Genetica (2015) 143:441–451 DOI 10.1007/s10709-015-9843-4
Genetic variability and phenotypic plasticity of metric thoracic traits in an invasive drosophilid in America Blanche Christine Bitner-Mathe´1 • Jean Robert David2,3,4
Received: 13 January 2015 / Accepted: 10 May 2015 / Published online: 29 May 2015 Ó Springer International Publishing Switzerland 2015
Abstract Thermal phenotypic plasticity of 5 metric thoracic traits (3 related to size and 2 to pigmentation) was investigated in Zaprionus indianus with an isofemale line design. Three of these traits are investigated for the first time in a drosophilid, i.e. thorax width and width of pigmented longitudinal white and black stripes. The reaction norms of white and black stripes were completely different: white stripes were insensitive to growth temperature while the black stripes exhibited a strong linear decrease with increasing temperatures. Thorax width exhibited a concave reaction norm, analogous but not identical to those of wing length and thorax length: the temperatures of maximum value were different, the highest being for thorax width. All traits exhibited a significant heritable variability and a low evolvability. Sexual dimorphism was very variable among traits, being nil for white stripes and thorax width, and around 1.13 for black stripes. The ratio thorax length to thorax width (an elongation index) was always [1, showing that males have a more rounded thorax at all temperatures. Black stripes revealed a significant increase of sexual dimorphism with increasing temperature. Shape
& Blanche Christine Bitner-Mathe´ [email protected]; [email protected] 1
Departamento de Gene´tica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Sala A2-109, Bloco A, Edifı´cio do Centro de Cieˆncias da Sau´de, Ilha do Funda˜o, Cidade Universita´ria, Rio de Janeiro, RJ 21941 902, Brazil
2
Laboratoire Evolution, Ge´nomes et Speciation (LEGS), CNRS, 91198 Gif sur Yvette, France
3
University Paris Sud 11, 91405 Orsay, France
4
De´partement Syste´matique et Evolution, UMR 5202 (OSEB), Muse´um National d’Histoire Naturelle, Paris, France
indices, i.e. ratios between size traits all exhibited a linear decrease with temperature, the least sensitive being the elongation index. All these results illustrate the complexity of developmental processes but also the analytical strength of biometrical plasticity studies in an eco-devo perspective. Keywords Drosophila Temperature Isofemale lines Sex dimorphism Zaprionus indianus Pigmentation Body size
Introduction In most ectotherm species, developmental temperature modifies the adult morphology, corresponding to a phenotypic plasticity, also called eco-devo (DeWitt and Scheiner 2004; Pigliucci and Preston 2004; Whitman 2009; Angilletta 2009). Because they are easily reared in the laboratory under well defined conditions, Drosophila species have been extensively investigated, especially for body size variation. A general observation is that size decreases with increasing developmental temperature. In a parallel way, it is known that in several Drosophila species, size varies genetically according to altitude and latitude, showing a decrease when moving from colder to warmer places (Bitner-Mathe´ et al. 1995; David and Capy 1988; Gilchrist et al. 2001; Gibert et al. 2004; David et al. 2006a). In other words, there is a parallelism between a physiological effect and a long term genetic adaptation. This parallelism, also observed in other insect species, has sometimes been called the temperature-size rule (Angilletta and Dunham 2003; Shelomi 2012). Why it is better to be smaller in a warmer environment still remains a matter of debate (Angilletta 2009). One possibility is that adaptation
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to cold in higher latitudes has imposed a selection on wing area in order to decrease the wing loading (Stalker 1980; Pe´tavy et al. 1997; Pitchers et al. 2013). Genetic correlation between wing size and other size-related traits may have resulted in an overall increase of size (David et al. 2011). Several other traits have been investigated in Drosophila and the response curves according to a thermal gradient, i.e. the reaction norms, have been established (Loeschcke et al. 1999; David et al. 2004b; Gibert et al. 2004; Yadav and Singh 2005; Rocha et al. 2008). For bristle numbers and ovariole number, concave curves have been observed, with a maximum in the middle of the thermal range. For body pigmentation, a decrease of the black melanin area on the abdomen is well documented in D. melanogaster and D. simulans. Interestingly, all these traits also exhibit more or less linear latitudinal clines. In the case of a concave norm, it is often argued that the maximum phenotype is akin to a physiological optimum. For body pigmentation, an adaptive explanation is related to the thermal budget hypothesis: it is better to be darker in a colder environment for a better absorption of the light radiations (Gibert et al. 2004). Up to now, almost all investigated species belong to the subgenus Sophophora. In this paper we investigate a more distantly related species, Zaprionus indianus (Gupta 1970) which, from a phylogenetic point of view, should be included into the Drosophila subgenus (Yassin et al. 2010; Yassin 2013). Z. indianus is a species of African origin (Tsacas 1985), which is also a powerful invader and has colonized recently the American continent (Vilela 1999; Yassin et al. 2008; Commar et al. 2012). All African Zaprionus belong to a single clade which comprises about 50 species (Yassin and David 2010). They are all remarkable by their color, i.e. the presence of longitudinal stripes on the head and the thorax. Each stripe is made of a median white stripe and is surrounded by two longitudinal black ones, resulting in a striking and easily recognized phenotype. However, the two types of stripes have independent developmental origins; the black stripes consist of pigmented cuticle, whereas the white stripes correspond to particular long, bent grooved trichomes that contain two cavities and reflect the light (Walt and Tobler 1978). Phenotypic plasticity was previously studied on several metric and meristic traits in an Indian population (Karan et al. 1999). Geographic genetic variation was investigated in Indian, African and South American populations and revealed latitudinal clines of size except for the recently introduced American populations (David et al. 2006b). Finally, a study of wing shape plasticity revealed a progressive elongation of the wing with decreasing growth temperature (Loh et al. 2008). In this paper we were primarily interested in the genetic variability and the plasticity of the longitudinal stripes,
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measured by their width on the thorax. Using an isofemale line design, we also investigated three other thoracic traits related to size, i.e. wing length, thorax length and thorax width, the plasticity of this last trait being analyzed for the first time in a drosophilid. We found that the reactions norms of white and black stripes were completely different, the white being practically insensitive to temperature. Sexual dimorphism was also very different, increasing with temperature for the black stripe, but without any change for the white one. The ratio thorax length to thorax width was consistently greater in female than in male, pointing to a more rounded thoracic shape in male.
Materials and methods Populations investigated Wild living adults of Zaprionus indianus were collected in December 2008 on the campus of the Federal University of Rio de Janeiro, using banana traps. Single females were isolated in culture vials to initiate isofemale lines (David et al. 2005). Ten of such lines were randomly taken and used in the present investigation. Experimental protocol For producing the first laboratory generation, wild collected females were isolated in culture vials at room temperature, 21–23 °C. After about 15 days, emerged adults were anesthetized and a group of 10 females and 10 males was established from each line. These groups were the parents of the experimental flies, which correspond to a second laboratory generation. After a maturation time of about 5 days, each parental group was transferred to a culture vial containing killed yeast, highly nutrient food that prevents crowding effects (Karan et al. 1999). This operation was repeated daily, and empty vials with eggs were transferred to one of 7 experimental temperatures, namely 14, 15, 17, 21, 25, 28 and 31 °C. Z. indianus is a tropical species and the viability at low temperatures is very poor, many individuals dying as larvae or pupae. Indeed, in a previous study analyzing the wing shape variability and using the same experimental protocol (Loh et al. 2008), it was impossible to get a sufficient progeny from all lines at 15 °C. In the present study, 2 or 3 vials of each line were set at the low temperatures (14 and 15 °C) in order to increase the adult number. A sufficient number was thus obtained for each of the 10 lines at 15 °C, but not at 14 °C, so that the results of 14 °C are not considered here, and only the data of 6 experimental temperatures are available. After their emergence, adults were transferred to a mild temperature, 17 or 21 °C, waiting to be measured.
