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Phenotypic plasticity of the aedeagus of Drosophila mediopunctata: Effect of the temperature
ARTICLE IN PRESS
Journal of Thermal Biology 30 (2005) 518–523 www.elsevier.com/locate/jtherbio
Phenotypic plasticity of the aedeagus of Drosophila mediopunctata: Effect of the temperature Carlos A.C. Andradea,b, Luciane M. Hatadania, Louis B. Klaczkoa, a
Departamento de Gene´tica e Evoluc- a˜o, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP, Cx. Postal 6109, Campinas 13083-070 SP, Brazil b Departamento de Biologia Marinha, Universidade Federal Fluminense, Nitero´i, RJ, Brazil Received 27 January 2005; accepted 28 May 2005
Abstract In this study, we analyzed the phenotypic plasticity of the male intromittent organ (aedeagus) of Drosophila mediopunctata genitalia in response to temperature. Size and shape of the aedeagus were examined with univariate statistics and geometric morphometrics. The following results emerged: firstly, flies raised at 16.5 1C had bigger aedeagi than those raised at 20 1C; secondly, significant differences in shape were also found between the two groups of flies, mainly in the apical area, located using the thin-plate spline interpolating function; thirdly, the coefficients of variation for the traits of the aedeagus were comparable to other traits in Drosophila and likewise to genital traits in other organisms. These findings were analyzed in an evolutionary context. r 2005 Elsevier Ltd. All rights reserved. Keywords: Sexual selection; Male genitalia evaluation; Geometric morphometrics; Genetic biodiversity
1. Introduction In several groups of animals with internal fertilization, the male genitalia have a fast and divergent evolution. This permits male genitalia traits to be frequently used as characters for defining species, as in Drosophila. In this genus the most utilized traits for species identification are on the male genitalia, especially in the intromittent organ, the aedeagus that show diagnosable differences (Vilela and Bachli, 1990). In the past two decades, the evolution of male genitalia has been studied in many groups of animals with internal fertilization (Eberhard, 1993, 1998; Arnqvist, 1997, 1998; Arnqvist and Thornhill, 1998; House and Simmons, 2003; Corresponding author. Tel.: +55 19 37881150; fax: +55 19 37886235. E-mail address: [email protected] (L.B. Klaczko).
Hosken and Stockley, 2004). There are three hypotheses, which attempt to explain the rapid and diverse evolution of this structure: lock-and-key (Shapiro and Porter, 1989; Arnqvist, 1997), pleiotropy (Mayr, 1963) and sexual selection (Eberhard, 1993; Arnqvist, 1997). The lock-and-key hypothesis deserves special attention since it predicts that genital traits should present relatively limited plasticity due to the nearly perfect adjustment between male and female genitalia (Shapiro and Porter, 1989; Arnqvist, 1997, 1998). Temperature is an abiotic factor, which affects several morphological characters of insects. In Drosophila, it has been repeatedly shown that it acts upon many traits, such as: length of the thorax and wing, and pigmentation in Drosophila melanogaster (Bitner-Mathe´ and Klaczko, 1999; Debat et al., 2003; Gibert et al., 2004; Hoffmann et al., 2003; Imasheva et al., 1996; Partridge et al., 1994) as well as in D. mediopunctata
0306-4565/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2005.05.011
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(Bitner-Mathe´ et al., 1995; Hatadani et al., 2004). In other organisms, such as in the chironomid Procladius choreus and in the mosquito Anopheles albimanus, a variation in the structure of the male genitalia has been detected, respectively, due to seasonality (Kobayashi, 1998) and the development temperature of the larvae (Hribar, 1996). Surprisingly, however, as far as we know, there are no papers on the effect of temperature on the aedeagus morphology of Drosophila.D. mediopunctata is a species belonging to the tripunctata group. It is a Neotropical species with a wide-geographical distribution. It has a long-maturation time, low fecundity and extended longevity (Klaczko, 1995). In this investigation, the main interest was in examining how the aedeagus of D. mediopunctata reacts with regard to two different temperatures. Due to the fact that we are dealing with a complex structure, small in size and with few anatomical points, we used not only univariate statistics but also geometric morphometrics to analyze the variation in size and shape of the aedeagus. A better understanding of this topic may contribute to support, or not, hypotheses that explain the evolution of male genitalia.
