences in relative activities. Major findings of the work are: (1) No additional allelic variation could be detected at any of the hexokinase loci by applying these techniques. The finding of abundant hidden genetic variation in XDH and est-5 does not extend to all enzyme loci. (2) Evidence from studies using pCMSA indicates that the hex-1 alleles 0.6, 0.8, 1.0 and 1.2 of the two species form a series of unit charge steps. Since the 0.94 allele of D. persimilis has mobility intermediate between 0.8 and 1.0, it is argued that routine electrophoretic techniques are sensitive to at least some conservative amino acid substitutions. (3) Strong correlations were found at the hex-1 locus between low allelic frequency, reduced relative activity and reduced stability to heat and urea denaturation. Since the three sibling species, D. pseudoobscura, D. persimilis and D. miranda, all appear to share a common high frequency allele (1.0) at that locus, these findings are taken as evidence that the observed allelic frequencies are a result of directional selection and mutation, rather than any form of balancing selection.     

Inheritance of isozyme variation and heterozygosity in Pinus ponderosa

Techniques are presented to detect 23 isozyme loci in the long-lived perennial plant, ponderosa pine. Meiotically derived megagametophyte from seeds is used to examine directly the segregation of allelic variants. Approximately seven seeds were initially examined for 12 enzymes from each of 47 trees from ten stands throughout the northern Rocky Mountain region. Additional seeds were also examined from selected families to confirm the inheritance of observed electrophoretic variants at 13 polymorphic loci and to estimate linkage relationships. Significant nonrandom segregation was consistently detected for three pairs of loci: ADH-1: AAT-2, ADH-1: PGI-1, and LAP-2: 6PG-1. Preliminary estimates of population parameters reveal a relatively high average heterozygosity (H=0.123). This is partitioned into a high amount of genetic variation within local stands, with only approximately 12% of the total heterozygosity resulting from genic differences between stands.Funds for this study were made available under the McIntire-Stennis program of the Forest, Range, and Conservation Experiment Station of the School of Forestry, University of Montana.     

Cloning and characterization of novel microsatellite DNA markers for the grass rockfish,Sebastes rastrelliger,and cross‐species amplification in 10 related Sebastes spp

Here we report development and characterization of seven polymorphic loci derived from grass rockfish (Sebastes rastrelliger) genomic DNA phagemid libraries enriched for microsatellite motifs. Within grass rockfish, average allelic diversity was 11.3 alleles per locus and average heterozygosity was 0.73. The seven loci also were surveyed in 10 related species of Sebastes, where allelic diversity ranged from highly polymorphic to monomorphic. Deviations from Hardy–Weinberg were nonsignificant in all but one species/locus combination suggesting low occurrence of null alleles. Significant linkage disequilibrium was detected among three loci, but these events were limited to a single species in each case.     

Molecular characterization of allelic variants of (GATA)n microsatellite loci in parthenogenetic lizards Darevskia unisexualis (Lacertidae)

Populations of parthenogenetic lizards of the genus Darevskia consist of genetically identical animals, and represent a unique model for studying the molecular mechanisms underlying the variability and evolution of hypervariable DNA repeats. As unisexual lineages, parthenogenetic lizards are characterized by some level of genetic diversity at microsatellite loci. We cloned and sequenced a number of (GATA)n microsatellite loci of Darevskia unisexualis. PCR products from these loci were also sequenced and the degree of intraspecific polymorphism was assessed. Among the five (GATA)n loci analysed, two (Du215 and Du281) were polymorphic. Cross-species analysis of Du215 and Du281 indicate that the priming sites at the D. unisexualis loci are conserved in the bisexual parental species, D. raddei and D. valentini. Sequencing the PCR products amplified from Du215 and Du281 and from monomorphic Du323 showed that allelic differences at the polymorphic loci are caused by microsatellite mutations and by point mutations in the flanking regions. The haplotypes identified among the allelic variants of Du281 and among its orthologues in the parental species provide new evidence of the cross-species origin of D. unisexualis. To our knowledge, these data are the first to characterize the nucleotide sequences of allelic variants at microsatellite loci within parthenogenetic vertebrate animals.     

