Precopulatory isolation mechanisms encompass geographic, ecological, seasonal, mechanical, and ethological isolation. The first three factors involve the spatial, temporal, or ecological separation of closely related species, while mechanical isolation is associated with anatomical differences in the male and female reproductive organs that hinder successful copulation. Among these mechanisms, ethological isolation is the most effective in restricting interspecific mating. The final two isolation mechanisms, namely (1) divergence in mating organ morphology and selective mating and (2) the development of species-specific behavioral patterns, are driven by sexual selection. Intrasexual competition for optimal mating partners leads to the emergence of distinct markers that facilitate the assessment of genetic compatibility between individuals. These markers are examined during courtship and can manifest as visual, acoustic, and/or chemical stimuli that are genetically determined.
4.1. The Effect of Sex Chromosomes and Autosomes on the Efficiency of Copulation
Our investigation focused on assessing the influence of
D. virilis X chromosomes and autosomes on copulation efficiency and the expression of courtship elements within the context of the
D. americana genome. The
D. americana genome includes the neo-X chromosome, which results from the fusion of the X and fourth chromosomes [
32,
33], and the neo-Y chromosome, which is represented by a fourth chromosome in addition to the normal Y chromosome in males [
34,
35,
36]. Two aspects related to the neo-X and neo-Y chromosomes should be considered when evaluating the impact of sex chromosomes and autosomes on courtship behavior and copulation. The first concern relates to the potential bias in the weight coefficients of
D. virilis X chromosomes and autosomes in males and females from the F
2 and backcrosses due to chromosome segregation disruption in F
1 individuals and selective elimination of certain genotypes in subsequent generations. In our study, as there were no significant differences in offspring survival between control crosses and F
1 hybrids, we can disregard the issue of estimate distortion due to shifts in chromosome population composition. The second issue arises from the rearrangement of the fourth chromosome in
D. americana, where it fuses with an X chromosome, resulting in a symmetrical distribution of the fourth chromosome of
D. virilis with its X or Y chromosomes. Chromosome segregation distortion is commonly observed in the second generation and offspring from backcrosses, while the overall proportion of chromosomes from both species in the offspring remains unchanged. In our proposed models, we determine the regression coefficients based on the weight coefficients of both species’ chromosomes, corresponding to their proportion in the offspring. The influence of segregation distortion in this case causes a shift in the estimates of homozygotes and heterozygotes for the fourth chromosome, favoring homozygotes by approximately 25%. Consequently, this suggests a contribution of this autosome to the obtained estimates of the X chromosome’s impact.
All estimates of the effect of the proportion of D. virilis chromosomes are presented in a standardized form, allowing for comparisons both within each experimental group and between the groups, for both sex chromosomes and autosomes. Given that copulation success tends to decrease with increasing heterospecificity in pairs, we hypothesized that, overall, the proportion of D. virilis chromosomes would have a negative impact on copulation latency and a positive impact on copulation duration in pairs with a D. virilis partner (either male or female). As anticipated, we observed the opposite pattern for pairs with a D. americana partner: the overall effect of D. virilis chromosomes on copulation latency was positive, while it was negative for copulation duration. Furthermore, we found that as the genetic similarity between mating partners increased, the latency decreased, and the copulation duration increased, suggesting more thorough insemination of the female.
Estimates based on the absolute values of the male genotype’s effect on copulation success predominantly indicate the influence of sex chromosomes, while the effect of the female genotype is primarily associated with autosomes. These differences can be explained by models of sex chromosome and autosomal evolution rates [
37,
38], which consider constraints on the expression of sex-specific genes, the accumulation of substitutions in males and females, and the degree of allele dominance. It is important to note that the rate of chromosome evolution corresponds to the fixation rate of alleles linked to these chromosomes, thereby influencing the overall contribution of chromosomes to diverging traits. Studies on Drosophila have shown at least equal rates of variability accumulation in males and females or a higher rate in males [
39,
40]. According to the models [
37,
38], we can expect an underrepresentation of differentially expressed genes located on the X-chromosome in females, regardless of the degree of their dominance, and the same underrepresentation in males if sex-limited genes are predominantly dominant. In other words, in pairs where the male has a constant genotype, Aut
D. virilis should consistently make a predominant contribution to copulation efficiency and the expression of courtship elements in females. Similarly, in pairs where the female has a constant genotype, X
D. virilis should make a predominant contribution in males, assuming there is significant recessiveness of alleles of genes differentially expressed in males. The contributions of X chromosomes in males and autosomes in females, being the most significant, determine the overall effects of chromosomes on copulation efficiency and courtship elements.
