**4. Sexual shape dimorphism examples in insect body and wing shape**

#### **4.1. Case 1 Body shape**

In coleopteran of the genus *Ceroglossus* (Carabidae) a phenomenon occurs which is completely opposed to that described above. *Ceroglossus* Solier is a genus endemic to *Nothofagus* forests of the extreme south of South America.

Studies of body shape in *Ceroglossus chilensis* have demonstrated that the similarity of males and females is directly associated with the sex ratio of this species [50]. Morphological sex dimorphism is much reduced and only visible under a microscope. However, in terms of geometric morphometrics the differences are visible in two body regions; the abdomen of females, whose variation has been reported to have an adaptive value due to the presence of eggs, and changes in the pronotum of the thorax in males, which has been attributed to intrasexual competition in this species[17,18,50].

#### *4.1.1. Methodology*

For the morphometric analyses a total of 116 specimens of *C. chilensis* were used from 2 populations (53 males and 63 females) of Santa Juana area in the Coast Range (37º10'S, 72º57'W) and near San Fabián de Alico in the Andes Foothills (36º37' S, 71º50'W), both localities in the Región del Bío-Bío. The geometric analysis considered exclusively variation in shape, and it was performed using a photograph in ventral view of males and females with an Olympus X- 715 digital camera; using the methodology of [51], we digitized 17 landmarks (LMs, anatomical homologous points) on every picture, by TpsDig 2.10 (Figure 2). All analyses were then run using MorphoJ software version 1.05a [53].

Once the Cartesian x-y coordinates were obtained for all landmarks, the shape information was extracted with a full Procrustes fit [4,9], taking into account the object symmetry of the structure. Procrustes superimposition is a procedure that removes the information of size, position and orientation to standardize each specimen according to centroid size. Due to the high difficulty to check the differences in sexual dimorphism in this group, the only way to differentiate was based on the presence of antennal careens located from the fifth to ninth Sexual Dimorphism Using Geometric Morphometric Approach 39

**Figure 2.** Location of the 17 landmarks in ventral view of *Ceroglossus chilensis* 

38 Sexual Dimorphism

of sexual dimorphism in *Drosophila* wings [36].

*Nothofagus* forests of the extreme south of South America.

intrasexual competition in this species[17,18,50].

**4.1. Case 1 Body shape** 

*4.1.1. Methodology* 

(SSD) in body size is considered to be one of the major determinants of mating success in many species [32-35]. Because larger males are generally more aggressive and more competitive than smaller males, larger males often attain greater reproductive success through intrasexual selection [14]. In contrast, sexual shape dimorphism (SShD) has been much less investigated [17-19,36]. From those studies that considered SShD, most have discussed it as a diagnostic trait for diverse purposes, such as sex identification or the analysis of ontogeny [37-40]. Nevertheless, some other authors have considered sexual dimorphism evolution covering only some aspects of a limited number of taxa, such as: the evolution of cranium in primates [41-44]; the proportions and dimensions in lizard bodies [45,46]; newts [47]; or in flies [48]; and variation of shape in insect heads [49]; and variation

**4. Sexual shape dimorphism examples in insect body and wing shape** 

In coleopteran of the genus *Ceroglossus* (Carabidae) a phenomenon occurs which is completely opposed to that described above. *Ceroglossus* Solier is a genus endemic to

Studies of body shape in *Ceroglossus chilensis* have demonstrated that the similarity of males and females is directly associated with the sex ratio of this species [50]. Morphological sex dimorphism is much reduced and only visible under a microscope. However, in terms of geometric morphometrics the differences are visible in two body regions; the abdomen of females, whose variation has been reported to have an adaptive value due to the presence of eggs, and changes in the pronotum of the thorax in males, which has been attributed to

For the morphometric analyses a total of 116 specimens of *C. chilensis* were used from 2 populations (53 males and 63 females) of Santa Juana area in the Coast Range (37º10'S, 72º57'W) and near San Fabián de Alico in the Andes Foothills (36º37' S, 71º50'W), both localities in the Región del Bío-Bío. The geometric analysis considered exclusively variation in shape, and it was performed using a photograph in ventral view of males and females with an Olympus X- 715 digital camera; using the methodology of [51], we digitized 17 landmarks (LMs, anatomical homologous points) on every picture, by TpsDig 2.10 (Figure