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Traits measured All measurements were done on anesthetized flies, using a binocular microscope equipped with an ocular micrometer. Micrometer units were then transformed into mm 9 100. For anesthesia, we found very convenient to use triethylamine instead of ether. With this product, anesthesia may last for more than 1 h, the fly remains in a natural position and does not move. On each individual, 3 linear dimensions of the mesothorax were measured: total wing length (WL), from the thoracic articulation to the tip, on a left side, lateral view; thorax length (TL) also a left side view from the neck to the tip of the scutellum; thorax width (TW), from a ventral view, as the distance between the bases of the two major posterior sternopleural bristles. From these dimensions, we calculated 3 shape indices, according to the following ratios (WL/TL; TL/TW; WL/ TW). The WL/TL ratio is inversely related to wing loading and considered as a flight parameter (Pe´tavy et al. 1997). It is known to decrease almost linearly with growth temperature (David et al. 2006a). The TL/TW ratio is an elongation index, rarely considered in a morphological studies (Chakir et al. 2008). The WL/TW ratio is also interesting and its biological significance is also an elongation parameter. Zaprionus species are remarkable by the presence of linear white stripes on the head and the thorax (Yassin and David 2010). Each stripe is bordered on each side by a line of black pigmentation, while the remaining thoracic tegument is pale yellow. These stripes are the same in female and male and it has been argued that they might be species visual recognition signal. On the thorax, there are 4 such stripes, and we measured the width of the left median stripe, at the level of the anterior dorsocentral macrochaeta. Two measurements were done: the width of the white stripe (WS), and the width of the total stripe (TS), including the black area around the WS. Then we estimated the breath of black pigmentation alone, as a difference: BS (black stripe) = TS - WS. The breath of these stripes is small: WS is around 0.04 mm and TS about 0.10 mm, so the measurement errors are important. However, since we had a fairly large sample (100 flies of each sex at each temperature) we will see that it has been possible to get valid conclusions on the phenotypic plasticity of these pigmented stripes.
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from the isofemale line design (David et al. 2005). Phenotypic plasticity, related to growth temperature was analyzed by considering the shape of the response curves, i.e. the reaction norms. We used the method of polynomial adjustments for analyzing the shapes and calculating characteristic values (David et al. 1997; Gibert et al. 2004).
Results Thoracic stripes The reaction norms of the width of the white stripe (WS) and of the black stripes (BS) are shown in Fig. 1, and the results of a 3 way ANOVA are in Table 1. A first conclusion is that the two traits vary significantly according to growth temperature, but in different ways. BS decreases almost linearly with temperature, and the temperature effect explains more than 33 % of the total variance. By contrast, temperature explains only 3 % of the variance of WS with no general linear trend (see Fig. 1). In both cases a significant genetic heterogeneity between lines is observed, as well as a line-temperature interaction. The difference between WS and BS is also evidenced by considering amount of variation observed in calculating the coefficients of variations (CVs) between temperatures which are 1.5 and 1.3 for WS and 10.5 and 10.6 for BS, in female and male respectively. The genetic variability among lines was further investigated (Table 2) by calculating the intraclass coefficient of correlation (ICC), which is akin to isofemale line heritability (David et al. 2005) and the genetic coefficient of variation (CVg), also called evolvability (Houle 1992). For
Data analysis The total data set (6000 linear measures) was investigated with SYSTATÓ v.13.0 (SPSS Inc.). Basic statistics: mean values, ANOVA, coefficients of variations, correlations, were calculated, as well as genetic parameters estimated
Fig. 1 Variation of the width of thoracic pigmented bands according to growth temperature. Triangles: males; circles: females. Open symbols (below) white stripe; filled symbols (above) black pigmented area
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Table 1 Results of ANOVA applied to the various traits investigated Source
df
Pigmentation WS
Size
Shape
BS
WL
TL
TW
WL/TL
WL/TW
TL/TW
13.17***
75.08***
72.67***
22.76***
1.52***
4.83***
6.87***
3.68***
2.32***
11.61***
0.02
0.10
4.25***
2.37***
6.87***
0.90
0.16
0.07
0.42
2.64 60.45
0.74 14.42
1.06* 16.58
2.60 48.54
Temperature
5
3.30***
33.65***
78.44***
34.59***
Sex
1
0.26*
22.12***
3.57***
2.90***
Line
9
14.05***
4.84***
0.35**
6.58***
1.00***
0.01
3.05***
3.03***
Sex 9 temp
5
Line 9 temp
45
Line 9 sex Line 9 sex 9 temp Error
0.40 11.78***
9
0.85
0.45
0.16
45 1080
2.08 67.29
1.23 33.69
0.96** 13.38
0.90** 10.84*** 0.76*** 1.48 41.64
0.27 10.16*** 0.70 11.72***
0.33
WS white stripe, BS black stripe, WL wing length, TL thorax length, TW thorax width; shape indices are presented as ratios. The table shows the percentage of the total variance explained by each source of variation *** P \ 0.001; ** P \ 0.01; * P \ 0.05
the intraclass correlations, there was no significant effect due to sex or temperature, and the average values are WS = 0.224 ± 0.047 (n = 12) and BS = 0.164 ± 0.036 (n = 12). These two values are not statistically different but both are significantly [0, confirming the significant genetic variability among lines (see also Table 2). Evolvability (CVg) was on average 6.61 ± 0.25 for WS and 10.95 ± 0.44 for BS: this difference is highly significant. The correlations between WS and BS were calculated at the within line, individual level and the between line, genetic level. Average values (not shown) were both negative but not significantly different from zero. This result confirms the developmental independence between the two kinds of pigmented stripes The correlations between pigmentation and size characters or shape indices were calculated, within each temperature, at the line level (Table 3). A clear conclusion is that WS was not correlated with size, while a slight but significant correlation was found for BS (Overall r = 0.29 ± 0.06, n = 6). This correlation was especially high with TW (average for both sexes: r = 0.44). In other words, size variations, and especially the width of the thorax, affect the width of BS (Fig. 2). With the shape indices, the most interesting result is a consistent negative correlation with BS and especially with the elongation index (TL/TW; average for both sexes: r = -0.28): a more elongated thorax is accompanied by narrower black stripes. Finally our data permit a comparison between sexes by considering two statistics, first the between sexes (F 9 M) correlation and second the sexual dimorphism as a female/male (F/M) ratio (Table 2). For each line the average values of female and male were always positively correlated with no temperature effect: the overall correlations are significantly different, i.e. 0.76 and 0.66 for WS and BS respectively.
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The F/M ratio revealed a very interesting difference. For WS, the ratio was always very close to one, meaning that both sexes were practically identical. This result may be compared with the very low dimorphism of body size, (see below). A contrasting result was obtained for BS with, firstly, an average value (1.13) much [1 and, second, a significant increase with temperature (Fig. 3). Size characters The data of each trait were submitted to a 3-way ANOVA, also shown in Table 1 as the percent of the total variance explained by each factor. WL is the most sensitive trait to temperature, and TW the least. Sex has a significant effect on WL and TL (smaller male) but not for TW. The heterogeneity among lines (genetic effects) is most important for TL and TW, and much less for WL, although, in all cases, the effects are highly significant. A significant interaction is also observed in the 3 traits between lines and temperatures, meaning the shapes of the reaction norms are variable among lines. The genetic variability was further analyzed by calculating the coefficients of intraclass correlation (ICC) for each temperature and trait, and average values are given in Table 4. There was no significant effect of temperature, sex or trait (ANOVA, not shown) and the overall average value is 0.26 ± 0.04, slightly greater than for the pigmented stripes, and in close agreement with previous data on an Indian population (Karan et al. 1999). Evolvability (CVg) was comprised between 0.95 and 1.62, being less for WL and maximum for TW and also greater in males than in females. The F 9 M correlation was variable among traits, least for WL (0.61) and greatest for TL (0.87). The F/M ratio was always close to 1, but different among traits. Values of 1.02 and 1.01 for wing and thorax length
