Experimental design
Fifteen isofemale lines (lines hereafter) of each species, derived from collections in the locality of Suyuque (San Luis province, Argentina), were employed in the experiments outlined below. In this area both species coexist in nearly equal proportions (45% of captured females were D. koepferae, 55% D. buzzatii). The advantages of the use of the isofemale line technique in quantitative evolutionary genetics have been thoroughly described in [30]. Briefly, the isofemale line technique is a convenient methodology for the analysis of quantitative traits under laboratory conditions. All experiments described below were conducted 24 generations after the foundation of isofemale lines.
Thirty first-instar larvae from each line were seeded in vials containing 6 ml. of media prepared with artificially fermented cactus (see [41] for details). Two different cactus species, Opuntia sulphurea and Trichocereus candicans which are commonly used as breeding substrates by D. buzzatii and D. koepferae in the locality sampled, were employed for the preparation of the semi-natural media. For each line, 4 replicated vials were run in each cactus type.
Five lines of each species were randomly chosen to generate interspecific hybrids. All possible combinations were attempted (25 potential crosses). F1 hybrids were produced by crossing 25 virgin females of a D. koepferae line with 50 males of a D. buzzatii line. The reciprocal cross invariably failed to produce viable progeny. Crosses were identified according to the number of the female parental line (D. koepferae) preceding the number of the male parental (D. buzzatii) line (e.g. the cross between D. koepferae line 48 and D. buzzatii line 55 was designated as 4855). Batches of 30 first-instar hybrid larvae were transferred to culture vials containing one of the two 'semi-natural' media. Four replicated vials were run per every combination of cactus and cross when the number of hatched larvae allowed it. The hybrid status of the descendants was ascertained by the cytological analysis of the polytene chromosomes of progeny larvae grown in vials run in parallel.
All cultures were maintained at 25 +/- 1°C with a 12:12 light/dark photoperiod until the emergence of adults. Adult flies were simultaneously collected and sexed under light CO2 anesthesia.
The aedeagus and both wings were dissected from 2 to 5 males emerged in each replicate. Aedeagi were mounted on slides and photographed with a digital camera mounted on a microscope at 400 × magnification. Wings were also mounted and ventral views of wing images were captured with a digital camera attached to a binocular microscope (25 ×) connected to a computer. In each image, we scored total wing length (WL) using TpsDig [55].
Morphological quantification
As shown in Figure 5 there are conspicuous differences in aedeagus morphology between D. buzzatii and D. koepferae that preclude the possibility of determining an adequate number of reliable homologous landmarks. However, the aedeagus is a flat quitinous organ that can be effectively described in shape and size in two dimensions when flattened under a cover slip. Consequently, we decided to employ an approach based on elliptic Fourier descriptors (EFDs) [56] as a proper resolution to the problem of shape quantification [47]. This is a type of analysis in which differences in x and y coordinates of the outline of the studied organ are fit separately as functions of arc length by Fourier analysis, so that the outline can be decomposed into a weighted sum of sine and cosine functions designated as harmonics. Outlines from digital images were used to obtain Fourier coefficients for a polynomial function of 30th degree which were computed with SHAPE v1.2 package, [57] using Elliptic Fourier analysis [56,58,59]. For the quantification of organs' shape we only considered the distal part of the aedeagus excluding the apodeme and the gonopods (Shaded areas in Figure 1. Thus, we simplified the studied contour by taking into account only the portion of the organ effectively involved in the penetration of female genitalia.
The area of each aedeagus (in pixels) was calculated from the digital images and considered as an estimator of the size of the organ. We performed a normalization of the descriptors based on the first harmonic ellipse that corresponds to the first Fourier approximation to the contour information (reviewed in [60]). Thus, size, orientation and starting position of the contours were standardized in accordance with the size and alignment of the major axis of the first ellipse, leading to representations of the organs that are only based on internal properties of the outlines (i.e. shape) [56].