Once the Cartesian x-y coordinates were obtained for all landmarks, the shape information was extracted with a full Procrustes fit [4,9], taking into account the object symmetry of the structure. Procrustes superimposition is a procedure that removes the information of size, position and orientation to standardize each specimen according to centroid size. Due to the high difficulty to check the differences in sexual dimorphism in this group, the only way to differentiate was based on the presence of antennal careens located from the fifth to ninth

2). All analyses were then run using MorphoJ software version 1.05a [53].

**Figure 3.** Canonical Variate Analysis (CVA) for the sexual shape dimorphism population of *Ceroglossus chilensis* \*each point represents a shape variable for female and male individuals in ventral view. The figure shows the first two CV components' axes with shape deformation images associated, and their antennal structure that is differentiation characteristic based on optic microscopy (careens presence in males).

segment [54], present "only in males" and observable under a dissecting microscope (Figure 3). Because of the symmetry of the structure, reflection is removed by including the original and mirror image of all configurations in the analysis and simultaneously superimposing all of them [55]. To examine the amount of symmetric variation and sexual shape dimorphism we used Procrustes ANOVA to assess studies on object symmetry. Differences between locations and sex were assessed using canonical variate analysis (CVA), a multivariate statistical method used to find the shape characters that best distinguish multiple groups of specimens

Sexual Dimorphism Using Geometric Morphometric Approach 41

Sampling: The study area was the farms named "El Guindo (36º50'12''W- 73º01'25''S) and Coyanmahuida" (36°49'28.66"S - 72°44'1.34"W) separated 20 km from one another in the province of Concepción, Biobío Region of Chile, where there are relict native forests with *Nothofagus obliqua* and *Peumus boldus*, among others. In order to determine intra- and interpopulation variation by means of geometric morphometrics, we used adults of *Synneuria* sp. (Lepidoptera, Geometridae). The individuals were collected by phototropic UV traps using an 800 watt electric generator; the light sources were placed over a white sheet to increase the luminosity. These traps were installed for a period of 4 hours in different sampling points. Finally we collected individuals which were processed, males and

The geometrical analysis, which considered variations attributed exclusively to shape, was performed using a photographic register of 63 males and 58 females of *Synneuria* sp., whose wings were each mounted in a fixed mould. The right wing of each was photographed with a Sony 10 DSC-H7 camera with directed fibre optics lighting, with which we constructed photographic matrixes using the TpsUtil 1.40 program [68]. We digitalized 18 morphological landmarks based upon the shape and vein pattern of the wing (Figure 4) for all individuals using the TpsDig 2.12 program (52). To determine if there are significant differences between male and female populations of *Synneuria*, a factorial variance analysis (ANOVA) was calculated base on the matrix of covariance between sexes generated by means of the

**Figure 4.** Location of 18 morphological landmarks in the right wing of *Syneuria* sp. (Benitez,

Neotropical Entomology unpublished data)

*4.2.1. Methodology* 

Procustes analysis.

females separated, wings cut, and mounted.

#### *4.1.2. Results*

The PCA plot for the symmetric component (individual variation) shows some differences between the populations analyzed. The first two PCs account for 53.643% (PC1 + PC2 + PC3 = 27.619% + 14.88% + 11.14) of the total shape variation and provide a reasonable approximation of the total amount of variation, with the other PC components that account each no more than 9.5% of the variation. The canonic analysis showed a clear differentiation of sexual shape dimorphism in both populations (Figure 3).

The Procrustes ANOVA for size does not show significant differences between populations and sex. Instead, Procrustes ANOVA for shape shows differences between populations (F = 3.79, P<0.0001) and high differences between sex (F = 11.76, P<0.0001). Besides, MANOVA tests, for both symmetric and asymmetric components, confirm these results (Pillay = 0.64, P<0.0001; Pillay = 0.31, P<0.0001 respectively).

