Insectes soc. 47 (2000) 354– 356 0020-1812/00/040354-03 $ 1.50+0.20/0 © Birkhäuser Verlag, Basel, 2000
Insectes Sociaux
Research article
Queen mating frequency and relatedness in young Atta sexdens colonies E.J. Fjerdingstad 1, * and J.J. Boomsma 2 1
Institute of Ecology, Biology Building, University of Lausanne, CH-1015 Lausanne, Switzerland, e-mail:
[email protected] Department of Population Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100 Copenhagen, Denmark, e-mail: JJ
[email protected] * Now at: Department of Zoology and Tropical Ecology, School of Tropical Biology, James Cook University, Townsville, Queensland 4811, Australia
2
Received 22 February 2000; revised 18 July 2000; accepted 31 July 2000.
Summary. Queen mating frequency is correlated with mature colony size across monogynous ant species as a whole. However, recent comparisons between Atta and Acromyrmex leafcutter ants found an opposite trend. We tested whether this result could be reproduced for Atta sexdens, using a new Atta colombica microsatellite marker to analyse queens and worker offspring from six young colonies. We found that all queens had mated multiply and used sperm from at least 2–3 males. This result is comparable to previous findings for Atta colombica and seems to confirm that queen mating frequency in Atta is lower than in Acromyrmex.
are Acromyrmex colonies. This apparent misfit with theory is intriguing, because studies on lower attines with very small colonies (Myrmicocrypta ednaella, Apterostigma collare and Cyphomyrmex longiscapus) have shown that queens are invariably single mated (Villesen et al., 1999). It is therefore of interest to test whether the trend reversal within the higher attines holds up when more Atta and Acromyrmex species are examined. Here we present the first genetic results on queen mating frequency in Panamanian Atta sexdens. Brasilian Atta sexdens queens were previously found to store on average four males’ worth of sperm (Kerr, 1961), but no genetic information was available.
Key words: Ants, Attini, mating frequency, polyandry. Materials and methods Introduction Two hypotheses predict a positive correlation between queen mating frequency and mature colony size in ants: (1) larger colonies may be more apparent to parasites and pathogens and may thus benefit more from an increased intracolonial genetic diversity (under certain life history assumptions, Sherman et al., 1988; Hamilton, 1987; Schmid-Hempel, 1994) and (2) queens of large colony species may benefit from mating multiply because it allows them to store more sperm and obtain a higher fecundity (Cole, 1983; Fjerdingstad and Boomsma, 1998). Across monogynous ant species, mature colony size and queen mating frequency were found to correlate positively (Boomsma and Ratnieks, 1996). Recent genetic results from higher attine ants have, however, shown that queen mating frequency in Panamanian Atta colombica (Fjerdingstad et al., 1998) was significantly lower than in sympatric populations of Acromyrmex octospinosus (Boomsma et al., 1999) and not higher than that of Acromyrmex echinatior (Bekkevold et al., 1999), even though Atta colonies are more than an order of magnitude larger than
We collected young, approximately one year old colonies of Atta sexdens near Puerto Caimito in the canal zone of Panama in 1993 and subjected DNA extracts of individual mother queens and offspring to microsatellite analysis. Three primer sets originally developed for Atta colombica were tested on the A. sexdens material. Two of the loci examined, Etta5-6TF and Etta7-8TF (Fjerdingstad et al., 1998), produced very small, monomorphic PCR products, suggesting that the microsatellite regions found in Atta colombica had been deleted in A. sexdens (Fjerdingstad, 1996). Our results for A. sexdens are therefore based on a single, not previously published, microsatellite locus, Etta1-2TF (Table 1). DNA extractions, PCR, gel electrophoresis and film interpretation were as in Fjerdingstad et al. (1998) except for the following modifications: DNA extractions were made in a volume of only 150 µl Chelex suspension, 1–3 µl of the Chelex extract was used in each PCR reaction, P-32 was used for internal labelling instead of S-35, and MgCl2 concentrations in the PCR mix were between 1.5 to 2 mM. Various PCR programs were tested involving annealing temperatures (Tm) between 50 C and 60°C. Most samples were amplified with a touch-down program: 3 min at 94 C, 10 cycles of 59 s at 94 C, 59 s at Tm = 55 C – 0,5 C/ cycle, 59 s at 72 C, then 30 cycles at Tm = 50 C, and a final 10 min at 72 C. As in Fjerdingstad et al. (1998) two known genotypes were run as standards for every 20 samples on each gel. We analysed an average of 14 worker offspring (range: 10 – 18) from each of six colonies. For four of the six colonies the queen was also
Insectes soc.