The variance-covariance matrix of the 120 (4 per harmonic) estimated EFDs coefficients was used as input in a principal components analysis. This procedure allowed us to summarize the information assessed in the coefficients and reduce the dimensionality of the variables [61] in a lower number of principal components (PC). Thus, the resulting scores of each PC of each specimen could be considered as reorganized uncorrelated morphological traits representing different aspects of total shape variation [57] that were used as shape descriptor variables in subsequent analyses. For the sake of simplicity and in order to avoid components accounting for possible biologically meaningless morphological variation only the first 5 components of both intraspecific and interspecific analyses were considered as shape variables in the ANOVAs.
We worked with two sets of PC scores. The first set of principal components were calculated from the matrix of coefficients derived form the analysis of the outlines of the genitalia of males of the parental lines employed in successful interspecific crosses and the hybrids. The second set was obtained separately for each species improving the assessment of intraspecific variation in aedeagus morphology by avoiding the noise resulting from conspicuous interspecific morphological differences in the estimation of the PCs. This set was used in the evaluation of intraspecific sources of shape variation. Preliminary analyses with this technique showed that it is repeatable and reliable in species discrimination [62].
Analysis of variance of aedeagus size and shape
Both interspecific and intraspecific size differences were investigated by means of ANOVAs, with Species (2 levels, fixed factor), Cactus (2 levels, fixed factor) and Line (nested in Species random factor) as main sources of variation. The variable was log-transformed to ensure homoscedasticity. According to our experimental design, in the ANOVAs for species, a significant cactus effect (C) may be interpreted as phenotypic plasticity, while significant differences among isofemale lines as due to genetic differences (since all lines were reared under the same conditions). Finally, a significant L × C interaction may be construed as an estimation of genotype by environment interaction (GEI) or more explicitly, that the response of isofemale lines is not independent of the rearing cactus.
Shape variation was assessed by means of MANOVAs using the scores of each PC as dependent variables and Cactus and Line as main sources of variation.
In the presence of allometry, a fraction of the changes in shape might be explained by changes in the size of the organ. We were interested to comparatively evaluate the amount of allometric change in genital morphology in these species. Thus, we calculated Pearson's product-moment correlation matrix among shape scores and aedeagus size in each species separately to explore the relationship between aedeagus shape and general size of the aedeagus (within organ allometry).
We also evaluated the allometric relationship between aedeagus size and wing length, a trait correlated with overall body size, which in the studied species are known to be affected by the rearing substrate [41]. Size data were log-transformed prior to all analysis.
The principal components scores obtained by means of the general assessment of interspecific shape variation of parental lines and F1 hybrids were used in the examination of morphological patterns of variation in the offspring of interspecific crosses. Size was analyzed by means of an ANOVA and shape with a MANOVA. In both cases the principal factor was Genotype (both parents and the hybrids) and in those crosses in which hybrid larvae could be reared in both cactus hosts, Cactus was also considered as a fixed factor.
Statistica 6.0 [63] was used for statistical analyses and in all cases the corresponding assumptions were properly tested.
Authors' contributions
IS and EH conceived the study and read the salivary gland squashes. IS and VC carried out the experimental crosses, egg collections, larval seeding, rearing and adult collection. IS dissected the male genitalia, performed the morphological quantification, statistical analyses, and wrote the first draft of this manuscript. IS and EH wrote the final version of this manuscript. VC mounted the wings on microscope slides and provided the wing measurements. VC and JF helped to draft the final version of this manuscript. All authors read and approved the final manuscript.
Acknowledgements
We especially thank D Roccatagliata and M Mudry for help during the early stages of this research. E. Soto helped during the data acquisition and made insightful comments during the writing of the manuscript and A. Korovaichuk greatly influenced the final draft. We also wish to thank to two anonymous reviewers for comments that helped to improve this paper. We also thank other members of our research group for helpful discussion. IS and VC are recipients of postgraduate scholarships of CONICET and Universidad de Buenos Aires, respectively. JJF and EH are members of Carrera del Investigador Científico of CONICET. This work was supported by grants of Universidad de Buenos Aires, ANPCyT and CONICET awarded to EH.
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