2. Materials and methods 2.1. Collection of flies, data gathering and repeatability Flies were collected in the Mata Santa Genebra (221440 4500 S, 471060 3300 W) (Santa Genebra Woods) in September of 2000. This is a semi-deciduous forest in the city of Campinas, Sa˜o Paulo, Brazil (Morelatto and Leita˜o-Filho, 1995). Each female was individually deposited in a tube with culture medium. From the progeny of each female, equal numbers of male and virgin females were set aside (about six couples from each progeny), and, after 10 days were placed in the same vial, from which first stage larvae were later collected. These larvae were distributed in tubes containing 10 ml of culture medium (8% yeast, 2% sugar, 2.5% agar, 0.003% nipagin), in a density of 10 larvae per tube, which were placed in two temperatures (16.5 and 20 1C). Males born from these tubes were separated and kept in wells of microtitration plates, having obtained 110 males (59 males at 16.5 1C and 51 males at 20 1C) at the end of the experiment. The aedeagus was isolated and prepared following the procedure outlined by Wheeler and Kambysellis (1966). The aedeagus is a very small, complex and threedimensional structure. Due to its rigid morphology it is possible, by means of gentle touches on the cover slip, to position it in a lateral or frontal view. However, corrections to the positioning must be made cautiously, since the aedeagus moves about in the balsam, for it has no support. In addition to this problem, there are no real
519
anatomical points on the organ. In a preliminary experiment we determined the best position for the analysis of the aedeagus testing the repeatability of measures taken in lateral and frontal positions. We found that measurement error was too big when the lateral position was used (repeatabilitieso0.26) and this approach was abandoned, since the frontal position gave reliable measures. After carefully placing the aedeagus in a frontal position, we sketched its outline using a camera lucida connected to a Zeiss optical microscope. For this study, having in mind the sources of error mentioned above and with the objective of raising precision in the measures, each specimen was drawn twice, with an interval of at least 1 month, after placing them put back into position. The concurrence of drawings was visually examined before taking the measurements. In only three cases, among 110 drawn specimens, was there evident discordance. For these, a third drawing was prepared in a totally independent way, and then compared to the prior drawings, and the discordant one was discarded. Both drawings of each aedeagus were scanned and saved to file. Next, Cartesian coordinates were taken from a total of 10 anatomical points (474 magnification, Fig. 1), using tpsDIG v1.20 (Rohlf, 1998); all points used represent maximums of curvatures or external points (types 2 and 3 of Bookstein, 1991). Twelve linear measures were taken, joining eight points, making up two superimposed trusses (AD, DF, FH, AH, AF, DH and CE, EG, GI, CI, CG, EI). To avoid redundancy, two points (B and J) were not used to obtain linear measures. The values of the linear measures were obtained from the Cartesian coordinates (using the software Distance 2.0, by Strauss, 1988). The averages of both drawings were used for all variables. Since we have two drawings for each specimen, it is possible to calculate the total final repeatability of the data in this work (Lessels and Boag, 1987). These values are shown in Table 1, and are all above 70%. 2.2. Geometric morphometrics and statistical tests Since the Cartesian coordinates of the landmarks are distributed in a Kendall’s space and their analysis is carried out in a linear space tangent to it (Bookstein, 1991), we tested the adequacy of this approximation with the tpsSmall v1.20 program (Rohlf, 2003). Next, using the program tpsRegr v1.25 (Rohlf, 1999), the anatomical points of the aedeagus were superposed with the Procrustes superposition technique. Thus, the anatomical points were translated, scaled, and rotated so as to minimize the squared and summed distances between the corresponding anatomical points (Bookstein, 1991; Slice, 1996). With this, the size factor was excluded, obtaining only the shape information. The variation of the shape of the aedeagus was analyzed,
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Fig. 1. Frontal view of the aedeagus of D. mediopunctata, lines show the linear measures taken.
Table 1 Repeatability of the analyzed variables of the aedeagus of Drosophila mediopunctata Variable
Repeatability
AD AF AH CE CG CI DF DH EG EI FH GI Centroid size Relative warp 1 Relative warp 2
0.85 0.87 0.86 0.86 0.83 0.94 0.81 0.96 0.71 0.77 0.83 0.87 0.91 0.92 0.91
therefore, for deformation patterns using the thin-plate spline method. To estimate the size of the aedeagus, the centroid size was calculated, which is defined as the square root of the sum of the squares of the distances between each anatomical point and the centroid (Bookstein, 1991). To test the effect of the temperature for the univariate measures and for the centroid size t-tests were carried out. For the global shape of the aedeagus, Goodall’s Ftest (Goodall, 1991) was used, as recommended by Rohlf (2000), as well as a non-parametric permutation test based on Goodall’s F statistic with 100,000 permutations. This test is independent of the restrictive
assumptions of Goodall’s F-test. Finally, to visualize the temperature effect on shape, we used the tpsRegr program obtaining figures for the mean configuration and for the two temperatures (with the respective shape changes exaggerated three times). All statistical analyses were carried out using the program Systat 8.0 or programs of the tps series (Rohlf, 1998, 2003).