#### **4.2. Case 2 Wing shape**

Within species, sexual dimorphism is a source of variation in life history (e.g., sexual size dimorphism and protandry), morphology (e.g., wing shape and colour pattern), and behaviour (e.g., chemical and visual signaling). Sexual selection and mating systems have been considered the primary forces driving the evolution of sexual dimorphism in insects especially in lepidoptera, and alternative hypotheses have been neglected [56]. Recent analyses demonstrate that many lepidopteran species exhibit female-biased sexual size dimorphism 73% of 48 species in Reference [57]. Size and shape differences are established during the larval period [58,59] by developmental and physiological mechanisms (e.g., number of larval instars and hormonal regulation). Because females of many species are capital breeders (i.e., they allocate larval resources for reproduction), and large size is related directly to fecundity [60-62], selection for large female body size appears to be driven by natural selection for increased fecundity [63]. Most of the morphological variations between males and females in moth and buterflies are due to the effects associated with the environment, whether phenotypic responses (plasticity) or particularly those which act during ontogenetic development [64-66]. Females are generally larger than males; this gives them adaptive advantages such as greater fecundity and better parental care [14,15,63,67].

In this section, sexual dimorphism was determined to be present in the wing shape of moths of the *Synneuria* genus, suggesting that the wing shape may be selected as a character to determine sex between lepidopteran species.

#### *4.2.1. Methodology*

40 Sexual Dimorphism

specimens

*4.1.2. Results* 

segment [54], present "only in males" and observable under a dissecting microscope (Figure 3). Because of the symmetry of the structure, reflection is removed by including the original and mirror image of all configurations in the analysis and simultaneously superimposing all of them [55]. To examine the amount of symmetric variation and sexual shape dimorphism we used Procrustes ANOVA to assess studies on object symmetry. Differences between locations and sex were assessed using canonical variate analysis (CVA), a multivariate statistical method used to find the shape characters that best distinguish multiple groups of

The PCA plot for the symmetric component (individual variation) shows some differences between the populations analyzed. The first two PCs account for 53.643% (PC1 + PC2 + PC3 = 27.619% + 14.88% + 11.14) of the total shape variation and provide a reasonable approximation of the total amount of variation, with the other PC components that account each no more than 9.5% of the variation. The canonic analysis showed a clear differentiation

The Procrustes ANOVA for size does not show significant differences between populations and sex. Instead, Procrustes ANOVA for shape shows differences between populations (F = 3.79, P<0.0001) and high differences between sex (F = 11.76, P<0.0001). Besides, MANOVA tests, for both symmetric and asymmetric components, confirm these results (Pillay = 0.64,

Within species, sexual dimorphism is a source of variation in life history (e.g., sexual size dimorphism and protandry), morphology (e.g., wing shape and colour pattern), and behaviour (e.g., chemical and visual signaling). Sexual selection and mating systems have been considered the primary forces driving the evolution of sexual dimorphism in insects especially in lepidoptera, and alternative hypotheses have been neglected [56]. Recent analyses demonstrate that many lepidopteran species exhibit female-biased sexual size dimorphism 73% of 48 species in Reference [57]. Size and shape differences are established during the larval period [58,59] by developmental and physiological mechanisms (e.g., number of larval instars and hormonal regulation). Because females of many species are capital breeders (i.e., they allocate larval resources for reproduction), and large size is related directly to fecundity [60-62], selection for large female body size appears to be driven by natural selection for increased fecundity [63]. Most of the morphological variations between males and females in moth and buterflies are due to the effects associated with the environment, whether phenotypic responses (plasticity) or particularly those which act during ontogenetic development [64-66]. Females are generally larger than males; this gives them adaptive advantages such as greater fecundity and better parental care [14,15,63,67].

In this section, sexual dimorphism was determined to be present in the wing shape of moths of the *Synneuria* genus, suggesting that the wing shape may be selected as a character to

of sexual shape dimorphism in both populations (Figure 3).

