Fig 1.
Graphical summary of the unusual reproduction system of V. emeryi and the experimental design.
Females and males are diploid and haploid, respectively, as indicated by the number of oval cartoon chromosomes, which are colored red and blue in queens (a) and males (b) in the parental generation (F0). V. emeryi reproduce clonally (via thelytoky and androgenesis), as well as sexually, with all possible offspring shown in F1. These reproductive modes produce four types of offspring: clonal queens by thelytoky (c), queens and workers by sexual reproduction (d and e), and androgenetically produced clonal males (f) that are identical to their fathers despite developing from queen-laid eggs [22] [20,21]. The experimental cross (encircled by a dashed line), which is genetically identical to a backcross, mated sexually produced queens (e) and clonal males (f) in the F1 generation and scored diploid male production in the F2 generation. The F2 offspring are not depicted in this diagram.
Table 1.
Offspring produced by sib-mated queens.
Fig 2.
Linkage mapping confirms the existence of two-locus CSD in V. emeryi.
Marker locations are shown by short dashes along the x-axis of the plot. A red line shows the permuted 95% significance level of the LOD score. This genome scan found two loci, named CsdQTL1 and CsdQTL2, on LG13 and LG14, respectively. These two loci and their interaction explained 98.0% of the observed phenotypic variance. The two-locus model obtained by QTL analysis is consistent with sex ratio data from the experimental crosses, which find a quarter of diploid males in the F2 offspring (Table 1).
Fig 3.
CsdQTL1 contains csd/fem with separate alleles in males and queen clones.
(A) Locus CsdQTL1 contained two tandem copies of tra homologs on the same scaffold. (B) Males and queens have different copies of both traA and traB, but the former gene is more diverse. Nodes with 100% bootstrap support are shown with double asterisks and nodes with 90–99% support with single astersks. These data suggest that the traA and and traB copies diverged before male and female clones separated from each other. As a result, each mating produces workers heterozygous at this locus, which was also consistent with linkage mapping and diploid male production data. The traA gene has higher diversity than traB and weak evidence of diversifying selection (S3 Fig), suggesting by analogy with honey bees, that this is the homolog of csd, while traB is the homolog of fem. Scale bar is in substitutions per site.
Table 2.
Genes located at CsdQTL2.
Fig 4.
Graphical summary of the results, with different CSD alleles color-coded.
QTL mapping (Fig 2) and F2 diploid male ratios (Table 1) both support a two-locus model of sex determiation in the workers. CsdQTL1, which has homologs of csd and fem, has diverged between male and female clones (Fig 3), assuring heterozygosity at that locus in F1 females. Both tra homologs are homozygous in clonal queens (F0), suggesting that if queens use the same sex determination mechanism as do workers, CsdQTL2 should be heterozygous. However, none of the exonic regions at CsdQTL2 are heterozygous in all of the queen samples. If this result is not a technical artifact, it suggests either thtat queens use a separate sex detemination mechanism, or that the sex determination mechanism in V. emeryi does not depend on heterozygosity in protein sequence at CsdQTL2. In any case, the molecular mechanism at CsdQTL2 remains a mystery.