6. METODOLOGIA
6.3. ENTRENAMIENTO Y VALIDACION
Seventy (83.3%) female brood were genotyped at all five loci, and 82 (97.6%) at two or more loci. For the male brood, 31(72%) were genotyped at five loci, and 43 (1(X)%) at two or more loci. Spermathecae were found for 66 (85.7%) females, 60 (90.9%) of which were inseminated. Sperm was successfully genotyped at loci Pal95 and Pal68 for 44 (73.3%) and 24 (40%) of these females, respectively. Sperm was not successfully genotyped at any other loci.
Of the 17 P. altematacoXomts examined in this thesis, one was found to contain no brood (nest 104) and another only male brood (nest 109). Kinship analysis of the female brood was performed on the remaining 15 nests. There, were between one and four sibgroups present within these colonies (2.34 ± 0.270, mean ± standard error) (Figures 5.2 and 5.3). In 11 nests there was no temporal overlapping of sibgroups, indicating that only one female had been laying eggs within these colonies at any one time (nests, 2, 33, 40, 44, 50, 70, 72 74, 83, 117, 122). Another nest contained two sibgroups which overlapped temporally (nest 62). These sibgroups combined could only have been produced by a single female as all candidate mothers were excluded except one: this females genotype was consistent with her being the mother of both sibgroups, suggesting that they were the result of double mating. Therefore, in a totd of 12 (80%) of the 15 nests examined, only one female had been producing female offspring at any one time. The three remaining nests contained sibgroups that overlapped, suggesting that the production of female offspring in these colonies was not completely monopolised by a single female at all times (nests 85, 123, 129).
Maternity assignment was successful for the youngest sibgroup in 14 of the 15 colonies, with nest 85 being the exception. Of the 14 colonies for which maternity assignment was successfully performed, 12 contained female brood presumed to be younger than 20 days (<20mg). Of these 12 colonies, 10 contained a single reproductive
Chapter 5. Reproductive skew in Parischnogaster alternata
122). Within these colonies the dominant females (assigned mothers) and subordinates (excluded candidates) were related on average to the eggs and small larvae by 0.59 ± 0.104 and 0.28 ± 0.072 respectively (Table 5.3). The estimate for subordinates was significantly different from mother-daughter relatedness of 0.5 lo) = -3.14 P<0.05), while the estimate for dominants was not io> = 3.05 P>0.05), suggesting that the correct females had been assigned as mothers of the young brood. In the passages that follow, examples are presented for several nests, of how maternity was assigned to brood. Details of maternity assignment for the remaining nests can be found in Appendix C. The skew results for these nests are presented in Section 5.3.2.
n 5 o o Vh O 1 2 3 4
Number of female brood sibgroups
Figure 5.2. Distribution of female brood sibgroups among 15 P. altemata colonies.
Chapter 5. Reproductive skew in Parischnogasteralternata 33 40 44 50 62 70 72 6 4 2 0 6 ■ 4 ■ 2 ■ 0 6 4 ■ 2 • 0 6 4 2 0 6 ■ 4 ■ 2 ■ 0 6 4 2 0 6 4 2 ' 0 6 4 2 0
nrni
74 83 85 117 122 123 129 6 - 4 - 2 - 0 - 6 - 4 - 2 - 0 - 6 - 4 ■ 2 - 0 ■ 6 • 4 ■ 2 • 0 ■ 6 ■ 4 ■ 2 ■ 0 ■ 6 4 2 0 6 4 2 0 '£Æà SL LL P SL LLFigure 5.3. Number of female brood in 15 P. altemata nests. C = colony number, E = eggs, SL = small larvae, LL = large larvae, P = pupae. The y-axis indicates the number of brood of each stage. Segment patterns indicate sibgroup membership. Within brood stages, older brood are stacked on younger brood.
Chapter 5. Reproductive skew in Parischnogasteralternata
Table 5.3. Mean within colony relatedness estimates and standard errors of adult females, dominants, and subordinates to different P. altemata brood classes. Included are tha P
values for significant differences from expected relatedness of 0.5 (mother-offspring). The probability of making any type I error due to multiple comparisons was controlled within male and female brood using Bonferroni tests by the Dunn-Sidak method (Sokal & Rohlf, 1995). NS = jR>0.05 (not significant), * = P<0.05, ** = P<0.01. Sample sizes are number of nests with numbers of individuals shown in parenthesis. The first number shown in parenthesis indicates the number of notional actors and the second indicates the number of notional recipients.