The observed influence of female autosomes on copulation efficiency, which is mediated by rapidly evolving courtship elements, aligns well with the expected effects according to evolutionary rate models. However, the impact of the male X chromosome is less evident. In the genus Drosophila, there is a notable scarcity of genes with a male-biased expression on the X chromosome, likely due to a transfer of such genes from the X chromosome to autosomes [
41]. The preferential dominance of alleles could explain the higher rate of mutation accumulation on autosomes in male-biased genes [
38]. However, it does not account for the selective reduction of copies of such genes on the X chromosome.
As previously mentioned, recessive alleles of male-biased genes are rapidly fixed on the X chromosome. This suggests that there is an accumulation of adaptively significant variability in the models of sex chromosomes and autosomal evolution. Furthermore, the conclusions regarding the impact of allele dominance and the difference in mutation rates between males and females on the rate ratios are also applicable to the accumulation of mutation load. It is important to note that recessive mutations predominantly contribute to the genetic variation of traits that determine fitness. These mutations can be categorized into three types of alleles:
- (i)
Recently emerged deleterious alleles that have low frequencies and lead to reduced fitness;
- (ii)
Neutral and slightly deleterious alleles that are in equilibrium;
- (iii)
Alleles with intermediate frequencies that are maintained by opposing or frequency-dependent selection [
42,
43].
Estimates of the variability proportion for individual fitness components, conducted on populations of
D. melanogaster, have revealed a substantial contribution, of at least 50%, from newly emerged alleles with deleterious effects [
44,
45,
46]. Experiments on inbred populations of Drosophila and Mimulus [
47,
48,
49] (method description: [
50]) were carried out to assess the relative influence of the contribution of neutral and slightly deleterious mutations. These experiments provide compelling evidence for the significant effect of alleles that constitute a pool of constant genetic variability and occur at intermediate frequencies. Hence, the accelerated accumulation of recessive alleles on the X chromosome, linked to detrimental or slightly deleterious effects approaching neutrality in terms of viability, leads to the degradation of male-biased X-linked genes in the presence of paralogs or functional homologs on autosomes.
It can be assumed that the demasculinization of the X chromosome would result in a reduction of its contribution to traits selectively expressed in males. However, this conclusion is not absolute. In
D. melanogaster,
D. simulans,
D. ananassae, and
D. mojavensis, as well as on Muller A elements, there is a consistent underrepresentation of male-biased genes on the X chromosome, which has persisted and evolved for millions of years, comprising 30–43% [
41]. The underrepresentation of these genes on the neo-X chromosome of
D. pseudoobscura, formed through the fusion of Muller A and Muller D elements 8–12 million years ago, is 37%. If there were constant negative selection and erosion of male-biased genes on the X chromosome, their proportion on the “old” X chromosomes would be significantly lower. However, certain factors seem to restrict the erosion of some male-biased genes on the X chromosome, resulting in the demasculinization process reaching a plateau at approximately 70% of the expected level. These factors likely include rapidly evolving genes that play a role in prezygotic isolation barriers and are directly affected by sexual selection. Previous studies on mating behavior variability in
D. virilis males with conspecific and heterospecific females (
D. americana texana and
D. novamexicana) demonstrated the prominent role of the X chromosome in male behavior during the tapping stage of courtship with heterospecific females [
16]. Apart from courtship traits, these factors may also involve morphological and physiological characteristics associated with fertilization.
For instance, a study investigating the contribution of sex chromosomes and autosomes to the species-specific shape of the copulatory apparatus in
D. virilis and
D. lummei males revealed a disproportionately higher contribution of the X chromosome to the variability of shape traits (partial eta-squared) than expected [
51].
4.2. Variability of the Courtship Structure
A comparative analysis of courtship structure variability in all tested variants with different degrees of conspecific and heterospecific pairs revealed a general similarity in structure.
The longest courtship elements were tapping and licking, which aligns with previously reported findings in closely related species of the
virilis group and other Drosophila species [
2,
27,
28,
52]. Typically, the courtship ritual is initiated with this pair of elements, followed by the addition of acoustic signals from both the male’s singing and the female’s response, usually coinciding with licking and tapping. The second pair of elements had a shorter duration compared to the first pair but exceeded the duration of the remaining elements [
28,
52,
53]. Elements such as following, circling, and copulation attempts were characterized by shorter durations and occurred later in the courtship sequence if present. Previous studies have shown that circling behavior is more typical in
D. americana compared to
D. virilis [
52]. Additionally,
D. americana males display simultaneous tapping and licking, while
D. virilis males initiate the second element somewhat later [
52].
