Genetica DOI 10.1007/s10709-015-9845-2
Interspecific hybridization does not affect the level of fluctuating asymmetry (FA) in the Drosophila bipectinata species complex Parul Banerjee1 • B. N. Singh1
Received: 17 December 2014 / Accepted: 13 May 2015 Ó Springer International Publishing Switzerland 2015
Abstract The Drosophila bipectinata species complex comprises of four very closely related species namely D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae. It was found that irrespective of the evolutionary divergence among the species, FA which is reflective of the developmental precision remains nearly same in four species. During the present study, the level of FA in different morphological traits was studied in interspecific hybrids and compared with that of parental species with the view that it would throw light on the degree of divergence between the parental species. If they have not diverged much, the interspecific hybrids may have a similar FA level, incompatibilities between their genomes being negligible. On the other hand, if there is substantial divergence, the level of FA may be higher due to incompatibility between the genomes of the parental species. The morphological traits taken were sternopleural bristle number and wing length in both males and females and ovariole number and sex-comb tooth number in females and males respectively. However, except in a few cases, we could not detect any significant differences in the level of FA in hybrids as compared to pure species. On the other hand, a number of abnormalities like poor viability, dystrophied ovaries, asymmetrical eyes etc., could be detected in hybrids from crosses involving D. pseudoananassae as one of the parents. Therefore, we conclude that specific developmental
Electronic supplementary material The online version of this article (doi:10.1007/s10709-015-9845-2) contains supplementary material, which is available to authorized users. & B. N. Singh [email protected]; [email protected] 1
Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India
pathways are more susceptible to developmental disturbances due to genomic incompatibilities than the large complex system bringing about developmental stability. Keywords Drosophila bipectinata species complex Fluctuating asymmetry Developmental stability Interspecific hybrids
Introduction The Drosophila bipectinata species complex comprises of four very closely related species namely D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae. This complex forms a part of the ananassae subgroup of the widely diverged melanogaster species group (Bock and Wheeler 1972). The members of this complex occur in the Oriental-Australian biogeographic zone and are sympatric over parts of their range of distribution (Okada 1979; Kopp and Barmina 2005; Matsuda et al. 2005). Phylogenetic relationship among them has been more or less uncovered through studies on polytene chromosomes, sexual isolation, isozymes, morphology of testes and seminal vesicles, divergence at certain nuclear and mitochondrial loci etc. (Bock 1971a, b, 1978; Jha and Rahman 1972; Yang et al. 1972; Tomimura et al. 2005; Kopp and Barmina 2005; Mishra and Singh 2006a; Kopp et al. 2006; Banerjee and Singh 2012) and it has been pointed out almost to the point of certainty that D. bipectinata, D. parabipectinata and D. malerkotliana are more closely related to each other than any one of them is, to D. pseudoananassae. Further, the level of Fluctuating Asymmetry (FA) i.e. subtle deviations from perfect bilateral symmetry in certain morphological traits was found to be similar in all the four species by us (Banerjee and Singh 2015a). Fluctuating
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asymmetry reflects the capacity of an organism to buffer the random noise generated during development (Ludwig 1932; Van Valen 1962). In every species, there is a system in place to cater to this and in the bipectinata complex we found that in terms of efficiency, the system is almost equal in all the four species. Thus, irrespective of their evolutionary divergence, developmental stability is similar in the members of the complex. In addition, the common attributes were that the level of FA varied in a trait and sex specific manner. Sexual traits were found to exhibit greater FA than the non sexual traits and males were more affected due to FA than the females in all the four species. Therefore, it could be safely said that not only is the efficiency of the stability system similar but the structure is also alike in the four species. However, they must have acquired distinct species status owing to substantial divergence that led to absolute reproductive isolation. Both premating and post mating isolating mechanisms have contributed to this and though sexual or premating isolation is not complete in any one species pair, post mating isolation in form of hybrid male sterility has nonetheless sated reproductive isolation (Bock 1978; Mishra and Singh 2006b, 2007; Banerjee and Singh 2012). The changes in the genes involved in mate recognition (in females), courtship rituals (in males) and hybrid male sterility have nevertheless not been affected in isolation and the divergence has been genome wide. This is evident in the amount of morphological differences found in the four species, as although the females cannot be told apart, the males are quite distinct with respect to abdominal tip pigmentation and arrangement of sex comb. Indeed, earlier attempts to derive a phylogenetic relationship among the members of this complex using isozymes, certain nuclear and mitochondrial genetic loci, even the degree of sexual isolation and certain aspects of remating behavior (Yang et al. 1972; Kopp and Barmina 2005; Banerjee and Singh 2012; Singh and Singh 2013, 2014a, b) have given very clear pictures regarding the relationship shared by the four species. Therefore, though our attempt to divide the four with respect to differences in FA has been futile (Banerjee and Singh 2015a), we decided to take our study on FA in this complex further and investigate FA in the interspecific hybrids. The rationale behind this was that, comparing the levels of FA in interspecific hybrids and their parents may throw light on the degree of divergence between the parental species. If they have not diverged much, the interspecific hybrids may have a similar FA level, incompatibilities between their genomes being negligible. In fact, hybrids may also show a lower level of FA, being more stable, in the sense that the level of heterozygosity is bound to be greater in them. It is known that heterozygosity promotes developmental stability (Lerner 1954; Soule 1979; Santos et al. 2005). On the other hand, if there is considerable divergence, the level of FA
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may be higher due to incompatibilities between the genomes of the parental species that may cause a greater developmental noise and also impinge on the processes bringing about developmental stability. There must be numerous genes involved in causing developmental stability, forming a complex circuitry and they could have diverged among the four species, without actually compromising on their effectiveness at buffering random developmental noise. However, such diverged systems when brought together in a hybrid may fail to function in a coordinated fashion thus reflected in enhanced FA. Such a possibility prompted us to undertake this study. We expected to come across newer insights regarding the relationship shared by each species pair revealed in differential levels of FA in their hybrids.
Materials and methods Drosophila stocks One strain of each of the four species was used in the present study. These strains are: D. bipectinata-PN 99 (mass culture established from flies collected from Pune, Maharashtra, India in the year 1999), D. parabipectinataMys (mass culture established from flies collected from Mysore, Karnataka, India, in the year 1988), D. malerkotliana-RC 91 (mass culture established from flies collected from Raichur, Karnataka, India in the year 1991) and D. pseudoananassae-KB284 (line established from a single female collected from Kuala Belalong Temburong District, Brunei, in the year 2003). Thus, the strains of D. bipectinata, D. parabipectinata and D. malerkotliana have all been established from flies collected from India, whereas, the D. pseudoananassae line was kindly provided by Prof. Artyom Kopp, University of California, Davis, USA. They have spent a number of generations in the laboratory and are being maintained on simple yeast-agar medium at approximately 24 °C following 12 h cycle of light and darkness. About 25 pairs are used as founders for the next generation. Hybridization regime For obtaining hybrids, reciprocal crosses were set up in food vials, using 7 days aged virgin flies as parents (20 pairs per vial). The parents were transferred to fresh food vials after 5 days. Thus, two replicates of each cross were made. When progeny emerged, males and females were sorted and kept in separate vials. They were aged for a few days (4–7 days) before scoring of the different morphological traits.
