View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Serveur académique lausannois Insect. Soc. DOI 10.1007/s00040-017-0543-1 Insectes Sociaux RESEARCH ARTICLE Camponotus fellah queens are singly mated Danielle P. Mersch1,2 · Christine La Mendola2 · Laurent Keller2 Received: 18 November 2016 / Revised: 17 January 2017 / Accepted: 20 January 2017 © International Union for the Study of Social Insects (IUSSI) 2017 Abstract The ant Camponotus fellah has been used Introduction in several behavioral and life history studies. An impor- tant factor that affects the genetic structure and division The genetic structure of ant colonies varies widely between of labour within a colony is whether queens are singly or species and can impact colony immunity, reproduc- multiply mated. To determine whether queens are singly tive output and division of labor (Crozier and Page 1985; mated in C. fellah, as is the case in some other Campono- Julian and Cahan 1999; Heinze and Keller 2000; Julian tus species, we developed nine polymorphic microsatellite and Fewell 2004; Rheindt et al. 2005; Oldroyd and Fewell markers and sequenced 16 workers each from 20 colo- 2007; Smith et al. 2008). Several studies have shown that nies at six loci. Data in all colonies were compatible with in colonies where queens are multiply mated workers from queen monoandry. All the workers of one of the colonies different patrilines vary in their tendency to specialize in had identical genotypes suggesting that they were clonally different tasks, thereby potentially affecting the efficiency produced or that the queen was inbred. We, therefore, geno- of division of labor and colony homeostasis (Robinson typed the mother queen as well as 31 more workers of the 1992; Rheindt et al. 2005; Schwander et al. 2005; Wier- same colony at the same six loci plus the three remaining nasz et al. 2008; Waddington et al. 2010). Increased within- loci. These data revealed that the queen was homozygous at colony genetic diversity due to multiple patrilines and/ eight of the nine loci and that she mated with a male hav- or matrilines may also improve resistance to parasites and ing a shared allele at all but one of the loci. Thus, the queen pathogens (Hughes and Boomsma 2004; Reber et al. 2008; was apparently not only inbred but also probably mated Schmidt et al. 2011) thereby enhancing the lifetime and with a brother. reproductive output of a colony. Thus, knowledge of the genetic makeup of colonies provides insights into what fac- Keywords Camponotus fellah · Microsatellites · tors might shape colony function. Monoandry · Ant · Genetic structure Studies in several species of the genus Camponotus (C. herculeanus, Gertsch et al. 1995; C. ligniperdus, Gertsch et al. 1995, Gadau et al. 1998; C. floridanus, Gadau et al. 1996; C. nawai, Satoh et al. 1997; C. consobrinus, Fraser et al. 2000; C. ocreatus, Goodisman and Hahn 2004 and * Danielle P. Mersch C. festinatus, Goodisman and Hahn 2005) revealed that [email protected] queens are generally singly mated with occasional instances * Laurent Keller of polyandry. A species of the genus Camponotus that has [email protected] not yet been investigated is C. fellah. This species has 1 MRC-Laboratory of Molecular Biology, Francis been extensively used to study nestmate recognition (Bou- Crick Avenue, Cambridge Biomedical Campus, lay et al. 2000a, b; Katzav-Gozansky et al. 2004; Katzav- Cambridge CB1 0QH, UK Gozansky et al. 2008), more recently to unravel the impact 2 Department of Ecology and Evolution, University of social isolation (Boulay et al. 1999; Koto et al. 2015) of Lausanne, 1015 Lausanne, Switzerland and to decipher the organizational principles of division Vol.:(0123456789)1 3 D. P. Mersch et al. of labor (Mersch et al. 2013; Greenwald et al. 2015; Kafsi and primer design. To determine which of the designed et al. 2016). The colonies exhibit pronounced division of primers were polymorphic we tested nine primers on two labor with workers organized in distinct social groups that workers from each of eight colonies (A–H). Six out of the specialize in different tasks (Mersch et al. 2013). The pro- nine microsatellites had five or more alleles in the workers pensity of a worker to join a social group, and preferentially tested (Table 1). We used these six microsatellites to assess execute some tasks might thus be dependent on a worker’s queen-mating frequency in 20 colonies using 16 workers patriline. We developed and tested a set of nine microsatel- per colony. However, because there was a very high level of lite loci to determine whether C. fellah queens are singly homozygosity in colony S, we increased the sample size to mated, or whether workers come from different fathers. 