3. Results 3.1. Univariate statistics Table 2 shows the averages with their standard errors and coefficients of variation for the 12 linear measures and centroid size of the aedeagus of D. mediopunctata. When comparing the variables in both temperatures, applying the t-test, statistically significant differences are noted for 10 measures (out of 14), which showed higher averages for the lower temperature (16.5 1C) than for the high temperature (20 1C), with the exception of EG. Another important information was the variation observed in each temperature that can be assessed by the coefficients of variation. Their averages were 7.173% and 7.773% for 16.5 and 20 1C, respectively, which are not significantly different (t-test ¼ 0.508; df ¼ 22; P ¼ 0:62). 3.2. Geometric morphometrics We found a positive correlation (r ¼ 1:00) between the tangent and Kendall’s shape spaces. This allowed us to use statistical tests based on the measures in the linear tangent space.
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Table 2 Averages (in mm), standard errors (SE), and coefficients of variation (CV) for the linear measures and centroid size of the aedeagus of Drosophila mediopunctata in two temperatures (16.5 and 20 1C) Variable
AD AF*** AH CE*** CG*** CI DF*** DH** EG* EI*** FH*** GI*** Centroid size***
16.5 1C
20 1C
x¯ SE
CV
x¯ SE
CV
0.17370.0008 0.19770.0009 0.17570.0009 0.04670.0007 0.07570.0006 0.07970.0006 0.06170.0006 0.10270.0008 0.04370.0006 0.07470.0006 0.05970.0007 0.04670.0007 0.21470.0008
0.036 0.036 0.037 0.119 0.057 0.059 0.081 0.056 0.105 0.059 0.090 0.111 0.032
0.17470.0008 0.18970.0009 0.17670.0009 0.03870.0006 0.07170.0008 0.07770.0009 0.05570.0007 0.09870.0010 0.04670.0007 0.07070.0006 0.05370.0006 0.03870.0007 0.20770.0010
0.034 0.035 0.034 0.113 0.075 0.079 0.096 0.073 0.106 0.064 0.087 0.126 0.033
t-test: *Po0:05; **Po0:001; ***Po0:0001.
Regarding centroid size, we observed it was significantly larger at 16.5 1C than at 20 1C (Table 2). This reiterates the observation with univariate statistics: flies raised at lower temperatures have larger aedeagi. Analyzing the global shape difference between both temperatures with Goodall’s F-test and its permutation version, we obtained highly statistically significant differences (Goodall’s F-test ¼ 25.40; df ¼ 16, 1728; Po0:0001; Ppermutation o0:0001). Fig. 2 shows the shape variation, exaggerated three times, as well as the mean configuration, using the thin-plate spline function. It is worthy mentioning that the important deformations are in the most apical region of the aedeagus (see points C, E, G, and I).
4. Discussion 4.1. Plasticity of the size and shape of the aedeagus The aedeagus of D. mediopunctata presented phenotypic plasticity regarding size. Flies raised at a lower temperature (16.5 1C) had larger aedeagi than those raised at a higher temperature (20 1C), this was shown by both the analysis of centroid size and univariate statistics. We also carried out a principal components analysis (data not shown) using the distance measures, which revealed the very same pattern found with geometric morphometrics. There were statistically significant differences in the first three components. The first principal component (PC1), which represents the latent variable size and was responsible for 51.5% of the total variation, was highly correlated with centroid size (r ¼ 0:84;
Fig. 2. Shape changes in the aedeagus of D. mediopunctata in two temperatures (16.5 and 20 1C). The mean configuration is shown in the center. Shape changes were exaggerated three times to make them more visible.