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Trait measurements Two non sexual and two sexual morphological traits were taken. These are sternopleural bristle number (SBN) and wing length (WL) in both males and females and ovariole number (ON) and sex comb tooth number (SCTN) in females and males respectively. The traits were scored on both right and left sides of hybrid progeny. Sternopleural bristles are found on sternopleuron of both males and females. They occur in two sets: the anterior bristles, which form an oblique row from the forecoxa towards the midline and the transverse bristles, which run in a thin line towards the centre of the fly, just anterior to the coxa of the middle leg. The anterior and transverse bristles were counted under a stereobinocular at 259 magnification and the total number of bristles on one side was taken as the sum of the anterior and transverse bristles. For measuring wing length, the wings of etherized flies were dissected out and mounted in insect’s saline (0.67 %). The distance between the anterior crossvein and the tip of the third longitudinal vein was taken as wing length. Measurements were done under compound microscope, at 109 magnification with the help of ocular micrometer (1 unit = 15 lm). For counting ovarioles, the ovaries of etherized hybrid females were dissected out in insect’s saline and subjected to 2 % aceto carmine stain for about 2 min. Thereafter, they were washed and mounted in 45 % acetic acid. Ovariole number was counted under a compound microscope at 109 magnification. Sex combs are found as stout bristles on the first two tarsal segments of the first leg of males. The first leg of etherized males were dissected out and mounted in insect’s saline. The number of teeth was counted under a compound microscope at 459 magnification. Total number of teeth was taken as the sum of the number of teeth on the first (C1 ? C2) and second C3 tarsal segments. Thus, apart from a few crosses in which getting progeny turned out to be very difficult, 50 sons and 50 daughters from each cross were scored. Testing normality of R–L distributions and estimation of measurement error Depending upon the mean and distribution of the differences in the right and left sides (R–L), of a trait in a population, departures from perfect bilateral symmetry have been given different names. They are Directional asymmetry (DA), Antisymmetry (AS) and Fluctuating asymmetry (FA) (Van Valen 1962). In directionally asymmetrical traits, there is a greater development of the trait on one side of the plane of symmetry and vice versa. Therefore, the mean of (R–L), in a population is not zero. However, the distribution around mean is normal (bell shaped). In traits showing antisymmetry, all individuals in
a population exhibit a greater development on one side of the plane of symmetry but which side it is, is not predetermined and is either right or left at random. Since half the individuals in a population have a larger right side and half have larger left side of the trait, the distribution is bimodal around a mean of zero. Bilateral traits in which mean of (R–L) is zero and the distribution is normal about the mean, exhibit FA i.e., there are subtle differences between the right and left sides in a population (Ludwig 1932; Van Valen 1962; Palmer and Strobeck 1986, 1992). While DA and AS have some underlying genetic basis i.e. some genes are directly responsible for causing asymmetry and are frequently adaptive, FA is purely due to developmental noise, and hence is best suited for gauging the level of developmental instability existing in a population. Sometimes, DA has also been found to differ between parents and hybrids and those exhibiting only FA in parents may liquidate for DA in the hybrids and this shift from FA to DA has been implicated to stress (hybridization may be one) in a population (Klingenberg et al. 1998; Schneider et al. 2003). However, this is not very often and it is better to just compare FA among parents and hybrids. Therefore, in such type of studies, appropriate tests need to be done to prove that the traits considered, exhibit FA and other forms of asymmetry are absent. In our previous study (Banerjee and Singh 2015a), undertaken for comparing the level of FA in certain morphological traits in the four species of the complex, the distribution of signed R–L exhibited normality. In addition, we found through one sample t-tests that the mean values of (R–L) did not deviate significantly from zero. Also, the skewness and kurtosis values did not depict any significant departure from normality. Therefore, it was proven that the traits considered only shows FA and other forms of asymmetries such as directional asymmetry or antisymmetry are absent. Further, for gauging measurement error (ME), a set of thirty individuals were measured twice in each group and mixed model 2-way ANOVA was applied on the replicates. The interaction term (side x individual) was significant in each case indicating that the difference in right and left side was more than difference between the two replicates. Therefore, we were observing true FA which was not an artifact of ME. In this study, data of the parental species was used for comparison and measurements of morphometric traits in hybrids was done by the same individual (PB). Therefore, for hybrids, repeat measurements were not made, considering that measurement error would be negligible. Trait size and trait FA For an individual, the trait size of a given trait was taken as the average of the values in the right and left sides
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(R ? L/2). Variances in trait size of single traits were compared among hybrids and respective parental species by using one-way ANOVA, followed by Bonferroni t tests, if significant difference occurred. Different indices have been used to describe the levels of FA in a sample (Palmer and Strobeck 2003), out of which, FA1 was used in the present study. FA1 is the absolute value of the difference in the trait size between the right and the left sides of the body. The variances in FA values of single traits were compared among hybrids and respective parental species through one-way ANOVA, followed by Bonferroni t tests. For getting a single FA value for all the traits in an individual, composite fluctuating asymmetry (CFA) was calculated. CFA is the summation of absolute FA values across traits in an individual. It was compared through one-way ANOVA among hybrids and respective parental species. Also mean CFA values of hybrids (both males and females) from all 12 possible crosses were compared through one-way ANOVA.
Results It was very difficult to get hybrids in crosses involving D. pseudoananassae, especially with D. pseudoananassae males. Very few progeny appeared in the crosses involving D. parabipectinata females and D. malerkotliana females with D. pseudoananassae males. Daughters from reciprocal crosses involving D. bipectinata 9 D. pseudoananassae were found to have dystrophied ovaries. Also, about 1–2 % of sons of D. pseudoaananassae females 9 D. bipectinata males and 50 % of sons of D. pseudoananassae females 9 D. parabipectinata males had asymmetrical eyes i.e. on one side eye was well formed while on the other, it exhibited various abnormalities. In some, on one side there was no eye (Singh and Banerjee 2015). The sons also had poor viability. In the later cross, there was also a marked sex ratio distortion. Therefore, it is clear that there is not only a strong sexual isolation, but also a robust post zygotic isolation in place. In crosses involving D. malerkotliana 9 D. pseudoananassae, though getting hybrids turned out to be as difficult, as in other crosses involving D. pseudoananassae, if not more, none of the sons were found to have the abnormal eye morphology. Whereas, most of the daughters were found to have dystrophied ovaries. Trait sizes of all the morphological traits varied significantly between hybrids and the respective parental species (Supplementary Tables 1–6). However, comparison of FA levels between hybrids and parents revealed FA levels to be different from parents in only few of the traits of some of the crosses (Tables 1, 2, 3, 4, 5 and 6). In hybrid daughters from reciprocal crosses involving D. bipectinata 9 D. pseudoananassae, the level of FA in ON
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differed significantly from parents (p \ 0.05; Table 1). Pair wise comparisons revealed the level of FA in ON to be statistically more in daughters of D. pseudoananassae females 9 D. bipectinata males than females of both the parental species, with the number of asymmetrical hybrid daughters being more than number of asymmetrical parental females (Fig. 1a). FA in SBN was found to be more in sons from reciprocal crosses involving D. parabipectinata 9 D. pseudoananassae than D. parabipectinata males (p \ 0.05; Table 2; Fig. 1b). ON FA in daughters of D. bipectinata females 9 D. parabipectinata males was found to be higher (p \ 0.05; Table 4; Fig. 1c) than females of both the parental species. ON FA was also found to be higher (p \ 0.05) in daughters of D. malerkotliana females 9 D. parabipectinata males than D. parabipectinata females (Table 6; Fig. 1d). In one case, hybrids exhibited lower FA as compared to parental species. Sons from reciprocal crosses involving D. bipectinata 9 D. malerkotliana were found to have lower WL FA than D. bipectinata males but the number of symmetrical individuals was comparable to D. malerkotliana males (p \ 0.05; Table 5; Fig. 1e). Sons of D. parabipectinata females 9 D. malerkotliana males were on the other hand found to have higher SCTN FA compared to the parental males with the number of asymmetrical individuals being slightly more than the same in the males of the parental species (p \ 0.05; Table 6; Fig. 1f). Supplementary Tables 7–12 compare CFA’s among hybrids and parents. Daughters of D. bipectinata 9 D. pseudoananassae were found to have higher CFA compared to females of the parental species (Fig. 2a). The daughters of D. parabipectinata 9 D. pseudoananassae were also found to have higher CFA as compared to the females of the parental species (Fig. 2b). Hybrid daughters of D. bipectinata 9 D. parabipectinata exhibited higher CFA than the females of the parental species (Fig. 2c). On the other hand, sons of D. bipectinata 9 D. parabipectinata were found to have lower CFA than males of the parental species (Fig. 2d). However, mean CFAs across hybrids (both males and females) from all twelve crosses were found to be similar (Fig. 3a, b).