The analysis of courtship behavior in heterospecific pairs revealed differences in the courtship elements employed by
D. virilis and
D. americana males. Typically,
D. americana males exhibited a complete courtship ritual, while
D. virilis males ceased courting
D. americana females at the tapping stage in one-third of the tests. This finding aligns with previous studies on heterospecific crosses involving sibling species of the
virilis group [
12,
28,
52]. Specifically, a significant proportion of
D. virilis males refused to court females of
D. lummei,
D. americana, and
D. littoralis after a brief tapping, whereas males of these species actively courted
D. virilis females. This asymmetric change in the courtship structure during reciprocal heterospecific tests with specific combinations of sibling species was characterized by a considerable reduction in the licking stage, a substantial decrease in the duration of all courtship elements, and a significant decline in the number of copulations in one direction. Conversely, a significant increase in the duration of key courtship elements was observed in the other direction. For instance, this pattern was observed in the courtship behavior of
D. virilis males towards females of
D. lummei,
D. americana,
D. novamexicana, and
D. littoralis, while the courtship ritual of
D. americana,
D. novamexicana, and
D. littoralis males towards
D. virilis females exemplified the second direction. These observations indicate the high significance of the chemical communication channel in species recognition during mate selection in
D. virilis.
Many Drosophilidae species possess contact chemoreceptors on the tarsi of their front legs, maxillary palps, and proboscis [
54]. During tapping and licking behaviors, males detect nonvolatile pheromones on the sternites’ surface of females through taste-receptor neurons [
55]. Studies have demonstrated that
D. virilis males can readily distinguish females of their own species from those of sibling species such as
D. americana americana,
D. americana texana, and
D. novamexicana. Conversely, males of
D. americana americana,
D. americana texana, and
D. novamexicana struggle to differentiate
D. virilis females from females of their own species during the tapping stage [
56]. Evidently, the chemical signal received by
D. virilis males either ceases or strongly inhibits courtship, as they recognize heterospecific females as alien. This results in a complete cessation of courtship or a significant reduction in the ritual, leading to a very low copulation frequency. On the other hand,
D. americana males do not perceive
D. virilis females as strangers; however, there is a breakdown in the signal exchange between partners, leading to a substantial increase in the duration of the tapping and licking stage. It is possible that in these instances,
D. virilis females do not recognize heterospecific males (“hesitation”), prompting the males to exert additional effort to ensure courtship culminates in copulation. Clearly, these differences in courtship behavior between
D. virilis and
D. americana are genetically determined. The presence or absence of species-specific alleles will result in corresponding phenotypic manifestations that influence the courtship-ritual structure, either aligning it with the conspecific variant (
D. virilis) or deviating from it.
In addition to the existing differences and disruptions in the courtship-ritual program between
D. virilis and
D. americana, there were also changes in the proportion of successfully copulating pairs. Among all experiments, the lowest percentage of copulating pairs was observed in heterospecific tests involving
D. virilis +
D. americana in both testing directions. It is noteworthy that during the designated observation period, the smallest number of copulations (only one copulation per 30 tested pairs) was consistently observed in the ♀
D. americana + ♂
D. virilis direction, resulting in all hybrids produced in this direction. On the other hand, in the reciprocal direction ♀
D. virilis + ♂
D. americana, approximately one-third of the pairs (11 pairs out of 30) successfully copulated. However, it is important to note that, despite this relatively higher copulation rate compared to the other reciprocal variant, the females did not produce viable offspring. This can be explained by the presence of postcopulatory isolation, as previously documented by various authors [
21,
22,
57].
It is important to note that the increased duration of nearly all courtship elements is attributed to disruptions in signal exchange through the acoustic communication channel [
29]. The acoustic duet between the female and male is known to play a crucial role in the successful progression of the courtship ritual in
D. virilis [
7] and any disturbance to this duet leads to changes in the courtship structure. For instance,
D. virilis males courting
D. americana females exhibited a reduction in the duration of mating songs, indicating decreased courtship intensity due to early recognition of the heterospecific female during the tapping stage. Conversely, courtship from a
D. americana male increased the duration of female singing in
D. virilis. These findings align with previous studies demonstrating that females exhibit active singing not only in conspecific tests but also in heterospecific tests where males engage in prolonged licking and singing [
7,
28]. Female singing is likely a stimulus that encourages males to persist in their courtship but does not directly contribute to the successful completion of the ritual. Based on the observation that
D. americana males attempted copulation after an extended courtship period in heterospecific tests, it can be concluded that the low percentage of successful copulations was determined by the final choice of the female during the last stage of courtship.