Po0:0001). The PC1 averages for flies raised at 16.5 1C were larger than those raised at 20 1C. Similarly, there were significant differences for the second and third principal components (shape variables). This reiterates the presence of size and shape differences. 4.2. Implications of the variation of traits of the male genitalia To characterize variability between species, TidonSklorz and Sene (1995) investigated the morphological
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variation of the aedeagus of the superspecies D. serido. They demonstrated that a certain region of the aedeagus contributes more to the variation of shape than the remaining measures, which contributed to distinguish three groups of flies. In another group of species, applying morphometric analysis, Kulikov et al. (2004) described the shape of the aedeagus among 11 morphologically similar species of the group of D. virilis. Thus, a fundamental step to understand the evolution of the male genitalia is to connect the intraspecific variation process with the interspecific diversification. Although the different hypotheses on the genitalia evolution attempt to explain the driving forces of this process, the effective causes have not yet been totally clarified (see Discussion in Arnqvist, 1997; Eberhard, 1993; Hosken and Stockley, 2004). We found that the aedeagus traits have levels of phenotypic variation (CV ¼ 7.05% and 7.68% for 16.5 and 20 1C, respectively) comparable to non-genitalic traits in this same species. For example, Bitner-Mathe´ et al. (1995) detected CVs of about 8% in a study with this species for the lengths of the femur, tibia, and wing. Moreover, our results are also comparable with those found in various studies on genital traits in other organisms (Arnqvist and Thornhill, 1998; Eberhard et al., 1998; Jennions and Kelly, 2002; House and Simmons, 2003). In the water strider G. incognitus, Arnqvist and Thornhill (1998) found that traits of the morphology of the genitalia are plastic and are sensitive to conditions of feeding stress. This finding, among others (e.g. the morphology of the genitalia was genetically correlated to measures of the size of the body and length of the leg), led them to reject the lock-and-key hypothesis. As a consequence, the pleiotropy and sexual selection hypotheses were both maintained. Our data show that the traits of the aedeagus suffered the influence of temperature, in other words, there is phenotypic plasticity for size as well as for shape, therefore reinforcing the evidence against the lock-and-key hypothesis. Finally, we can conclude that size and shape of the aedeagus of D. mediopunctata suffered a clear influence of temperature. However, the shape of the aedeagus presented an interesting result: in a specific sector (the most apical area), there was change impinged by the temperature. Such change was captured by the morphometric methods. Since the male genitalia operates inside that of the female, it is necessary to search for more information regarding shape and function of the mating structures of the female. This step is essential to better understand how the male genitalia evolve.
Acknowledgments It is a pleasure to thank Wilma N. de Souza for technical assistance, and Edward R. Hogan for review-
ing the English version of the manuscript. This work was supported by Grants from: Programa Institucional de Capacitac- a˜o Docente e Te´cnica (PICDT/Capes); Conselho Nacional de Desenvolvimento Te´cnico e Cientı´ fico (CNPq); Fundac- a˜o de Amparo a` Pesquisa de Sa˜o Paulo (FAPESP); and Coordenac- a˜o de Aperfeic- oamento de Pessoal de Nı´ vel Superior (CAPES).
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ARTICLE IN PRESS C.A.C. Andrade et al. / Journal of Thermal Biology 30 (2005) 518–523 House, C.M., Simmons, L.W., 2003. Genital morphology and fertilization success in the dung beetle Onthophagus taurus: an example of sexually selected male genitalia. Proc. R. Soc. Lond. B 270, 447–455. Hribar, L.J., 1996. Larval rearing temperature affects morphology of Anopheles albimanus (Diptera: Culicidae) male genitalia. J. Am. Mosquito Control Assoc. 12, 295–297. Imasheva, A.G., Loeschcke, V., Zhivotovsky, L.A., Lazebny, O.E., 1996. Effects of extreme temperatures on the phenotypic variation and developmental stability in Drosophila melanogaster and Drosophila buzzatii. Biol. J. Linn. Soc. 61, 117–126. Jennions, M.D., Kelly, C.D., 2002. Geographical variation in male genitalia in Brachyrhaphis episcopi (Poeciliidae): is it sexually or naturally selected? Oikos 97, 79–86. Klaczko, L.B., 1995. Population genetics of D. mediopunctata. In: Levine, L.I. (Ed.), Genetics of Natural Populations: The Continuing Importance of Theodosius Dobzhansky. Columbia University Press, New York, pp. 140–153. Kobayashi, T., 1998. Seasonal changes in body size and male genital structures of Procladius choreus. (Diptera: Chironomidae: Tanypodinae). Aquatic Insects 20, 165–172. Kulikov, A.M., Melnikov, A.I., Gornostaev, N.G., Lazebny, O.E., Mitrofanov, V.G., 2004. Morphological analysis of male mating organ in the Drosophila virilis species group: a multivariate approach. J. Zool. Syst. Evol. Res. 42, 135–144. Lessels, C.M., Boag, P.T., 1987. Unrepeatable repeatabilities: a commom mistake. Auk 104, 116–121. Mayr, E., 1963. Animal Species and Evolution. Harvard University Press, Cambridge, MA. Morelatto, P.C., Leita˜o-Filho, H.F. (Eds.), 1995. Ecologia e preservac- a˜o de uma floresta tropical urbana: Reserva de Santa Genebra. Unicamp, Campinas.
