Karyological characterization of the endemic Iberian rock lizard, Iberolacerta monticola (Squamata, Lacertidae): insights into sex chromosome evolution.

Rock lizards of the genus Iberolacerta constitute a promising model to examine the process of sex chromosome evolution, as these closely related taxa exhibit remarkable diversity in the degree of sex chromosome differentiation with no clear phylogenetic segregation, ranging from cryptic to highly heteromorphic ZW chromosomes and even multiple chromosome systems (Z1Z1Z2Z2/Z1Z2W). To gain a deeper insight into the patterns of karyotype and sex chromosome evolution, we performed a cytogenetic analysis based on conventional staining, banding techniques and fluorescence in situ hybridization in the species I. monticola, for which previous cytogenetic investigations did not detect differentiated sex chromosomes. The karyotype is composed of 2n = 36 acrocentric chromosomes. NORs and the major ribosomal genes were located in the subtelomeric region of chromosome pair 6. Hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes and interstitially in 5 chromosome pairs. C-banding showed constitutive heterochromatin at the centromeres of all chromosomes, as well as clear pericentromeric and light telomeric C-bands in several chromosome pairs. These results highlight some chromosomal markers which can be useful to identify species-specific diagnostic characters, although they may not accurately reflect the phylogenetic relationships among the taxa. In addition, C-banding revealed the presence of a heteromorphic ZW sex chromosome pair, where W is smaller than Z and almost completely heterochromatic. This finding sheds light on sex chromosome evolution in the genus Iberolacerta and suggests that further comparative cytogenetic analyses are needed to understand the processes underlying the origin, differentiation and plasticity of sex chromosome systems in lacertid lizards.

The estimation of parental genotypes by the analysis of a fixed number of their offspring.

When genotypes are cryptic in adult organisms but visible in young life-stages, it is possible to estimate the genotypes in parents after the examination of a fixed number of their offspring. Thus, in Drosophila, males collected in the wild can be crossed to females of a homozygous laboratory stock, and the identification for genotype of seven larvae is largely enough to guarantee a correct diagnosis of the two alleles of the father (P = 0.984). On the other hand, when collected females were inseminated in nature, it should be possible to determine the constitution both the female and her mate, after the analysis of a sufficient number of their offspring. Nevertheless, exact formulas for the probabilities of correct diagnoses of parental genotypes in this case have not yet been published. In this paper we derive such formulas, and illustrate their application. The conclusion we draw from our calculations is that, unless 13 larvae are examined among the offspring, the probability of misdiagnosis is over the 0.05 level. Of course, the errors produced become greater as smaller offspring numbers are analyzed. But in the case of one locus with two alleles, we have derived formulas to correct the observed number of matings and estimate its actual number in the sample, for any offspring sample size. After these corrections, we have been able to calculate estimates of relative mating frequencies in the sampled population, together with their associated errors, which can be subsequently used in studies of adult fitness components, mainly in those dealing with sexual selection.

The Evolutionary History of DROSOPHILA BUZZATII. Xii. the Genetic Basis of Sterility in Hybrids between D. BUZZATII and Its Sibling D. SERIDO from Argentina.

The genetic basis of hybrid sterility has been investigated in backcross segmental hybrids between two sibling species, Drosophila buzzatii and D. serido. Asynapsis of homologous bands in hybrid polytene chromosomes has been used to identify the D. serido chromosome segments introgressed into the D. buzzatti genome. All the investigated chromosomes contain male sterility factors. For autosomes, sterility is produced when an introgressed D. serido chromosome segment, or combination of segments, reaches a minimum size. On the other hand, any introgressed X chromosome segment from D. serido, irrespective of its size, produces either male hybrid sterility or inviability.

The theoretical distribution of lengths of intact chromosome segments around a locus held heterozygous with backcrossing in a diploid species.

When two different isogenic lines of a diploid species (or two different species) are crossed, the resulting F1 individuals should be heterozygous at all the loci fixed for different alleles in the two strains (in the limit, at all the loci of the genome). If one of these loci is then held heterozygous for several generations of repeated backcrossing to the same strain, the average length of intact chromosome segments (with reference to the original parental chromosome) on both sides of the selected locus, or, equivalently, the average length of segments surrounding that locus which are still heterozygous (with reference to the fully heterozygous F1 chromosome), may diminish, but cannot increase. Several authors have derived equations to predict this average. We show that the most widely used criterion, developed by R.A. Fisher, leads to serious overestimations of the true parametric values, when applied to early generation analyses, with the corresponding errors in the interpretation of experimental results. We then derive the exact equations both for the average and standard deviation of the lengths of intact chromosome segments surrounding a locus held heterozygous after any number of generations of backcrossing. Our results are in close agreement with those found by a former author, although involving a rather different approach.

