A long-term study on seasonal changes of gametic disequilibrium between allozymes and inversions in Drosophila subobscura

Seasonal variation (spring, early summer, late summer, and autumn) of gametic disequilibrium between gene arrangements (OST and O3+4) of the O chromosome and Lap, Pept-1, and Acph allozyme loci, located inside these inversions, has been recorded in a natural population of Drosophila subobscura during seven years over a 15-year period. The length of the study allowed us to investigate the temporal variation of the allozyme-inversion associations by statistical methods of time series analysis. Cyclic seasonal changes of allozyme-inversion associations for both Lap and Pept-1 are detected in the natural population. In both cases, the patterns of seasonal change are due to the seasonal change of frequency of Lap and Pept-1 allozymes occurring exclusively within the OST gene arrangement. In contrast, the allozyme frequencies at these loci within the O3+4 gene arrangement are stable along seasons. The patterns of temporal variation of allozyme-inversion associations for Lap and Pept-1 in the natural population are contrasted with those previously published that correspond to gene arrangements of the O chromosome and nucleotide polymorphism at the rp49 region located inside these inversions, suggesting that natural selection is operating on these allozyme-inversion associations.     

Genetic coadaptation in the chromosomal polymorphism of Drosophila subobscura II. Changes of gametic disequilibrium in experimental populations

Experimental populations were examined for temporal changes of gametic disequilibria between allozyme loci (Lap and Pept-1) and gene arrangements of the O chromosome of Drosophila subobscura (O _st and O ₃₊₄₊₇) under several environmental conditions. In the foundation of the experimental populations a genetic perturbation was carried out in order to test the relevance of the current hypotheses used to explain the allozyme-inversion associations observed in natural populations. Differential changes of gametic disequilibria were detected over generations under the different environmental conditions. Mere mechanical or stochastic factors cannot explain the results and natural selection is probably the major agent generating the detected gametic associations. The observations are interpreted as a proof of coadaptation of D. subobscura inversions.     

Non-random associations between Lap and Pept-1 loci and gene arrangements of chromosome O in D. subobscura — experiments in laboratory populations

In this paper the well-known non-random associations between Lap and Pept-1 loci and gene arrangements of chromosome O are studied in laboratory populations of D. subobscura. An increase of the frequency of the allele Lap ^1.00, towards an equilibrium point (0.70), was found to be associated with an increase of the gene arrangement O₃₊₄. This is an accordance to the associations found in natural populations. On the contrary no such an increase was observed in populations polymorphic for Lap and Pep-1 loci but homokaryotypic for gene arrangement O₃₊₄₊₈ differing in the initial allele frequencies at these loci. Although epistatic selection cannot be completely ruled out, our results are better explained under the assumption of neutrality.     

Geographic pattern of allozyme and inversion polymorphism on chromosome O of Drosophila subobscura and its evolutionary origin

The chromosome O of Drosophila subobscura was studied with respect to genetic variability at three enzyme loci (Odh, Me, and Lap-4) and with respect to inversion polymorphism. Population samples were taken from seven localities along a north-south gradient from Sweden and Scotland to Tunisia.The chromosomal analysis revealed clinal frequency changes for gene arrangements from north to south. With the enzyme loci Odh and Me allele frequencies are similar throughout the distribution range. Both loci are located outside the common inversion complex O₃₊₄. On the other hand, frequency changes parallel to those of the gene arrangements were observed for the alleles of the Lap-locus. Nonrandom associations between Lap-alleles and the superimposed gene arrangements O_ST, O₃₊₄, O₃₊₄₊₈, and O₃₊₄₊₂₃ were found. These gene arrangements differ from each other with respect to allele frequencies at the Lap-locus but for a given gene arrangement the relative frequencies of Lap-alleles remain relatively constant along the north-south gradient. Thus allele frequencies at the Lap-locus can be predicted from inversion frequencies.These observations can be interpreted in such a way that the pattern of allozyme variation within gene arrangements is due to founder effects caused by the unique origin of inversions. The gene blocks in the different inversions seem to represent more or less separated gene pools. In polymorphic populations the coexistence of genetically differentiated inversions presumably gives rise to heterotic interaction.     