Discussion Studies comparing FA levels between parental species and their hybrids have always helped in understanding the relationship between the species pair in question. Often two aspects are kept in mind while doing such type of studies. Firstly, if two species are not very widely diverged or two populations (of the same species) have diverged much but have yet not achieved the species status, then in their F1 hybrids developmental stability improves (FA is lowered),
Genetica Table 1 Analysis of variance for comparison of trait FA (R–L) among D.bipectinata, D. pseudoananassae and their hybrids
Sex–trait Female—SBN
Source of variation
SS
df
Total
153.355
199
2.295
3
0.765
151.060
196
0.771
61.955
199
0.455
3
Between groups Within groups Female—WL
Total Between groups Within groups
Female—ON
Total Between groups Within groups
Male—SBN
Total
Male—SCTN
196
0.314
199
–
26.935
3
8.978
276.940
196
1.413
73.619
167
–
0.894
3
0.298
Within groups
72.725
164
0.443
Total
73.708
167
4.163
3
1.388
69.546 198.286
164 167
0.424 –
10.735
3
3.578
187.551
164
1.144
Within groups Total Between groups Within groups
0.993
– 0.152
61.500
Between groups
F
–
303.875
Between groups Male—WL
MS
0.483
6.354*
0.672
– 3.272#
3.129*
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05 #
Table 2 Analysis of variance for comparison of trait FA (R–L) among D. parabipectinata, D. pseudoananassae and their hybrids
Significant difference in WL FA between the parental males
Sex—Trait Female—SBN
Source of variation Total
183 3
Within groups
63.245
180
Total
61.995
183
0.895
3
Within groups Total Between groups Within groups Male—SBN
Total Between groups
Male—WL
– 0.298
61.100
180
0.339
253.777
183
–
10.475
3
3.492
243.302
180
1.352
91.776
195
–
4.836
3
1.612
192
0.453
Total
74.995
195
1.377
3
0.459
73.618 224.097
192 195
0.383 –
13.407
3
4.469
210.690
192
1.097
Between groups Within groups
0.118
0.351
86.939
Within groups Total
F
– 0.0416
Within groups Between groups Male—SCTN
MS
0.125
Between groups Female—ON
df
63.370
Between groups Female—WL
SS
0.878
2.583
3.560*
– 1.197
4.073#
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05 #
Significant difference in SCTN FA between the parental males
owing to hybrid vigour. There is also a possibility that increased heterozygosity in the hybrids will not affect FA and hence developmental stability. On the other hand,
going beyond the F1 generation to the F2 and even F3 generations in hybridization (possible in intraspecific hybridizations or when both the sexes of the interspecific
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Genetica Table 3 Analysis of variance for comparison of trait FA (R–L) among D. malerkotliana, D. pseudoananassae and their hybrids
Sex–Trait Female—SBN
Source of variation Total
SS
159
1.004
3
0.335
Within groups
58.740
156
0.377
Total
51.994
159
0.264
3
Between groups Within groups Female—ON
Total Within groups Total
Male—WL
156
0.332
159
–
9.335
3
3.112
229.640
156
1.472
74.589
157
–
0.714
3
0.238
Within groups
73.875
154
0.480
Total
59.190
157
1.135
3
0.378
58.055 174.715
154 157
0.377 –
2.320
3
0.773
172.395
154
1.119
Between groups Male—SCTN
Within groups Total Between groups Within groups
0.889
– 0.0846
51.740
Between groups
F
–
238.975
Between groups Male—SBN
MS
59.744
Between groups Female—WL
df
0.255
2.114
0.496
– 1.003
0.691
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number
Table 4 Analysis of variance for comparison of trait FA (R–L) among D. bipectinata, D. parabipectinata and their hybrids
Sex—Trait Female—SBN
Source of variation Total
Female—ON
199 3
0.220
Within groups
69.160
196
0.353
Total
65.955
199
–
Between groups Within groups
1.575 64.380
3 196
0.525 0.328
344.395
199
30.175
3
314.220
196
71.995
199
0.375
3
0.125
Within groups
71.620
196
0.365
Total
84.875
199
–
4.175
3
Total Within groups Total Between groups
Male—WL
Between groups Within groups Male—SCTN
MS
0.660
Between groups Male—SBN
df
69.820
Between groups Female—WL
SS
Total Between groups Within groups
F
– 0.623
1.598
– 10.058
6.274*
1.603 –
1.392
80.700
196
0.412
242.000
199
–
9.480
3
3.160
232.520
196
1.186
0.342
3.380#
2.664
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05 #
Significant difference in WL FA between the parental males
hybrids are fertile), one can unearth the effect of the breakdown of coadapted complexe(s) of genes. The meiotic products in the F1 hybrids (both the crossovers and the non crossovers), being almost at a par are equally
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represented in the F2 and F3, which is called breakdown of coadapted complex of genes. This often leads to decreased developmental stability (higher FA) in the F2 and F3 generations of hybrids. Therefore, improved stability in the
Genetica Table 5 Analysis of variance for comparison of trait FA (R–L) among D. bipectinata, D. malerkotliana and their hybrids
Sex—Trait Female—SBN
Source of variation Total
199 3
0.165
Within groups
65.460
196
0.334
Total
63.755
199
0.415
3
Within groups Total Between groups Within groups Male—SBN
Total Between groups
Male—WL
63.340
196
0.323
302.480
199
–
9.720
3
3.240
292.760
196
1.494
90.195
199
–
0.495
3
0.165
196
0.458
Total
83.595
199
4.015
3
1.338
79.580 196.480
196 199
0.406 –
5.520
3
1.840
190.960
196
0.974
Between groups Within groups
0.494
– 0.138
89.700
Within groups Total
F
–
Within groups Between groups Male—SCTN
MS
0.495
Between groups Female—ON
df
65.955
Between groups Female—WL
SS
0.428
2.169
0.361
– 3.296*
1.889
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05
Table 6 Analysis of variance for comparison of trait FA (R–L) among D. parabipectinata, D. malerkotliana and their hybrids
Sex—Trait Female—SBN
Source of variation
SS
df
Total
158.195
199
1.135
3
0.378
157.060 77.875
196 199
0.801 –
Between groups Female—WL
Within groups Total Between groups Within groups
Female—ON
Total Between groups Within groups
Male—SBN
Total Between groups
Male—WL
0.855
3
0.285
77.020
196
0.393
343.595
199
13.575
3
330.020
196
1.684
80.555
199
–
1.975
3
0.658
196
0.401
Total
65.155
199
–
Total Between groups Within groups
0.472
0.725
– 4.525
78.580
Within groups
F
–
Within groups Between groups Male—SCTN
MS
0.375
3
0.125
64.780
196
0.331
254.395
199
15.135
3
5.045
239.260
196
1.221
2.687*
1.642
0.378
– 4.133*
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.001
F1 but reduced stability in further generations indicates that the pair in question has evolved their own independent coadapted complex of genes and this indeed gives a very precise picture of the degree of their divergence.
Literature is replete with studies concerning FA in hybrids (and comparison with parents) and the results are a mixed bag (Jackson 1937a, b; Felly 1980; Zaharov 1981; Graham and Felly 1985; Leary et al. 1985; Ferguson 1986;
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Fig. 1 Number of symmetrical (FA = 0) and asymmetrical individuals (FA = 1, 2, 3, 4, 5 etc.) in pure species and their hybrids. a ovariole number (ON) in females, b sternopleural bristle number (SBN) in females, c ovariole number (ON) in females, d ovariole
number (ON) in females, e wing Length (WL) in males, f sex comb tooth number (SCTN) in males. bi: D. bipectinata, pa: D. parabipectinata, ma: D. malerkotliana, ps: D. pseudoananassae. Note: Species represented first is the female parent
Lamb and Avise 1987; Ross and Robertson 1990; lamb et al. 1990; Markow and Ricker 1991; Graham 1992; Blows and Sokolowaki 1995; Hutchinson and Cheverud 1995; Alibert et al. 1997; Andersen et al. 2002, 2006, 2008; Rego et al. 2006; Carreira et al. 2008; Vishalakshi and Singh 2009; Demontis et al. 2010; Trotta et al. 2010; Novicic et al. 2011). Alibert and Auffray (2003) said that 71 % published work show an increase in FA in hybrids (of different genera, or species) from parents. In some of the studies, natural hybrid populations (mostly insects, fish,
frogs and lizards) were examined. It has been postulated by Graham and Felly (1985) that the age of the hybrid zone determines whether hybrids will have a greater or lesser FA than the parental populations. If they have existed for a long while, coadapted gene complexes having had time to evolve, would rescue any dire effect on FA. If they have not, FA would be enhanced despite increased heterozygosity. However, in this case we are considering Drosophila species that never hybridize in nature and therefore must never have got the chance to evolve
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Genetica Fig. 2 Comparison of mean Composite Fluctuating Asymmetry (CFAs) among pure species and their hybrids. a D. bipectinta, D. pseudoananassae and their daughters, b D. parabipectinata, D. pseudoananassae and their daughters, c D. bipectinata, D. parabipectinata and their daughters, d D. bipectinata, D. parabipectinta and their sons. bi: D. bipectinata, pa: D. parabipectinata, ma: D. malerkotliana, ps: D. pseudoananassae. Note: Species represented first is the female parent
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Fig. 3 Comparison of mean Composite Fluctuating Asymmetry (CFA) among hybrids. a Daughters, b sons, of all the 12 crosses. bi: D. bipectinata, pa: D. parabipectinata, ma: D. malerkotliana, ps: D. pseudoananassae. Note: Species represented first is the female parent