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Partridge, L., Barrie, B., Fowler, K., French, V., 1994. Evolution and development of body size and cell size in Drosophila melanogaster in response to temperature. Evolution 48, 1269–1276. Rohlf, F.J., 1998. TpsDig, version 1.20. Department of Ecology and Evolution, State University of New York at Stony Brook, Available from http://life.bio.sunysb.edu/morph. Rohlf, F.J., 1999. TpsRegr, version 1.25. Department of Ecology and Evolution, State University of New York at Stony Brook, Available from http://life.bio.sunysb.edu/ morph. Rohlf, F.J., 2000. Statistical power comparisons among alternative morphometric methods. Am. J. Phys. Anthrop. 111, 463–478. Rohlf, F.J., 2003. TpsSmall, version 1.20. Department of Ecology and Evolution, State University of New York at Stony Brook, Available from http://life.bio.sunysb.edu/morph. Shapiro, A.M., Porter, A.H., 1989. The lock-and-key hypothesis evolutionary and biosystematic interpretation of insect genitalia. Ann. Rev. Entomol. 34, 231–245. Slice, D.E., 1996. Three-dimensional generalized resistant fitting and the comparison of least squares and resistant fit residuals. In: Marcus, L.F., Corti, M., Loy, A., Naylor, G., Slice, D.E. (Eds.), Advances in Morphometrics. Plenum, New York, pp. 179–200. Tidon-Sklorz, R., Sene, F.M., 1995. Evolution of the Buzzatii cluster (Drosophila repleta species group) in middle South America. Evol. Biol. 8y9, 71–85. Vilela, C.R., Bachli, G., 1990. Taxonomic studies on Neotropical species of seven genera of Drosophilidae (Diptera). Mitt. Scheiz. Ent. Ges. 63 (Supp.), 1–332. Wheeler, M.R., Kambysellis, M.P., 1966. Notes on the Drosophilidae (Diptera) of Samoa. Univ. Texas Publs. 6615, 533–565.
Journal of Thermal Biology 30 (2005) 518–523 www.elsevier.com/locate/jtherbio
Phenotypic plasticity of the aedeagus of Drosophila mediopunctata: Effect of the temperature Carlos A.C. Andradea,b, Luciane M. Hatadania, Louis B. Klaczkoa, a
Departamento de Gene´tica e Evoluc- a˜o, Instituto de Biologia, Universidade Estadual de Campinas, UNICAMP, Cx. Postal 6109, Campinas 13083-070 SP, Brazil b Departamento de Biologia Marinha, Universidade Federal Fluminense, Nitero´i, RJ, Brazil Received 27 January 2005; accepted 28 May 2005
Abstract In this study, we analyzed the phenotypic plasticity of the male intromittent organ (aedeagus) of Drosophila mediopunctata genitalia in response to temperature. Size and shape of the aedeagus were examined with univariate statistics and geometric morphometrics. The following results emerged: firstly, flies raised at 16.5 1C had bigger aedeagi than those raised at 20 1C; secondly, significant differences in shape were also found between the two groups of flies, mainly in the apical area, located using the thin-plate spline interpolating function; thirdly, the coefficients of variation for the traits of the aedeagus were comparable to other traits in Drosophila and likewise to genital traits in other organisms. These findings were analyzed in an evolutionary context. r 2005 Elsevier Ltd. All rights reserved. Keywords: Sexual selection; Male genitalia evaluation; Geometric morphometrics; Genetic biodiversity
1. Introduction In several groups of animals with internal fertilization, the male genitalia have a fast and divergent evolution. This permits male genitalia traits to be frequently used as characters for defining species, as in Drosophila. In this genus the most utilized traits for species identification are on the male genitalia, especially in the intromittent organ, the aedeagus that show diagnosable differences (Vilela and Bachli, 1990). In the past two decades, the evolution of male genitalia has been studied in many groups of animals with internal fertilization (Eberhard, 1993, 1998; Arnqvist, 1997, 1998; Arnqvist and Thornhill, 1998; House and Simmons, 2003; Corresponding author. Tel.: +55 19 37881150; fax: +55 19 37886235. E-mail address: [email protected] (L.B. Klaczko).