WS white stripe, BS black stripe, F female, M male
1.002 ± 0.009 1.111 ± 0.007
WS
BS
Sexual dimorphism (F/M ratio)
0.95 0.85
12.50
M
WS
11.99
F
7.70 8.67
F
0.14*
M
M
0.09 ns
F
BS
F 9 M correlation
BS
WS
Genetic coefficient of variation (CVg)
BS
0.43*** 0.59***
F M
Intraclass correlation coefficient (ICC) WS
15 °C
Temperature
Sex
Trait
1.137 ± 0.013
1.019 ± 0.007
0.76
0.79
11.73
11.75
5.83
6.20
0.32***
0.33***
0.14*
0.29***
17 °C
1.101 ± 0.011
1.009 ± 0.010
0.69
0.73
10.94
12.00
6.36
7.21
0.08
0.20**
0.27***
0.33***
21 °C
1.134 ± 0.014
1.014 ± 0.008
0.54
0.63
10.76
12.09
5.94
6.36
0.06
0.23***
0.18**
0.09
25 °C
1.148 ± 0.011
1.007 ± 0.007
0.61
0.63
8.66
11.65
5.73
6.20
0.02
-0.04
0.08
0.12*
28 °C
Table 2 Genetic variability, sexual dimorphism and female 9 male correlation for the two pigmentation traits at various growth temperatures
1.241 ± 0.027
0.993 ± 0.005
0.54
0.82
7.66
9.69
6.44
6.72
0.31***
0.23**
0.10*
0.08
31 °C
1.126 ± 0.007
1.007 ± 0.004
0.66 ± 0.05
0.76 ± 0.05
10.38 ± 0.76
11.53 ± 0.37
6.50 ± 0.45
6.73 ± 0.25
0.15 ± 0.05
0.17 ± 0.05
0.23 ± 0.08
0.22 ± 0.06
Mean ± SE
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respectively confirm previous results (David et al. 2006b). Interestingly, TW was on average identical in both sexes. The reaction norms of the 3 size traits as a function of growth temperature are shown in Fig. 4. In all cases they are not linear, exhibit a maximum in within the thermal range and can be described by a quadratic polynomial. With such adjustments, we calculated characteristic values, i.e. the coordinates of the maximum and a curvature (g2) parameter. Average values are given in Table 5. As expected maximum values are very different between traits; a significant difference between sexes is found only for WL. The position of the temperature of maximum value (TMV) is significantly variable among traits, with the lowest value for the wing (both sexes average 17.3 °C) and the highest for thorax width (22.6°). Curvature values are different but this is explained by the variations in means of the traits. Shape indices The results of a 3-way ANOVA are shown in Table 1. For all 3 indices, significant effects of temperature, sex and line, as well as the temperature-line interaction are significant. The thorax elongation index (TL/TW) is the least sensitive, with a greater error term. The genetic variability was further analyzed by considering the intraclass correlation and evolvability (Table 6). The overall intraclass correlation (0.31) was slightly greater than for the traits themselves. Evolvability was more stable and not influenced by sex or temperature. Overall average value (1.26) is slightly [1 and is very close to that found for the traits themselves (1.17). The reaction norms of the 3 shape indices are given in Fig. 5. In all cases, monotonically decreasing norms were observed, and linear adjustments are shown. Males have always a slightly lesser value than females, but the sexual Table 3 Genetic correlation between size traits and pigmentation
Trait
Fig. 2 Relationship between thorax width and stripe width: below, white stripe; above, black stripe. Triangles: males; circles: females. Data from all temperature are pooled. Note the lack of correlation for the white stripe but the positive correlation for the black pigmentation
dimorphism is small i.e. 1.009, 1.022 and 1.013 for WL/ TL, WL/TW and TL/TW respectively. The sensitivity to temperature may be considered by estimating the amount of variation observed between 15 and 31 °C, scaled to the mean of each trait. The amount of variation is 8.5, 11.2 and 2.8 % for WL/TL, WL/TW and TL/TW respectively. The elongation index, TL/TW, is much less sensitive to growth temperature than the two others.
Discussion The present work analyzed the thermal phenotypic plasticity of five different metric traits in a cosmopolitan tropical drosophilid. Three of the traits (thorax width and widths of the longitudinal stripes) are investigated for the
WS
BS
F
M
F
M
WL
0.07 ± 0.18
-0.12 ± 0.15
0.12 ± 0.12
0.29 ± 0.10
TL
0.05 ± 0.20
-0.23 ± 0.13
0.11 ± 0.13
0.31 ± 0.07
TW
-0.05 ± 0.20
-0.27 ± 0.16
0.39 ± 0.11
0.49 ± 0.06
Size trait mean
-0.09 ± 0.06 (n = 6)
0.29 ± 0.06 (n = 6)
WL/TL
0.05 ± 0.11
0.15 ± 0.09
-0.06 ± 0.13
-0.10 ± 0.15
WL/TW
0.07 ± 0.09
-0.18 ± 0.14
-0.29 ± 0.09
-0.36 ± 0.12
0.02 ± 0.10
0.07 ± 0.12
-0.25 ± 0.04
-0.30 ± 0.02
TL/TW Shape indices mean
0.03 ± 0.05 (n = 6)
-0.23 ± 0.05 (n = 6)
Each value is the mean of 6 values obtained at the 6 growth temperatures WS white stripe, BS black stripe, WL wing length, TL thorax length, TW thorax width; shape indices are presented as ratios. F female, M male
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Fig. 3 Variation of sexual dimorphism (female/male ratio) of black stripes according to growth temperature in the 10 isofemale lines investigated. In spite of a broad irregularity, a significant increasing trend is observed
first time. The results allowed a broad diversity of analyses and comparisons among traits and temperatures. We discuss here the most interesting results. The thermal developmental range of Zaprionus indianus Our results clearly show that Z. indianus can be reared between 15 and 31 °C, while the thermal range of Drosophila melanogaster is 12–31 °C (Pe´tavy et al. 2001; David et al. 2004a). The greater sensitivity to cold of Zaprionus, correlated with a male complete sterility at 15 °C (Araripe et al. 2004) is certainly a major feature explaining that, for the moment, the species is restricted to tropical and subtropical countries. The diversity of the shapes of the reaction norms Since we used 6 different growth temperatures, the data can be ordered along a thermal gradient and the response curves are the reaction norms. For WL, the reaction norm is mostly decreasing, with a maximum close to the lower developmental threshold. This is why many investigators consider that ‘‘body size’’ decreases with increasing Table 4 Genetic variability of size traits, sexual dimorphisms (F/M ratio) and females-male (F 9 M) correlation
temperature (Angilletta 2009; Shelomi 2012). Indeed this is sometimes called the temperature-size rule (Angilletta and Sears 2004). Our data on thorax length confirm what was already clear in D. melanogaster (Morin et al. 1999; David et al. 2006a) that the reaction norm shows a maximum value at a higher temperature than WL and thus a clear concave shape. The new data on TW still enforce this conclusion, since the TMV is still higher than for TL: the temperature size rule is clearly not valid for different size-related traits (David et al. 2006a). It was previously shown that different parts of the wing exhibited different reaction norms with very different thermal maxima (Moreto et al. 1998). An analogous situation is now found for the thorax. This illustrates the difficulty to define an overall size in any organism. As usual in taxonomy, shape variations are described by morphological indices or ratio (Ba¨chli et al. 2004). The 3 calculated indices all produced similar, almost linearly decreasing norms. The decreasing WL/TL has been observed in all drosophilids investigated so far, it is inversely proportional to wing loading and might be the direct target of natural selection responsible of body size latitudinal lines (Stalker 1980; Pe´tavy et al. 1997; Loeschcke et al. 1999). The WL/TW ratio has less obvious biological significance, but also exhibits a sharp decrease. The TL/TW describes the elongation of the thorax. In D. melanogaster, a study at a single temperature (25 °C) (Chakir et al. 2008) produced a value of 1.55. In Zaprionus, the same index is 1.72–1.75: in other words, the thorax is relatively much longer. It is interesting to note that this thorax shape index is only poorly affected by temperature variations, contrasting with the highly significant effect of temperature for an elongation index of the wing in this same species (Loh et al. 2008). The two pigmentation traits, which were the main focus of this paper, also produced linear norms, but with very different shapes. The black area exhibited a highly significant, almost linear decrease with temperature. Similar results are observed in both sexes, but pigmentation is much less in males, and SD fairly high, with a F/M ratio of 1.13, contrasting with the fact that both sexes have almost