coadapted complexes. In other cases, where closely related Drosophila species which do not hybridize in nature were involved; too the results have been conflicting. For example, Markow and Ricker (1991) found that only hybrid daughters but not sons of D. melanogaster 9 D. simulans exhibited enhanced FA. In addition, a number of phenodeviants could also be spotted among the hybrid daughters. Rego et al. (2006), who studied FA in wing size and shape in the species pair D. subobscura 9 D. madeirensis demonstrated that though FA did not appreciably increase in the hybrids, proper bilateral development was severely compromised in them. In the species pair D. buzzatii 9 D. kopeferae Carreira et al. (2008) found no significant
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increase in FA in hybrids as compared to the parental species. In our lab Vishalakshi and Singh (2009) studied hybrids of the sibling species pair D. ananassae and D. pallidosa and since F1 hybrids of both sexes are fully viable and fertile, they went beyond F1 to check FA in F2 and F3 generations. This aided in testing whether FA is affected due to breakdown of coadapted gene complexes (that must have evolved in each species separately) in the F2 and F3 generations due to free recombination in F1. However, they found no significant differences in the level of FA in F1, F2, or F3 generations compared to the parental species demonstrating that neither breakdown of coadapted complexes nor enhanced heterozygosity has any influence
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on developmental stability of hybrids. They went further in saying that the hybrids are as stable as their parents. Our study, however, is different from any of the above, as it involves four species sharing different phylogenetic relationships among themselves and which never hybridize in nature. To boot, the hybrid males are sterile. So neither is there any hybrid zone in nature, having their own coadapted complexes, nor is it a question of hybrids of a single pair (we have six pairs). Also, we had no scope of checking the effect of breakdown of coadapted complexes having no option but to stop at the F1 generation, hybrid sons being sterile. However, coadaptation per se is more the attribute of a species and through interracial (intraspecific) hybridization studies it is often checked whether different populations or races within a species have evolved their own coadapted complexes (Singh 1972; Andersen et al. 2002, 2008; Novicic et al. 2011). Among the members of this complex, D. bipectinata which is most widely distributed and genetically more variable has been used for investigations concerning coadaptation. In our laboratory, different geographic populations of D. bipectinata were hybridized up to the F2 and F3 generations and increased wing length in the F1 but reduced wing length in further generations indicated the occurrence of coadaptation (Banerjee and Singh 1998). Yet, when it is a question of two distinct species, studying coadaptation is not very relevant as it is almost certain that they have undergone independent coadaptation. Then, it becomes more important to study the level of genomic incompatibility which may well be reflected in the level of FA in the F1 interspecific hybrids. Thus, we hoped that the incongruity/congruity between the genomes of each pair would be reflected in the level of FA. Yet, unlike our expectations, the hybrids betrayed only a little effect on the level of FA if at all any. In the species pair D. bipectinata–D. parabipectinata, while the hybrid daughters exhibited higher CFA, hybrid sons exhibited lower CFA as compared to parents. Recently, we studied sexual isolation in the members of this complex and based on asymmetrical patterns of mating preference, it was proposed that D. parabipectinata is derived from D. bipectinata (Banerjee and Singh 2012). Therefore, being an ancestral-derived species pair they must be closer to each other than any other pair in this complex. Hybridization affects the level of FA in opposite directions in the two sexes, in the sons, increased heterozygosity may have lead to reduced CFA and on the other hand, in the daughters incompatibilities must have had a more pronounced effect than increased heterozygosity, leading to raised CFA (that was mainly due to increased FA in the ovariole number). The hybrids from species pair D. bipectinata–D. malerkotliana (derived from a common ancestor; Banerjee and Singh 2012), did not show much difference in the level of
FA in any traits except WL in males. In the pair D. parabipectinata–D. malerkotliana too level of FA in hybrids and parents was almost matching. In fortification of earlier findings, both in our lab and elsewhere (Bock 1978; Mishra and Singh 2006a; Banerjee and Singh 2014) that D. pseudoananassae is distantly related to the other three species of the complex, it was difficult to cross D. pseudoananassae with them, especially when D. pseudoananassae males were involved. Earlier, we postulated that the courtship rituals of D. pseudoananassae must have diverged to such an extent that the females of the other three species recognize it with difficulty. It may also be possible that the females of the other three species have evolved a hostile reproductive tract environment, killing sperm from the males of a distantly related species like D. pseudoananassae (Banerjee and Singh 2014). The progeny produced from such difficult crosses also exhibited several abnormalities. Most of the daughters from reciprocal crosses involving D. bipectinata 9 D. pseudoananassae and D. malerkotliana 9 D. pseudoananassae were found to have dystrophied ovaries. It has always been said that among the hybrids of this complex, the females are fertile and the males are sterile. Yet, females with dystrophied ovaries cannot be fertile which demonstrates that fertility of hybrid daughters is not hundred percent at least in some crosses. Therefore, we planned to investigate this further and check the fertility of hybrid daughters from all the crosses. We found that daughters having D. pseudoananassae as one of their parents had drastically reduced fertility compared to the parental females (Banerjee and Singh 2015b). In the crosses D. pseudoananassae females 9 D. bipectinata males and D. pseudoananassae females 9 D. parabipectinata males, the hybrid sons were found to have severely compromised eye symmetry and poor viability. While on one side the eyes were properly developed, the other side had gradation in abnormalities. In the reciprocal crosses however, the sons were normal. Since no daughters were found to be affected and sons of only one of the reciprocal crosses were affected, it is evident that ‘X’ chromosome is involved in causing abnormal development of eyes. The X chromosome of the maternal species, D. pseudoananassae (inherited by the sons in both the crosses), is perhaps incompatible with the autosomes of the paternal species D. bipectinata and D. parabipectinata, which is causing the left–right signaling during eye formation to go awry (Singh and Banerjee 2015). However, while abnormalities and asymmetries are quite evident, subtle asymmetries in hybrids are not very different from the parental species (CFA of hybrid daughters involving D. bipectinata 9 D. pseudoananassae and D. parabipectinata 9 D. pseudoananassae are only slightly greater than respective parental females). In addition, hybrid sons and
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daughters from all twelve crosses were found to have comparable CFAs. In conclusion, interspecific hybrid sons and daughters especially those involving D. pseudoananassae and the other three species of the complex show certain abnormalities like dystrophied ovaries and asymmetrical eyes, even poor viability. These clearly indicate that they are developmentally unstable. Yet, this fact is concealed when FA is compared across the hybrids and parents. Therefore the system taking care of correcting the subtle asymmetries arising due to developmental noise is not affected or affected little in the hybrids. Nevertheless, they are certainly not as stable as their parents, as specific developmental pathways like those involved in development of proper symmetrical eyes and normal ovaries do get affected due to hybridization. There is incompatibility between the genomes of D. pseudoananassae and the other three species and genes required in specific developmental pathways like the formation of eyes and ovary have diverged to such an extent that they cannot work in a coordinated fashion in the hybrid background. On the other hand, the system bringing about developmental stability (to buffer random noises of development), is very large involving a number of genes forming a complex circuitry, so even if there are minor incompatibilities, the system as a whole remains impervious and thus the effect is not very pronounced. Therefore, the system responsible for developmental stability is so robust and perhaps also conserved that irrespective of genomic incompatibilities or the level of heterozygosity, it carries out its function well, so well that the level of FA remains unaffected in the hybrids. Acknowledgments Financial assistance in the form of Meritorious Fellowship to PB and UGC-BSR Faculty Fellowship Award to BNS, from the University Grants Commission, New Delhi is gratefully acknowledged. We thank Prof M Matsuda, Japan and Prof. A. Kopp, USA for kindly providing the stocks of the Drosophila bipectinata species complex. We also thank the two anonymous reviewers for their helpful suggestions that helped to improve the manuscript. Conflict of interest of interest.