Hosken and Stockley, 2004). There are three hypotheses, which attempt to explain the rapid and diverse evolution of this structure: lock-and-key (Shapiro and Porter, 1989; Arnqvist, 1997), pleiotropy (Mayr, 1963) and sexual selection (Eberhard, 1993; Arnqvist, 1997). The lock-and-key hypothesis deserves special attention since it predicts that genital traits should present relatively limited plasticity due to the nearly perfect adjustment between male and female genitalia (Shapiro and Porter, 1989; Arnqvist, 1997, 1998). Temperature is an abiotic factor, which affects several morphological characters of insects. In Drosophila, it has been repeatedly shown that it acts upon many traits, such as: length of the thorax and wing, and pigmentation in Drosophila melanogaster (Bitner-Mathe´ and Klaczko, 1999; Debat et al., 2003; Gibert et al., 2004; Hoffmann et al., 2003; Imasheva et al., 1996; Partridge et al., 1994) as well as in D. mediopunctata
0306-4565/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2005.05.011
ARTICLE IN PRESS C.A.C. Andrade et al. / Journal of Thermal Biology 30 (2005) 518–523
(Bitner-Mathe´ et al., 1995; Hatadani et al., 2004). In other organisms, such as in the chironomid Procladius choreus and in the mosquito Anopheles albimanus, a variation in the structure of the male genitalia has been detected, respectively, due to seasonality (Kobayashi, 1998) and the development temperature of the larvae (Hribar, 1996). Surprisingly, however, as far as we know, there are no papers on the effect of temperature on the aedeagus morphology of Drosophila.D. mediopunctata is a species belonging to the tripunctata group. It is a Neotropical species with a wide-geographical distribution. It has a long-maturation time, low fecundity and extended longevity (Klaczko, 1995). In this investigation, the main interest was in examining how the aedeagus of D. mediopunctata reacts with regard to two different temperatures. Due to the fact that we are dealing with a complex structure, small in size and with few anatomical points, we used not only univariate statistics but also geometric morphometrics to analyze the variation in size and shape of the aedeagus. A better understanding of this topic may contribute to support, or not, hypotheses that explain the evolution of male genitalia.
2. Materials and methods 2.1. Collection of flies, data gathering and repeatability Flies were collected in the Mata Santa Genebra (221440 4500 S, 471060 3300 W) (Santa Genebra Woods) in September of 2000. This is a semi-deciduous forest in the city of Campinas, Sa˜o Paulo, Brazil (Morelatto and Leita˜o-Filho, 1995). Each female was individually deposited in a tube with culture medium. From the progeny of each female, equal numbers of male and virgin females were set aside (about six couples from each progeny), and, after 10 days were placed in the same vial, from which first stage larvae were later collected. These larvae were distributed in tubes containing 10 ml of culture medium (8% yeast, 2% sugar, 2.5% agar, 0.003% nipagin), in a density of 10 larvae per tube, which were placed in two temperatures (16.5 and 20 1C). Males born from these tubes were separated and kept in wells of microtitration plates, having obtained 110 males (59 males at 16.5 1C and 51 males at 20 1C) at the end of the experiment. The aedeagus was isolated and prepared following the procedure outlined by Wheeler and Kambysellis (1966). The aedeagus is a very small, complex and threedimensional structure. Due to its rigid morphology it is possible, by means of gentle touches on the cover slip, to position it in a lateral or frontal view. However, corrections to the positioning must be made cautiously, since the aedeagus moves about in the balsam, for it has no support. In addition to this problem, there are no real
519
anatomical points on the organ. In a preliminary experiment we determined the best position for the analysis of the aedeagus testing the repeatability of measures taken in lateral and frontal positions. We found that measurement error was too big when the lateral position was used (repeatabilitieso0.26) and this approach was abandoned, since the frontal position gave reliable measures. After carefully placing the aedeagus in a frontal position, we sketched its outline using a camera lucida connected to a Zeiss optical microscope. For this study, having in mind the sources of error mentioned above and with the objective of raising precision in the measures, each specimen was drawn twice, with an interval of at least 1 month, after placing them put back into position. The concurrence of drawings was visually examined before taking the measurements. In only three cases, among 110 drawn specimens, was there evident discordance. For these, a third drawing was prepared in a totally independent way, and then compared to the prior drawings, and the discordant one was discarded. Both drawings of each aedeagus were scanned and saved to file. Next, Cartesian coordinates were taken from a total of 10 anatomical points (474 magnification, Fig. 1), using tpsDIG v1.20 (Rohlf, 1998); all points used represent maximums of curvatures or external points (types 2 and 3 of Bookstein, 1991). Twelve linear measures were taken, joining eight points, making up two superimposed trusses (AD, DF, FH, AH, AF, DH and CE, EG, GI, CI, CG, EI). To avoid redundancy, two points (B and J) were not used to obtain linear measures. The values of the linear measures were obtained from the Cartesian coordinates (using the software Distance 2.0, by Strauss, 1988). The averages of both drawings were used for all variables. Since we have two drawings for each specimen, it is possible to calculate the total final repeatability of the data in this work (Lessels and Boag, 1987). These values are shown in Table 1, and are all above 70%. 2.2. Geometric morphometrics and statistical tests Since the Cartesian coordinates of the landmarks are distributed in a Kendall’s space and their analysis is carried out in a linear space tangent to it (Bookstein, 1991), we tested the adequacy of this approximation with the tpsSmall v1.20 program (Rohlf, 2003). Next, using the program tpsRegr v1.25 (Rohlf, 1999), the anatomical points of the aedeagus were superposed with the Procrustes superposition technique. Thus, the anatomical points were translated, scaled, and rotated so as to minimize the squared and summed distances between the corresponding anatomical points (Bookstein, 1991; Slice, 1996). With this, the size factor was excluded, obtaining only the shape information. The variation of the shape of the aedeagus was analyzed,
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Fig. 1. Frontal view of the aedeagus of D. mediopunctata, lines show the linear measures taken.
