LAUSR

Significance A defining goal in genetics is linking variation in DNA sequence to trait evolution between populations and, ultimately, species. Genome sequencing efficiently captures such variation but typically in millions of tiny fragments that omit haplotype or linkage information. We present “haplotagging,” a simple, rapid linked-read sequencing technique that allows high-throughput sequencing without sacrificing haplotype information. We validated this affordable approach for whole-genome haplotyping in large populations. We used haplotagging to investigate the rise of a novel hybrid morph in parallel hybrid zones of two comimetic Heliconius butterfly species in Ecuador. Our results reveal that strikingly parallel divergences in their genomes produced coordinated shifts in haplotype frequencies across the hybrid zone, giving rise to comimetic hybrid morphs in each species. Genetic variation segregates as linked sets of variants or haplotypes. Haplotypes and linkage are central to genetics and underpin virtually all genetic and selection analysis. Yet, genomic data often omit haplotype information due to constraints in sequencing technologies. Here, we present “haplotagging,” a simple, low-cost linked-read sequencing technique that allows sequencing of hundreds of individuals while retaining linkage information. We apply haplotagging to construct megabase-size haplotypes for over 600 individual butterflies (Heliconius erato and H. melpomene), which form overlapping hybrid zones across an elevational gradient in Ecuador. Haplotagging identifies loci controlling distinctive high- and lowland wing color patterns. Divergent haplotypes are found at the same major loci in both species, while chromosome rearrangements show no parallelism. Remarkably, in both species, the geographic clines for the major wing-pattern loci are displaced by 18 km, leading to the rise of a novel hybrid morph in the center of the hybrid zone. We propose that shared warning signaling (Müllerian mimicry) may couple the cline shifts seen in both species and facilitate the parallel coemergence of a novel hybrid morph in both comimetic species. Our results show the power of efficient haplotyping methods when combined with large-scale sequencing data from natural populations.