The evolution of sexual isolation during speciation depends on a joint change in male sexual traits and female preference for those traits 
. Theoretical work has identified several genetic conditions favouring this process, such as sex linkage and spatial clustering of genes underlying species-specific sexual signalling systems, 
. Our empirical knowledge of the genetics underlying male secondary sexual traits is increasing 
, but the genetics underlying female choice mechanisms, causing biases in male fertilization success 
, remain largely unexplored. Sex linkage of the genes underlying female choice mechanisms should lead to increased potential rate of sequence divergence in response to selection 
and favours processes such as reinforcement 
and good genes sexual selection 
, but not Fisherian runaway selection 
. The spatial clustering of mate choice genes affects inter-taxon recombination during periods of contact and gene exchange and can mitigate the homogenising effects of gene flow 
. Therefore taxa whose female choice genes are more sex-linked and/or more highly clustered are expected to be more prone to speciation, other things being equal.
Biases in male reproductive success may be caused by multiple female choice components (; Text S1
) that together can have an overriding influence on reproductive isolation between populations 
. From a mechanistic point of view, female mate choice is the product of the interplay between neurological and physiological processes, which in turn are regulated by gene expression patterns during courtship and mating. An important step in understanding the link between mate choice and speciation is therefore to understand how female gene expression patterns affect reproductive isolation. Gene expression studies do not rely on pre-existing genetic divergence to reveal mechanistic associations between genes and traits, making them ideal for identifying genes that are potential targets for future divergence. Establishing the identity of the genes underlying plastic female responses to males belonging to their own population versus other populations can be used to make predictions of (i) the potential for future divergence, (ii) the rate by which divergence may proceed, and (iii) what evolutionary processes are likely to be driving their evolution.
Mechanisms of mate choice influencing sexual isolation.
from outside sub-Saharan Africa (cosmopolitan or M strain) are thought to have diverged from southern African strains during their spread around the world as human commensals within the last 10,000 years 
. They are genetically depauperate and the majority of their genetic variation is thought to be a subset of that found in Africa 
, although a number of fixed differences exist (10) and mean FST
between European M strain and Zimbabwean (Z strain) D. melanogaster
is 0.23 
. Lineages such as these that are at an early stage of evolving reproductive isolation may serve as important model systems for studying speciation through divergence in sexual signalling systems. There is partial reproductive isolation between populations of Drosophila melanogaster
from Zimbabwe (Z strain) and from the rest of the world (M strain) 
; M strain females show no apparent pre-copulatory preferences for M males but Z strain females prefer Z males. Sperm-egg incompatibilities also exist when females from ‘strong Z’ isofemale lines (those with strong sexual preference for Z males) mate with M strain males, but not vice versa 
. In this study we use mate choice experiments and gene expression analysis in Z strain female D. melanogaster
to examine three key components influencing speciation: (i) which of the known mate choice mechanisms in Drosophila
are likely to be involved in Z female discrimination against M males (i.e. that play a role in relation to sexual isolation), (ii) the degree of sex linkage of the candidate mate choice genes involved in sexual isolation; and (iii) the physical clustering of these genes. Thus, we view female discrimination of males belonging to other populations than their own as a composite trait (i.e. the result of a joint action of several mate choice mechanisms) and use gene expression analysis to establish a candidate set of genes underlying this composite trait. The candidate set of genes is subsequently used to evaluate the potential for evolution of stronger sexual isolation (i.e. stronger discrimination against males not belonging to their own population).
By identifying genes differentially expressed between Z strain females mated to preferred Z males versus less preferred M males, we find that (i) tissue specificity patterns of the identified candidate genes indicate the action of multiple mate choice mechanisms involved in sexual isolation, (ii) candidate mate choice genes involved in sexual isolation cluster disproportionately on the X chromosome, and (iii) they form several tight physical clusters on the X chromosome and the autosomes. These conditions are expected to lead to faster evolution than would be the case with few genes involved in mate choice and little X linkage, plus a greater possibility for divergence in sympatry in certain genomic regions. We therefore conclude that mate choice may act as an even more powerful engine of speciation than previously realized.