Hidden effects of X chromosome introgressions on spermatogenesis in Drosophila simulans x D. mauritiana hybrids unveiled by interactions among minor genetic factors.

One of the most frequent outcomes of interspecific hybridizations in Drosophila is hybrid male sterility. Genetic dissection of this reproductive barrier has revealed that the number of responsible factors is very high and that these factors are frequently engaged in complex epistatic interactions. Traditionally, research strategies have been based on contrasting introgressions of chromosome segments that produce male sterility with those that allow fertility. Few studies have investigated the phenotypes associated with the boundary between fertility and sterility. In this study, we cointrogressed three different X chromosome segments from Drosophila mauritiana into D. simulans. Hybrid males with these three segments are usually fertile, by conventional fertility assays. However, their spermatogenesis shows a significant slowdown, most manifest at lower temperatures. Each of the three introgressed segments retards the arrival of sperm to the seminal vesicles. Other small disturbances in spermatogenesis are evident, which altogether lead to an overall reduction in the amount of motile sperm in their seminal vesicles. These results suggest that a delay in the timing of spermatogenesis, which might be brought about by the cumulative action of many different factors of minor segment, may be the primary cause of hybrid male sterility.

Isolation and characterization of polymorphic microsatellite markers in Iberolacerta monticola, and cross-species amplification in Iberolacerta galani and Zootoca vivipara.

Fourteen polymorphic microsatellite loci are described for the Iberian rock lizard, Iberolacerta monticola. Genetic variation in a sample of 20 individuals from Piornedo (northwestern Spain) was quantified both by the number of alleles per locus, which ranged from six to 13, and by the expected frequency of heterozygotes under random mating (heterozygosity), which ranged from 0.761 to 0.902. Single locus and global exclusion probabilities were also computed, and indicate a high power of these markers for paternity assignments and mating system studies of I. monticola. All the analysed loci were also polymorphic in Iberolacerta galani, but only seven in Zootoca vivipara.

Location of X-linked polygenic effects causing sterility in male hybrids of Drosophila simulans and D. mauritiana.

There is general agreement that hybrid male sterility in Drosophila is caused by changes at several (perhaps many) factors, most of them located on the X chromosome. These factors have been generally considered as major genes, each one of them able to bring about sterility by itself. However, the evidence on this last point is not conclusive. In principle, the possibility that they correspond to located polygenic effects instead of genes with a large effect cannot be excluded. This paper shows that some of the factors that cause male sterility in D. simulans/D. mauritiana hybrids, located by recombination on the X chromosome, are indeed 'effective factors', or located polygenic effects. Some of the consequences of this finding are explored.

The evolutionary history of D. buzzatii. XXII. Chromosomal and genic sterility in male hybrids of Drosophila buzzatii and Drosophila koepferae.

The genetic basis of sterility in F1 male hybrids of Drosophila buzzatii and D. koepferae has been investigated in two steps. (1) By successive backcrossing of hybrid females to either parental species. (2) By assessment of the effects on male fertility of selected segments of polytene chromosomes from the donor species on a background entirely derived from the recipient species. The length of introgressed segments producing sterility was progressively reduced through repeated backcrosses. This procedure sometimes led to an approximate mapping of major genes of hybrid sterility (genic sterility) on the polytene chromosome map. At other times it was found that sterility was produced only when the introgressed segment exceeded a certain threshold size (chromosomal sterility). The contribution of the autosomes to hybrid sterility seems to be mainly of the chromosomal type. The evidence concerning the X chromosome is equivocal. No fertile males were found following introgression with any of the investigated segments of this chromosome. These results are compatible both with the presence of at least six major genes of hybrid sterility (genic sterility) and with the existence of a rather small threshold size for the chromosome segments producing sterility (chromosomal sterility). The role of the Y chromosome was not investigated in this study.

The evolutionary history of Drosophila buzzatii. IX. High frequencies of new chromosome rearrangements induced by introgressive hybridization.