Notional actor Notional recipient Mean relatedness ± standard error
Sample size P value (F test)
Female brood
Adult females All female brood 0.39 ± 0.039 15 (68, 83)
Dominants All female brood 0.53 ± 0.126 10(10,42) NS
Subordinates All female brood 0.35 ± 0.075 10 (34, 42) NS Adult females Female eggs and
small larvae
0.36 ± 0.081 13 (56, 33) Dominants Female eggs and
small larvae
0.59 ± 0.104 10(10, 26) NS Subordinates Female eggs and
small larvae
0.28 ± 0.072 10 (32, 26) *
Male brood
Adult females All male brood 0.31 ± 0.020 15 (70, 43)
Dominants All male brood 0.49 ± 0.045 9 (9, 28) NS
Subordinates All male brood 0.23 ± 0.030 9 (32, 28) **
Adult females Male eggs and small larvae
0.31 ± 0.052 5 (27, 9)
Dominants Male eggs and
small larvae
0.52 ± 0.034 7 (7, 12) NS Subordinates Male eggs and
small larvae
Chapter 5. Reproductive skew in Parischnogasteralternata
(i) Nests for which all potential mothers o f the youngest brood were collected (nests 33,40, 50, 123)
Nest 33
In this nest there was a single female sibgroup consisting of six brood that ranged in age from small larvae to pupae. From the maternal genotypes, only two of the five collected adult females could have produced this sibgroup. Sperm alleles were amplified for both of these females at locus Pal68. Only female 1.D.32 had the paternal allele for the brood sibgroup. As all potential mothers were collected, 1.D.32 was the mother of all the female brood. Please refer to Appendix C for maternity assignment details on nests 40, 50 and
123.
(ii) Nests fo r which all potential mothers o f the youngest brood were not collected but maternity assignment was facilitated by the availability o f sperm genotypes(nests 2, 44, 62, 74, 83,217, 122)
Nest 44
This nest contained a single sibgroup consisting of two large female larvae (>20mg). Three of four adult females collected had maternal genotypes that matched the female brood. However, one of these females was not inseminated (1.D.45) and therefore could not have been the mother. Of the remaining two females both had been genotyped for Sperm alleles at locus Pal95. Indvidual 42 did not have the correct paternal allele for the sibgroup and therefore was not its mother. Individual 41did have the correct sperm allele at locus Pal95 and therefore may have been the mother of the female brood. An uncollected individual was last seen on the nest 35 days prior to collection. It is assumed that pupation occurs in P. altematalS days on average following egg deposition as it does
in P. nigricans serrei (Section 5.2.4.1). The larvae were therefore likely to be younger
Chapter 5. Reproductive skew in Parischnogasteralternata
this nest contained four male brood whose genotypes suggested that they could have been brothers of the female brood. Three of these male brood were younger than 20 days (<20mg) and could only have been produced by I.D.41, thus providing further evidence that this female was also the mother of the female progeny.
Nest 62
This nest contained two temporally overlapping sibgroups, each composed of two female brood (Figure 5.3). Four adult females were collected. Three of these could not have produced any of the brood. The remaining female, I.D. 59, had a maternal genotype that matched both brood sibgroups. Furthermore, the sperm of this individual had been amplified at locus Pal95 and found to consist of two alleles. These sperm alleles matched the paternal alleles of the two sibgroups. However, this result was confounded by the fact that one of the sperm alleles also matched one of the maternal alleles, i.e., the genotype of I.D.59 at locus Pal95 was 174/174 while its sperm alleles were 174/194, therefore the sperm allele 174 may have been due to maternal contamination. However, small brood from each sibgroup were present within the nest. An egg of 0.22mg from one sibship and a larva of 0.3 Img from the other. Prior to collection a joiner had been seen on the nest on one occasion only 17 days earlier and another individual had not been seen for 29 days. Based on the age (weight) of these young brood it was very unlikely that either of these females had produced them. Therefore there were no other candidates other than I.D.59 that could have produced the egg and the young larva. Therefore, I.D.59 was multiply mated and was the mother of both female brood sibgroups.
