In spring 2005 and 2006 we collected complete colonies of six different Temnothorax species: T. nylanderi (Förster, 1850) and T. affinis (Mayr, 1855) in Sommerhausen (Würzburg, Germany), T. crassispinus (Karavejev, 1926) in Unterisling (Regensburg, Germany), T. unifasciatus (Latreille, 1798) in Waldenhausen (Germany) and Gargnano (Lago di Garda, Italy) and T. recedens (Nylander, 1856) and T. lichtensteini (Bondroit, 1918) Gargnano (Lago di Garda, Italy). While T. nylanderi and T. crassispinus are closely related sibling species [38], the other four species are phylogenetically more distantly related (Additional file 4).
Temnothorax colonies were collected from their nests in decaying branches on the ground and, in T. unifasciatus, T. lichtensteini and T. recedens, also from crevices in stone walls. The colonies were transferred into small plastic boxes (10 cm × 10 cm × 3 cm) with a regularly moistened plaster floor and kept in incubators under artificial climate conditions with the temperature gradually being raised from spring (10°C night/20°C day) to summer (17°C night/28°C day) conditions [39, 40]. Twice per week, colonies were provided with water, honey, and pieces of cockroaches.
Mixed-species colony set up
In 2005, colonies of T. nylanderi, T. crassispinus and T. unifasciatus, with a sufficient amount of larvae in each colony, were chosen for the mixed-species experiment (N = 45 colonies; Table 1). In 2006, the same mixed-species colonies were set up with additional mixed colonies plus T. recedens and colonies without a queen (N = 165 colonies; Table 1). The number of worker pupae in T. recedens colonies was restricted; therefore, only five mixed colonies with T. recedens worker pupae were set up (Table 1). Mixed colonies were set up in early summer, when most larvae had developed into pupae. We transferred 50 worker pupae of the same species into a nest with either a queen from a different species in mixed colonies or a con-specific queen in control colonies (Table 1). To obtain the required sample size of 50 pupae, worker pupae were taken from five different con-specific colonies. No larvae or eggs were added to the colonies. To allow worker pupae to fully develop, we placed 30 marked adult workers from the colony of queen origin into each nest and removed them four weeks later after most of the pupae had developed into adult workers.
Several experimental colonies, in which the transferred worker pupae did not develop into adults (2005: 18 of 45 colonies; 2006, 8 of 100 colonies), had to be excluded from the study.
Worker ovary activation
In 2005, worker ovary activation was investigated in all colony set ups. In 2006, we investigated worker ovary activation in all colonies without a queen, all colonies with T. recedens workers and a queen from a different species, and five randomly chosen colonies of each of the remaining colony set ups (total N = 145 colonies; Table 1). The colonies were frozen six weeks after the transferred worker pupae had developed into adult worker, and workers and queens were dissected to assess their ovary activation [41]. Workers having elongated ovaries (> 1 mm) with viable, oval eggs similar in shape and color to those found in the ovaries of queens were classified as "fertile".
For statistical analyses, two sample permutation tests were used to assess the difference of numbers of fertile workers per colony between groups of control colonies and mixed colonies and between control colonies and colonies without a queen.
Male-production by workers
The remaining 65 colonies of the 2006 set up were kept in incubators (gradual decrease of temperature to 0°C night/10°C day for 15 weeks and gradual increase again to 17°C night/28°C day thereafter) until hibernated brood had developed in 2007 (Tables 1 and 2). From May to August 2007 all freshly enclosed adult males were collected and frozen at -20°C for further analyses. After all male pupae had enclosed, all colonies were frozen and queens were dissected to determine their ovarian status.
T. nylanderi, T. crassispinus and T. unifasciatus males are of dark brown pigmentation. T. recedens males have a pale pigmentation and could easily be distinguished from males of the other three species by inspecting their coloration. T. nylanderi, T. crassispinus and T. unifasciatus males are morphologically similar and thus were distinguished by electrophoresis of the glucose-6-phosoate isomerase [GPI; [27, 38]] or sequencing the mitochondrial cytochrome b (Cyt b) gene.
Allozyme analyses
Electrophoresis of glucose-6-phosoate isomerase for Temnothorax ants has been described previously [27]. Electromorphs were named according to their migration velocities in the gel (fast f; medium m; slow s). T. crassispinus and T. nylanderi are fixed almost completely for the electromorphs m and f, respectively [27, 38] and T. nylanderi occasionally exhibits the electromorph s [27]. In T. unifasciatus, 32 of 36 workers from 20 colonies were homozygous for the electromorph s and 4 were heterozygous with electromorph genotype sm [see also [42]]. Queens were analyzed when necessary.
The gasters of individual workers and queens were homogenized in 20 μl Tris-EDTA pH 7.0 buffer. Proteins were separated by 90 min electrophoresis at 10 V/cm and 20 mA on 10 cm × 8 cm × 0.75 mm 7.5% polyacrylamide slab gels using a Tris-glycine pH 8.3 buffer. The enzyme was stained using standard histochemical techniques [43].
