A total of 606 males were analyzed in this study (252 D. buzzatii, 294 D. koepferae and 60 interspecific hybrids).
The total number of principal components explaining a significant proportion of shape variation was 13 in D. koepferae and 12 in D buzzatii (results not shown). The cumulative contribution of the first 5 principal components of the elliptic Fourier descriptors (EFDs) of the genital outlines accounted for over 74% and 77% of total shape variance in D. koepferae and D. buzzatii, respectively and nearly 84% of shape variation in the interspecific analysis (Table 1). The proportion of morphological variation summarized by each PC is illustrated in Figure 1.
Parental species size and shape variation
We detected significant differences in aedeagus size between species but more notably between flies reared in different cacti (Table 2a). However, in a nested ANOVA design as ours, the random factors Line in Species and Line by Cactus are the error terms of the fixed factors (Species, Cactus and their interaction). Large values of these terms may provide unreliable results in the testing of the fixed factors. Consequently we performed "a posteriori" contrasts of the Species by Cacti interaction to confirm the general results. Post hoc comparisons showed that D. buzzatii male flies reared in Opuntia had larger male genitalia than those grown in T. candicans (Tukey, p = 0.028) while in D. koepferae differences between flies grown in different cactus media were not significant (Tukey, p = 0.69). As visually observed, D. buzzatii and D. koepferae significantly differed in their genitalic shape and presented morphological variation not only among lines within species but also interacting with the breeding substrate (Table 2a).
The results of intraspecific ANOVAs also revealed important differences (Table 2b). In D. buzzatii the Cactus by Line interaction was significant and accounted for a relatively high percentage (12.1%) of phenotypic variance in aedeagus size. However, only the Line factor was significant in D. koepferae, neither the Cactus effect nor the Cactus by Line interaction were significant.
In summary, according to our experimental design, based on the isofemale line technique , shows that aedeagus size is not only phenotypically plastic, but also that substantial heterogeneity exists among lines in their plastic response, suggesting that plasticity has a genetic basis in D. buzzatii. In D. koepferae, in contrast, our results show that variation in aedeagus size has a genetic component, devoid of any plastic response in relation to the breeding substrate. According to the results of the MANOVAs, variability among lines in aedeagus shape was significant in both species (Table 2a, b). The proportion of total shape variation explained by the interaction Cactus by Line also differed between species. Approximately 9% of total shape variance was explained by the Cactus by Line interaction in D. koepferae. This interaction was significant for PC3 which is related with variation in thickness in both dorsal and ventral median portions of the organ (Figure 1). Conversely, in D. buzzatii, the Cactus by Line interaction was significant for PC1, which describes changes in the process of the ventral margin (Figure 1) and accounts for an important proportion (30%) of the explained morphological variance.
Correlation analysis between aedeagus size and shape also revealed important interspecific differences. On one hand, aedeagus shape was strongly correlated with size in D. buzzatii (more than 16% of the total shape variation was allometric, Table 3). Conversely, none of the 5 principal shape variables in D. koepferae were significantly associated with genital size (Table 3).
We also studied the relationship between variables describing size of male genitalia and wing length. These variables were not significantly correlated in D. koepferae (r = 0.13, p = 0.07), whereas in D. buzzatii, we detected a significant allometric relationship (r = 0.32, p D. buzzatii as suggested by a coefficient of allometry not significantly different from 1 (slope value of linear adjusted function = 0.82; 95% confidence interval values [0.42 to 1.23]).
Aedeagus morphology in interspecific hybrids
Four interspecific crosses, out of 25 attempted, (crosses 4855, 4853, 8832 and 3512) yielded enough hybrid progeny as to perform the present study. These results are in agreement with previous studies reporting strong premating isolation between D. buzzatii and D. koepferae . In order to asses hybrid male fertility, and prior to dissection, hybrid males were aged for 1 week and placed for 5 days in vials with several mature virgin females of D. buzzatii or D. koepferae. In all cases hybrid males failed to produce offspring even though copulation attempts were observed in the vials. Hybrid progeny obtained in crosses 4853 and 8832 could only be tested in vials prepared with the medium prepared with fermenting Opuntia due to low numbers of hybrid larvae, while in the other crosses the yield of hybrid progeny was high enough to be reared in both cactus media.
Regarding size, differences among genotypes (hybrids plus both parental lines) were significant in all crosses (Table 4). In Figure 2 we illustrate size differences among crosses and genotypes reared in Opuntia, the rearing substrate where all crosses were able to be tested. F1 hybrid males from crosses 4853 and 8832 reared in Opuntia vials presented intermediate values that differed significantly from both parental strains (p F2,69 = 4, 49, p D. buzzatii line in Opuntia, while differences between hybrids and the D. buzzatii parent were not significant in Trichocereus. In all cases, D. koepferae presented the largest genitalia in both cacti. In one cross (3512) mean genitalic size in hybrids was significantly lower than the male parental D. buzzatii line (p = 0.025, Tukey's test). Based on the correlation matrix, only PC1 scores were correlated with organ size in hybrids accounting for 50.4% of shape variation (Table 3). Unfortunately, the low number of hybrids and the high proportion of individuals with improperly unfolded wings precluded the analysis of allometric relationships between wing size and the variable describing aedeagus size.
Significant shape differences among genotypes were detected in all crosses (Table 4). Post hoc comparisons showed that all genotypes differed from each other in the shape of the genitalia at least in PC1 shape scores (p
In figure 3 we present a plot of the first two principal components describing shape variation (PC1 and PC2). The species differentiate themselves along the first shape axis (PC1) and the hybrid scores fall within the parental values. As can be observed the mean PC1 values of D. buzzatii lines involved in successful interspecific crosses tended to be negative and those of D. koepferae positive. However, hybrids failed to present intermediate values for both shape variables simultaneously. For instance, hybrids of cross 8832 had shape scores for both PC1 and PC2 that placed them in the morphological space closer to the D. koepferae parent (Line 88) than to D. buzzatii (Line 32). On the contrary, in cross 3512 a hybrid genital morphology was more similar to D. buzzatii for PC1 (Line12) but the mean for PC2 was more extreme than any of the parental lines. Suggestively, as explained above, hybrids in 3512 also presented smaller genitalia than both parental lines.
In order to evaluate the degree of resemblance of the morphology of hybrids to each parental line, we calculated the Euclidean distance to the morphological centroid of each parental strain using the shape (PCs) scores of each individual hybrid. As a rough index of morphological dissimilarity, hybrids would show equal mean distances to the centroids of both parental clouds of points if they have intermediate aedeagus morphology. Expression dominance of one genome over the other would produce phenotypes resembling more closely one parental strain or the other. Morphological dominant expression was tested with an ANOVA in which the variable was the Euclidean distance of each hybrid male to the centroid of each parental species (mean parental shape) with Cross and Parents as fixed factors. The ANOVA revealed significant differences among crosses though it should be noted that hybrid resemblance to parental strains were not independent of the cross (F3,110=17.59; p 4).