Computational protocol: Sexual Dimorphism and Allometric Effects Associated With the Wing Shape of Seven Moth Species of Sphingidae (Lepidoptera: Bombycoidea)

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[…] Specimens used in this study come from the Entomological Collection of Embrapa Cerrados. In it are catalogued 142 species of Sphingidae, which represents 77% of the estimated richness for the group in Brazil (). Females are much rarer than males in entomological collections, possibly due to the patrolling behavior of males (). Thus, we chose only species that were represented in the collection by both sexes to further analyses. Among these species were Enyo ocypete (Linnaeus 1758) (Macroglossinae: Dilophonotinini), Erinnyis ello (Linnaeus 1758) (Macroglossinae: Dilophonotinini), Erinnyis oenotrus (Cramer 1780) (Macroglossinae: Dilophonotinini), Isognathus menechus (Boisduval [1875]) (Macroglossinae: Dilophonotinini), Pseudosphinx tetrio (Linnaeus 1771) (Macroglossinae: Dilophonotinini), Eumorpha anchemolus (Cramer 1779) (Macroglossinae: Philampelini), and Xylophanes chiron (Cramer 1777) (Macroglossinae: Macroglossini).To obtain the morphological data, the right fore and hindwings of all individuals of these species were photographed using a Sony Cyber-shot camera (DSC-W650 16.1 megapixels) mounted on a tripod (Sony VCT-R100, New York city, NY). When the right wing was damaged, the left wing was used and then flipped (e.g., , ). Each specimen was fixed on a mount board at a distance of 8 cm from the camera lens and no zoom effects were used, so wing size could be estimated. To distinguish males and females, we first observed the frenulum, which is a brush of bristles in females and a single bristle in males. In addition to this, we also moved the scales of the last abdominal segment with the aid of a brush to verify the presence of anal buds in females or valve in males ().To evaluate the wing shape, 16 type 1 landmarks on the extremities and junctions of veins were digitalized using program TpsDig 1.18 (). The following parts of the wing were not sampled: the region bounded by the costal margin and the Radial 3 vein (Rs3) and part of the region bounded by Radial 3 vein (Rs3) to its insertion in Radial 4 (Rs4), part of the region bounded by the inner edge and the anal vein (2A + 3A) in the forewing; the costal margin and subcostal and radial veins (SC + R) and part of the region bounded by the inner margin and the anal vein (2A) on the hindwing (details in ). Landmarks in these regions were not chosen, as it would be difficult to ensure homologies between points on those curvatures (landmark type 2), and complex explanations derived from them would be less reliable (). Fig. 1. To make the veins more visible without damaging the entomological material, xylene was applied along the wings. The anatomic landmarks were obtained from the dorsal face of the forewings and the ventral face of the hindwings (), so that the entire wing could be photographed and the overlap between them did not hinder full visualization of the structures.The variables that describe wing shapes (uniform components and partial warps) were acquired using TPSRELW v.1.18 (). This method involves superimposition, centralization, and minimization of the Euclidean distances between anatomical landmarks to compare the shapes of the structures (). This method eliminates any information related to the position and orientation of the points (i.e., rotation, translation, and mathematic scale).Additionally, the software also provides a multivariate isometric wing size estimator called the centroid size, defined as the square root of the sum of the squares of the distances from each anatomic landmark to the center of mass of each configuration (). The resulting variable is used as a single measurement that accounts for the multivariable nature of the wing size in all subsequent analyzes. This procedure removes only the effects of isometric size. Therefore, the configurations keep both shape variations unrelated to size and allometric variations. [...] Wing shape variation of each species was assessed by discriminant analysis of the uniform components and partial warps scores, using specimens grouped by sex for acquiring the canonical variables that summarize the set of original variables in each grouping (i.e., males and females). We also performed an analysis of correct classifications by stepwise through klaR package () of the software R 2.13.1 to verify the ability of discriminant analysis to classify individuals in males and females. Additionally, to evaluate the presence of size sexual dimorphism, we performed a t-test on centroid size of males and females.Then, allometric effects in wing shape of each species were measured with simple regression analyses between the canonical variate as dependent variables and the log-transformed values of centroide size as independent variables (see ). In this analysis, we also calculated the slopes of the regressions (b) and whether these slopes statistically differed from 1. A calculated b = 1 would indicate isometry, thus, a lack of allometry (). The normalized residuals of these regressions (normalized difference between the Y value predicted for the straight line equation and the actual Y value; ) were used for assessing the nonallometric variation components in shape variation. For this, deformations in wing shape between sexes were accessed, by performing another set of multiple regressions (i.e., for each species) between the uniform components and partial warps and the normalized residuals, obtained in the first set of regressions, using the software TPSREGR v. 1.31 (). Changes in shape, after the effects of size are removed, are deviations from allometry. Dimorphism unrelated to allometry could be explained by adaptation for differences in foraging and breeding activities. Therefore, to verify whether after the effect of size was removed there were significant differences between the wing shapes of males and females, a t-test for each species was performed separately. […]

Pipeline specifications

Software tools TpsDIG, TpsRelw, TpsRegr
Application Macroscope & basic digital camera imaging
Organisms Homo sapiens, Ilex paraguariensis