Table 1 Repeatability of the analyzed variables of the aedeagus of Drosophila mediopunctata Variable
Repeatability
AD AF AH CE CG CI DF DH EG EI FH GI Centroid size Relative warp 1 Relative warp 2
0.85 0.87 0.86 0.86 0.83 0.94 0.81 0.96 0.71 0.77 0.83 0.87 0.91 0.92 0.91
therefore, for deformation patterns using the thin-plate spline method. To estimate the size of the aedeagus, the centroid size was calculated, which is defined as the square root of the sum of the squares of the distances between each anatomical point and the centroid (Bookstein, 1991). To test the effect of the temperature for the univariate measures and for the centroid size t-tests were carried out. For the global shape of the aedeagus, Goodall’s Ftest (Goodall, 1991) was used, as recommended by Rohlf (2000), as well as a non-parametric permutation test based on Goodall’s F statistic with 100,000 permutations. This test is independent of the restrictive
assumptions of Goodall’s F-test. Finally, to visualize the temperature effect on shape, we used the tpsRegr program obtaining figures for the mean configuration and for the two temperatures (with the respective shape changes exaggerated three times). All statistical analyses were carried out using the program Systat 8.0 or programs of the tps series (Rohlf, 1998, 2003).
3. Results 3.1. Univariate statistics Table 2 shows the averages with their standard errors and coefficients of variation for the 12 linear measures and centroid size of the aedeagus of D. mediopunctata. When comparing the variables in both temperatures, applying the t-test, statistically significant differences are noted for 10 measures (out of 14), which showed higher averages for the lower temperature (16.5 1C) than for the high temperature (20 1C), with the exception of EG. Another important information was the variation observed in each temperature that can be assessed by the coefficients of variation. Their averages were 7.173% and 7.773% for 16.5 and 20 1C, respectively, which are not significantly different (t-test ¼ 0.508; df ¼ 22; P ¼ 0:62). 3.2. Geometric morphometrics We found a positive correlation (r ¼ 1:00) between the tangent and Kendall’s shape spaces. This allowed us to use statistical tests based on the measures in the linear tangent space.
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Table 2 Averages (in mm), standard errors (SE), and coefficients of variation (CV) for the linear measures and centroid size of the aedeagus of Drosophila mediopunctata in two temperatures (16.5 and 20 1C) Variable
AD AF*** AH CE*** CG*** CI DF*** DH** EG* EI*** FH*** GI*** Centroid size***
16.5 1C
20 1C
x¯ SE
CV
x¯ SE
CV
0.17370.0008 0.19770.0009 0.17570.0009 0.04670.0007 0.07570.0006 0.07970.0006 0.06170.0006 0.10270.0008 0.04370.0006 0.07470.0006 0.05970.0007 0.04670.0007 0.21470.0008
0.036 0.036 0.037 0.119 0.057 0.059 0.081 0.056 0.105 0.059 0.090 0.111 0.032
0.17470.0008 0.18970.0009 0.17670.0009 0.03870.0006 0.07170.0008 0.07770.0009 0.05570.0007 0.09870.0010 0.04670.0007 0.07070.0006 0.05370.0006 0.03870.0007 0.20770.0010
0.034 0.035 0.034 0.113 0.075 0.079 0.096 0.073 0.106 0.064 0.087 0.126 0.033
t-test: *Po0:05; **Po0:001; ***Po0:0001.