WL
TL
TW
F
M
F
M
F
M
ICC
0.16 ± 0.02
0.26 ± 0.05
0.27 ± 0.05
0.42 ± 0.09
0.18 ± 0.03
0.29 ± 0.06
CVg
0.95 ± 0.09
1.18 ± 0.16
1.12 ± 0.16
1.45 ± 0.26
1.24 ± 0.07
1.62 ± 0.13
F/M
1.020 ± 0.001
1.011 ± 0.002
0.998 ± 0.002
F9M
0.608 ± 0.042
0.867 ± 0.008
0.714 ± 0.078
Each value is the mean of the 6 studied temperatures ICC coefficient of intraclass correlation, CVg coefficient of genetic variation, WL wing length, TL thorax length, TW thorax width
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Fig. 4 Reaction norms of the 3 size-related traits according to growth temperature. a–c Mean value after a quadratic adjustment; triangles: males; circles: females. d Reaction norms of female wing length for 10 isofemale lines
Table 5 Characteristic values of the norms of reactions for the 3 size-related traits measured on the thorax n
MV ± SE
12 ± SE
TMV ± SE
312.28 ± 0.67
-0.190 ± 0.017
16.92 ± 0.62
Traits
Sex
WL
F M
8
306.87 ± 0.93
-0.214 ± 0.017
17.62 ± 0.56
TL
F
9
139.20 ± 0.43
-0.067 ± 0.007
20.61 ± 0.35
M
9
138.59 ± 0.64
-0.089 ± 0.011
21.19 ± 0.37
TW
F
9
79.66 ± 0.30
-0.027 ± 0.004
22.34 ± 0.30
M
10
80.04 ± 0.40
-0.032 ± 0.008
22.79 ± 0.33
8
MV maximum value, 12 curvature, TMV temperature of maximum value. n number of lines used for the calculation of a quadratic adjustment; notice that this number is generally \10, some lines did not produce a valid adjustment, i.e. a maximum outside the thermal range. WL wing length, TL thorax length, TW thorax width, F female, M male
identical sizes. The black pigmentation corresponds to a deposit of black melanin in the cuticle and its synthesis is well known (Wittkopp et al. 2003). In D. melanogaster female, the plasticity of abdomen pigmentation is a most
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investigated trait and an almost linear decrease with increasing temperature is well documented (Gibert et al. 2004). Similar phenomenon is also observed in D. mediopunctata (Rocha et al. 2008). This is probably a general process, but the place where black melanin is deposited is extremely variable due to developmental regulation. For the white pigmented stripes, the reaction norm was an almost horizontal line, with no sexual dimorphism. This stripe has a very different origin, since it corresponds to a shape modification of the cellular trichomes on the thorax: a flattened non cylindrical shape results in the reflection of the light (Walt and Tobler 1978). Other drosophilids have shining, light reflecting area on the head (for example in the D. nasuta complex of the immigrans group of Drosophila or in the montium group of Sophophora). The role of these reflecting areas is not known but might be involved in sexual behavior. In Zaprionus, the female exhibits a special sexual rejection behavior which is a rapid, left right oscillation of the body. Possibly the longitudinal bands might help to visualize and interpret this signal. In the butterfly Bicyclus anynana, visual, mate recognition
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Table 6 Analysis of the shape indices expressed as ratios
WL/TL
WL/TW
TL/TW
F
M
F
M
F
M
ICC
0.36 ± 0.04
0.36 ± 0.04
0.24 ± 0.05
0.18 ± 0.04
0.37 ± 0.04
0.35 ± 0.05
CVg
1.08 ± 0.11
1.09 ± 0.13
1.25 ± 0.09
1.22 ± 0.11
1.21 ± 0.06
1.16 ± 0.10
F/M
1.009 ± 0.001
1.022 ± 0.002
1.013 ± 0.001
F9M
0.799 ± 0.044
0.635 ± 0.051
0.821 ± 0.025
Each value is the mean of the 6 studied temperatures WL wing length, TL thorax length, TW thorax width, ICC coefficient of intraclass correlation, CVg coefficient of genetic variation, F/M female–male ratio, F 9 M female-male correlation
signals are better known. These visual traits are almost non-plastic, while other pigmented areas are very plastic with respect to developmental temperature (San Martin et al. 2011). The magnitude of plasticity Different characters are more or less plastic and one way to compare the magnitude of plasticity, getting rid of the difference in mean values, is to calculate a between temperature coefficient of variation (David et al. 2004a; Gibert et al. 2004). With this method, the metric traits analyzed in this work may be classified as follow, in decreasing order from 1.7 to 1.2: TL [ WL = BS [ TW [ WS. For the 3 shape indices, a decrease is also observed, with the following order WL/TW [ WL/TL [ TL/TW. The various traits react differently to temperature not only by revealing different reaction norm shapes but also by the magnitude of their reactivity. This certainly corresponds to complex, but not known, developmental regulations. Genetic variability and evolvability As usual with a full-sib, isofemale line design (David et al. 2005), we found significant genetic differences among lines for all traits. This genetic variability may be quantified by calculating an intraclass correlation coefficient. However, due to the small number of investigated lines (10) this coefficient is not precise so that a significant variability between temperatures was never observed. More interesting results were however obtained by considering the genetic CV, which is the between line standard deviation divided by the mean. This CVg reveals the capacity for a trait to evolve and has been called evolvability (Houle 1992). An interesting result, which has been found for the 3 linear dimensions as well as the 3 ratios, is that evolvability changed with temperature (see Table 1). The reaction norms showed a lesser value in the middle of the thermal range and greater values at extreme temperatures. This is a
strong argument in favor of the hypothesis that extreme environments might increase the evolving capacity of a population by revealing a cryptic genetic variation (Hoffmann and Parsons 1997; Gibson and Dworkin 2004). A similar observation was also done in D. melanogaster (David et al. 2006c) and D. ananassae (Sisodia and Singh 2009). The two pigmentation traits did not provide such a clear pattern, since genetic CVs were higher at low temperature and then decreased, with only a slight increase at high temperature. Sexual dimorphism (SD) Zaprionus indianus is a species in which males and females are very similar in their external phenotype. Sexual dimorphism (SD), expressed as a female/male ratio, is 1.02 for wing length and 1.01 for thorax length (David et al. 2006c). We found here that the difference practically disappeared when TW was considered. Also SD in Zaprionus is not affected by developmental temperature. These results are very different from those on D. melanogaster, where the female is on average 15 % bigger than the male when either wing or thorax length are considered (Chakir et al. 2008), and where SD increases according to growth temperature from 1.12 up to 1.17 between 12 and 31 °C (David et al. 2011). When the shape indices are considered in Zaprionus, a significant sex difference was always observed, with lesser values in male (Fig. 5). The main difference concerns the elongation index (TL/TW) for which SD is consistently[1, meaning that the thorax is more rounded in male than in female; a similar observation has been made in D. melonogaster (Chakir et al. 2008). Shape variation between female and male are however not a rule. For example, in Zaprionus, the wing shape (expressed as a width/length ratio) show a clear increase with developmental temperature but remains the same in both sexes (Loh et al. 2008). For the pigmentation traits, we got again contrasting results. For the white stripe width, males and females are
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much greater value for BS. We may also recall that, for the abdominal bristle number, SD was found to be very variable between successive segments, with averages ranging from 1.0 to 0.5 (Araripe et al. 2004). All these results suggest that all quantitative traits are affected by sex, but that, according to the trait, the SD ratio may be extremely variable, revealing a diversity of genetic mechanism, in sex interactions. In conclusion, investigating thermal phenotypic plasticity appears a powerful means for discriminating the developmental bases of various quantitative traits. Our present state of knowledge suggests that, in an evolutionary, developmental and phylogenetic (Evo-Devo) perspective, many more species should be investigated and compared; among them, Z. indianus might become a reference model. Acknowledgments We thank Amir Yassin for thoughtful comments on early drafts. This work was supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Comite Franc¸ais d’´Evaluation de la Coope´ation Universitaire avec le Bre´sil (COFECUB). This paper results from a French–Brazilian cooperation programme on Zaprionus.