Seasonal changes in chromosomal inversion polymorphism in a <Emphasis Type="Italic">Drosophila subobscura</Emphasis> natural population from a southeastern European continental refugium of the last glaciation period

Chromosomal inversion polymorphism was studied in a natural population of Drosophila subobscura from the Djerdap gorge of the Danube River, which is one of southeastern European continental refugia during the last glaciation period. The existence of changes in chromosomal inversion polymorphism during different periods of time was tested. The observed high abundance of chromosomal arrangements and lower IFR value allowed this population to be classified with central European populations. It was characterized by the O₇ and O_{3 + 4 + 5} arrangements, not found in other populations from the region analyzed. Within the region studied, the E_{1 + 2 + 9 + 12} chromosomal arrangement was found, apart from this population from a warm habitat, only in one population from a Mediterranean refugium. Many similarities have been observed between the population from the Djerdap gorge and other populations from the region analyzed, which indicates that postglacial migration may have started from this southeastern European refugium. Seasonal changes in the frequencies of the E_st and O_{3 + 4 + 7} chromosomal arrangements, E_st and O_{3 + 4 + 7}, being more frequent in spring and in summer, respectively, were found. The changes in the J chromosome (J_st/J_st), E chromosome (E_st/E_{1 + 2 + 9} and E₈/E_{1 + 2 + 9}), and O chromosome (O_st/O_{3 + 4 + 22}; O_{3 + 4}/O_{3 + 4}; and O_{3 + 4 + 1}/O_{3 + 4 + 7}), whose karyotypic combination frequencies were associated with seasonal changes, were also found. These changes are most probably associated with seasonal climatic changes in the population analyzed, which confirms that the chromosomal inversions in D. subobscura are adaptive. The text was submitted by the author in English.     

Seasonal cycles of allozyme-by-chromosomal-inversion gametic disequilibrium in Drosophila subobscura

Allozyme loci are frequently found non randomly associated to the chromosomal inversions in which they are included in Drosophila. Two opposite views compete to explain strong allozyme-by-inversion gametic disequilibria: they result from natural selection or, conversely, merely represent remnants of associations accidentally established at the origin of inversions. Empirical efforts aimed at deciding between adaptive and historical scenarios have focused on the spatial distribution of disequilibria. Yet, the evolutionary significance of these associations remains uncertain. I report here the results of a time-series analysis of the seasonal variation of alleles at six allozyme loci (Acph, Lap, Pept-1, Ao, Mpi, and Xdh) in connection with the O chromosomal polymorphisms of D. subobscura. The findings were: (1) in the segment I of the O chromosome, Lap and Pept-1 allozymes changed seasonally in a cyclical fashion within the ST gene arrangement, but they changed erratically within the 3 + 4 gene configuration; (2) the frequencies of Lap1.11 and Pept-1(0.40) within ST dropped to their lowest values in early and late summer, respectively, when the seasonal level of the ST arrangement is lowest. Furthermore, Lap1.11 and Pept-1(0.40) covary with ST only within these seasons, yet in a fashion inconsistent with these alleles having a major influence on the dynamics of the inversion; (3) seasonal cycling of alleles within inversions were not detected at Acph, Ao, Mpi, and Xdh, yet these loci are nearly monomorphic at the study population, and/or their sampled series were shorter than those for Lap and Pept-1; and (4) simply monitoring allozyme frequencies separately for each inversion proved to be superior, for evidencing the seasonal cycles of the disequilibria, to the use of the D' coefficient of association. Observed seasonal cycles of allozymes within inversions likely reflect natural selection.     

Temporal changes of chromosomal polymorphism in natural populations of Drosophila subobscura

The behaviour of the chromosomal polymorphism of D. subobscura was analyzed in relation to temporal changes, daily, seasonal and annual. Firstly, chromosome analyses were carried out over a year in a natural population. Samples were taken at monthly intervals in Tibidabo, a locality close to Barcelona. In all the months except January, the number of individuals captured was enough to carry out a chromosome analysis of that population. The A, E and O chromosomes showed a great uniformity in the frequencies of gene arrangement over the year. However, significant changes occur in the frequencies of J and U chromosomes. The J₁ and U₁₊₂₊₈ arrangements showed a similar tendency, with two maxima, in June and February, and a minimum in September. These changes seem to be unrelated to the cyclical climatic changes. A chromosome analysis of the subsamples captured during the day, in the May sample, was done. In no case was the change in chromosome frequencies statistically significant. The behaviour of the U_st arrangement must be noted, the frequency of which decreased through the day. Finally, the data for the Tibidabo population were compared with samples captured in nearby localities over a period of 21 years. Significant differences were found in the frequencies of all the chromosomes, with the exception of J. The most differentiated population was the last sampled, from 1982. In this population the st arrangements tended to decrease in the A, E and O chromosomes and A₂, E_1+2+9+12 and O₃₊₄₊₇ increase, respectively.     