Mitochondrial analyses
When males could not be distinguished by electrophoresis, we in addition sequenced the cytochrome b (Cyt b) gene. DNA was extracted from the gasters of males using the CTAB method (1%) as previously described [44]. The mitochondrial cytochrome b (Cyt b) gene was analyzed using the primers CbI (CB-J-10933) and 16Sar (LR-N-13398) [45]. The 20 μl PCR reaction mixture consisted of 1 μl DNA, 0.125 mM dNTPs, 0.25 μM of each primer, 11.1 μl dd H2O, 2 μl 10× PCR buffer (MBI), 2.5 mM MgCl2 and 1 μl of 1 unit/μl Taq Polymerase. Genes were amplified at an annealing temperature of 48°C with 38 cycles. PCR products were separated by electrophoresis on a 1% ethidiumbromide-stained agarose gel (TAE buffer) for 30 min at 100 mA and then purified with High Pure PCR cleanup Micro Kit (Roche). Cycle sequencing was carried out with 3 μl of purified PCR-Product using ABI-Cycle sequencing Kit Version 1.1. Single-stranded PCR products were sequenced using an ABI PRISM 310 automatic sequencer (Perkin-Elmer, Applied Biosystems). The first 450 base pairs of the Sequences representing the Cyt b gene were read and aligned with Sequencing Analysis Software version 3.4 (Perkin-Elmer, Applied Biosystems).
Cuticular hydrocarbons of queens and workers from different species of Temnothoraxants
To estimate the chemical distances between the four species of Temnothorax ants used for the mixed-species colony set ups, queens and workers from T. nylanderi, T. crassispinus, T. unifasciatus, and T. recedens were analyzed. For the identification of queen specific signals, queens and workers from two additional species, T. affinis and T. lichtensteini, were included in the analysis. From each species the queens of 5 to 10 unmanipulated colonies plus 1 to 3 workers from each of the colonies were chemically analyzed. All colonies were collected in spring 2006 (see above). T. unifasciatus colonies were used only from the population in Italy.
Chemical Analysis
Hydrocarbons were extracted four to five weeks after colonies had been subjected to artificial summer condition (17°C night/28°C day; see above). Workers were frozen and hydrocarbons were obtained through solvent extraction by individually immersing each worker for 10 min in 20 μl pentane. After evaporation of the solvent, the residues were re-dissolved in 15 μl pentane, of which 2 μl were injected into an Agilent Technologies 6890N gas chromatograph. Hydrocarbons of queens were obtained through SPME (Solid Phase Micro Extraction) which gives qualitative and quantitative similar results [46]. A 30 μm polydimethylsiloxane fiber was gently rubbed for 10 min against the gaster of the immobilized queen and injected into the injection port of the same gas chromatograph as above. The gas chromatograph was equipped with a flame ionization detector and a HP-5 capillary column (30 m × 0.32 mm × 0.25 μm, J&W Scientific, USA). The injector was split/splitless and the carrying gas was helium at 1 ml/min. The same temperature program was used for the solvent and the solid phase micro extraction with the temperature initially held at 70°C for 1 min, increased from 70°C to 180°C at 30°C/min, from 180°C to 310°C at 5°C/min, and held constant at 310°C for 5 min.
For identification of the peaks, the pooled extracts of 30 workers of each species were injected into a combined gas chromatography and mass spectrometry (GC-MS; Agilent Technologies 6890N) equipped with a RH- 5 ms+ fused silica capillary column (30 m × 0.25 mm × 0.25 μm, J&W Scientific, USA). The injector was split/splitless (250°C) with the purge valve opened after 60 sec and the carrying gas was helium at 1 ml/min. Temperature was held constant for 1 min at 60°C, increased from 60°C to 300°C at 5°C/min and held constant for 10 min at 300°C. The electron impact mass spectra (EI-MS; Agilent 5973 inert mass selective detector) were recorded with an ionization voltage of 70 eV, a source temperature of 230°C and an interface temperature of 315°C. We identified n-alkanes by comparing mass spectra with data from a commercial MS library (NIST, Gaithersburg, MD, USA). Methyl-alkanes were identified by diagnostic ions, standard MS databases (see above), and by determining Kovats indices by the method of Carlson et al. [47]. MSD ChemStation Software (Agilent Technologies, Palo Alto, CA, USA) for Windows was used for data acquisition.
For statistical analysis of the chemical distance between the four species involved in the mixed-species experiment, we included peaks consistently present in queens and workers of all four species, plus peaks with a relative area of more than 1% that were present in at least 50% of individuals in a group of workers or queens within each species. Standardized peak areas were transformed by square root. Principle coordinate (PCO) analyses based on Gower's centered matrix was used to visualize the patterns of differences in the multivariate chemical structure among groups [48–50]. Euclidean distance matrix was analyzed based on centroids of groups calculated from principle coordinates. PCO and Euclidean distance analyses were performed using the program PCO [49].
For the identification of queen specific signals we analyzed each species separately and included peaks consistently present in the groups of queens and workers within each species. Standardized peak areas were transformed by using the formula: Zij = log[Xi, j/g(Xj)], with Xi, j being the standardized peak area i for the sample j, and g(Xj) the geometric mean of all peaks of the sample j [51]. For multivariate analyses, the number of variables was reduced by principle component analysis (PCA). The factor scores obtained by PCA were used in a subsequent discriminant analyses (DA) to determine whether groups could be distinguished on the basis of their cuticular profiles. Wilks' λ significance and the percentage of correct assignments were used to evaluate the validity of the discriminant function. We used Mann-Whitney U-tests to compare percentages of single compounds between groups and adjusted p-values for multiple comparisons using Bonferroni's method. PCA and DA analyses were performed using Statistica 6.0.