References
Fig. 5 Reaction norms of shape ratios according to growth temperature. WL/TL: wing to thorax length; WL/TW: wing length to thorax width; TL/TW: thorax length to thorax width. Linear regressions are shown. Triangles: males; circles: females. Note the 3 ratios have very different values. Sexual dimorphism is more pronounced for TL/TW
almost identical, with a SD value close to 1.01, a result similar to that of TL. The width of the black stripe is positively correlated with TW but very different among sexes, with an overall SD value close to 1.13. In a previous study (David et al. 2006c), it was shown that the sternopleural bristle number in Zaprionus exhibited a SD of 1.07, i.e. much more than for size. Here we still find a
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Angilletta MJ (2009) Thermal adaptation: a theoretical and empirical synthesis. Oxford University Press, New York Angilletta MJ, Dunham AE (2003) The temperature size rule in ectotherms: simple evolutionary explanations may not be general. Am Nat 162:332–342 Angilletta MJ, Sears MW (2004) Evolution of thermal reaction norms for growth rate and body size in ectotherms: an introduction to the symposium. Integr Comp Biol 44:401–402 Araripe LO, Klaczko LB, Moreteau B, David JR (2004) Male sterility thresholds in a tropical cosmopolitan drosophilid, Zaprionus indianus. J Therm Biol 29:73–80 Ba¨chli G, Vilela CR, Escher SA, Saura A (2004) The Drosophilidae (Diptera) of Fennoscandia and Denmark. Brill, Leiden Bitner-Mathe´ BC, Pexioto AA, Klaczko LB (1995) Morphological variation in a natural population of Drosophila mediopunctata: altitudinal cline, temporal changes, and influence of chromosomal inversions. Heredity 75:54–61 Chakir M, Negoua H, Moreteau B, David JR (2008) Quantitative morphometrical analysis of a North African population of Drosophila melanogaster: sexual dimorphism, and comparison with European populations. J Genet 87:373–382 Commar LS, Galego LGC, Ceron CR, Carareto CMA (2012) Taxonomic and evolutionary analysis of Zaprionus indianus and its colonization of Palearctic and Neotropical regions. Genet Mol Biol 35:395–406 David JR, Capy P (1988) Genetic variation of Drosophila melanogaster natural populations. Trends Genet 4:106–111 David JR, Gibert P, Gravot E, Petavy G, Morin JP, Karan D, Moreteau B (1997) Phenotypic plasticity and developmental temperature in Drosophila: analysis and significance of reaction norms of morphometrical traits. J Therm Biol 22:41–451 David JR, Gibert P, Moreteau B (2004a) Evolution of reaction norms. In: DeWitt TJ, Scheiner S (eds) Phenotypic plasticity, functional
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Genetic variability and phenotypic plasticity of metric thoracic traits in an invasive drosophilid in America Blanche Christine Bitner-Mathe´1 • Jean Robert David2,3,4
Received: 13 January 2015 / Accepted: 10 May 2015 / Published online: 29 May 2015 Ó Springer International Publishing Switzerland 2015
Abstract Thermal phenotypic plasticity of 5 metric thoracic traits (3 related to size and 2 to pigmentation) was investigated in Zaprionus indianus with an isofemale line design. Three of these traits are investigated for the first time in a drosophilid, i.e. thorax width and width of pigmented longitudinal white and black stripes. The reaction norms of white and black stripes were completely different: white stripes were insensitive to growth temperature while the black stripes exhibited a strong linear decrease with increasing temperatures. Thorax width exhibited a concave reaction norm, analogous but not identical to those of wing length and thorax length: the temperatures of maximum value were different, the highest being for thorax width. All traits exhibited a significant heritable variability and a low evolvability. Sexual dimorphism was very variable among traits, being nil for white stripes and thorax width, and around 1.13 for black stripes. The ratio thorax length to thorax width (an elongation index) was always [1, showing that males have a more rounded thorax at all temperatures. Black stripes revealed a significant increase of sexual dimorphism with increasing temperature. Shape
& Blanche Christine Bitner-Mathe´ [email protected]; [email protected] 1
Departamento de Gene´tica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Avenida Carlos Chagas Filho, 373, Sala A2-109, Bloco A, Edifı´cio do Centro de Cieˆncias da Sau´de, Ilha do Funda˜o, Cidade Universita´ria, Rio de Janeiro, RJ 21941 902, Brazil
2
Laboratoire Evolution, Ge´nomes et Speciation (LEGS), CNRS, 91198 Gif sur Yvette, France
3
University Paris Sud 11, 91405 Orsay, France
4
De´partement Syste´matique et Evolution, UMR 5202 (OSEB), Muse´um National d’Histoire Naturelle, Paris, France
indices, i.e. ratios between size traits all exhibited a linear decrease with temperature, the least sensitive being the elongation index. All these results illustrate the complexity of developmental processes but also the analytical strength of biometrical plasticity studies in an eco-devo perspective. Keywords Drosophila Temperature Isofemale lines Sex dimorphism Zaprionus indianus Pigmentation Body size
Introduction In most ectotherm species, developmental temperature modifies the adult morphology, corresponding to a phenotypic plasticity, also called eco-devo (DeWitt and Scheiner 2004; Pigliucci and Preston 2004; Whitman 2009; Angilletta 2009). Because they are easily reared in the laboratory under well defined conditions, Drosophila species have been extensively investigated, especially for body size variation. A general observation is that size decreases with increasing developmental temperature. In a parallel way, it is known that in several Drosophila species, size varies genetically according to altitude and latitude, showing a decrease when moving from colder to warmer places (Bitner-Mathe´ et al. 1995; David and Capy 1988; Gilchrist et al. 2001; Gibert et al. 2004; David et al. 2006a). In other words, there is a parallelism between a physiological effect and a long term genetic adaptation. This parallelism, also observed in other insect species, has sometimes been called the temperature-size rule (Angilletta and Dunham 2003; Shelomi 2012). Why it is better to be smaller in a warmer environment still remains a matter of debate (Angilletta 2009). One possibility is that adaptation
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to cold in higher latitudes has imposed a selection on wing area in order to decrease the wing loading (Stalker 1980; Pe´tavy et al. 1997; Pitchers et al. 2013). Genetic correlation between wing size and other size-related traits may have resulted in an overall increase of size (David et al. 2011). Several other traits have been investigated in Drosophila and the response curves according to a thermal gradient, i.e. the reaction norms, have been established (Loeschcke et al. 1999; David et al. 2004b; Gibert et al. 2004; Yadav and Singh 2005; Rocha et al. 2008). For bristle numbers and ovariole number, concave curves have been observed, with a maximum in the middle of the thermal range. For body pigmentation, a decrease of the black melanin area on the abdomen is well documented in D. melanogaster and D. simulans. Interestingly, all these traits also exhibit more or less linear latitudinal clines. In the case of a concave norm, it is often argued that the maximum phenotype is akin to a physiological optimum. For body pigmentation, an adaptive explanation is related to the thermal budget hypothesis: it is better to be darker in a colder environment for a better absorption of the light radiations (Gibert et al. 2004). Up to now, almost all investigated species belong to the subgenus Sophophora. In this paper we investigate a more distantly related species, Zaprionus indianus (Gupta 1970) which, from a phylogenetic point of view, should be included into the Drosophila subgenus (Yassin et al. 2010; Yassin 2013). Z. indianus is a species of African origin (Tsacas 1985), which is also a powerful invader and has colonized recently the American continent (Vilela 1999; Yassin et al. 2008; Commar et al. 2012). All African Zaprionus belong to a single clade which comprises about 50 species (Yassin and David 2010). They are all remarkable by their color, i.e. the presence of longitudinal stripes on the head and the thorax. Each stripe is made of a median white stripe and is surrounded by two longitudinal black ones, resulting in a striking and easily recognized phenotype. However, the two types of stripes have independent developmental origins; the black stripes consist of pigmented cuticle, whereas the white stripes correspond to particular long, bent grooved trichomes that contain two cavities and reflect the light (Walt and Tobler 1978). Phenotypic plasticity was previously studied on several metric and meristic traits in an Indian population (Karan et al. 1999). Geographic genetic variation was investigated in Indian, African and South American populations and revealed latitudinal clines of size except for the recently introduced American populations (David et al. 2006b). Finally, a study of wing shape plasticity revealed a progressive elongation of the wing with decreasing growth temperature (Loh et al. 2008). In this paper we were primarily interested in the genetic variability and the plasticity of the longitudinal stripes,
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measured by their width on the thorax. Using an isofemale line design, we also investigated three other thoracic traits related to size, i.e. wing length, thorax length and thorax width, the plasticity of this last trait being analyzed for the first time in a drosophilid. We found that the reactions norms of white and black stripes were completely different, the white being practically insensitive to temperature. Sexual dimorphism was also very different, increasing with temperature for the black stripe, but without any change for the white one. The ratio thorax length to thorax width was consistently greater in female than in male, pointing to a more rounded thoracic shape in male.
Materials and methods Populations investigated Wild living adults of Zaprionus indianus were collected in December 2008 on the campus of the Federal University of Rio de Janeiro, using banana traps. Single females were isolated in culture vials to initiate isofemale lines (David et al. 2005). Ten of such lines were randomly taken and used in the present investigation. Experimental protocol For producing the first laboratory generation, wild collected females were isolated in culture vials at room temperature, 21–23 °C. After about 15 days, emerged adults were anesthetized and a group of 10 females and 10 males was established from each line. These groups were the parents of the experimental flies, which correspond to a second laboratory generation. After a maturation time of about 5 days, each parental group was transferred to a culture vial containing killed yeast, highly nutrient food that prevents crowding effects (Karan et al. 1999). This operation was repeated daily, and empty vials with eggs were transferred to one of 7 experimental temperatures, namely 14, 15, 17, 21, 25, 28 and 31 °C. Z. indianus is a tropical species and the viability at low temperatures is very poor, many individuals dying as larvae or pupae. Indeed, in a previous study analyzing the wing shape variability and using the same experimental protocol (Loh et al. 2008), it was impossible to get a sufficient progeny from all lines at 15 °C. In the present study, 2 or 3 vials of each line were set at the low temperatures (14 and 15 °C) in order to increase the adult number. A sufficient number was thus obtained for each of the 10 lines at 15 °C, but not at 14 °C, so that the results of 14 °C are not considered here, and only the data of 6 experimental temperatures are available. After their emergence, adults were transferred to a mild temperature, 17 or 21 °C, waiting to be measured.
