Divergent mitonuclear coadaptation could facilitate speciation. We investigate this possibility in two hybridizing species of warblers, Setophaga occidentalis and S. townsendi, in western North America. Inland S. townsendi harbor distinct mitochondrial DNA haplotypes from those of S. occidentalis. These populations also differ in several nuclear DNA regions. Coastal S. townsendi demonstrate mixed mitonuclear ancestry from S. occidentalis and inland S. townsendi. Of the few highly-differentiated chromosomal regions between inland S. townsendi and S. occidentalis, a 1.2 Mb gene block on chromosome 5 is also differentiated between coastal and inland S. townsendi. Genes in this block are associated with fatty acid oxidation and energy-related signaling transduction, thus linked to mitochondrial functions. Genetic variation within this candidate gene block covaries with mitochondrial DNA and shows signatures of divergent selection. Spatial variation in mitonuclear ancestries is correlated with climatic conditions. Together, these observations suggest divergent mitonuclear coadaptation underpins cryptic differentiation in this species complex.

23Mitochondrial (mtDNA) and nuclear (nDNA) genes interact to govern metabolic 24 pathways of mitochondria. When differentiated populations interbreed at secondary 25 contact, incompatibilities between mtDNA of one population and nDNA of the other 26 could result in low fitness of hybrids. Hermit Warblers (S. occidentalis abbreviated as 27 HEWA) and inland Townsend's Warblers (Setophaga townsendi, abbreviated as i-28 TOWA) exhibit distinct mtDNA haplotypes and a few nDNA regions of high 29 differentiation, whereas coastal TOWA (c-TOWA) displays a mix of these genetic 30 patterns consistent with ancient hybridization of HEWA and i-TOWA. Of the few highly-31 differentiated nDNA regions between i-TOWA and HEWA, two of these regions (on 32 chromosome 5 and Z, respectively) are also differentiated between c-TOWA and i-33 TOWA, similar to the mtDNA pattern. These two nDNA regions are associated with 34 mitochondrial fatty acid metabolism. Moreover, these nDNA regions are correlated with 35 mtDNA ancestries among sites, a pattern consistent with mito-nuclear co-adaptation. 36Such mito-nuclear coevolution might be driven by climate-related selection, because the 37 mito-nuclear ancestry is correlated with climatic conditions among sampling sites. These 38 results suggest that cryptic differentiation in this species complex has been shaped by 39 climate-correlated adaptation associated with mito-nuclear fatty acid metabolism. 40 41

Setophaga townsendi is a species of wood warbler (family Parulidae) in northwestern North America that has geographic structure in the mitochondrial and nuclear genomes: while interior populations have differentiated mitonuclear ancestry from the sister species S. occidentalis, coastal populations have a mix of inland and S. occidentalis mitonuclear ancestries. This coastal to inland transition in genomic ancestry raises the possibility of similar geographic structure in phenotypic traits, especially those involved in mate choice. Using qualitative and multivariate approaches, we investigated whether there is a sharp transition between coastal and inland populations in both song and in nuclear DNA. We find there is a shallow geographic cline in Type I song but not in Type II song. Nuclear DNA shows a gradient between coast and inland. There is little correlation between variation in song and the isolation-by-distance pattern in the nuclear DNA. Learned songbird song is shaped by both genetic and cultural processes. There has been a debate on whether song learning promotes or slows down population differentiation. By comparing the within-species variation in song and genetic structures, we can expand our understanding of the dynamic interplay between mating signals and population differentiation.