Apparent absence of genetic differences among species of Teladorsagia (Nematoda: Trichostrongylidae)

The technique of allozyme electrophoresis was applied to three species of Teladorsagia present in sheep to determine the extent of genetic variation among species. Thirty-four enzyme loci were established of which 22 were invariant and 12 were shown to be polymorphic. No fixed allelic differences were detected among the species and the level of polymorphism was within the range found commonly between populations of a single species. Therefore, the genetic data support breeding data and existing morphological evidence that T. circumcincta, T. davtiani and T. trifurcata as currently recognized do in fact belong to a single species.     

Inheritance of isozyme variation and heterozygosity in Pinus ponderosa.

Techniques are presented to detect 23 isozyme loci in the long-lived perennial plant, ponderosa pine. Meiotically derived megagametophyte from seeds is used to examine directly the segregation of allelic variants. Approximately seven seeds were initially examined for 12 enzymes from each of 47 trees from ten stands throughout the northern Rocky Mountain region. Additional seeds were also examined from selected families to confirm the inheritance of observed electrophoretic variants at 13 polymorphic loci and to estimate linkage relationship. Significant norandom segregation was consistently detected for three pairs of loci: ADH-1:AAT-2, ADH-1:PGI-1, and LAP-2:6PG-1. Preliminary estimates of population parameters reveal a relatively high average heterozygosity (H = 0.123). This is partitioned into a high amont of genetic variation within local stands, with only approximately 12% of the total heterozygosity resulting from genic difference between stands.     

Mating is rare within as well as between clades of the human pathogen Candida albicans

Candida albicans is a diploid yeast that can undergo mating and a parasexual cycle, but is apparently unable to undergo meiosis. Characterization of the population structure of C. albicans has shown that reproduction is largely clonal and that mating, if it occurs, is rare or limited to genetically related isolates. Because molecular typing has delineated distinct clades in C. albicans, we have tested whether recombination was common within clades, but rare between clades. Two hundred and three C. albicans isolates have been subjected to multilocus sequence typing (MLST) and the haplotypes at heterozygous MLST genotypes characterized. The C. albicans isolates were distributed among nine clades, of which five corresponded to those previously identified by Ca3 fingerprinting. In each of these clades with more than 10 isolates, polymorphic nucleotide positions located on between 3 and 4 of the six loci were in Hardy-Weinberg disequilibrium. Moreover, each of these polymorphic sites contained excess heterozygotes. This was confirmed by an expanded analysis performed on a recently published MLST dataset for 1044 isolates. On average, 66% of polymorphic positions in the individual clades were in significant excess of heterozygotes over the five clades. These data indicate that mating within clades as well as self-fertilization are both limited and that C. albicans clades do not represent a collection of cryptic species. The study of haplotypes at heterozygous loci performed on our dataset indicates that loss of heterozygosity events due to mitotic recombination is moderately common in natural populations of C. albicans. The maintenance of substantial heterozygosity despite relatively frequent loss of heterozygosity could result from a selective advantage conferred by heterozygosity.     

Mating is rare within as well as between clades of the human pathogen Candida albicans.

Candida albicans is a diploid yeast that can undergo mating and a parasexual cycle, but is apparently unable to undergo meiosis. Characterization of the population structure of C. albicans has shown that reproduction is largely clonal and that mating, if it occurs, is rare or limited to genetically related isolates. Because molecular typing has delineated distinct clades in C. albicans, we have tested whether recombination was common within clades, but rare between clades. Two hundred and three C. albicans isolates have been subjected to multilocus sequence typing (MLST) and the haplotypes at heterozygous MLST genotypes characterized. The C. albicans isolates were distributed among nine clades, of which five corresponded to those previously identified by Ca3 fingerprinting. In each of these clades with more than 10 isolates, polymorphic nucleotide positions located on between 3 and 4 of the six loci were in Hardy-Weinberg disequilibrium. Moreover, each of these polymorphic sites contained excess heterozygotes. This was confirmed by an expanded analysis performed on a recently published MLST dataset for 1044 isolates. On average, 66% of polymorphic positions in the individual clades were in significant excess of heterozygotes over the five clades. These data indicate that mating within clades as well as self-fertilization are both limited and that C. albicans clades do not represent a collection of cryptic species. The study of haplotypes at heterozygous loci performed on our dataset indicates that loss of heterozygosity events due to mitotic recombination is moderately common in natural populations of C. albicans. The maintenance of substantial heterozygosity despite relatively frequent loss of heterozygosity could result from a selective advantage conferred by heterozygosity.     