Analysis of inbreeding in a colonizing population of Drosophila subobscura

An analysis of the effects of inbreeding on the genetic structure of a colonizing population of Drosophila subobscura has been carried out. Species of Drosophila, particularly D. subobscura, may have lethal alleles associated with chromosomal inversions and our aim was to assess the extent to which the genome is balanced in this way. The frequencies of chromosomal inversions were compared between a large population and a set of 72 lines that were maintained by brother-sister mating for 10 generations. Fisher's matrix method was used to calculate the expected homozygosity in these inbred lines for 5 allozyme loci (Aph, Hk-1, Lap, Odh, and Pept-1) used as markers of large chromosomal segments. Furthermore, the expected rates of fixation corresponding to these allozyme loci were also calculated. The results show that the amount of homozygosis observed did not differ significantly from expectations (with the corresponding loss of lines as a consequence of the reduction in viability). However, two deviations from strict neutrality were observed: there was a heterozygote excess at the Lap locus, and the frequency of the O ₅ inversion (always associated with a lethal gene in colonizing populations) was higher than expected.     

Differential flight activity among karyotypes: daily and weather-induced changes in chromosomal inversion polymorphism in natural populations of Drosophila subobscura

The question is posed whether karyotype frequencies in the active fraction of Drosophila subobscura populations vary consistently in the course of a day, or in accordance with weather changes. Significant daily variation occurred in all chromosomes with the exception of the sex chromosome. The proportions of the karyotypes U_st/1+2 and I_st/st decreased from early to late morning and increased during the evening. I_st/1 displayed a complementary behaviour. The homokaryotypes O_st/st and E_st/st decreased both during the morning and the evening. Weather disturbances caused a remarkable variation in the frequencies of the standard gene arrangements.     

Genetic and environmental factors in the resistance of Drosophila subobscura adults to high temperature shock

Two replicate selection experiments to increase and decrease heat shock resistance of Drosophila subobscura adults were carried out maintaining control lines. In the present paper, the chromosomal-inversion and enzymatic polymorphism variation with selection is analyzed. The results indicate an erratic variation of chromosomal arrangement frequencies for practically all the chromosomes in the selected lines, showing a loss of the less frequent arrangements especially in sensitive lines. Only the A chromosome and the O+4 arrangement show a behaviour that may not be due to random effects, which points to the possible existence of heat shock factor(s) in these chromosomes. Similarly, an erratic variation of allele frequencies is observed for all the enzymes studied (Aph, Pept-1) except for the Hk-1 enzyme. We cannot establish the possible participation of this locus in heat shock resistance from the results obtained up to now. A significant decrease in heterozygosity is detected in sensitive lines from chromosomal-inversion polymorphism.     

Seasonal changes of chromosomal inversion polymorphism in a Drosophila subobscura natural population from southeastern European continental refugium of the last glaciation period

Chromosomal inversion polymorphism was studied in a natural population of Drosophila subobscura from Djerdap gorge of Danube river, that represents one of southeastern European continental refugiums during last glaciation period. The existence for changes of chromosomal inversion polymorphism during different periods of time was tested. Observed very high abundance of chromosomal arrangements and lower IFR value grouped this population to central European populations. It was characterized with O7 and O(3+4+5) arrangements, not found in other populations from the region analyzed. Except in this population from warmer habitat, observed E(1+2+9+12) chromosomal arrangement was found, in the region analyzed, only in one population from Mediterranean refugial area. Many similarities have been observed between population from Djerdap gorge and other populations from the region analyzed, indicating that process of post-glacial migration may have started from this southeastern European refugium area. Seasonal changes in the frequencies for Est and O(3+4+7) chromosomal arrangements where Est was more frequent during the spring period and O(3+4+7) during the summer period were found. The changes for J-chromosome (Jst/Jst), E-chromosome (Est/E(1+2+9); E(8)/E(1+2+9) and O-chromosome (Ost/O(3+4+22); O(3+4)/O(3+4); O(3+4+1)/O(3+4+7) whose karyotypic combination frequencies were associated with seasonal changes were also found. These changes most probably represent associations with seasonal climatic changes in the population analyzed, supporting adaptivity of chromosomal inversions in D. subobscura.     

