Myrmecological News 15 53-61 Vienna, May 2011 Queen number and raiding behavior in the ant genus Myrmecocystus (Hymenoptera: Formicidae) Bert HÖLLDOBLER, Bernd GRILLENBERGER & Jürgen GADAU Abstract An experimental field study demonstrates that mature colonies of Myrmecocystus mimicus WHEELER, 1908 raid neigh- boring conspecific small colonies without preceding territorial tournament actions. We also report a total of 17 complete brood raids that did not originate from territorial tournaments, collected during 10 field research seasons. The number of captured brood and booty varied greatly: 6 - 137 larvae, 9 - 152 pupae, 0 - 4 callows, 0 - 23 honeypots. We also observed raiding ants transporting liquid food in their crops when they left the raided nest (49 - 409). Most likely, this food was solicited from honeypots inside the foreign nest. In general, the captured booty during these raids is consider- ably smaller than that retrieved during raids that originated from tournaments. The socio-genetic analyses provided evidence that workers eclosing from raided brood become part of the work force of the raider colony. This was shown for Myrmecocystus mimicus and M. depilis FOREL, 1901. In M. depilis, we confirm previous findings by KRONAUER & al. (2003) of interspecific raiding (i.e., M. depilis raids M. mimicus but not vice versa). In addition, we provide genetic evidence for facultative polygyny in M. mimicus, and obligatory monogyny and occasional polyandry in M. depilis. Key words: Myrmecocystus mimicus, Myrmecocystus depilis, intraspecific raiding, territoriality, polygyny, polyandry. Myrmecol. News 15: 53-61 (online 10 March 2011) ISSN 1994-4136 (print), ISSN 1997-3500 (online) Received 25 September 2010; revision received 24 November 2010; accepted 25 November 2010 Prof. Dr. Bert Hölldobler, University of Würzburg, Behavioral Physiology and Sociobiology, Biocenter, 97074 Würzburg, Germany; Arizona State University, School of Life Sciences, and Center for Social Dynamics and Complexity, Tempe, AZ 85287, USA. Dr. Bernd Grillenberger, University of Würzburg, Behavioral Physiology and Sociobiology, Biocenter, 97074 Würzburg, Germany. Assoc. Prof. Dr. Jürgen Gadau (contact author), Arizona State University, School of Life Sciences, and Center for Social Dynamics and Complexity, Tempe, AZ 85287, USA. E-mail: [email protected] Introduction The honey ant species of the genus Myrmecocystus owe Both species are active during the day and forage on ter- their trivial name to a special storage caste, the so-called mites and dead insects, harvest floral nectar, and collect honeypots or repletes. The crop in the gaster of these in- honeydew from hemipterous plant-sap feeding insects dividuals is filled with sugar solutions and proteins (CON- (SNELLING 1976). To defend these patchy and unpredict- WAY 1977, 1990) and, depending on the species, in the most able food sources, M. mimicus and M. depilis establish extended condition the gaster can reach sizes of a pea to a spatio-temporal territories by conducting ritualized tour- cherry. During times of scarcity, the honeypots regurgitate naments (HÖLLDOBLER & LUMSDEN 1980, HÖLLDOBLER the contents of their crops to colony members. This food 1981). If there is a substantial imbalance in strength (col- storage strategy is a special adaptation to life in arid re- ony size) between opposing colonies, the stronger colony gions. The genus Myrmecocystus has been the focus of sev- interferes with the foraging activity of the weaker colony eral behavioral (e.g., HÖLLDOBLER 1976, HÖLLDOBLER & by engaging the latter in tournament interactions directly at LUMSDEN 1980, HÖLLDOBLER 1981, BARTZ & HÖLLDOB- its nest area. These interference actions can still further es- LER 1982, LUMSDEN & HÖLLDOBLER 1983, HÖLLDOBLER calate to all-out attacks, whereby the stronger colony raids 1986, LLOYD & al. 1989), ecological (e.g., HÖLLDOBLER the smaller nest, enslaves the worker brood and callows, 1982, DUNCAN & LIGHTON 1994, CHEW 1995, BEDIR pillages the honeypots and kills or drives off the queen 1998, SANDERS & GORDON 2000, COLE & al. 2001), and (HÖLLDOBLER 1976, 1981). Such intraspecific raids also genetic studies (e.g., KRONAUER & GADAU 2002, KRON- occur without preceding territorial interactions. Mature col- AUER & al. 2003). onies literally scout for small incipient colonies in their Myrmecocystus mimicus WHEELER, 1908 and M. depilis neighborhood and raid them. FOREL, 1901 are sister species and belong to the subgenus We present here the quantitative raiding data gathered Endiodioctes (see SNELLING 1976, KRONAUER & al. 2004). during 10 field research seasons. These behavioral data are Ross H. Crozier Memorial Volume compared with previous genetic results obtained by em- Overall foragers of 12 M. mimicus colonies and 25 M. de- ploying microsatellites and mitochondrial DNA sequences pilis colonies were collected in 2002 and 11 colonies of (KRONAUER & al. 2004) and new genetic data reported in M. mimicus and 24 M. depilis in 2003. Of each colony