The authors declare that they have no conflict
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Rego C, Matos M, Santos M (2006) Symmetry breaking in interspecific Drosophila hybrids is not due to developmental noise. Evolution 60:746–761 Ross KG, Robertson JL (1990) Developmental stability, heterozygosity and fitness in two introduced fire ants (Solenopsis invicta and S. richteri) and their hybrids. Heredity 64:93–103 Santos M, Iriarte PF, Cespedes W (2005) Genetics and geometry of canalization and developmental stability in Drosophila subobscura. BMC Evol Biol 5:7 Schneider SS, Leamy LJ, Lewis LA, De Grandi-Hoffman G (2003) The influence of hybridization between African and European honeybees, Apis mellifera, on asymmetries in wing size and shape. Evolution 57:2350–2364 Singh BN (1972) The lack of evidence for coadaptation in geographic populations of Drosophila ananassae. Genetica 43:582–588 Singh BN, Banerjee P. (2015) Sex specific asymmetry in eye development in interspecific hybrids in the Drosophila bipectinata species complex. J Genet 94 (in press) Singh A, Singh BN (2013) Studies on remating behaviour in the Drosophila bipectinata species complex: intra- and interspecific variations. Behav Proc 96:79–87 Singh A, Singh BN (2014a) Studies on remating behaviour in the Drosophila bipectinata species complex. Evidence for sperm displacement in D. bipectinata. Curr Sci 107:511–515 Singh A, Singh BN (2014b) Sperm displacement in the Drosophila bipectinata species complex: evidence for interspecific variations. Behav Proc 113:105–109 Soule ME (1979) Heterozygosity and developmental stability: another look. Evolution 33:396–401 Tomimura Y, Matsuda M, Tobari YN (2005) Chromosomal phylogeny and geographical divergence in Drosophila bipectinata complex. Genome 48:487–502 Trotta V, Pertoldi C, Rudoy A, Manenti T, Cavicchi S, Guerra D (2010) Thermal plasticity of wing size and shape in Drosophila melanogaster, D. simulanas and their hybrids. Clim Res 43:71–79 Van Valen L (1962) A study of fluctuating asymmetry. Evolution 16:125–142 Vishalakshi C, Singh BN (2009) Fluctuating asymmetry is hybrids of sibling species, Drosophila ananassae and D. pallidosa is trait and sex specific. J Hered 100:181–191 Yang SY, Wheeler L, Bock IR (1972) Isozyme variations and phylogenetic relationships in Drosophila bipectinata species complex. University of Texas Publications 7213:213–227 Zaharov VM (1981) Fluctuating asymmetry as an index of developmental homeostasis. Genetica 13:241–256
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Interspecific hybridization does not affect the level of fluctuating asymmetry (FA) in the Drosophila bipectinata species complex Parul Banerjee1 • B. N. Singh1
Received: 17 December 2014 / Accepted: 13 May 2015 Ó Springer International Publishing Switzerland 2015
Abstract The Drosophila bipectinata species complex comprises of four very closely related species namely D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae. It was found that irrespective of the evolutionary divergence among the species, FA which is reflective of the developmental precision remains nearly same in four species. During the present study, the level of FA in different morphological traits was studied in interspecific hybrids and compared with that of parental species with the view that it would throw light on the degree of divergence between the parental species. If they have not diverged much, the interspecific hybrids may have a similar FA level, incompatibilities between their genomes being negligible. On the other hand, if there is substantial divergence, the level of FA may be higher due to incompatibility between the genomes of the parental species. The morphological traits taken were sternopleural bristle number and wing length in both males and females and ovariole number and sex-comb tooth number in females and males respectively. However, except in a few cases, we could not detect any significant differences in the level of FA in hybrids as compared to pure species. On the other hand, a number of abnormalities like poor viability, dystrophied ovaries, asymmetrical eyes etc., could be detected in hybrids from crosses involving D. pseudoananassae as one of the parents. Therefore, we conclude that specific developmental
Electronic supplementary material The online version of this article (doi:10.1007/s10709-015-9845-2) contains supplementary material, which is available to authorized users. & B. N. Singh [email protected]; [email protected] 1
Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005, India
pathways are more susceptible to developmental disturbances due to genomic incompatibilities than the large complex system bringing about developmental stability. Keywords Drosophila bipectinata species complex Fluctuating asymmetry Developmental stability Interspecific hybrids
Introduction The Drosophila bipectinata species complex comprises of four very closely related species namely D. bipectinata, D. parabipectinata, D. malerkotliana and D. pseudoananassae. This complex forms a part of the ananassae subgroup of the widely diverged melanogaster species group (Bock and Wheeler 1972). The members of this complex occur in the Oriental-Australian biogeographic zone and are sympatric over parts of their range of distribution (Okada 1979; Kopp and Barmina 2005; Matsuda et al. 2005). Phylogenetic relationship among them has been more or less uncovered through studies on polytene chromosomes, sexual isolation, isozymes, morphology of testes and seminal vesicles, divergence at certain nuclear and mitochondrial loci etc. (Bock 1971a, b, 1978; Jha and Rahman 1972; Yang et al. 1972; Tomimura et al. 2005; Kopp and Barmina 2005; Mishra and Singh 2006a; Kopp et al. 2006; Banerjee and Singh 2012) and it has been pointed out almost to the point of certainty that D. bipectinata, D. parabipectinata and D. malerkotliana are more closely related to each other than any one of them is, to D. pseudoananassae. Further, the level of Fluctuating Asymmetry (FA) i.e. subtle deviations from perfect bilateral symmetry in certain morphological traits was found to be similar in all the four species by us (Banerjee and Singh 2015a). Fluctuating
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asymmetry reflects the capacity of an organism to buffer the random noise generated during development (Ludwig 1932; Van Valen 1962). In every species, there is a system in place to cater to this and in the bipectinata complex we found that in terms of efficiency, the system is almost equal in all the four species. Thus, irrespective of their evolutionary divergence, developmental stability is similar in the members of the complex. In addition, the common attributes were that the level of FA varied in a trait and sex specific manner. Sexual traits were found to exhibit greater FA than the non sexual traits and males were more affected due to FA than the females in all the four species. Therefore, it could be safely said that not only is the efficiency of the stability system similar but the structure is also alike in the four species. However, they must have acquired distinct species status owing to substantial divergence that led to absolute reproductive isolation. Both premating and post mating isolating mechanisms have contributed to this and though sexual or premating isolation is not complete in any one species pair, post mating isolation in form of hybrid male sterility has nonetheless sated reproductive isolation (Bock 1978; Mishra and Singh 2006b, 2007; Banerjee and Singh 2012). The changes in the genes involved in mate recognition (in females), courtship rituals (in males) and hybrid male sterility have nevertheless not been affected in isolation and the divergence has been genome wide. This is evident in the amount of morphological differences found in the four species, as although the females cannot be told apart, the males are quite distinct with respect to abdominal tip pigmentation and arrangement of sex comb. Indeed, earlier attempts to derive a phylogenetic relationship among the members of this complex using isozymes, certain nuclear and mitochondrial genetic loci, even the degree of sexual isolation and certain aspects of remating behavior (Yang et al. 1972; Kopp and Barmina 2005; Banerjee and Singh 2012; Singh and Singh 2013, 2014a, b) have given very clear pictures regarding the relationship shared by the four species. Therefore, though our attempt to divide the four with respect to differences in FA has been futile (Banerjee and Singh 2015a), we decided to take our study on FA in this complex further and investigate FA in the interspecific hybrids. The rationale behind this was that, comparing the levels of FA in interspecific hybrids and their parents may throw light on the degree of divergence between the parental species. If they have not diverged much, the interspecific hybrids may have a similar FA level, incompatibilities between their genomes being negligible. In fact, hybrids may also show a lower level of FA, being more stable, in the sense that the level of heterozygosity is bound to be greater in them. It is known that heterozygosity promotes developmental stability (Lerner 1954; Soule 1979; Santos et al. 2005). On the other hand, if there is considerable divergence, the level of FA
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may be higher due to incompatibilities between the genomes of the parental species that may cause a greater developmental noise and also impinge on the processes bringing about developmental stability. There must be numerous genes involved in causing developmental stability, forming a complex circuitry and they could have diverged among the four species, without actually compromising on their effectiveness at buffering random developmental noise. However, such diverged systems when brought together in a hybrid may fail to function in a coordinated fashion thus reflected in enhanced FA. Such a possibility prompted us to undertake this study. We expected to come across newer insights regarding the relationship shared by each species pair revealed in differential levels of FA in their hybrids.
Materials and methods Drosophila stocks One strain of each of the four species was used in the present study. These strains are: D. bipectinata-PN 99 (mass culture established from flies collected from Pune, Maharashtra, India in the year 1999), D. parabipectinataMys (mass culture established from flies collected from Mysore, Karnataka, India, in the year 1988), D. malerkotliana-RC 91 (mass culture established from flies collected from Raichur, Karnataka, India in the year 1991) and D. pseudoananassae-KB284 (line established from a single female collected from Kuala Belalong Temburong District, Brunei, in the year 2003). Thus, the strains of D. bipectinata, D. parabipectinata and D. malerkotliana have all been established from flies collected from India, whereas, the D. pseudoananassae line was kindly provided by Prof. Artyom Kopp, University of California, Davis, USA. They have spent a number of generations in the laboratory and are being maintained on simple yeast-agar medium at approximately 24 °C following 12 h cycle of light and darkness. About 25 pairs are used as founders for the next generation. Hybridization regime For obtaining hybrids, reciprocal crosses were set up in food vials, using 7 days aged virgin flies as parents (20 pairs per vial). The parents were transferred to fresh food vials after 5 days. Thus, two replicates of each cross were made. When progeny emerged, males and females were sorted and kept in separate vials. They were aged for a few days (4–7 days) before scoring of the different morphological traits.