Patterns of puffing activity and chromosomal polymorphism in Drosophila subobscura I. J. and U chromosomes

A study of the puffing patterns of the J_st, J₁, U_1–2, U_1–2–8 and U_st chromosomal arrangements of Drosophila subobscura, from different geographical origins, has been carried out. Twenty-eight puffs were observed, 10 on the J chromosome, and 18 on the U chromosome. No differences, whether qualitative or quantitative, have been found between the puffing pattern of the J chromosome, whether from the same of different geographical background. In the U chromosome, the U_1–2 and U₁₊₂₊₈ arrangements show the same puffing pattern, and neither quantitative nor qualitative differences were found. However, the puffing pattern of these chromosomes alters considerably in the U_st arrangement of the K228 laboratory strain.     

Associations of Alleles of the Esterase-1 Locus with Gene Arrangements of the Left Arm of the Second Chromosome in DROSOPHILA ROBUSTA

Evidence of strong associations of Est-1 alleles with the 2L, 2L1 and 2L3 gene arrangements of the left arm of the second chromosome in D. robusta is presented. Each gene arrangement is polymorphic for three to four Est-1 alleles. The allele frequencies differ in the 2L3 and 2L arrangements; the allele Est-1^.92 is 8% in the 2L3 arrangement (n=203)—this allele is 82% in the 2L arrangement (n=203); the allele Est-1^1.0 is 66% and 14.8% in the 2L3 and 2L arrangements, respectively. There are no differences in allele frequencies in 2L3 arrangements from any of the widely separated seven different populations; similarly the allele frequencies in the 2L arrangement are alike in all five widely separated populations studied. The allele frequencies in the 2L1 arrangement are intermediate to those observed in the 2L3 and the 2L arrangements and show north-south clinal change. These associations between Est-1 alleles and gene arrangements of the left arm of the second chromosome are due to natural selection favoring different allele frequencies in different gene arrangements, as a result of epistatic interactions between the Est-1 locus and the loci on the gene arrangements. As expected, we observe that the proportion of heterozygotes is greater in the inversion heterokaryotypes than in the homokaryotypes.     

Evolutionary genetics of Drosophila ananassae. I. Effect of selection on body size and inversion frequencies

Adaptive importance of inversion polymorphism has been discussed in Drosophila species at several levels but no study has been carried out demonstrating the individual and combined effects of polymorphic inversions on the fitness of flies through bi‐directional selection. Therefore, artificial bi‐directional selection for thorax length in Drosophila ananassae was carried out for 10 generations. Both, Tukey test for selection difference and regression coefficients of offspring on mid‐parent are highly significant. The realized heritability (h²) in males of both high and low selection lines is more or less similar but is more pronounced in low line females, which suggests the asymmetrical response. This asymmetry in selection is discussed in the light of evidence provided by the study of chromosome inversion frequencies in different selection lines at different generations of selection. Interestingly, chromosome inversion frequency changes towards homozygosity for different gene arrangements in different selection lines. Tests of correlations at G₆ and G₁₀ among different gene arrangements as well as with mean thorax length suggest that 2L‐ST gene arrangement is negatively correlated, while 3L‐ST gene arrangement is positively correlated with thorax length. Furthermore, the present study shows the significant effects of 3L‐ST and 2L + 3L (positive correlation) on thorax length, while 3R‐ST and 2L + 3R show significant effect (negative correlation) on thorax length, which was not evident in the previous study. Present results also suggest how polymorphic inversions and their combinations affect the body size differently in different selection lines. These results suggest that thorax length in D. ananassae is under polygenic control and inversion polymorphism plays crucial role in maintaining body size by modifying genotypic frequency under various selection pressures.     

Multilocus analysis of nucleotide variation in Drosophila madeirensis, an endemic species of the Laurisilva forest in Madeira

Drosophila madeirensis is an endemic species of Madeira that inhabits the island Laurisilva forest. Nucleotide variation in D. madeirensis is analysed in six genomic regions and compared to that previously reported for the same regions in Drosophila subobscura, an abundant species in the Palearctic region that is closely related to D. madeirensis. The gene regions analysed are distributed along the O₃ inversion. The O₃ arrangement is monomorphic in D. madeirensis, and it was present in ancestral populations of D. subobscura but went extinct in this species after the origin of the derived O_ST and O₃₊₄ arrangements. Levels of nucleotide polymorphism in D. madeirensis are similar to those present in the O_ST and O₃₊₄ arrangements of D. subobscura, and the frequency spectrum is skewed towards rare variants. Purifying selection against deleterious nonsynonymous mutations is less effective in D. madeirensis. Although D. madeirensis and D. subobscura coexist at present in Madeira, no clear evidence of introgression was detected in the studied regions.     