P<0.0001; Pillay = 0.31, P<0.0001 respectively).

determine sex between lepidopteran species.

**4.2. Case 2 Wing shape** 

Sampling: The study area was the farms named "El Guindo (36º50'12''W- 73º01'25''S) and Coyanmahuida" (36°49'28.66"S - 72°44'1.34"W) separated 20 km from one another in the province of Concepción, Biobío Region of Chile, where there are relict native forests with *Nothofagus obliqua* and *Peumus boldus*, among others. In order to determine intra- and interpopulation variation by means of geometric morphometrics, we used adults of *Synneuria* sp. (Lepidoptera, Geometridae). The individuals were collected by phototropic UV traps using an 800 watt electric generator; the light sources were placed over a white sheet to increase the luminosity. These traps were installed for a period of 4 hours in different sampling points. Finally we collected individuals which were processed, males and females separated, wings cut, and mounted.

The geometrical analysis, which considered variations attributed exclusively to shape, was performed using a photographic register of 63 males and 58 females of *Synneuria* sp., whose wings were each mounted in a fixed mould. The right wing of each was photographed with a Sony 10 DSC-H7 camera with directed fibre optics lighting, with which we constructed photographic matrixes using the TpsUtil 1.40 program [68]. We digitalized 18 morphological landmarks based upon the shape and vein pattern of the wing (Figure 4) for all individuals using the TpsDig 2.12 program (52). To determine if there are significant differences between male and female populations of *Synneuria*, a factorial variance analysis (ANOVA) was calculated base on the matrix of covariance between sexes generated by means of the Procustes analysis.

**Figure 4.** Location of 18 morphological landmarks in the right wing of *Syneuria* sp. (Benitez, Neotropical Entomology unpublished data)

#### *4.2.2. Results*

The morphological variation among moths determined by Procrustes ANOVA indicates that variation in shape between sexes is highly significant (Table 1).

Sexual Dimorphism Using Geometric Morphometric Approach 43

**Figure 6.** 3-D dispersion graph of shape variables by sex and locality in moths of the genus *Synneuria*. The points in the dark area indicate populations of males from El Guindo and Coyanmahuida,

respectively, and in the clear area females from El Guindo and Coyanmahuida. \*\* Each point within the

volumetric sector indicates a variable with different shape.


**Table 1.** Two-way ANOVA for differences in shape of *Synneuria* sp using the first relative warp as dependent variable.

The relative warp plot shows some differences between sexes within each population analyzed (Figure 5). The first three Rws account for 91.74% of the total shape variation and provide a reasonable approximation of the total amount of variation, and the other Rw components account each for no more than 5% of variation. In order to visualize the variation in wing shape graphically we took the mean of the first three relative warps. We found different morphotypes for males and females of Coyanmahuida and Guindo (Figure 6).

**Figure 5.** Morphological deformation grids showing distributions of shape for males and females of *Synneuria* sp. in the different localities, CN: Coyanmahuida and EG: El Guindo.

*4.2.2. Results* 

dependent variable.

The morphological variation among moths determined by Procrustes ANOVA indicates that

Effect SS df MS F p Sex **0.001526 1 0.001526 4.237204 0.018961**  Locality 0.000061 1 0.000061 0.169012 0.684121 Sex x Locality 0.000490 1 0.000490 1.360991 0.253206

**Table 1.** Two-way ANOVA for differences in shape of *Synneuria* sp using the first relative warp as

**Figure 5.** Morphological deformation grids showing distributions of shape for males and females of

*Synneuria* sp. in the different localities, CN: Coyanmahuida and EG: El Guindo.

morphotypes for males and females of Coyanmahuida and Guindo (Figure 6).

The relative warp plot shows some differences between sexes within each population analyzed (Figure 5). The first three Rws account for 91.74% of the total shape variation and provide a reasonable approximation of the total amount of variation, and the other Rw components account each for no more than 5% of variation. In order to visualize the variation in wing shape graphically we took the mean of the first three relative warps. We found different

variation in shape between sexes is highly significant (Table 1).