Development and characterization of 10 novel microsatellite markers from Chital deer (Cervus axis) and their cross–amplification in other related species

Ten polymorphic microsatellite loci were isolated and characterized from Chital deer (Cervus axis). These loci show high levels of allelic diversity with four to eight alleles per locus in the 22 individuals of the free‐ranging population of Chital deer in Nehru Zoological Park, Hyderabad. In addition, we found that all the loci show cross–amplification in closely related as well as distantly related deer species. The amplification of these markers in different genera further indicates that these can be applied to a wide range of endangered deer species for their population genetics studies and conservation management.     

Genetic diversity and gene flow in the morphologically variable, rare endemics Begonia dregei and Begonia homonyma (Begoniaceae)

Begonia dregei and B. homonyma (Begoniaceae), rare plants endemic to coastal forests of eastern South Africa, are two closely related species with high levels of variation among populations in the shape of leaves. Distribution of genetic variation and genetic relatedness were investigated in 12 populations of B. dregei and seven of B. homonyma using polyacrylamide gel electrophoresis. Twelve of the 15 enzyme loci examined were polymorphic, but only seven loci were polymorphic within at least one population. Genetic diversity measures indicated that the among-population gene differentiation represents >90% of the total genetic component in both species considered individually or combined. This indicated restricted gene flow, consistent with the limited dispersal abilities of Begonia generally and the ancient separation of isolated forest patches. Genetic distances among populations are much higher than usually found within species. Allozyme data provide no support for the recognition of B. dregei and B. homonyma as distinct species.     

Enzymatic survey of four populations of Atherina boyeri based on electrophoresis and the occurrence of a microsporidiosis

Biochemical genetics of a Atherinid species, Atherina boyeri were undertaken to determine variability. Among the 17 loci studied, 7 were polymorphic, of which three were retained for a more detailed comparison of populations inhabiting the French Mediterranean coastal lagoons. A microsporidiosis is present in these fishes and is limited to certain lagoons. Comparison of allelic frequencies at the polymorphic loci between fish populations of infected and healthy lagoons yielded no significant differences. Several hypotheses are discussed and attempt to agree parasite distribution with genetical results.     

Microsatellite variation and differentiation in North Atlantic eels

We screened 11 populations of American, European, and Icelandic eels (Anguillidae) for allelic variation and genetic divergence at six polymorphic microsatellite loci. Within either of the two recognized Anguilla species in the North Atlantic (rostrata in the Americas, anguilla in Europe), population genetic structure was statistically significant but weak; fully 95% of the total genetic variation was present within geographic locales rather than distributed among them. The two Anguilla species also overlap greatly in allelic frequencies, so the available data proved ineffective for addressing hypotheses about the possible hybrid origins of some Icelandic eels. The overlapping microsatellite profiles contrast with nearly diagnostic species differences documented previously in allozymes and mtDNA. This and similar empirical findings in several other species support theoretical concerns that homoplasy (convergent evolution) in allelic states can compromise the utility of rapidly mutating microsatellite loci for certain types of microevolutionary questions regarding gene flow and species differences.     

Variation of allozyme loci in endemic gammarids of Lake Baikal

The outstandingly rich and highly endemic fauna of Lake Baikal includes more than 200 species of gammarids (Crustacea, Amphipoda), most of which occur only in Baikal. Allelic variation at 23 enzyme loci has been studied for the first time in four species of Baikalian gammarids. The levels of polymorphism are similar to those observed in other invertebrates, including gammarids: c. 20–25% of the loci are polymorphic with 2.53 to 3.3 alleles and mean heterozygosity of 0.048 to 0.105 per polymorphic locus. The genotype frequences in local populations in most cases markedly deviate from Hardy-Weinberg equilibrium with a deficiency of heterozygotes. Populations were shown to be strongly subdivided even over a range of less than 100 m. On the larger scale genetic differences among the populations do not show any strong correlation with geographical separation. Genetic distances among species were found to be rather low: from 0.159 between closely related, but morphologically clearly distinct species of the genus Spinacanthus to 0.546 between the representatives of different genera. We discuss these patterns of population structure with respect to the problem of the origin of endemic faunas in ancient lakes. Our results suggest that speciation occurred rapidly and relatively recently. Genetic distances within and among species support models of speciation based on the founder effect or differentiation of a peripheral population rather than on divergence of large subpopulations. The rate of morphological evolution in Baikalian gammarids is very high compared with their biochemical evolution.     