Regarding centroid size, we observed it was significantly larger at 16.5 1C than at 20 1C (Table 2). This reiterates the observation with univariate statistics: flies raised at lower temperatures have larger aedeagi. Analyzing the global shape difference between both temperatures with Goodall’s F-test and its permutation version, we obtained highly statistically significant differences (Goodall’s F-test ¼ 25.40; df ¼ 16, 1728; Po0:0001; Ppermutation o0:0001). Fig. 2 shows the shape variation, exaggerated three times, as well as the mean configuration, using the thin-plate spline function. It is worthy mentioning that the important deformations are in the most apical region of the aedeagus (see points C, E, G, and I).
4. Discussion 4.1. Plasticity of the size and shape of the aedeagus The aedeagus of D. mediopunctata presented phenotypic plasticity regarding size. Flies raised at a lower temperature (16.5 1C) had larger aedeagi than those raised at a higher temperature (20 1C), this was shown by both the analysis of centroid size and univariate statistics. We also carried out a principal components analysis (data not shown) using the distance measures, which revealed the very same pattern found with geometric morphometrics. There were statistically significant differences in the first three components. The first principal component (PC1), which represents the latent variable size and was responsible for 51.5% of the total variation, was highly correlated with centroid size (r ¼ 0:84;
Fig. 2. Shape changes in the aedeagus of D. mediopunctata in two temperatures (16.5 and 20 1C). The mean configuration is shown in the center. Shape changes were exaggerated three times to make them more visible.
Po0:0001). The PC1 averages for flies raised at 16.5 1C were larger than those raised at 20 1C. Similarly, there were significant differences for the second and third principal components (shape variables). This reiterates the presence of size and shape differences. 4.2. Implications of the variation of traits of the male genitalia To characterize variability between species, TidonSklorz and Sene (1995) investigated the morphological
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variation of the aedeagus of the superspecies D. serido. They demonstrated that a certain region of the aedeagus contributes more to the variation of shape than the remaining measures, which contributed to distinguish three groups of flies. In another group of species, applying morphometric analysis, Kulikov et al. (2004) described the shape of the aedeagus among 11 morphologically similar species of the group of D. virilis. Thus, a fundamental step to understand the evolution of the male genitalia is to connect the intraspecific variation process with the interspecific diversification. Although the different hypotheses on the genitalia evolution attempt to explain the driving forces of this process, the effective causes have not yet been totally clarified (see Discussion in Arnqvist, 1997; Eberhard, 1993; Hosken and Stockley, 2004). We found that the aedeagus traits have levels of phenotypic variation (CV ¼ 7.05% and 7.68% for 16.5 and 20 1C, respectively) comparable to non-genitalic traits in this same species. For example, Bitner-Mathe´ et al. (1995) detected CVs of about 8% in a study with this species for the lengths of the femur, tibia, and wing. Moreover, our results are also comparable with those found in various studies on genital traits in other organisms (Arnqvist and Thornhill, 1998; Eberhard et al., 1998; Jennions and Kelly, 2002; House and Simmons, 2003). In the water strider G. incognitus, Arnqvist and Thornhill (1998) found that traits of the morphology of the genitalia are plastic and are sensitive to conditions of feeding stress. This finding, among others (e.g. the morphology of the genitalia was genetically correlated to measures of the size of the body and length of the leg), led them to reject the lock-and-key hypothesis. As a consequence, the pleiotropy and sexual selection hypotheses were both maintained. Our data show that the traits of the aedeagus suffered the influence of temperature, in other words, there is phenotypic plasticity for size as well as for shape, therefore reinforcing the evidence against the lock-and-key hypothesis. Finally, we can conclude that size and shape of the aedeagus of D. mediopunctata suffered a clear influence of temperature. However, the shape of the aedeagus presented an interesting result: in a specific sector (the most apical area), there was change impinged by the temperature. Such change was captured by the morphometric methods. Since the male genitalia operates inside that of the female, it is necessary to search for more information regarding shape and function of the mating structures of the female. This step is essential to better understand how the male genitalia evolve.
Acknowledgments It is a pleasure to thank Wilma N. de Souza for technical assistance, and Edward R. Hogan for review-
ing the English version of the manuscript. This work was supported by Grants from: Programa Institucional de Capacitac- a˜o Docente e Te´cnica (PICDT/Capes); Conselho Nacional de Desenvolvimento Te´cnico e Cientı´ fico (CNPq); Fundac- a˜o de Amparo a` Pesquisa de Sa˜o Paulo (FAPESP); and Coordenac- a˜o de Aperfeic- oamento de Pessoal de Nı´ vel Superior (CAPES).
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