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Trait measurements Two non sexual and two sexual morphological traits were taken. These are sternopleural bristle number (SBN) and wing length (WL) in both males and females and ovariole number (ON) and sex comb tooth number (SCTN) in females and males respectively. The traits were scored on both right and left sides of hybrid progeny. Sternopleural bristles are found on sternopleuron of both males and females. They occur in two sets: the anterior bristles, which form an oblique row from the forecoxa towards the midline and the transverse bristles, which run in a thin line towards the centre of the fly, just anterior to the coxa of the middle leg. The anterior and transverse bristles were counted under a stereobinocular at 259 magnification and the total number of bristles on one side was taken as the sum of the anterior and transverse bristles. For measuring wing length, the wings of etherized flies were dissected out and mounted in insect’s saline (0.67 %). The distance between the anterior crossvein and the tip of the third longitudinal vein was taken as wing length. Measurements were done under compound microscope, at 109 magnification with the help of ocular micrometer (1 unit = 15 lm). For counting ovarioles, the ovaries of etherized hybrid females were dissected out in insect’s saline and subjected to 2 % aceto carmine stain for about 2 min. Thereafter, they were washed and mounted in 45 % acetic acid. Ovariole number was counted under a compound microscope at 109 magnification. Sex combs are found as stout bristles on the first two tarsal segments of the first leg of males. The first leg of etherized males were dissected out and mounted in insect’s saline. The number of teeth was counted under a compound microscope at 459 magnification. Total number of teeth was taken as the sum of the number of teeth on the first (C1 ? C2) and second C3 tarsal segments. Thus, apart from a few crosses in which getting progeny turned out to be very difficult, 50 sons and 50 daughters from each cross were scored. Testing normality of R–L distributions and estimation of measurement error Depending upon the mean and distribution of the differences in the right and left sides (R–L), of a trait in a population, departures from perfect bilateral symmetry have been given different names. They are Directional asymmetry (DA), Antisymmetry (AS) and Fluctuating asymmetry (FA) (Van Valen 1962). In directionally asymmetrical traits, there is a greater development of the trait on one side of the plane of symmetry and vice versa. Therefore, the mean of (R–L), in a population is not zero. However, the distribution around mean is normal (bell shaped). In traits showing antisymmetry, all individuals in
a population exhibit a greater development on one side of the plane of symmetry but which side it is, is not predetermined and is either right or left at random. Since half the individuals in a population have a larger right side and half have larger left side of the trait, the distribution is bimodal around a mean of zero. Bilateral traits in which mean of (R–L) is zero and the distribution is normal about the mean, exhibit FA i.e., there are subtle differences between the right and left sides in a population (Ludwig 1932; Van Valen 1962; Palmer and Strobeck 1986, 1992). While DA and AS have some underlying genetic basis i.e. some genes are directly responsible for causing asymmetry and are frequently adaptive, FA is purely due to developmental noise, and hence is best suited for gauging the level of developmental instability existing in a population. Sometimes, DA has also been found to differ between parents and hybrids and those exhibiting only FA in parents may liquidate for DA in the hybrids and this shift from FA to DA has been implicated to stress (hybridization may be one) in a population (Klingenberg et al. 1998; Schneider et al. 2003). However, this is not very often and it is better to just compare FA among parents and hybrids. Therefore, in such type of studies, appropriate tests need to be done to prove that the traits considered, exhibit FA and other forms of asymmetry are absent. In our previous study (Banerjee and Singh 2015a), undertaken for comparing the level of FA in certain morphological traits in the four species of the complex, the distribution of signed R–L exhibited normality. In addition, we found through one sample t-tests that the mean values of (R–L) did not deviate significantly from zero. Also, the skewness and kurtosis values did not depict any significant departure from normality. Therefore, it was proven that the traits considered only shows FA and other forms of asymmetries such as directional asymmetry or antisymmetry are absent. Further, for gauging measurement error (ME), a set of thirty individuals were measured twice in each group and mixed model 2-way ANOVA was applied on the replicates. The interaction term (side x individual) was significant in each case indicating that the difference in right and left side was more than difference between the two replicates. Therefore, we were observing true FA which was not an artifact of ME. In this study, data of the parental species was used for comparison and measurements of morphometric traits in hybrids was done by the same individual (PB). Therefore, for hybrids, repeat measurements were not made, considering that measurement error would be negligible. Trait size and trait FA For an individual, the trait size of a given trait was taken as the average of the values in the right and left sides
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(R ? L/2). Variances in trait size of single traits were compared among hybrids and respective parental species by using one-way ANOVA, followed by Bonferroni t tests, if significant difference occurred. Different indices have been used to describe the levels of FA in a sample (Palmer and Strobeck 2003), out of which, FA1 was used in the present study. FA1 is the absolute value of the difference in the trait size between the right and the left sides of the body. The variances in FA values of single traits were compared among hybrids and respective parental species through one-way ANOVA, followed by Bonferroni t tests. For getting a single FA value for all the traits in an individual, composite fluctuating asymmetry (CFA) was calculated. CFA is the summation of absolute FA values across traits in an individual. It was compared through one-way ANOVA among hybrids and respective parental species. Also mean CFA values of hybrids (both males and females) from all 12 possible crosses were compared through one-way ANOVA.
Results It was very difficult to get hybrids in crosses involving D. pseudoananassae, especially with D. pseudoananassae males. Very few progeny appeared in the crosses involving D. parabipectinata females and D. malerkotliana females with D. pseudoananassae males. Daughters from reciprocal crosses involving D. bipectinata 9 D. pseudoananassae were found to have dystrophied ovaries. Also, about 1–2 % of sons of D. pseudoaananassae females 9 D. bipectinata males and 50 % of sons of D. pseudoananassae females 9 D. parabipectinata males had asymmetrical eyes i.e. on one side eye was well formed while on the other, it exhibited various abnormalities. In some, on one side there was no eye (Singh and Banerjee 2015). The sons also had poor viability. In the later cross, there was also a marked sex ratio distortion. Therefore, it is clear that there is not only a strong sexual isolation, but also a robust post zygotic isolation in place. In crosses involving D. malerkotliana 9 D. pseudoananassae, though getting hybrids turned out to be as difficult, as in other crosses involving D. pseudoananassae, if not more, none of the sons were found to have the abnormal eye morphology. Whereas, most of the daughters were found to have dystrophied ovaries. Trait sizes of all the morphological traits varied significantly between hybrids and the respective parental species (Supplementary Tables 1–6). However, comparison of FA levels between hybrids and parents revealed FA levels to be different from parents in only few of the traits of some of the crosses (Tables 1, 2, 3, 4, 5 and 6). In hybrid daughters from reciprocal crosses involving D. bipectinata 9 D. pseudoananassae, the level of FA in ON
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differed significantly from parents (p \ 0.05; Table 1). Pair wise comparisons revealed the level of FA in ON to be statistically more in daughters of D. pseudoananassae females 9 D. bipectinata males than females of both the parental species, with the number of asymmetrical hybrid daughters being more than number of asymmetrical parental females (Fig. 1a). FA in SBN was found to be more in sons from reciprocal crosses involving D. parabipectinata 9 D. pseudoananassae than D. parabipectinata males (p \ 0.05; Table 2; Fig. 1b). ON FA in daughters of D. bipectinata females 9 D. parabipectinata males was found to be higher (p \ 0.05; Table 4; Fig. 1c) than females of both the parental species. ON FA was also found to be higher (p \ 0.05) in daughters of D. malerkotliana females 9 D. parabipectinata males than D. parabipectinata females (Table 6; Fig. 1d). In one case, hybrids exhibited lower FA as compared to parental species. Sons from reciprocal crosses involving D. bipectinata 9 D. malerkotliana were found to have lower WL FA than D. bipectinata males but the number of symmetrical individuals was comparable to D. malerkotliana males (p \ 0.05; Table 5; Fig. 1e). Sons of D. parabipectinata females 9 D. malerkotliana males were on the other hand found to have higher SCTN FA compared to the parental males with the number of asymmetrical individuals being slightly more than the same in the males of the parental species (p \ 0.05; Table 6; Fig. 1f). Supplementary Tables 7–12 compare CFA’s among hybrids and parents. Daughters of D. bipectinata 9 D. pseudoananassae were found to have higher CFA compared to females of the parental species (Fig. 2a). The daughters of D. parabipectinata 9 D. pseudoananassae were also found to have higher CFA as compared to the females of the parental species (Fig. 2b). Hybrid daughters of D. bipectinata 9 D. parabipectinata exhibited higher CFA than the females of the parental species (Fig. 2c). On the other hand, sons of D. bipectinata 9 D. parabipectinata were found to have lower CFA than males of the parental species (Fig. 2d). However, mean CFAs across hybrids (both males and females) from all twelve crosses were found to be similar (Fig. 3a, b).