Nest 117
Two female brood sibgroups were present within this nest (Figure 5.3). The oldest sibgroup consisted of five brood that ranged in age from small larvae (<20 mg) to pupae. The youngest sibgroup consisted of an individual egg. Six adult females were collected from this nest. None of these females could have produced the oldest sibgroup. Only two of the adult females had maternal genotypes that could have produced the youngest
Chapter 5. Reproductive skew in Parischnogasteralternata
sibgroup (the egg). Sperm was successfully genotyped for both of these females (I.D. 128 & I.D. 129) at locus P al95. Only I.D. 129 had the sperm allele at this locus to match the egg. Sperm had also been genotyped for this female at locus 168. The sperm allele at this locus also matched the brood, strongly suggesting that I.D. 129 was the mother of the egg. The older sibgroup which also contained small brood was probably produced by a long term resident which had last been seen on the nest 15 days before collection. This suggested that a change over in reproductive tenure had recently taken place as a result of the death of the previous dominant. Therefore, skew was not calculated amongst the young brood in this nest. Maternity assignment details on nests 2, 74, 83 and 122 are provided in Appendix C.
(Hi) Nests for which mothers were assigned with less confidence (70, 72, 129)
Nest 72
There were four female brood in this nest which formed four sibgroups (Figure 5.3). Four adult females were collected. Sperm was amplified for three of these, indicating that these indivduals were not the mothers of any of the brood. The maternal genotype of the remaining female (I.D.71) did not match the genotypes of the young brood. Therefore, the mothers of the brood had not been collected. Two long term residents had last been seen on the nest 15 and 26 days prior to collection. The two youngest brood weighed 6.94mg and 11.5mg. Therefore the resident which had last been seen on the nest 15 days before collection was assigned as the mother of one of the brood. The remaining progeny was a mystery, as it is unlikely that a small larva of 11.5mg was produced by a female that had last been seen on the nest 26 days before collection (Pupation occurs in P. nigricans serrei
approximately 28 days following egg deposition (Turillazzi, 1985b)). No females that could have produced this brood had been seen on the nest. However, this brood had to have been laid by an uncollected female. Therefore, it was assigned to an uncollected female which allowed group size to be set at six and skew among the small brood to be
Chapter 5. Reproductive skew in Parischnogasteralternata
estimated. Details on maternity assignment for nests 70 and 129 can be found in Appendix C.
(iv) A Nest for which mothers could not be assigned (nest 85)
Nest 85
This nest contained eight female brood that formed four sibgroups, three of which overlapped (Figure 5.3). Seven adult females were collected from this nest. Six of these females could not have produced any of the sibgroups. One of the seven females (I.D.96) could have produced one of the sibgroups that consisted of a single larva of 34.3mg. I.D.96 also had the sperm allele to match this larva at locus 195. A long term resident had last been seen on the nest 12 days prior to collection and two other females 37 days prior to collection. These females were probably the mothers of the three remaining unassigned sibgroups. Skew could not be calculated for this nest.
5.3.2 Skew among female brood
Assignment of maternity was successfully performed for 14 colonies. Of these colonies, nine contained two or more female eggs and small larvae (<20mg). Reproductive skew was calculated for these nests using the computer program Skew 1.1.1 (Krieger & Keller, 1997) (Section 5.2.5). Reproductive skew amongst the young brood for these colonies was 1.0 in seven of the nine nests examined, i.e., within seven nests, a single female had produced all of the young female brood (Table 5.4).
In two, colonies skew was less than one. Skew exceeded the 95% upper confidence limits of the expected skew under the assumption of random reproduction in only two of the nine colonies (nests 70 & 83). However, the skew indices were calculated using very small samples (between two and eight brood per nest). The fact that the observed skew was 1.0 in seven of the nine colonies suggests that skew in P. altemata probably is nearly always one (Table 5.4). To investigate this further, an analysis was performed to determine the maximum likelihood proportion of females produced by the dominants as
Chapter 5. Reproductive skew in Parischnogasteralternata
opposed to the subordinates (Section 5.2.4.1). The analysis was performed for the seven nests that contained a minimum of two young female brood and for which the genotypes of the dominants were known (nests 33, 40, 50, 62, 70, 83, 123). Unfortunately, two nests for which skew was less than 1.0 could not be used in the analysis because the genotypes of the dominants were not known (nests 72, 129). The likelihood curve shows that on average, the small female brood were likely to have all been produced by a single dominant female on each nest (Figure 5.4). Therefore, in the seven nests used in the analysis, skew amongst the small female brood was most likely to be 1.0.