Introgression of a chromosome segment from Drosophila serido into the genome of its sibling D. buzzatii brought about the release of mutator potential in the hybrids. Mutator activity was determined by examining the frequency of new chromosomal rearrangements, that appeared only in the progeny of hybrid individuals. Mutation frequency was 30 times greater in the progeny of hybrid males than in that of hybrid females. There was a remarkable influence of the D. buzzatii genetic background on the frequency of production of these new rearrangements. The appearance of a new rearrangement did not depend on the genotype of the larva that bore it, but only on that of its hybrid progenitor. Among the new rearrangements there were inversions, translocations, and duplications. The number of translocations was significantly lower than that of inversions or duplications; this last type was the most frequently recorded. The distribution of the aberrations among the four major autosomes seemed to be homogeneous, although the total number of breakpoints was significantly greater in chromosome 4 than in the others. No rearrangement was found on the X chromosome. Breakpoints within three of the four affected autosomes were not randomly distributed.

The evolutionary history of Drosophila buzzatii. XXI. Cumulative action of multiple sterility factors on spermatogenesis in hybrids of D. buzzatii and D. koepferae.

The genetic basis of sterility in male hybrids of Drosophila buzzatii and D. koepferae has been investigated by assessment of the effects on spermatogenesis of substituting separate chromosome segments of the recipient species with the homologous material from the donor species, either in heterozygous (autosomes) or hemizygous (X chromosome) condition, after successive backcrossing of hybrid females to either parental species. Introgressed segments were identified by the characteristic asynapsis of the polytene chromosomes in their heterospecific regions. Except for one case, the introgression of chromosome segments either from autosome 3, 4, or 5 brings about sterility only when the introgressed segment exceeds a minimum size (threshold size). Segments of equal size frequently produce similar abnormalities, whose severity increases with the size of the introgressed segment. Apparently, throughout these autosomes of D. buzzatii and D. koepferae there are many non-allelic, minor sterility genes, whose individual segregation cannot be recognized phenotypically, and which act cumulatively on the same characteristics of spermatogenesis, each contributing a small effect to the phenotype. Accordingly, these genes should be considered as polygenes, and the type of sterility they bring about should be properly designated polygenic sterility.

Evolutionary history of the human endogenous retrovirus family ERV9.

Several distinct families of endogenous retrovirus-like elements (ERVs) exist in the genomes of primates. Despite the important evolutionary consequences that carrying these intragenomic parasites may have for their hosts, our knowledge about their evolution is still scarce. A matter of particular interest is whether evolution of ERVs occurs via a master lineage or through several lineages coexisting over long periods of time. In this work, the paleogenomic approach has been applied to the study of the evolution of ERV9, one of the human endogenous retrovirus families mobilized during primate evolution. By searching the GenBank database with the first 676 bp of the ERV9 long terminal repeat, we identified 156 different element insertions into the human genome. These elements were grouped into 14 subfamilies based on several characteristic nucleotide differences. The age of each subfamily was roughly estimated based on the average sequence divergence of its members from the subfamily consensus sequence. Determination of the sequential order of diagnostic substitutions led to the identification of four distinct lineages, which retained their capacity of transposition over extended periods of evolution. Strong evidence for mosaic evolution of some of these lineages is presented. Taken altogether, the available data indicate that the possibility of ERV9 still being active in the human lineage can not be discarded.

Genetic mapping of the Adh locus in the repleta group of Drosophila by in situ hybridization.

A biotinylated probe of the Adh (alcohol dehydrogenase) gene of Drosophila melanogaster was used for in situ hybridization on polytene chromosomes of D. mojavensis and D. buzzatii, two species of the repleta group of the genus Drosophila. Hybridization showed that the Adh gene maps at the G1a band of the third chromosome. This is in accordance with a previous result obtained through the use of interspecific hybrid asynapsis as a cytological marker and establishes the limits of the precision of this method.

Structural features of the mdg1 lineage of the Ty3/gypsy group of LTR retrotransposons inferred from the phylogenetic analyses of its open reading frames.