Discussion Studies comparing FA levels between parental species and their hybrids have always helped in understanding the relationship between the species pair in question. Often two aspects are kept in mind while doing such type of studies. Firstly, if two species are not very widely diverged or two populations (of the same species) have diverged much but have yet not achieved the species status, then in their F1 hybrids developmental stability improves (FA is lowered),
Genetica Table 1 Analysis of variance for comparison of trait FA (R–L) among D.bipectinata, D. pseudoananassae and their hybrids
Sex–trait Female—SBN
Source of variation
SS
df
Total
153.355
199
2.295
3
0.765
151.060
196
0.771
61.955
199
0.455
3
Between groups Within groups Female—WL
Total Between groups Within groups
Female—ON
Total Between groups Within groups
Male—SBN
Total
Male—SCTN
196
0.314
199
–
26.935
3
8.978
276.940
196
1.413
73.619
167
–
0.894
3
0.298
Within groups
72.725
164
0.443
Total
73.708
167
4.163
3
1.388
69.546 198.286
164 167
0.424 –
10.735
3
3.578
187.551
164
1.144
Within groups Total Between groups Within groups
0.993
– 0.152
61.500
Between groups
F
–
303.875
Between groups Male—WL
MS
0.483
6.354*
0.672
– 3.272#
3.129*
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05 #
Table 2 Analysis of variance for comparison of trait FA (R–L) among D. parabipectinata, D. pseudoananassae and their hybrids
Significant difference in WL FA between the parental males
Sex—Trait Female—SBN
Source of variation Total
183 3
Within groups
63.245
180
Total
61.995
183
0.895
3
Within groups Total Between groups Within groups Male—SBN
Total Between groups
Male—WL
– 0.298
61.100
180
0.339
253.777
183
–
10.475
3
3.492
243.302
180
1.352
91.776
195
–
4.836
3
1.612
192
0.453
Total
74.995
195
1.377
3
0.459
73.618 224.097
192 195
0.383 –
13.407
3
4.469
210.690
192
1.097
Between groups Within groups
0.118
0.351
86.939
Within groups Total
F
– 0.0416
Within groups Between groups Male—SCTN
MS
0.125
Between groups Female—ON
df
63.370
Between groups Female—WL
SS
0.878
2.583
3.560*
– 1.197
4.073#
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05 #
Significant difference in SCTN FA between the parental males
owing to hybrid vigour. There is also a possibility that increased heterozygosity in the hybrids will not affect FA and hence developmental stability. On the other hand,
going beyond the F1 generation to the F2 and even F3 generations in hybridization (possible in intraspecific hybridizations or when both the sexes of the interspecific
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Genetica Table 3 Analysis of variance for comparison of trait FA (R–L) among D. malerkotliana, D. pseudoananassae and their hybrids
Sex–Trait Female—SBN
Source of variation Total
SS
159
1.004
3
0.335
Within groups
58.740
156
0.377
Total
51.994
159
0.264
3
Between groups Within groups Female—ON
Total Within groups Total
Male—WL
156
0.332
159
–
9.335
3
3.112
229.640
156
1.472
74.589
157
–
0.714
3
0.238
Within groups
73.875
154
0.480
Total
59.190
157
1.135
3
0.378
58.055 174.715
154 157
0.377 –
2.320
3
0.773
172.395
154
1.119
Between groups Male—SCTN
Within groups Total Between groups Within groups
0.889
– 0.0846
51.740
Between groups
F
–
238.975
Between groups Male—SBN
MS
59.744
Between groups Female—WL
df
0.255
2.114
0.496
– 1.003
0.691
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number
Table 4 Analysis of variance for comparison of trait FA (R–L) among D. bipectinata, D. parabipectinata and their hybrids
Sex—Trait Female—SBN
Source of variation Total
Female—ON
199 3
0.220
Within groups
69.160
196
0.353
Total
65.955
199
–
Between groups Within groups
1.575 64.380
3 196
0.525 0.328
344.395
199
30.175
3
314.220
196
71.995
199
0.375
3
0.125
Within groups
71.620
196
0.365
Total
84.875
199
–
4.175
3
Total Within groups Total Between groups
Male—WL
Between groups Within groups Male—SCTN
MS
0.660
Between groups Male—SBN
df
69.820
Between groups Female—WL
SS
Total Between groups Within groups
F
– 0.623
1.598
– 10.058
6.274*
1.603 –
1.392
80.700
196
0.412
242.000
199
–
9.480
3
3.160
232.520
196
1.186
0.342
3.380#
2.664
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05 #
Significant difference in WL FA between the parental males
hybrids are fertile), one can unearth the effect of the breakdown of coadapted complexe(s) of genes. The meiotic products in the F1 hybrids (both the crossovers and the non crossovers), being almost at a par are equally
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represented in the F2 and F3, which is called breakdown of coadapted complex of genes. This often leads to decreased developmental stability (higher FA) in the F2 and F3 generations of hybrids. Therefore, improved stability in the
Genetica Table 5 Analysis of variance for comparison of trait FA (R–L) among D. bipectinata, D. malerkotliana and their hybrids
Sex—Trait Female—SBN
Source of variation Total
199 3
0.165
Within groups
65.460
196
0.334
Total
63.755
199
0.415
3
Within groups Total Between groups Within groups Male—SBN
Total Between groups
Male—WL
63.340
196
0.323
302.480
199
–
9.720
3
3.240
292.760
196
1.494
90.195
199
–
0.495
3
0.165
196
0.458
Total
83.595
199
4.015
3
1.338
79.580 196.480
196 199
0.406 –
5.520
3
1.840
190.960
196
0.974
Between groups Within groups
0.494
– 0.138
89.700
Within groups Total
F
–
Within groups Between groups Male—SCTN
MS
0.495
Between groups Female—ON
df
65.955
Between groups Female—WL
SS
0.428
2.169
0.361
– 3.296*
1.889
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.05
Table 6 Analysis of variance for comparison of trait FA (R–L) among D. parabipectinata, D. malerkotliana and their hybrids
Sex—Trait Female—SBN
Source of variation
SS
df
Total
158.195
199
1.135
3
0.378
157.060 77.875
196 199
0.801 –
Between groups Female—WL
Within groups Total Between groups Within groups
Female—ON
Total Between groups Within groups
Male—SBN
Total Between groups
Male—WL
0.855
3
0.285
77.020
196
0.393
343.595
199
13.575
3
330.020
196
1.684
80.555
199
–
1.975
3
0.658
196
0.401
Total
65.155
199
–
Total Between groups Within groups
0.472
0.725
– 4.525
78.580
Within groups
F
–
Within groups Between groups Male—SCTN
MS
0.375
3
0.125
64.780
196
0.331
254.395
199
15.135
3
5.045
239.260
196
1.221
2.687*
1.642
0.378
– 4.133*
SBN sternopleural bristle number, WL wing length, ON ovariole number, SCTN sex comb tooth number * p \ 0.001
F1 but reduced stability in further generations indicates that the pair in question has evolved their own independent coadapted complex of genes and this indeed gives a very precise picture of the degree of their divergence.
