A test of Haldane’s rule in Neodiprion sawflies and implications for the evolution of postzygotic isolation in haplodiploids

Haldane’s rule—a pattern in which hybrid sterility or inviability is observed in the heterogametic sex of an interspecific cross—is one of the most widely obeyed rules in nature. Because inheritance patterns are similar for sex chromosomes and haplodiploid genomes, Haldane’s rule may apply to haplodiploid taxa, predicting that haploid male hybrids will evolve sterility or inviability before diploid female hybrids. However, there are several genetic and evolutionary mechanisms that may reduce the tendency of haplodiploids to obey Haldane’s rule. Currently, there are insufficient data from haplodiploids to determine how frequently they adhere to Haldane’s rule. To help fill this gap, we crossed a pair of haplodiploid hymenopteran species (Neodiprion lecontei and Neodiprion pinetum) and evaluated the viability and fertility of female and male hybrids. Despite considerable divergence, we found no evidence of reduced fertility in hybrids of either sex, consistent with the hypothesis that hybrid sterility evolves slowly in haplodiploids. For viability, we found a pattern opposite of Haldane’s rule: hybrid females, but not males, had reduced viability. This reduction was most pronounced in one direction of the cross, possibly due to a cytoplasmic-nuclear incompatibility. We also found evidence of extrinsic postzygotic isolation in hybrids of both sexes, raising the possibility that this form or reproductive isolation tends to emerge early in speciation in host-specialized insects. Our work emphasizes the need for more studies on reproductive isolation in haplodiploids, which are abundant in nature, but under-represented in the speciation literature.

Methods
Adult sex ratio data were collected from laboratory rearing logs from 2013-2017 during times when both species and hybrids were being reared in the lab. Hand-written data were transcribed into an excel file and checked for accuracy before analysis in R. 

Embryo viability data were collected from laboratory rearing logs and experimental notebooks for an interspecific cross in which we recorded egg number and counted hatchlings 48 hours after hatching started. Hand-written data were transcribed into an excel file and checked for accuracy before analysis in R. 

Female fertility data were collected from laboratory rearing logs and experimental notebooks in which we recorded whether or not eggs were laid and total egg number laid by pure-species and hybrid females. Hand-written data were transcribed into an excel file and checked for accuracy before analysis in R.  

Male fertility data were recorded in experimental notebooks as sperm were observed under a microscope. These hand-written data were transcribed into an excel file and checked for accuracy before analysis in R. We also used laboratory rearing notebooks to verify that hybrid males produced viable female offspring. 

Male behavioral sterility data were recorded in experimental notebooks during mating assays and then transcribed into an excel file and checked for accuracy before  analysis in R.