Genetic divergence in the snorkel snail,Rhiostoma housei,a species complex in Thailand (Caenogastropoda: Cyclophoridae)

Allozyme variation was examined in 292 samples of the snorkel snail, Rhiostoma housei, collected from 12 localities within four regions of Thailand. Ten allozyme loci were screened across all the samples using horizontal starch gel electrophoresis, and eight of these loci, from eight enzyme systems, were found to be polymorphic. Within these eight loci the expected heterozygosity (H_exp) was moderate to high ranging from 0.015 to 0.148 with an overall mean of 0.081 ± 0.041, whilst a high genetic heterogeneity among samples was found (F_st = 0.798). R. housei exhibited a weak pattern of isolation by distance over the entire tested species range in Thailand. The high F_st and moderate to high H_exp suggest gene flow amongst populations is partially restricted resulting in fragmentation into local gene pools. Based on allele frequencies and an allozyme dendrogram, six groups were identified, and these entities may represent separate biological species. The large values of genetic distances and fixed allelic differences obtained for each combination of allopatric samples imply the presence of cryptic species under the name R. housei.     

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC₄ strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac⁺), lactococcin-producing (Lap⁺), lactococcin-resistant (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g of DNA. PC₄ protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac^–Lap^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r and Lac⁺ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC₄ transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.     

Allozyme Genotype-Environment Relationships. I. Variation in Natural Populations of DROSOPHILA PACHEA

Remarkable geographic and seasonal uniformities in gene and genotype frequencies were found for four enzyme loci. Principal component analysis revealed two patterns of allele frequency changes in three of the allozyme loci (Est-b, Est-c and Acph) and one pattern in the Mdh-d locus. These patterns accounted for 90% to 100% of the variability at these loci. Significant positive correlations were obtained between several of the components and the variability in the non-saponifiable and alkaloid fractions of the senita cactus host plant. The genetic variance within a single locality (Guaymas, Mexico) was as great as the variance between all localities sampled. This is interpreted as a local founder effect. The temporary nature of the rotting cactus breeding site, the delayed maturation and the differential maturation rate of females and males are three factors proposed to cause forced outbreeding and counter the local founder effect. A combination of selection resulting from a direct interaction of the genotype and the environment, in addition to migration, is proposed to maintain the uniform gene frequencies.     

Fitness of Allozyme Variants in DROSOPHILA PSEUDOOBSCURA. I. Selection at the PGM-1 and ME-2 Loci

We have studied in Drosophila pseudoobscura the effect of allozyme variation on seven fitness components: female fecundity, egg hatchability, egg-to-adult survival under near-optimal and under competitive conditions, rate of development under near-optimal and under competitive conditions, and mating capacity of males. Three genotypes at each of two loci, Pgm-1 and Me-2, have been studied in various combinations. These two loci are highly polymorphic in natural populations of D. pseudoobscura. Statistically significant differences involving one or more genotypes exist for all components of fitness. No single genotype is best for all fitness components; rather the relative fitnesses of genotypes are reversed when different parameters are considered, or when they are studied in different environmental conditions. Also, the average egg-to-adult survival and rate of development are better when different genotypes are reared together than when they occur in pure culture. Four different modes of selection have been uncovered by our experiments. These forms of selection may account for the persistence of the two allozyme polymorphisms in nature, and for previously observed seasonal fluctuations of the allelic frequencies in natural populations.     

Seasonal and interannual variations of sockeye salmon (<Emphasis Type="Italic">Oncorhynchus nerka</Emphasis>) microsatellite DNA in two Kamchatka lake-river systems

Seasonal and interannual variations in the sockeye salmon populations from two lake-river systems of the East and West Kamchatka were studied. Stability of allele and genotypic frequencies of six microsatellite DNA loci in the adjacent generations and spawning populations of the sockeye salmon of the Bol’shaya River was confirmed experimentally. The pairwise intersample differentiation (F _st) of the local sockeye salmon populations from the southwestern Kamchatka coast (Ozernaya and Bol’shaya Rivers) was almost 7 times higher than the corresponding values for the spawning populations of the Bol’shaya River sockeye salmon of the adjacent years; 15 times, for the adjacent Bol’shaya River sockeye salmon generations; and four times, for the seasonal races within the Kamchatka River.