47 workers and also genotyped the queen using one of her legs. In this colony we also genotyped all individuals at the three remaining microsatellites (7275, 7207, 7300). Materials and methods We extracted DNA from each individual using the Bio- Sprint 96 DNA Blood Kit for tissue sample from QIA- Collection of ants GEN and eluted the DNA in a final volume of 200 µl. We performed PCR amplifications for each ant sample in a Adult workers were collected from 20 lab-reared colonies total volume of 20 μl. To reduce the amount of work and (A–T) by opening the nest and then collecting workers ran- sequencing, we multiplexed primers 1284, 6826 and 5134 domly both near the brood and outside the nest. Each lab in one reaction, and primers 3186, 3675, 4380 in the other. colony was established from a single queen collected dur- Each reaction consisted of 4 μl of genomic DNA, 4 μl of ing a mating flight in 2007 on the campus of the Tel Aviv 10 × PCR buffer (QIAGEN), 1.92 μl of 25 MgClmM 2, University, Tel Aviv, Israel, which is within the natural 0.32 μl of 25 mM dNTPs, 1 μl of each forward and reverse range of the species (Ionescu-Hirsch 2009). primer (10 μM), 0.3 µl of 5 U/µl Taq polymerase (QIA- GEN), 3.2 µl of 5 × enhancers (Qsolution from QIAGEN), Genetic analysis and 0.26 µl H2O. The PCR consisted of an initial denatur- ing step of 5 min at 95 °C, followed by 35 cycles that each PCR primers were developed for C. fellah microsatellite consisted of 30 s of denaturation at 95 °C, 30 s of annealing loci from a partial genomic library that was derived from at 55 °C, and 60 s elongation at 72 °C. A final elongation 10 µg of genomic DNA extracted with the BioSprint 96 step was performed at 72 °C for 10 min. We analyzed PCR robot (QIAGEN #900852) from one worker, and sent to products with the ABIprism3100 DNA sequencer and the Ecogenics (http://www.ecogenics.ch/) for 454 sequencing GeneMapper Software (Applied Biosystems). Table 1 Characteristics of the nine microsatellite loci from Camponotus fellah colonies Locus Primer sequence (5′–3′) (F: forward; R: reverse) Ta (°C) Repeat type Size of cloned No. of alleles Amplicon size range amplicon (bp) 1284 F: GTC GGA ATG TTG CAC CTA CG 53.8 (AG) 226 5 230–237 R: CAG ACC GCG AGA GGA GAT AC 3186 F: AAT TGA ACG TTT CGT CCG CC 51.8 (AC) 190 8 194–206 R: AGT CTT TAC GCC CTC CTA CG 3675 F: TTG TAG AGC GAC GAC GAG AG 53.8 (CGT) 203 8 221–252 R: TGA GAG GTG AGA GCA CGA AG 4380 F: TGG CTA CAG TTG TTG TGC AG 54.8 (AC) 115 10 126–148 R: TTT GTC GCG GCT CGT AAA TC 5134 F: TAC AAT CGA GTG GAC GGG AG 53.8 (CGA) 247 9 246–299 R: CAG ATG AAA GGC GAA CGG TG 6826 F: GTA CGC GGT CAG AAT TAC GC 53.8 (AG) 176 14 178–207 R: ACC TGA TTT CTG GTT GGC AC 7207 F: AGG CAT TCG ATA CTG TCG GC 53.8 (TGC) 163 2 184–187 R: TAC GAC AAG GAT GCT ACC GC 7275 F: CGC ATC GGG AGC ATA AAC AG 53.8 (CA) 102 5 118–123 R: AAA GCT GGC ATG CAG TTA CG 7300 F: AAT TTG CGG AGA ATC CGT GG 51.8 (CT) 185 3 201–205 R: CAT CGG CAA AGT CGT CCT TC All forward primers are preceded with an 18-bp M13-tag: 5′-TGT AAA ACG ACG GCC AGT -3′ 1 3 Camponotus fellah queens are singly mated Data analysis Table 2 Summary of all worker genotypes and inferred male and queen genotypes We estimated linkage disequilibrium between each pair Colony Locus Worker Inferred male Inferred queen of loci using the Genepop software (Raymond and Rous- genotypes (# set 1995; Rousset 2008). For each colony, we determined workers) the number of worker patrilines. This was simplified by the A 3675 224::230 (7) 224 221::230 fact that all colonies were lab raised with all workers being 221::224 (9) the offspring of a single queen. Further, in ants, males are 1284 230::236 (8) 236 230::231 haploid because of the haplodiploid sex determination sys- 231::236 (8) tem of Hymenoptera. Thus, at each locus, a worker inherits 4380 128::128 (6) 128 128::132 one allele from her father and one of the two alleles of the 128::132 (10) queen. If two males share the same alleles at all genotyped 5134 261::261 (9) 261 246::261 246::261 (7) loci, it is impossible to distinguish their offspring. The the- 6826 188::188 (7) 188 188::190 oretical probability of such non-detection errors was esti- 188::190 (9) mated as: 3186 197::197 (11) 197 197::203 197::203 (5) 2 a qi B 3675 221::221 (7) 221 221::228 221::228 (5) 221::224 (2) b with qi as the frequency of the i allele (Boomsma and Ratnieks 1996).