Vol. 47, 2000
Research article
355
Table 1. Basic information on the Etta 1-2TF locus used for the genetic analysis of Atta sexdens colonies. The effective number of alleles was calculated using the allele frequencies obtained through Relatedness 4.2c (Goodnight, 1996), weighting colonies equally Locus
Cloned core sequence of Atta colombica
Sequence of PCR primers
Size of cloned allele (bp)
No. of alleles in Atta sexdens
Effective no. of alleles in Atta sexdens
Etta1-2TF
(AT)5(TG)43TCAG(TA)6
5¢-GTATTGTTCGATGAGAAATAGAGC-3¢
191
7
5.9
5¢-CGGCTGACGTGTTGAATC-3¢
resulted in a lower estimate (RIBD = 0.29 ± 0.082 (SE), 95% C.I.: 0.079–0.501; standard error obtained by jack-knifing over colonies; Table 2). However, the concommitant estimate of the population-wide inbreeding coefficient F was negative and of a considerable magnitude, even though non-significant (F = – 0.12, t = 2.38, P = 0.06 for a two-tailed test, NS). Such a deviation from Hardy-Weinberg proportions will have affected the relatedness estimate, as negative inbreeding coefficients tend to induce underestimates of relatedness (Pamilo, 1993; Boomsma and Van der Have, 1998). We estimated the relatedness incorporating the observed negative inbreeding coefficient according to Pamilo’s (1993) equation 3 based on the pedigree observed effective paternity frequency and obtained an estimate of r = 0.29. Although it is not fully clear to which extent this equation is appropriate for negative inbreeding coefficients, the inferred value is the same as the value obtained through IBD analysis (r = 0.29). Therefore there seems to be little evidence that the obtained IBD estimate indicates a higher queen mating frequency than the one estimated through pedigree analysis. Mate number and relatedness in A. sexdens thus seems to be similar to what was found for Atta colombica (average number of mates per queen: 2.6, average relatedness = 0.52, Fjerdingstad et al., 1998) and Acromyrmex echinatior (2.53 and 0.40–0.45, Bekkevold et al., 1999) but lower than those of the sympatric Acromyrmex octospinosus (6.1 and 0.33, Boomsma et al., 1999). Analysing more offspring per colony might lead to higher absolute estimates of Atta sexdens queen
available. We found a total of seven alleles. The effective number of alleles was 5.9 (Table 2), estimated as the reciprocal of the sum of squared allele frequencies. The probability that two fathers carried the same alleles at our marker locus and thus could not be distinguished (non-detection error, Pamilo, 1993; Boomsma and Ratnieks, 1996) was 17%, calculated as the sum of squared allele frequencies (Pamilo, 1993). On the basis of queen and offspring genotypes, we reconstructed the number of mates per queen (as in Fjerdingstad et al., 1998). All offspring genotypes were compatible with the observed or inferred maternal genotype and thus consistent with the hypothesis of monogyny. Through pedigree analysis, by examining the proportional representation of different paternal males in each colony, we also estimated the genetically effective mating frequency of queens (cf. Starr, 1984; Boomsma and Ratnieks, 1996) and thereby nestmate relatedness (cf. Page, 1986). In addition we estimated nestmate relatedness through identity-by-descent analysis of the allelic and genotypic patterns within colonies as compared to the background population (Queller and Goodnight, 1989) and estimated the inbreeding coefficient of the population (F) using the program RELATEDNESS 4.2c (Goodnight, 1996).
Results and discussion Our genetic analysis showed that Atta sexdens queens used sperm from 2.7 males (range 2–3) on average (Table 2). The harmonic mean effective mating frequency (me) was 2.3 (Table 2), a value which changed little (m e, p = 2.6) when applying Pamilo’s (1993) probabilistic correction for errors due to sample size. These values translated into average nestmate relatedness estimates of R = 0.47 (range 0.43–0.51) and Rp = 0.44 (Table 2). The identity-by-descent (IBD) analysis
Table 2. Absolute and effective number of patrilines (mates/queen) detected through microsatellite analysis and the inferred relatedness among colony-members. N = number of offspring analysed per queen,  y 2i = sum of squared paternity contributions, me = effective paternity frequency (Starr, 1984,  p 2i = sample size corrected  y 2i (cf. Pamilo, 1993), me, p and Rp = the estimates of average effective paternity and relatedness based on  p 2i , RIBD = regression relatedness based on analysis of the allelic and genotypic similarity of colony-members as compared to the background population (Queller and Goodnight, 1989; Goodnight, 1996) Colony
N
Queen genotyped?
Patrilines
 y2i
me
Ry
 p 2i
AsexC AsexE AsexF AsexJ AsexK AsexL
15 10 18 12 16 14
Yes No Yes Yes No Yes
2 3 2 3 3 3
0.52 0.44 0.52 0.39 0.37 0.38
1.92 2.27 1.91 2.57 2.72 2.65
0.51 0.47 0.51 0.44 0.43 0.44
0.47 0.38 0.50 0.33 0.32 0.33
Average
14
2.7
0.44
2.3
0.47
0.39
me,p
Rp
RIBD 0.39 0.24 0.39 0.29 0.25 0.19
2.6
0.44
0.29
356
E.J. Fjerdingstad and J.J. Boomsma
Atta sexdens queen mating frequency
mating frequency since more rare fathers would be detected. The relatedness estimates and the estimates of effective paternity should, however, not change much since rare fathers contribute little to intracolonial genetic diversity. Our results thus corroborate the earlier finding that Acromyrmex absolute and especially effective queen mating frequencies are at least as high as those of Atta queens. More precise conclusions await studies on more species. Further investigations could suitably address also the small-colony genera Trachymyrmex and Sericomyrmex that appear to phylogenetically link lower and higher attines (Wetterer et al., 1998). Such work is in progress (P. Villesen and J.J. Boomsma, in prep.). Acknowledgments We thank the Carlsberg Foundation for financial support, the Smithsonian Tropical Research Institute and INRENARE for endorsing our field work in Panama, Jes Pedersen for discussion, and Chris Starr and one anonymous reviewer for constructive comments. Pekka Pamilo and Laurent Keller kindly provided lab space for EJF during part of this project.
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