The increasing amount of data generated in recent years has opened the way to exhaustive studies of the relationships among different members of the Ty3/gypsy group of LTR retrotransposons, a widespread group of eukaryotic transposable elements. Former research led to the identification of several independent lineages within this group. One of the worse represented of them is that of mdg1, integrated so far only by the Drosophila retrotransposons mdg1 and 412. Our exhaustive database searches indicate the existence of three other Drosophila members of this lineage. Two of them correspond to elements already known, namely, Stalker and blood, but the third one is a new element, which we have called Pilgrim. This element is well represented within the D. melanogaster genome, as revealed by our Southern blot analysis of different strains. The case of Stalker is particularly remarkable, since its phylogenetic relationships clearly point to the mosaic origin of its genome. Finally, our analysis of the evolution of a small ORF preserved within the 5' leader region of these elements indicates different evolutionary rates, presumably as a result of distinct selective constraints.

Amplification and phylogenetic relationships of a subfamily of blood, a retrotransposable element of Drosophila.

To get a better understanding of the effect of interelement selection on the variation of long terminal repeat retrotransposon families, we have investigated the evolutionary history of blood in the Drosophila melanogaster species complex. We carried out a PCR approach to amplify the 5' untranslated region from blood in the four species of the complex. This procedure revealed two main classes of size variants. Phylogenetic analyses of nucleotide sequences from these variants and blood elements from the Drosophila Genome Projects database show that elements are grouped according to their size, so that they probably correspond to two subfamilies. These two subfamilies arose prior to the split of the complex, and several facts indicate that the expansion of one of them is leading to the competitive exclusion of the other, at least from the euchromatic regions of the genome.

On the difficulties of discriminating between major and minor hybrid male sterility factors in Drosophila by examining the segregation ratio of sterile and fertile sons in backcrossing experiments.

The observation of segregation ratios of sterile and fertile males in offspring samples from backcrossed hybrid females is, in principle, a valid method to unveil the genetic basis of hybrid male sterility in Drosophila. When the female parent is heterozygous (hybrid) for a sterility factor with major effects, equal proportions of fertile and sterile sons are expected in her offspring. However, intact (not recombined) chromosome segments of considerable length are expected to give segregation ratios that can not be easily differentiated from the 1:1 ratio expected from a single factor. When the phenotypic character under analysis can be determined by combinations of minor factors from the donor species spanning a certain chromosome length, very large offspring samples may be needed to test this alternative hypothesis against the null hypothesis of a single major factor. This is particularly the case of hybrid male sterility determinants in Drosophila.

A polygenic basis of hybrid sterility may give rise to spurious localizations of major sterility factors.

A simple model is presented to illustrate how an underlying generalized polygenic basis of hybrid sterility is expected to lead to spurious localizations of factors with major effects, when a conventional experiment of recombination mapping is carried out. The model shows that a major gene will be detected at roughly the same distance from each of the chromosome markers used in the experiment. These expectations are contrasted with the results from several experiments on hybrid male sterility in Drosophila, which claimed to have mapped single sterility factors. It is concluded that, except for one report, all the evidence presented so far on the genetic basis of hybrid male sterility in Drosophila is in fact compatible with the generalized polygenic model.

A three-locus system of interspecific incompatibility underlies male inviability in hybrids between Drosophila buzzatii and D. koepferae.

In hybrids between the sibling species D. buzzatii and D. koepferae, both sexes are more or less equally viable in the F1. However, backcross males to D. buzzatii are frequently inviable, apparently because of interspecific genetic incompatibilities that are cryptic in the F1. We have performed a genetic dissection of the effects of the X chromosome from D. koepferae. We found only two cytological regions, termed hmi-1 and hmi-2, altogether representing 9% of the whole chromosome, which when introgressed into D. buzzatii cause inviability of hybrid males. Observation of the pattern of asynapsis of polytene chromosomes (incomplete pairing, marking introgressed material) in females and segregation analyses were the technique used to infer the X chromosome regions responsible for this hybrid male inviability. The comparison of these results with those previously obtained with the same technique for hybrid male sterility in this same species pair indicate that in the X chromosome of D. koepferae there are at least seven times more regions that produce hybrid male sterility than hybrid male inviability. We have also found that the inviability brought about by the introgression of hmi-1 is suppressed by the cointrogression of two autosomal sections from D. koepferae. Apparently, these three regions conform to a system of species-specific complementary factors involved in an X-autosome interaction that, when disrupted in backcross hybrids by recombination with the genome of its sibling D. buzzatii, brings about hybrid male inviability.