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Traits measured All measurements were done on anesthetized flies, using a binocular microscope equipped with an ocular micrometer. Micrometer units were then transformed into mm 9 100. For anesthesia, we found very convenient to use triethylamine instead of ether. With this product, anesthesia may last for more than 1 h, the fly remains in a natural position and does not move. On each individual, 3 linear dimensions of the mesothorax were measured: total wing length (WL), from the thoracic articulation to the tip, on a left side, lateral view; thorax length (TL) also a left side view from the neck to the tip of the scutellum; thorax width (TW), from a ventral view, as the distance between the bases of the two major posterior sternopleural bristles. From these dimensions, we calculated 3 shape indices, according to the following ratios (WL/TL; TL/TW; WL/ TW). The WL/TL ratio is inversely related to wing loading and considered as a flight parameter (Pe´tavy et al. 1997). It is known to decrease almost linearly with growth temperature (David et al. 2006a). The TL/TW ratio is an elongation index, rarely considered in a morphological studies (Chakir et al. 2008). The WL/TW ratio is also interesting and its biological significance is also an elongation parameter. Zaprionus species are remarkable by the presence of linear white stripes on the head and the thorax (Yassin and David 2010). Each stripe is bordered on each side by a line of black pigmentation, while the remaining thoracic tegument is pale yellow. These stripes are the same in female and male and it has been argued that they might be species visual recognition signal. On the thorax, there are 4 such stripes, and we measured the width of the left median stripe, at the level of the anterior dorsocentral macrochaeta. Two measurements were done: the width of the white stripe (WS), and the width of the total stripe (TS), including the black area around the WS. Then we estimated the breath of black pigmentation alone, as a difference: BS (black stripe) = TS - WS. The breath of these stripes is small: WS is around 0.04 mm and TS about 0.10 mm, so the measurement errors are important. However, since we had a fairly large sample (100 flies of each sex at each temperature) we will see that it has been possible to get valid conclusions on the phenotypic plasticity of these pigmented stripes.
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from the isofemale line design (David et al. 2005). Phenotypic plasticity, related to growth temperature was analyzed by considering the shape of the response curves, i.e. the reaction norms. We used the method of polynomial adjustments for analyzing the shapes and calculating characteristic values (David et al. 1997; Gibert et al. 2004).
Results Thoracic stripes The reaction norms of the width of the white stripe (WS) and of the black stripes (BS) are shown in Fig. 1, and the results of a 3 way ANOVA are in Table 1. A first conclusion is that the two traits vary significantly according to growth temperature, but in different ways. BS decreases almost linearly with temperature, and the temperature effect explains more than 33 % of the total variance. By contrast, temperature explains only 3 % of the variance of WS with no general linear trend (see Fig. 1). In both cases a significant genetic heterogeneity between lines is observed, as well as a line-temperature interaction. The difference between WS and BS is also evidenced by considering amount of variation observed in calculating the coefficients of variations (CVs) between temperatures which are 1.5 and 1.3 for WS and 10.5 and 10.6 for BS, in female and male respectively. The genetic variability among lines was further investigated (Table 2) by calculating the intraclass coefficient of correlation (ICC), which is akin to isofemale line heritability (David et al. 2005) and the genetic coefficient of variation (CVg), also called evolvability (Houle 1992). For
Data analysis The total data set (6000 linear measures) was investigated with SYSTATÓ v.13.0 (SPSS Inc.). Basic statistics: mean values, ANOVA, coefficients of variations, correlations, were calculated, as well as genetic parameters estimated
Fig. 1 Variation of the width of thoracic pigmented bands according to growth temperature. Triangles: males; circles: females. Open symbols (below) white stripe; filled symbols (above) black pigmented area
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Table 1 Results of ANOVA applied to the various traits investigated Source
df
Pigmentation WS
Size
Shape
BS
WL
TL
TW
WL/TL
WL/TW
TL/TW
13.17***
75.08***
72.67***
22.76***
1.52***
4.83***
6.87***
3.68***
2.32***
11.61***
0.02
0.10
4.25***
2.37***
6.87***
0.90
0.16
0.07
0.42
2.64 60.45
0.74 14.42
1.06* 16.58
2.60 48.54
Temperature
5
3.30***
33.65***
78.44***
34.59***
Sex
1
0.26*
22.12***
3.57***
2.90***
Line
9
14.05***
4.84***
0.35**
6.58***
1.00***
0.01
3.05***
3.03***
Sex 9 temp
5
Line 9 temp
45
Line 9 sex Line 9 sex 9 temp Error
0.40 11.78***
9
0.85
0.45
0.16
45 1080
2.08 67.29
1.23 33.69
0.96** 13.38
0.90** 10.84*** 0.76*** 1.48 41.64
0.27 10.16*** 0.70 11.72***
0.33
WS white stripe, BS black stripe, WL wing length, TL thorax length, TW thorax width; shape indices are presented as ratios. The table shows the percentage of the total variance explained by each source of variation *** P \ 0.001; ** P \ 0.01; * P \ 0.05
the intraclass correlations, there was no significant effect due to sex or temperature, and the average values are WS = 0.224 ± 0.047 (n = 12) and BS = 0.164 ± 0.036 (n = 12). These two values are not statistically different but both are significantly [0, confirming the significant genetic variability among lines (see also Table 2). Evolvability (CVg) was on average 6.61 ± 0.25 for WS and 10.95 ± 0.44 for BS: this difference is highly significant. The correlations between WS and BS were calculated at the within line, individual level and the between line, genetic level. Average values (not shown) were both negative but not significantly different from zero. This result confirms the developmental independence between the two kinds of pigmented stripes The correlations between pigmentation and size characters or shape indices were calculated, within each temperature, at the line level (Table 3). A clear conclusion is that WS was not correlated with size, while a slight but significant correlation was found for BS (Overall r = 0.29 ± 0.06, n = 6). This correlation was especially high with TW (average for both sexes: r = 0.44). In other words, size variations, and especially the width of the thorax, affect the width of BS (Fig. 2). With the shape indices, the most interesting result is a consistent negative correlation with BS and especially with the elongation index (TL/TW; average for both sexes: r = -0.28): a more elongated thorax is accompanied by narrower black stripes. Finally our data permit a comparison between sexes by considering two statistics, first the between sexes (F 9 M) correlation and second the sexual dimorphism as a female/male (F/M) ratio (Table 2). For each line the average values of female and male were always positively correlated with no temperature effect: the overall correlations are significantly different, i.e. 0.76 and 0.66 for WS and BS respectively.