Literature is replete with studies concerning FA in hybrids (and comparison with parents) and the results are a mixed bag (Jackson 1937a, b; Felly 1980; Zaharov 1981; Graham and Felly 1985; Leary et al. 1985; Ferguson 1986;
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Fig. 1 Number of symmetrical (FA = 0) and asymmetrical individuals (FA = 1, 2, 3, 4, 5 etc.) in pure species and their hybrids. a ovariole number (ON) in females, b sternopleural bristle number (SBN) in females, c ovariole number (ON) in females, d ovariole
number (ON) in females, e wing Length (WL) in males, f sex comb tooth number (SCTN) in males. bi: D. bipectinata, pa: D. parabipectinata, ma: D. malerkotliana, ps: D. pseudoananassae. Note: Species represented first is the female parent
Lamb and Avise 1987; Ross and Robertson 1990; lamb et al. 1990; Markow and Ricker 1991; Graham 1992; Blows and Sokolowaki 1995; Hutchinson and Cheverud 1995; Alibert et al. 1997; Andersen et al. 2002, 2006, 2008; Rego et al. 2006; Carreira et al. 2008; Vishalakshi and Singh 2009; Demontis et al. 2010; Trotta et al. 2010; Novicic et al. 2011). Alibert and Auffray (2003) said that 71 % published work show an increase in FA in hybrids (of different genera, or species) from parents. In some of the studies, natural hybrid populations (mostly insects, fish,
frogs and lizards) were examined. It has been postulated by Graham and Felly (1985) that the age of the hybrid zone determines whether hybrids will have a greater or lesser FA than the parental populations. If they have existed for a long while, coadapted gene complexes having had time to evolve, would rescue any dire effect on FA. If they have not, FA would be enhanced despite increased heterozygosity. However, in this case we are considering Drosophila species that never hybridize in nature and therefore must never have got the chance to evolve
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Genetica Fig. 2 Comparison of mean Composite Fluctuating Asymmetry (CFAs) among pure species and their hybrids. a D. bipectinta, D. pseudoananassae and their daughters, b D. parabipectinata, D. pseudoananassae and their daughters, c D. bipectinata, D. parabipectinata and their daughters, d D. bipectinata, D. parabipectinta and their sons. bi: D. bipectinata, pa: D. parabipectinata, ma: D. malerkotliana, ps: D. pseudoananassae. Note: Species represented first is the female parent
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Fig. 3 Comparison of mean Composite Fluctuating Asymmetry (CFA) among hybrids. a Daughters, b sons, of all the 12 crosses. bi: D. bipectinata, pa: D. parabipectinata, ma: D. malerkotliana, ps: D. pseudoananassae. Note: Species represented first is the female parent
coadapted complexes. In other cases, where closely related Drosophila species which do not hybridize in nature were involved; too the results have been conflicting. For example, Markow and Ricker (1991) found that only hybrid daughters but not sons of D. melanogaster 9 D. simulans exhibited enhanced FA. In addition, a number of phenodeviants could also be spotted among the hybrid daughters. Rego et al. (2006), who studied FA in wing size and shape in the species pair D. subobscura 9 D. madeirensis demonstrated that though FA did not appreciably increase in the hybrids, proper bilateral development was severely compromised in them. In the species pair D. buzzatii 9 D. kopeferae Carreira et al. (2008) found no significant
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increase in FA in hybrids as compared to the parental species. In our lab Vishalakshi and Singh (2009) studied hybrids of the sibling species pair D. ananassae and D. pallidosa and since F1 hybrids of both sexes are fully viable and fertile, they went beyond F1 to check FA in F2 and F3 generations. This aided in testing whether FA is affected due to breakdown of coadapted gene complexes (that must have evolved in each species separately) in the F2 and F3 generations due to free recombination in F1. However, they found no significant differences in the level of FA in F1, F2, or F3 generations compared to the parental species demonstrating that neither breakdown of coadapted complexes nor enhanced heterozygosity has any influence
Genetica
on developmental stability of hybrids. They went further in saying that the hybrids are as stable as their parents. Our study, however, is different from any of the above, as it involves four species sharing different phylogenetic relationships among themselves and which never hybridize in nature. To boot, the hybrid males are sterile. So neither is there any hybrid zone in nature, having their own coadapted complexes, nor is it a question of hybrids of a single pair (we have six pairs). Also, we had no scope of checking the effect of breakdown of coadapted complexes having no option but to stop at the F1 generation, hybrid sons being sterile. However, coadaptation per se is more the attribute of a species and through interracial (intraspecific) hybridization studies it is often checked whether different populations or races within a species have evolved their own coadapted complexes (Singh 1972; Andersen et al. 2002, 2008; Novicic et al. 2011). Among the members of this complex, D. bipectinata which is most widely distributed and genetically more variable has been used for investigations concerning coadaptation. In our laboratory, different geographic populations of D. bipectinata were hybridized up to the F2 and F3 generations and increased wing length in the F1 but reduced wing length in further generations indicated the occurrence of coadaptation (Banerjee and Singh 1998). Yet, when it is a question of two distinct species, studying coadaptation is not very relevant as it is almost certain that they have undergone independent coadaptation. Then, it becomes more important to study the level of genomic incompatibility which may well be reflected in the level of FA in the F1 interspecific hybrids. Thus, we hoped that the incongruity/congruity between the genomes of each pair would be reflected in the level of FA. Yet, unlike our expectations, the hybrids betrayed only a little effect on the level of FA if at all any. In the species pair D. bipectinata–D. parabipectinata, while the hybrid daughters exhibited higher CFA, hybrid sons exhibited lower CFA as compared to parents. Recently, we studied sexual isolation in the members of this complex and based on asymmetrical patterns of mating preference, it was proposed that D. parabipectinata is derived from D. bipectinata (Banerjee and Singh 2012). Therefore, being an ancestral-derived species pair they must be closer to each other than any other pair in this complex. Hybridization affects the level of FA in opposite directions in the two sexes, in the sons, increased heterozygosity may have lead to reduced CFA and on the other hand, in the daughters incompatibilities must have had a more pronounced effect than increased heterozygosity, leading to raised CFA (that was mainly due to increased FA in the ovariole number). The hybrids from species pair D. bipectinata–D. malerkotliana (derived from a common ancestor; Banerjee and Singh 2012), did not show much difference in the level of
FA in any traits except WL in males. In the pair D. parabipectinata–D. malerkotliana too level of FA in hybrids and parents was almost matching. In fortification of earlier findings, both in our lab and elsewhere (Bock 1978; Mishra and Singh 2006a; Banerjee and Singh 2014) that D. pseudoananassae is distantly related to the other three species of the complex, it was difficult to cross D. pseudoananassae with them, especially when D. pseudoananassae males were involved. Earlier, we postulated that the courtship rituals of D. pseudoananassae must have diverged to such an extent that the females of the other three species recognize it with difficulty. It may also be possible that the females of the other three species have evolved a hostile reproductive tract environment, killing sperm from the males of a distantly related species like D. pseudoananassae (Banerjee and Singh 2014). The progeny produced from such difficult crosses also exhibited several abnormalities. Most of the daughters from reciprocal crosses involving D. bipectinata 9 D. pseudoananassae and D. malerkotliana 9 D. pseudoananassae were found to have dystrophied ovaries. It has always been said that among the hybrids of this complex, the females are fertile and the males are sterile. Yet, females with dystrophied ovaries cannot be fertile which demonstrates that fertility of hybrid daughters is not hundred percent at least in some crosses. Therefore, we planned to investigate this further and check the fertility of hybrid daughters from all the crosses. We found that daughters having D. pseudoananassae as one of their parents had drastically reduced fertility compared to the parental females (Banerjee and Singh 2015b). In the crosses D. pseudoananassae females 9 D. bipectinata males and D. pseudoananassae females 9 D. parabipectinata males, the hybrid sons were found to have severely compromised eye symmetry and poor viability. While on one side the eyes were properly developed, the other side had gradation in abnormalities. In the reciprocal crosses however, the sons were normal. Since no daughters were found to be affected and sons of only one of the reciprocal crosses were affected, it is evident that ‘X’ chromosome is involved in causing abnormal development of eyes. The X chromosome of the maternal species, D. pseudoananassae (inherited by the sons in both the crosses), is perhaps incompatible with the autosomes of the paternal species D. bipectinata and D. parabipectinata, which is causing the left–right signaling during eye formation to go awry (Singh and Banerjee 2015). However, while abnormalities and asymmetries are quite evident, subtle asymmetries in hybrids are not very different from the parental species (CFA of hybrid daughters involving D. bipectinata 9 D. pseudoananassae and D. parabipectinata 9 D. pseudoananassae are only slightly greater than respective parental females). In addition, hybrid sons and
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daughters from all twelve crosses were found to have comparable CFAs. In conclusion, interspecific hybrid sons and daughters especially those involving D. pseudoananassae and the other three species of the complex show certain abnormalities like dystrophied ovaries and asymmetrical eyes, even poor viability. These clearly indicate that they are developmentally unstable. Yet, this fact is concealed when FA is compared across the hybrids and parents. Therefore the system taking care of correcting the subtle asymmetries arising due to developmental noise is not affected or affected little in the hybrids. Nevertheless, they are certainly not as stable as their parents, as specific developmental pathways like those involved in development of proper symmetrical eyes and normal ovaries do get affected due to hybridization. There is incompatibility between the genomes of D. pseudoananassae and the other three species and genes required in specific developmental pathways like the formation of eyes and ovary have diverged to such an extent that they cannot work in a coordinated fashion in the hybrid background. On the other hand, the system bringing about developmental stability (to buffer random noises of development), is very large involving a number of genes forming a complex circuitry, so even if there are minor incompatibilities, the system as a whole remains impervious and thus the effect is not very pronounced. Therefore, the system responsible for developmental stability is so robust and perhaps also conserved that irrespective of genomic incompatibilities or the level of heterozygosity, it carries out its function well, so well that the level of FA remains unaffected in the hybrids. Acknowledgments Financial assistance in the form of Meritorious Fellowship to PB and UGC-BSR Faculty Fellowship Award to BNS, from the University Grants Commission, New Delhi is gratefully acknowledged. We thank Prof M Matsuda, Japan and Prof. A. Kopp, USA for kindly providing the stocks of the Drosophila bipectinata species complex. We also thank the two anonymous reviewers for their helpful suggestions that helped to improve the manuscript. Conflict of interest of interest.
The authors declare that they have no conflict
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