Error 0.010086 28 0.000360

**Figure 6.** 3-D dispersion graph of shape variables by sex and locality in moths of the genus *Synneuria*. The points in the dark area indicate populations of males from El Guindo and Coyanmahuida, respectively, and in the clear area females from El Guindo and Coyanmahuida. \*\* Each point within the volumetric sector indicates a variable with different shape.

#### **4.3. Discussion Case 1 and 2**

The adoption of new techniques to determine variation in shape of both animals and plants is currently a widely discussed issue [69,70]. Geometric morphometrics can unify methodologies to quantify and visualize shape in all the ways that are possible.

Sexual Dimorphism Using Geometric Morphometric Approach 45

This revision is intended to provide a wide view of GM use in some of the diverse study areas of sexual dimorphism in insects, confirming that by using the new tools that define shape as a differentiation characteristic it is possible to determine variations at minimum scales, which can be explained by means of sexual selection. Furthermore, by using geometric morphometric, besides identifying variations regarding sex, the researcher may determine relations between anatomic points of shape, in order to identify asymmetry patterns and generate hypotheses about the group development stability, [55,77]. Therefore, it is worth noting that in recent years research efforts have increased exponentially, and GM gains attention every day as a usefull tool for quantitative integration in morphology study due to its easy, inexpensive and fast application. Consequently, scientists are taking steps to combine these advanced techniques of morphometry study, to unify methodologies with molecular and genetic studies, in order to get results with total evidence within the analysis

To Dr. Viviane Jerez and Dr. Luis Parra of Zoology Department, Universidad de Concepción. Chile, for their comments and cooperation to generate the articles that were the core of the chapter and Ms. María Raquel Lazo de la Vega for her careful revision of the

[1] Adams C, Rohlf J, Slice D. Geometric morphometrics: ten years of progress following

[2] Bookstein FL. A hundred years of morphometrics. Acta Zoologica Academiae

[3] Adams D, Funk DJ. Morphometric inferences on sibling species and sexual dimorphism in *Neochlamisus bebbianae* leaf beetles: Multivariate applications of the thin-plate spline.

[5] Kendall DG. The diffusion of shape. Advances in Applied Probability 1977;9 428-430. [6] Bookstein F, Chernoff B, Elder R, Humphries J, Smith G, Strauss R. Morphometrics in evolutionary biology. Michigan: The Academy of Natural Sciences of Philadelphia;

**5. Conclusions** 

itself.

**Author details** 

Hugo A. Benítez

language.

**6. References** 

1985.

**Acknowledgement** 

*Faculty of Life Sciences, University of Manchester, Manchester, UK Instituto de Alta Investigación, Universidad de Tarapacá, Chile* 

the "revolution". Italian Journal of Zoology 2004;71 5-16.

[4] Dryden I, Mardia K. Statistical Shape Analysis. Chichester: Wiley; 1998.

Scientiarum Hungaricae 1998;44 7-59.

Systematic Biology 1997;46(1) 180-194.

For case 1, geometric morphometric was capable of detecting variation between species that are not clearly visible on plain sight, but rather at a sexual selection level between species. However, small variations on body shape could mark the difference in both populations, and these were proven according to Procustes distances and also by means of variance analyses. It is worth noting that the populations studied were subject to climate differences based on the different mountain ranges from which individuals were collected.

Although these differences are not obvious, individuals of the Coast Range had less thickened bodies than those of the Andes foothills. It has been reported that a climate with high relative humidity and constant temperatures promotes a thinner, subelytral cavity; this result was therefore expected for the Coast Range. The individuals of the Andes foothills had more visible morphological variations, which may be a consequence of the instability of the environment in this area (more variation in temperature, leading to thicker subelytral cavities). However, in spite of the climatic differences between populations there were not large morphological differences in the sexual dimorphism between populations. We may infer from this that gene flow has not been interrupted between them [17].

Regarding the case of Lepidoptera wing, it was very similar to the findings in beetles, but in this case differences were determined by means of small variations in wing shape associated with venation and flying styles of males and females [18].