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The F/M ratio revealed a very interesting difference. For WS, the ratio was always very close to one, meaning that both sexes were practically identical. This result may be compared with the very low dimorphism of body size, (see below). A contrasting result was obtained for BS with, firstly, an average value (1.13) much [1 and, second, a significant increase with temperature (Fig. 3). Size characters The data of each trait were submitted to a 3-way ANOVA, also shown in Table 1 as the percent of the total variance explained by each factor. WL is the most sensitive trait to temperature, and TW the least. Sex has a significant effect on WL and TL (smaller male) but not for TW. The heterogeneity among lines (genetic effects) is most important for TL and TW, and much less for WL, although, in all cases, the effects are highly significant. A significant interaction is also observed in the 3 traits between lines and temperatures, meaning the shapes of the reaction norms are variable among lines. The genetic variability was further analyzed by calculating the coefficients of intraclass correlation (ICC) for each temperature and trait, and average values are given in Table 4. There was no significant effect of temperature, sex or trait (ANOVA, not shown) and the overall average value is 0.26 ± 0.04, slightly greater than for the pigmented stripes, and in close agreement with previous data on an Indian population (Karan et al. 1999). Evolvability (CVg) was comprised between 0.95 and 1.62, being less for WL and maximum for TW and also greater in males than in females. The F 9 M correlation was variable among traits, least for WL (0.61) and greatest for TL (0.87). The F/M ratio was always close to 1, but different among traits. Values of 1.02 and 1.01 for wing and thorax length
WS white stripe, BS black stripe, F female, M male
1.002 ± 0.009 1.111 ± 0.007
WS
BS
Sexual dimorphism (F/M ratio)
0.95 0.85
12.50
M
WS
11.99
F
7.70 8.67
F
0.14*
M
M
0.09 ns
F
BS
F 9 M correlation
BS
WS
Genetic coefficient of variation (CVg)
BS
0.43*** 0.59***
F M
Intraclass correlation coefficient (ICC) WS
15 °C
Temperature
Sex
Trait
1.137 ± 0.013
1.019 ± 0.007
0.76
0.79
11.73
11.75
5.83
6.20
0.32***
0.33***
0.14*
0.29***
17 °C
1.101 ± 0.011
1.009 ± 0.010
0.69
0.73
10.94
12.00
6.36
7.21
0.08
0.20**
0.27***
0.33***
21 °C
1.134 ± 0.014
1.014 ± 0.008
0.54
0.63
10.76
12.09
5.94
6.36
0.06
0.23***
0.18**
0.09
25 °C
1.148 ± 0.011
1.007 ± 0.007
0.61
0.63
8.66
11.65
5.73
6.20
0.02
-0.04
0.08
0.12*
28 °C
Table 2 Genetic variability, sexual dimorphism and female 9 male correlation for the two pigmentation traits at various growth temperatures
1.241 ± 0.027
0.993 ± 0.005
0.54
0.82
7.66
9.69
6.44
6.72
0.31***
0.23**
0.10*
0.08
31 °C
1.126 ± 0.007
1.007 ± 0.004
0.66 ± 0.05
0.76 ± 0.05
10.38 ± 0.76
11.53 ± 0.37
6.50 ± 0.45
6.73 ± 0.25
0.15 ± 0.05
0.17 ± 0.05
0.23 ± 0.08
0.22 ± 0.06
Mean ± SE
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respectively confirm previous results (David et al. 2006b). Interestingly, TW was on average identical in both sexes. The reaction norms of the 3 size traits as a function of growth temperature are shown in Fig. 4. In all cases they are not linear, exhibit a maximum in within the thermal range and can be described by a quadratic polynomial. With such adjustments, we calculated characteristic values, i.e. the coordinates of the maximum and a curvature (g2) parameter. Average values are given in Table 5. As expected maximum values are very different between traits; a significant difference between sexes is found only for WL. The position of the temperature of maximum value (TMV) is significantly variable among traits, with the lowest value for the wing (both sexes average 17.3 °C) and the highest for thorax width (22.6°). Curvature values are different but this is explained by the variations in means of the traits. Shape indices The results of a 3-way ANOVA are shown in Table 1. For all 3 indices, significant effects of temperature, sex and line, as well as the temperature-line interaction are significant. The thorax elongation index (TL/TW) is the least sensitive, with a greater error term. The genetic variability was further analyzed by considering the intraclass correlation and evolvability (Table 6). The overall intraclass correlation (0.31) was slightly greater than for the traits themselves. Evolvability was more stable and not influenced by sex or temperature. Overall average value (1.26) is slightly [1 and is very close to that found for the traits themselves (1.17). The reaction norms of the 3 shape indices are given in Fig. 5. In all cases, monotonically decreasing norms were observed, and linear adjustments are shown. Males have always a slightly lesser value than females, but the sexual Table 3 Genetic correlation between size traits and pigmentation
Trait
Fig. 2 Relationship between thorax width and stripe width: below, white stripe; above, black stripe. Triangles: males; circles: females. Data from all temperature are pooled. Note the lack of correlation for the white stripe but the positive correlation for the black pigmentation
dimorphism is small i.e. 1.009, 1.022 and 1.013 for WL/ TL, WL/TW and TL/TW respectively. The sensitivity to temperature may be considered by estimating the amount of variation observed between 15 and 31 °C, scaled to the mean of each trait. The amount of variation is 8.5, 11.2 and 2.8 % for WL/TL, WL/TW and TL/TW respectively. The elongation index, TL/TW, is much less sensitive to growth temperature than the two others.
Discussion The present work analyzed the thermal phenotypic plasticity of five different metric traits in a cosmopolitan tropical drosophilid. Three of the traits (thorax width and widths of the longitudinal stripes) are investigated for the
WS
BS
F
M
F
M
WL
0.07 ± 0.18
-0.12 ± 0.15
0.12 ± 0.12
0.29 ± 0.10
TL
0.05 ± 0.20
-0.23 ± 0.13
0.11 ± 0.13
0.31 ± 0.07
TW
-0.05 ± 0.20
-0.27 ± 0.16
0.39 ± 0.11
0.49 ± 0.06
Size trait mean
-0.09 ± 0.06 (n = 6)
0.29 ± 0.06 (n = 6)
WL/TL
0.05 ± 0.11
0.15 ± 0.09
-0.06 ± 0.13
-0.10 ± 0.15
WL/TW
0.07 ± 0.09
-0.18 ± 0.14
-0.29 ± 0.09
-0.36 ± 0.12
0.02 ± 0.10
0.07 ± 0.12
-0.25 ± 0.04
-0.30 ± 0.02
TL/TW Shape indices mean
0.03 ± 0.05 (n = 6)
-0.23 ± 0.05 (n = 6)
Each value is the mean of 6 values obtained at the 6 growth temperatures WS white stripe, BS black stripe, WL wing length, TL thorax length, TW thorax width; shape indices are presented as ratios. F female, M male
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447
Fig. 3 Variation of sexual dimorphism (female/male ratio) of black stripes according to growth temperature in the 10 isofemale lines investigated. In spite of a broad irregularity, a significant increasing trend is observed
first time. The results allowed a broad diversity of analyses and comparisons among traits and temperatures. We discuss here the most interesting results. The thermal developmental range of Zaprionus indianus Our results clearly show that Z. indianus can be reared between 15 and 31 °C, while the thermal range of Drosophila melanogaster is 12–31 °C (Pe´tavy et al. 2001; David et al. 2004a). The greater sensitivity to cold of Zaprionus, correlated with a male complete sterility at 15 °C (Araripe et al. 2004) is certainly a major feature explaining that, for the moment, the species is restricted to tropical and subtropical countries. The diversity of the shapes of the reaction norms Since we used 6 different growth temperatures, the data can be ordered along a thermal gradient and the response curves are the reaction norms. For WL, the reaction norm is mostly decreasing, with a maximum close to the lower developmental threshold. This is why many investigators consider that ‘‘body size’’ decreases with increasing Table 4 Genetic variability of size traits, sexual dimorphisms (F/M ratio) and females-male (F 9 M) correlation
temperature (Angilletta 2009; Shelomi 2012). Indeed this is sometimes called the temperature-size rule (Angilletta and Sears 2004). Our data on thorax length confirm what was already clear in D. melanogaster (Morin et al. 1999; David et al. 2006a) that the reaction norm shows a maximum value at a higher temperature than WL and thus a clear concave shape. The new data on TW still enforce this conclusion, since the TMV is still higher than for TL: the temperature size rule is clearly not valid for different size-related traits (David et al. 2006a). It was previously shown that different parts of the wing exhibited different reaction norms with very different thermal maxima (Moreto et al. 1998). An analogous situation is now found for the thorax. This illustrates the difficulty to define an overall size in any organism. As usual in taxonomy, shape variations are described by morphological indices or ratio (Ba¨chli et al. 2004). The 3 calculated indices all produced similar, almost linearly decreasing norms. The decreasing WL/TL has been observed in all drosophilids investigated so far, it is inversely proportional to wing loading and might be the direct target of natural selection responsible of body size latitudinal lines (Stalker 1980; Pe´tavy et al. 1997; Loeschcke et al. 1999). The WL/TW ratio has less obvious biological significance, but also exhibits a sharp decrease. The TL/TW describes the elongation of the thorax. In D. melanogaster, a study at a single temperature (25 °C) (Chakir et al. 2008) produced a value of 1.55. In Zaprionus, the same index is 1.72–1.75: in other words, the thorax is relatively much longer. It is interesting to note that this thorax shape index is only poorly affected by temperature variations, contrasting with the highly significant effect of temperature for an elongation index of the wing in this same species (Loh et al. 2008). The two pigmentation traits, which were the main focus of this paper, also produced linear norms, but with very different shapes. The black area exhibited a highly significant, almost linear decrease with temperature. Similar results are observed in both sexes, but pigmentation is much less in males, and SD fairly high, with a F/M ratio of 1.13, contrasting with the fact that both sexes have almost
