Thelytoky is a rare phenomenon resulting in worker-derived female offspring from unfertilized eggs. In the Cape honeybee, A. m. capensis, this mode of parthenogenesis is tightly linked to some other reproductive traits like an early onset of egg-laying (Anderson 1963; Ruttner and Hesse 1981; Moritz and Hillesheim 1985; Plettner et al. 1993) and the production of queen-like amounts of the queen mandibular gland pheromones (Hemmling et al. 1979; Crewe and Velthuis 1980; Moritz et al. 2002; Wossler 2002). Thus, under natural conditions with intra-colonial selection, thelytokous individuals producing the strongest queen-like pheromone signal with the highest amounts of 9-ODA will establish themselves as pseudoqueen and suppress the reproduction of other workers (Moritz et al. 1996, 2000, 2004; Härtel et al. 2011; Okosun et al. 2015). To avoid this intra-colonial arms race in our experiment, every laying A. m. capensis worker was tested individually, which is essential to eventually reveal a Mendelian segregation. We here show that workers produced by a multiply mated A. m. capensis queen heterozygous at the th locus (arrhenotoky; thelytoky) reproduce either by arrhenotoky or thelytoky. The two modes of parthenogenesis segregate well in a Mendelian 1:1 ratio predicted by a single recessive locus control.

In contrast, Chapman et al. (2015) reported that thelytokous parthenogenesis in A. m. capensis is under multi-locus control. However, a careful analysis of their dataset revealed their results largely confirm our findings and thus also the one locus model for worker thelytoky. The contrasting interpretation of the same dataset results from using the number and genotypes of the actual laying workers within a colony instead of the number of the overall offspring produced in the colony to assess the mode of inheritance of worker parthenogenesis. Using the overall number of female and male offspring produced within the whole colony will suffer from the introduction of a strong bias as not every worker lays exactly the same number of offspring in the colony and worker reproduction can be inhibited or suppressed by intra-colonial selection (Moritz et al. 1996, 2000, 2004; Härtel et al. 2011). Furthermore, re-counting the offspring of an individual will inflate the estimate for the allelic version present in the reproductive worker and hence is a pseudo-replication. If the actual number of reproductive individuals is considered, a distinct 1:1 segregation of both modes of parthenogenesis within colonies showing both arrhenotokous and thelytokous parthenogenesis can be observed. Only two experimental colonies (colony. 9 and 20) of Chapman et al. (2015) do not support the single locus model. According to the crossing scheme for colony 9 (Capensis × Scutellata) and the backcross scheme for colony 20 ((Capensis × Scutellata) × Scutellata) and under single locus control, these colonies should not produce any thelytokous offspring. Whereas in colony 9 (F1 generation) just a single individual did not match the single locus mode by reproducing via thelytoky, 13 individuals in colony 20 (F2 generation) produce female offspring when they should not according to the single locus model. Although we cannot exclude this outcome to be due to an incomplete penetrance of the thelytoky trait or due to a multi-locus control, we suspect this outcome to be most likely caused by insufficient control of the breeding individuals. In particular, the genotype of the putative A. m. scutellata drone 20 seems to be questionable. If this drone had carried the thelytoky allele instead of the arrhenotoky allele, the entire dataset is in agreement with the one locus model. Indeed, there is reason for concern because this drone carries an allele otherwise only found in the A. m. capensis lineage. Hence, we have to conclude that the choice of individuals, supposed to have a certain genotype at the th-locus, just based on their geographical origin may be insufficient at best. It ignores the possibility that the putative thelytoky allele may have been present in the Douglas (A. m. scutellata) region due to introgression of thelytoky alleles from the parasitic clone present there (Neumann et al. 2011). It is crucial to test the incidence of the thelytoky/arrhenotoky alleles in the mother colony, and it is crucial to confirm that the chosen drone is offspring of the queen heading the colony. Furthermore, the choice of breeding individuals by geography ignores the occurrence of the arrhenotoky allele in the A. m. capensis population at the Cape of Good Hope. Although its frequency might be low, two independent studies found a queen that carries the arrhenotoky allele at the th-locus (Beekman et al. 2009; this study). Therefore, it might be possible that the A. m. capensis and A. m. scutellata queens and drones used for crossing may have carried either the arrhenotoky or the thelytoky allele respectively.

Taken together, we see little merit in rejecting the single-locus inheritance of thelytoky in A. m. capensis. First, because it requires the fewest assumptions it remains the most parsimonious explanation following Occam’s razor ‘Pluralitas non est ponenda sine neccesitate’. Only if it is falsified by evidence it should be refused. Second, the single locus control is further supported by the lack of amphitoky (intermediate mode of parthenogenesis) of laying workers in both studies discussed here, as well as in Ruttner (1988) and Lattorff et al. (2005). Third, the clear-cut 1:1 segregation pattern and the occurrence of both types of parthenogenesis within a single patriline rule out epigenetic mechanisms or paternal factors determining the mode of parthenogenesis in A. m. capensis. Also, maternal effects other than the alleles of the Mendelian locus can safely be excluded, because otherwise there should be no polymorphism at all among the workers tested in our study. Moreover, a single locus control has also been suggested in other thelytokous Hymenoptera. The allelic origin of asexual reproduction in the parasitoid wasp Lysiphlebus fabarum appears to be controlled by a single locus (Sandrock and Vorburger 2011).

However, we also found that the previously claimed genomic region around gemini on chromosome 13 (Lattorff et al. 2005, Jarosch et al. 2011) does not harbour the locus switching between thelytoky and arrhenotoky in the current study. Nevertheless, although gemini is not the genetic switch for thelytoky in this experimental population, both gemini and the 9-bp deletion (tae1) may be involved in controlling the reproductive capacity in female honeybees which is tightly interwoven in the complex phenotype of the ‘thelytoky syndrome’ (Lattorff and Moritz 2013). The detection of the actual ‘thelytoky switch’ will require a concise mapping population coupled with a much higher marker coverage than possible with microsatellite techniques, which is now entirely feasible using SNP marker-based methods (e.g. Stolle and Moritz 2013).