DOI: 10.5061/dryad.b2rbnzsd8
title: Evolution of male genitalia in the Drosophila repleta species group (Diptera: Drosophilidae)

description: The Drosophila repleta group comprises more than one hundred species that
 inhabit several environments in the Neotropics and use different hosts as
 rearing and feeding resources. Rather homogeneous in their external
 morphology, they are generally distinguished by the male genitalia, their
 fastest evolving morphological trait, constituting an excellent model to
 study patterns of genital evolution in the context of a continental
 adaptive radiation. Although much is known about the evolution of animal
 genitalia at population level, surveys on macroevolutionary scale of this
 phenomenon are scarce. Herein, in order to elucidate the macroevolutionary
 patterns of genital evolution through deep time and large continental
 scales, a suite of phylogenetic comparative methods was used in this group
 for the first time. Our results indicate that male genital size and some
 aspects of shape have been evolving by speciational evolution, probably
 due to the microevolutionary processes involved in species mate
 recognition. In contrast, several features of the aedeagus shape seemed to
 have evolved stochastically and thereby in a gradual fashion, with
 heterogeneous evolutionary phenotypic rates among clades, however. In
 general, the tempo of the evolution of aedeagus morphology was constant
 from the origin of the group until the Pliocene, when it accelerated in
 some clades that diversified mainly in this period. The incidence of novel
 ecological conditions in the tempo of aedeagus evolution and the
 relationship between species mate recognition and speciation in the
 Drosophila repleta group are discussed.
description: <strong>Data collect</strong> Aedeagi from adult males of 51
 species of the repleta group were obtained via direct dissection, from the
 laboratory photographic collection or images available on bibliography.
 Aedeagus morphology in species of the repleta group shows low
 intraspecific but high interspecific variability (Barrios-Leal et al.,
 2017; Stefanini et al., 2018) . Thus, one aedeagus per species was
 quantified. When dissected, male genitalia were mounted on microscope
 slides and flattened with cover slips with DPX (Sigma-Aldrich) mountant.
 Slides were photographed at 400 x magnification with a camera mounted on a
 microscope following Soto et al. (2007).   <strong>Morphological
 quantification</strong> Size and shape components of
 morphology were analyzed separately. The original size of different
 individuals coming from different sources was recovered by scaling the
 original photos to the same pixel resolution using the scales provided.
 Aedeagal morphology was quantified in lateral view with an open outline
 approach which uses sliding semilandmarks (Bookstein, 1996) to quantify
 two or three-dimensional homologous curves and surfaces (Gunz et al.,
 2005, Gunz and Mitteroecker, 2013; Schlager, 2017). To implement this
 analytic approach, the outlines were first extracted from jpg images and
 transformed into closed outlines using Momocs package version 2.0-6
 (Bonhomme et al., 2014). The closed outlines retrieved were standardized
 by using 800 (x,y) coordinates also with Momocs package. Then, three
 landmarks (i.e., discrete anatomical loci or geometric features
 recognizable in all specimens of the sample) were located on outlines of
 aedeagus, namely, the landmarks one and three at each side of the base and
 landmark one at the tip, with the def_ldk function of Momocs package. The
 three resultant inter-landmark segments represent the outlines of the
 lateral dorsal margin, ventral dorsal margin and the base of the
 intromittent part of aedeagus. The base contains non-relevant information
 because it is an artificial linear segment retrieved from the manipulation
 of the images. Therefore, the segment corresponding to the base (the
 segment between landmarks one and three) was deleted with the aim to
 obtain the open outlines that only carry the biological information.
 Subsequently, using the curve length as an index of geometric accuracy
 (Macleod, 1999), 169 semilandmarks were determined as the optimum number
 to reproduce the perimeter of inter-landmark boundary curves of the
 samples (i.e., 89 semilandmarks corresponding to the lateral dorsal margin
 segment and 80 corresponding to the lateral ventral margin segment). The
 optimal number of coordinates necessary to represent a given curve is the
 minimum number of coordinates that has at least the 99% of the length of
 the original curve perimeter (Macleod, 1999). Then, the spacing of the
 semilandmarks is optimized by allowing them to slide along the curves.
 Herein, the bending energy criteria of sliding was used to the detriment
 of the Procrustes distance criteria, which can induce distortions of the
 original curve (Gunz & Mitteorocker, 2013). Semilandmarks are used
 to represent homologous curves and surfaces by sets of points,
 establishing a geometric homology between corresponding semilandmarks
 across the sample (Bookstein, 1996). This geometric correspondence is
 established by the sliding of the semilandmarks along the curves, which
 reduces the effect of the arbitrary initial spacing (i.e., shape
 variation) of the arbitrary initial equidistant spacing (Gunz &
 Mitteorocker, 2013). One issue of the sliding semilandmarks approach was
 that the adjustment of the semilandmarks positions takes place along
 tangents (i.e., linear trajectories) to the boundary outline and not on
 the boundary outline curve itself and this can lead to serious technical
 artifacts (see Macleod, 2013 for a comprehensive revision of this
 morphometric technique). In the version of the sliding semilandmarks
 procedure implemented here, this issue has been satisfactorily solved by
 the addition of one step that comprises the reprojection on the original
 curve of slid semilandmarks on the tangent (Gunz et al., 2005; Schlager,
 2013; Schlager, 2017). Differences in size, rotation and position were
 removed by performing Generalized Procrustes Analysis (GPA; Gower, 1975)
 on the open outlines using only the three fixed landmarks. The size proxy
 of each outline extracted in the GPA analysis (i.e., Centroid size, Cs)
 was retained and used as an estimate of aedeagus size for subsequent
 analyses. Optimization of the number of semilandmarks, the slide of those
 on tangent to the curves, reprojection on the curves and GPA were
 performed using Morpho package version 2.8 (Schlager, 2017) in R
 environment. Several functions needed to fully implement this sliding
 semilandmarks approach were taken from
 https://github.com/millacarmona/gmorphometrics/blob/main/Functions_MillaCarmona.R. The Procrustes coordinates obtained were used to perform a Principal Component Analysis (PCA), with the prcomp function of the stats package of R environment (R Core Team 2016). The PCA permitted to summarize and reduce the dimensionality of the shape information described by the coefficients. The resulting scores of each Principal Component (PC) could be considered as reorganized and uncorrelated morphological descriptors representing different aspects of total shape variation (Zeldtich et al., 2012). After applying the Broken-Stick method (Jackson, 1993) implemented in the R package adephylo (Jombart et al., 2010), three PCs (representing about 85% of the total Shape variation) were retained for the subsequent statistical analyses of shape variation.