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.

How species evolve reproductive isolation in the species-rich Amazon basin is poorly understood in vertebrates. Here, we sequenced a reference genome and used a genome-wide sample of SNPs to analyze a hybrid zone between two highly cryptic species of Hypocnemis warbling-antbirds-the Rondonia warbling-antbird (H. ochrogyna) and Spix's warbling-antbird (H. striata)-in a headwater region of southern Amazonia. We found that both species commonly hybridize, producing F 1 s and a variety of backcrosses with each species but we detected only one F 2-like hybrid. Patterns of heterozygosity, hybrid index, and interchromosomal linkage disequilibrium in hybrid populations closely match expectations under strong postzygotic isolation. Hybrid zone width (15.4 km) was much narrower than expected (211 km) indicating strong selection against hybrids. A remarkably high degree of concordance in cline centers and widths across loci, and a lack of reduced interspecific F st between populations close to versus far from the contact zone, suggest that genetic incompatibilities have rendered most of the genome immune to introgression. These results support intrinsic postzygotic isolation as a driver of speciation in a moderately young cryptic species pair from the Amazon and suggest that species richness of the Amazon may be grossly underestimated.

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