For a number of authors, the variation in wing shape does not provide sufficient evidence to conclude that this is only a product of sexual dimorphism. It is frequently argued that individual variation in shape may be strongly dependent on environmental conditions [3,71]. However, our study showed a significant difference in wing shape between sexes, both within and between localities. Therefore, we conclude that the differences found here are analytical for the species and areas studied.

The differences among the individual configurations of each sex were captured using mathematical functions varying according to the position of each landmark in the wing image. These differences were located in landmarks 5, 6, 7 and 8, respectively. The geometric variation detected showed that the landmarks located on the base of the radial veins were key characters to distinguish different wing morphotypes among populations and sexes. The crucial attributes for the group would benefit the dispersion, migration and sexual selection; in males for the nuptial flight, territoriality and sexual selection, and in females primarily as a characteristic flight behaviour in the search for host plants e.g. [72-75]. Therefore, selection would act on wing shape to optimize flight characteristics [76].

### **5. Conclusions**

44 Sexual Dimorphism

between them [17].

[76].

**4.3. Discussion Case 1 and 2** 

The adoption of new techniques to determine variation in shape of both animals and plants is currently a widely discussed issue [69,70]. Geometric morphometrics can unify

For case 1, geometric morphometric was capable of detecting variation between species that are not clearly visible on plain sight, but rather at a sexual selection level between species. However, small variations on body shape could mark the difference in both populations, and these were proven according to Procustes distances and also by means of variance analyses. It is worth noting that the populations studied were subject to climate differences

Although these differences are not obvious, individuals of the Coast Range had less thickened bodies than those of the Andes foothills. It has been reported that a climate with high relative humidity and constant temperatures promotes a thinner, subelytral cavity; this result was therefore expected for the Coast Range. The individuals of the Andes foothills had more visible morphological variations, which may be a consequence of the instability of the environment in this area (more variation in temperature, leading to thicker subelytral cavities). However, in spite of the climatic differences between populations there were not large morphological differences in the sexual dimorphism between populations. We may infer from this that gene flow has not been interrupted

Regarding the case of Lepidoptera wing, it was very similar to the findings in beetles, but in this case differences were determined by means of small variations in wing shape associated

For a number of authors, the variation in wing shape does not provide sufficient evidence to conclude that this is only a product of sexual dimorphism. It is frequently argued that individual variation in shape may be strongly dependent on environmental conditions [3,71]. However, our study showed a significant difference in wing shape between sexes, both within and between localities. Therefore, we conclude that the differences found here

The differences among the individual configurations of each sex were captured using mathematical functions varying according to the position of each landmark in the wing image. These differences were located in landmarks 5, 6, 7 and 8, respectively. The geometric variation detected showed that the landmarks located on the base of the radial veins were key characters to distinguish different wing morphotypes among populations and sexes. The crucial attributes for the group would benefit the dispersion, migration and sexual selection; in males for the nuptial flight, territoriality and sexual selection, and in females primarily as a characteristic flight behaviour in the search for host plants e.g. [72-75]. Therefore, selection would act on wing shape to optimize flight characteristics

with venation and flying styles of males and females [18].

are analytical for the species and areas studied.

methodologies to quantify and visualize shape in all the ways that are possible.

based on the different mountain ranges from which individuals were collected.

This revision is intended to provide a wide view of GM use in some of the diverse study areas of sexual dimorphism in insects, confirming that by using the new tools that define shape as a differentiation characteristic it is possible to determine variations at minimum scales, which can be explained by means of sexual selection. Furthermore, by using geometric morphometric, besides identifying variations regarding sex, the researcher may determine relations between anatomic points of shape, in order to identify asymmetry patterns and generate hypotheses about the group development stability, [55,77]. Therefore, it is worth noting that in recent years research efforts have increased exponentially, and GM gains attention every day as a usefull tool for quantitative integration in morphology study due to its easy, inexpensive and fast application. Consequently, scientists are taking steps to combine these advanced techniques of morphometry study, to unify methodologies with molecular and genetic studies, in order to get results with total evidence within the analysis itself.