Table 5.4. The reproductive skew (5, calculated from Krieger & Keller (1997)) and whether it exceeds the 95%, 99% and 99.9% upper confidence limits of the expected skew under the assumption of random reproduction, for nine P. altemata colonies. NS = P>0.05, ** =P<0.01, *** = P<0.001. Nest number Skew Expected skew Number of potential reproductives Number of breeders Number of female eggs and small larvae (<20mg) Significance level 33 1.0 0.68 5 1 2 NS 40 1.0 0.50 2 1 2 NS 50 1.0 0.38 3 1 3 NS 62 1.0 0.47 4 1 3 NS 70 1.0 0.45 5 1 4 ** 72 0.67 0.72 6 2 2 NS 83 1.0 0.45 10 1 8 *** 123 1.0 0.33 2 1 3 NS 129 0.64 0.54 5 2 2 NS Mean± s.e. 0.92 ± 0.051 0.50 ± 0.043 4.67 ± 0.816 1.22 ± 0.147 3.22 ± 0.641
Chapter 5. Reproductive skew in Parischnogasteralternata 0.8- 1 0 .4 -
!
0.2- 0 0.2 0.4 0.6 0.8Fraction of females produced by dominant
Figure 5.4. Likelihood curve showing the proportion of females likely to be daughters of the dominants, averaged over all nests (n = 7).
5.3.3 M aternity of male o ffsp rin g
Maternity assignment of male brood was not possible using the genotypic data that was available (Section 5.2.4.2). Instead, maternity of male brood was investigated using a maximum-likelihood approach (Arévalo gr aA, 1998; Hastings et a l, 1998; Sumner et al.,
2002). Dominants were identified from the maternity assignment as those individuals that laid the largest proportion of female eggs and small larvae (Section 5.3.1). The remaining adult females were designated as subordinates, as all were capable of laying male eggs. The male eggs and small larvae were then subjected to a maximum-likelihood analysis to determine the likely proportion of males produced by the reproductive dominants (Arévalo
et a l, 1998; Hastings é ta l, 1998; Sumner et a l, 2002). The analysis was performed on
those colonies for which a dominant had been identified, and male eggs and small larvae were genotyped (nests, 33, 44, 50, 62, 70, 83, 122). These colonies contained between one and three male eggs and small larvae per nest. As sample sizes were small, colony-
Chapter 5. Reproductive skew in Parischnogasteralternata
specific likelihoods were multiplied together to give an overall population likelihood. In total, the sample size consisted of only 12 male brood. The maximum likelihood curve for male production is displayed in Figure 5.5. The curve suggests that for the seven nests analysed, all male eggs and small larvae were laid by the female that had laid most or all of the female eggs.
0.8
I
0.6 •C) O S 0.4!
0.2 0.6 0.8 0.2 0.4Fraction of males produced by dominant
Figure 5.5. Likelihood curve showing the proportion of males likely to be sons of the dominants, averaged over all nests (n = 7).
Dominant females and subordinates were related on average to the male eggs and small larvae by 0.52 ± 0.034 and 0.25 ± 0.058 respectively (Table 5.3). The estimate for subordinates was significantly different from mother-son relatedness of 0.5 {t^Q Q^ (z)?) “ ' 6.69 P<0.01), while the estimate for dominants was not ^2)7) = 1 21 P>0.05). This supports the result of the maximum-likelihood analysis by suggesting that dominant females were the mothers of the young brood.
Chapter 5. Reproductive skew in Parischnogasteralternata
In both nests that contziined a single female sibgroup and a minimum of three male brood, all of the male brood had genotypes that were consistent with having been produced by a single female (nest 33, 5 male brood, and nest 83, 6 male brood). Other nests containing a minimum of three young male progeny (<20mg) were also examined (nests 44, 50, 109 and 129). The genotypes of the young male brood in these nests were consistent with having been produced by a single female in three of the four nests. The exception was nest 109 which contained three male eggs that must have been produced by at least two females (this nest contained no female brood). This indicates that although male production is predominantly monopolized by a single female, as indicated by the maximum-likelihood analysis, occasionally deviations from this pattern do occur.