this paper. The analysis of changes in genetic patterns of between 10 and 50 individuals were collected directly at the colony populations due to raiding has to take into account entrance to avoid contamination with stray foragers of for- that in mature Myrmecocystus mimicus colonies occasion- eign colonies. Workers were immediately conserved in 95% ally more than one queen can be found (R. Mendez, pers. ethanol. comm.). Most likely, these are instances of primary poly- On average, 20 individuals per colony (range 10 - 44) gyny originating from pleometrotic colony founding, com- were analyzed using the Myrmecocystus microsatellite mar- mon in M. mimicus. Often, however, in such founding as- kers developed by KRONAUER & GADAU (2002). After test- sociations the queen number is reduced to monogyny dur- ing the variability of these markers in M. mimicus and M. ing colony development (BARTZ & HÖLLDOBLER 1982). depilis with five individuals from five colonies of each spe- The situation is different for M. depilis. We never found cies (n = 50), we decided that the following markers would pleometrotic founding groups in this species and we could provide a good resolution for our questions (M. mimicus: never enforce pleometrotic colony founding in the labora- Mm1, Mm2, Mm3, Mm4 and Mm5; M. depilis: Mm1, tory. Mm2, Mm4 and Mm5). DNA extraction was performed following a standard chloroform-phenol protocol employed Material and methods by GADAU & al. (1996). The quality and amount of DNA The study site for collecting raiding data for Myrmecocys- was assessed on 0.8% agarose minigels. The extracted DNA tus mimicus was located in a mesquite-acacia community solution was diluted 1:10 with purified water (HPLC-grade). near Portal, Arizona, and Rodeo, New Mexico. Field data The PCR conditions are based on the recommended proto- on raiding were collected during the summer months of 10 cols from LI-COR® and KRONAUER & GADAU (2002), years spanning 1984 to 2003. Unfortunately, many raiding but have been slightly modified. They are similar for all events we witnessed were useless for this paper, because we loci and species, and differed only in the concentration of encountered them when they were already in progress primer and template in the master mix. The general reac- and therefore it was impossible to determine the amount tion solution in a 10µl volume was: 1× PCR-buffer (10 mM of brood captured during one raid. However, over a total Tris-HCL, 50 mM KCL, 0.08% Nonidet P40), 0.2 mM of 10 field seasons we were able to observe 17 raids in their dNTPs, 1.75 mM MgCl2, 0.5 U of Taq DNA Polymerase entirety. These raids occurred without any signs of preced- (MBI Fermentas), the suitable concentration of forward and ing tournaments. reverse Primer (ranging from 0.05 to 0.10 pmol/µl) and 1 or To test whether raiding behavior is initiated by scouts 2 µl of the 1:10 diluted DNA solution. The reaction was that discovered colonies within the home range of a resi- performed in a "Biometra® T1 Thermocycler", an "Eppen- dent colony, we conducted experiments with artificial in- dorf® Mastercycler", or "Eppendorf® Mastercycler gra- cipient nests. To accomplish this, we excavated two ma- dient" thermocycler with the following parameters: 3 min ture colonies with the aid of a backhoe. From these colo- at 94°C, followed by 30 cycles of 94°C for 1 min, 50°C nies, small colonies consisting of approximately 100 to 200 for 1 min, 72°C for 1.5 min, and a final extension time of workers, 30 larvae, 20 pupae and 5 - 10 honeypots, were 5 min at 72°C (KRONAUER & GADAU 2002). housed in a test-tube nest. Each tube measured 15 × 2.2 cm, The PCR products were analyzed using a LI-COR® with water supply in the tube trapped at the bottom by 4300 DNA Analyser and the SAGA Generation 2 (LI- cotton plugs. The tubes were closed with a cork stopper COR®, 2001) software. The SAGA Generation 2 (LI- through which a plastic tube (diameter 0.6 cm) reached COR®, 2001) software is supposed to be able to score into the tube nest and served as nest entrance. After two the molecular weight of the samples down to the scale of days of acclimatization, the artificial incipient nests were one base pair. But as the results are influenced by factors buried 6 to 10 cm deep in the soil with the exit tube reach- like gel irregularities, the variation between different gels is ing the surface. The exit tube opening was surrounded by not negligible. Therefore, as a confirmation of the soft- sand and little stone pebbles. Five such test nests each were ware's results we run some samples from different gels placed in the surroundings of four mature Myrmecocystus on a control gel to confirm allele identities. GENEPOP mimicus nests in distances from four to 20 meters in ar- ON THE WEB (Raymond & Rousset 2004) was em- bitrarily chosen directions. We checked four times a day for ployed to determine inbreeding coefficients and linkage dis- a period of 18 days whether or not there was still activity equilibrium for the derived queen genotypes.