Fully informative microsatellite markers for the tetraploid plant Eritrichium nanum (Boraginaceae)

We characterized eight polymorphic, codominant nuclear microsatellite loci in the tetraploid plant Eritrichium nanum. The different allelic configurations occurring under tetrasomic inheritance were fully resolved at all loci. Two natural populations showed high observed heterozygosities, which were in agreement with Hardy-Weinberg expectations. There was no evidence of genetic linkage disequilibrium for any pair of loci. The results suggest that these microsatellite markers are useful for mating system and population genetic analyses in high-alpine E. nanum.     

Electrophoretic identification and phylogenetic relationships of indigenous tilapiine species of Zimbabwe

In order to characterize Zimbabwean tilapias 34 populations of seven species, two in the genus Tilapia and five in Oreochromis , were analysed using horizontal starch gel electrophoresis. Twenty-seven loci were polymorphic while five were monomorphic but had fixed allelic differences among some species and six were monomorphic for the same allele in all species. No fixed allelic differences were found between O. mossambicus and O.mortimeri , and only one locus distinguished O. andersonii from O. mossambicus and O. mortimeri . Pairwise comparisons of O. mossambicus or O. mortimeri populations of genetic distances frequently showed a greater degree of difference within the species than was evident in some O. mossambicus O. mortimeri pairs. This study failed to distinguish O. mortimeri as a distinct species from O. mossambicus . The Tilapia species formed distinct clusters from Oreochromis in both consensus eighbourjoining and distance Wagner topologies. Oreochromis ossambicus, O. mortimeri and O. andersonii clustered together consistently. Although some differences from the taxonomic arrangement of Trewavas are discussed, the principal relationships revealed in our analysis are consistent with most recent studies.     

The effect of selection on esterase allozymes in a barley population

Changes in gene and genotypic frequencies at four esterase loci were monitored over 25 generations in Composite Cross V, an experimental population of barley, to obtain experimental evidence concerning the balance of forces responsible for: (1) the marked differences in allelic frequencies among barleys from different ecogeographical regions of the world; and (2) the extensive allelic variation found within local populations of barley. Analyses of the highly significant changes in allelic frequencies which occurred in CCV showed they were due to directional selection favoring particular alleles and not to mutation, migration or genetic drift. The results show that intense balancing selection, featuring consistent excesses of heterozygotes, also occurred in CCV. It is concluded that among the factors of neo-Darwinian evolution, natural selection plays the predominant role in determining the observed patterns of allelic variation in the barley species as a whole.     

Molecular karyotype analysis and mapping of housekeeping genes to chromosomes of selected species complexes of Leishmania.

The molecular karyotypes for 20 reference strains of species complexes of Leishmania were determined by contour-clamped homogeneous electric field (CHEF) electrophoresis. Determination of number/position of chromosome-sized bands and chromosomal DNA locations of housekeeping genes were the two criteria used for differentiating and classifying the Leishmania species. We have established two gel running conditions for optimal separation of chromosomes, which resolved DNA molecules as large as 2,500 kilobase pairs (kb). Chromosomes were polymorphic in number (22-30) and size (200-2,500 kb) of bands among members of five complexes of Leishmania. Although each stock had a distinct karyotype, in general the differences found between strains and/or species within each complex were not clear enough for parasite identification. However, each group showed a specific number of size-concordant DNA molecules, which allowed distinction among the Leishmania complex parasites. Clear differences between the Old and New world groups of parasites or among some New World Leishmania species were also apparent in relation to the chromosome locations of beta-tubulin genes. Based on these results as well as data from other published studies the potential of using DNA karyotype for identifying and classifying leishmanial field isolates is discussed.     

Pattern of Inheritance of Microsatellite Loci in the Squid Loligo pealeii (Mollusca: Cephalopoda)

Six microsatellite loci are described for the squid Loligo pealeii. All loci exhibit some degree of allelic diversity. The pattern of inheritance was tested for 3 loci through an analysis of the filial genotypes from a female-male mating. At all 3 loci, the ratios of the filial genotypes conformed to the ratios expected by Mendelian inheritance. The hypervariable loci will be useful in studies on sexual selection in this species, whereas the relatively less variable loci will be useful to address questions of population structure. Received March 13, 2000; accepted May 22, 2000.