WL
TL
TW
F
M
F
M
F
M
ICC
0.16 ± 0.02
0.26 ± 0.05
0.27 ± 0.05
0.42 ± 0.09
0.18 ± 0.03
0.29 ± 0.06
CVg
0.95 ± 0.09
1.18 ± 0.16
1.12 ± 0.16
1.45 ± 0.26
1.24 ± 0.07
1.62 ± 0.13
F/M
1.020 ± 0.001
1.011 ± 0.002
0.998 ± 0.002
F9M
0.608 ± 0.042
0.867 ± 0.008
0.714 ± 0.078
Each value is the mean of the 6 studied temperatures ICC coefficient of intraclass correlation, CVg coefficient of genetic variation, WL wing length, TL thorax length, TW thorax width
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Fig. 4 Reaction norms of the 3 size-related traits according to growth temperature. a–c Mean value after a quadratic adjustment; triangles: males; circles: females. d Reaction norms of female wing length for 10 isofemale lines
Table 5 Characteristic values of the norms of reactions for the 3 size-related traits measured on the thorax n
MV ± SE
12 ± SE
TMV ± SE
312.28 ± 0.67
-0.190 ± 0.017
16.92 ± 0.62
Traits
Sex
WL
F M
8
306.87 ± 0.93
-0.214 ± 0.017
17.62 ± 0.56
TL
F
9
139.20 ± 0.43
-0.067 ± 0.007
20.61 ± 0.35
M
9
138.59 ± 0.64
-0.089 ± 0.011
21.19 ± 0.37
TW
F
9
79.66 ± 0.30
-0.027 ± 0.004
22.34 ± 0.30
M
10
80.04 ± 0.40
-0.032 ± 0.008
22.79 ± 0.33
8
MV maximum value, 12 curvature, TMV temperature of maximum value. n number of lines used for the calculation of a quadratic adjustment; notice that this number is generally \10, some lines did not produce a valid adjustment, i.e. a maximum outside the thermal range. WL wing length, TL thorax length, TW thorax width, F female, M male
identical sizes. The black pigmentation corresponds to a deposit of black melanin in the cuticle and its synthesis is well known (Wittkopp et al. 2003). In D. melanogaster female, the plasticity of abdomen pigmentation is a most
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investigated trait and an almost linear decrease with increasing temperature is well documented (Gibert et al. 2004). Similar phenomenon is also observed in D. mediopunctata (Rocha et al. 2008). This is probably a general process, but the place where black melanin is deposited is extremely variable due to developmental regulation. For the white pigmented stripes, the reaction norm was an almost horizontal line, with no sexual dimorphism. This stripe has a very different origin, since it corresponds to a shape modification of the cellular trichomes on the thorax: a flattened non cylindrical shape results in the reflection of the light (Walt and Tobler 1978). Other drosophilids have shining, light reflecting area on the head (for example in the D. nasuta complex of the immigrans group of Drosophila or in the montium group of Sophophora). The role of these reflecting areas is not known but might be involved in sexual behavior. In Zaprionus, the female exhibits a special sexual rejection behavior which is a rapid, left right oscillation of the body. Possibly the longitudinal bands might help to visualize and interpret this signal. In the butterfly Bicyclus anynana, visual, mate recognition
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449
Table 6 Analysis of the shape indices expressed as ratios
WL/TL
WL/TW
TL/TW
F
M
F
M
F
M
ICC
0.36 ± 0.04
0.36 ± 0.04
0.24 ± 0.05
0.18 ± 0.04
0.37 ± 0.04
0.35 ± 0.05
CVg
1.08 ± 0.11
1.09 ± 0.13
1.25 ± 0.09
1.22 ± 0.11
1.21 ± 0.06
1.16 ± 0.10
F/M
1.009 ± 0.001
1.022 ± 0.002
1.013 ± 0.001
F9M
0.799 ± 0.044
0.635 ± 0.051
0.821 ± 0.025
Each value is the mean of the 6 studied temperatures WL wing length, TL thorax length, TW thorax width, ICC coefficient of intraclass correlation, CVg coefficient of genetic variation, F/M female–male ratio, F 9 M female-male correlation
signals are better known. These visual traits are almost non-plastic, while other pigmented areas are very plastic with respect to developmental temperature (San Martin et al. 2011). The magnitude of plasticity Different characters are more or less plastic and one way to compare the magnitude of plasticity, getting rid of the difference in mean values, is to calculate a between temperature coefficient of variation (David et al. 2004a; Gibert et al. 2004). With this method, the metric traits analyzed in this work may be classified as follow, in decreasing order from 1.7 to 1.2: TL [ WL = BS [ TW [ WS. For the 3 shape indices, a decrease is also observed, with the following order WL/TW [ WL/TL [ TL/TW. The various traits react differently to temperature not only by revealing different reaction norm shapes but also by the magnitude of their reactivity. This certainly corresponds to complex, but not known, developmental regulations. Genetic variability and evolvability As usual with a full-sib, isofemale line design (David et al. 2005), we found significant genetic differences among lines for all traits. This genetic variability may be quantified by calculating an intraclass correlation coefficient. However, due to the small number of investigated lines (10) this coefficient is not precise so that a significant variability between temperatures was never observed. More interesting results were however obtained by considering the genetic CV, which is the between line standard deviation divided by the mean. This CVg reveals the capacity for a trait to evolve and has been called evolvability (Houle 1992). An interesting result, which has been found for the 3 linear dimensions as well as the 3 ratios, is that evolvability changed with temperature (see Table 1). The reaction norms showed a lesser value in the middle of the thermal range and greater values at extreme temperatures. This is a
strong argument in favor of the hypothesis that extreme environments might increase the evolving capacity of a population by revealing a cryptic genetic variation (Hoffmann and Parsons 1997; Gibson and Dworkin 2004). A similar observation was also done in D. melanogaster (David et al. 2006c) and D. ananassae (Sisodia and Singh 2009). The two pigmentation traits did not provide such a clear pattern, since genetic CVs were higher at low temperature and then decreased, with only a slight increase at high temperature. Sexual dimorphism (SD) Zaprionus indianus is a species in which males and females are very similar in their external phenotype. Sexual dimorphism (SD), expressed as a female/male ratio, is 1.02 for wing length and 1.01 for thorax length (David et al. 2006c). We found here that the difference practically disappeared when TW was considered. Also SD in Zaprionus is not affected by developmental temperature. These results are very different from those on D. melanogaster, where the female is on average 15 % bigger than the male when either wing or thorax length are considered (Chakir et al. 2008), and where SD increases according to growth temperature from 1.12 up to 1.17 between 12 and 31 °C (David et al. 2011). When the shape indices are considered in Zaprionus, a significant sex difference was always observed, with lesser values in male (Fig. 5). The main difference concerns the elongation index (TL/TW) for which SD is consistently[1, meaning that the thorax is more rounded in male than in female; a similar observation has been made in D. melonogaster (Chakir et al. 2008). Shape variation between female and male are however not a rule. For example, in Zaprionus, the wing shape (expressed as a width/length ratio) show a clear increase with developmental temperature but remains the same in both sexes (Loh et al. 2008). For the pigmentation traits, we got again contrasting results. For the white stripe width, males and females are
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much greater value for BS. We may also recall that, for the abdominal bristle number, SD was found to be very variable between successive segments, with averages ranging from 1.0 to 0.5 (Araripe et al. 2004). All these results suggest that all quantitative traits are affected by sex, but that, according to the trait, the SD ratio may be extremely variable, revealing a diversity of genetic mechanism, in sex interactions. In conclusion, investigating thermal phenotypic plasticity appears a powerful means for discriminating the developmental bases of various quantitative traits. Our present state of knowledge suggests that, in an evolutionary, developmental and phylogenetic (Evo-Devo) perspective, many more species should be investigated and compared; among them, Z. indianus might become a reference model. Acknowledgments We thank Amir Yassin for thoughtful comments on early drafts. This work was supported by Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de Nı´vel Superior (CAPES) and Comite Franc¸ais d’´Evaluation de la Coope´ation Universitaire avec le Bre´sil (COFECUB). This paper results from a French–Brazilian cooperation programme on Zaprionus.
References
Fig. 5 Reaction norms of shape ratios according to growth temperature. WL/TL: wing to thorax length; WL/TW: wing length to thorax width; TL/TW: thorax length to thorax width. Linear regressions are shown. Triangles: males; circles: females. Note the 3 ratios have very different values. Sexual dimorphism is more pronounced for TL/TW
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