Genetic evidence for hybrid trait speciation in heliconius butterflies

Abstract

Homoploid hybrid speciation is the formation of a new hybrid species without change in chromosome number. So far, there has been a lack of direct molecular evidence for hybridization generating novel traits directly involved in animal speciation. Heliconius butterflies exhibit bright aposematic color patterns that also act as cues in assortative mating. Heliconius heurippa has been proposed as a hybrid species, and its color pattern can be recreated by introgression of the H. m. melpomene red band into the genetic background of the yellow banded H. cydno cordula. This hybrid color pattern is also involved in mate choice and leads to reproductive isolation between H. heurippa and its close relatives. Here, we provide molecular evidence for adaptive introgression by sequencing genes across the Heliconius red band locus and comparing them to unlinked wing patterning genes in H. melpomene, H. cydno, and H. heurippa. 670 SNPs distributed among 29 unlinked coding genes (25,847bp) showed H. heurippa was related to H. c. cordula or the three species were intermixed. In contrast, among 344 SNPs distributed among 13 genes in the red band region (18,629bp), most showed H. heurippa related with H. c. cordula, but a block of around 6,5kb located in the 3' of a putative kinesin gene grouped H. heurippa with H. m. melpomene, supporting the hybrid introgression hypothesis. Genealogical reconstruction showed that this introgression occurred after divergence of the parental species, perhaps around 0.43Mya. Expression of the kinesin gene is spatially restricted to the distal region of the forewing, suggesting a mechanism for pattern regulation. This gene therefore constitutes the first molecular evidence for adaptive introgression during hybrid speciation and is the first clear candidate for a Heliconius wing patterning locus.

Figure 1. Relative likelihood of association between H. heurippa and H. m. melpomene versus H. c. cordula for (A) genes unlinked to color pattern and (B) across the HmB red color pattern locus.

Likelihood values are plotted for each variable position, where positive likelihood values indicate a SNP position at which H. heurippa and H. m. melpomene are more similar, and negative values a position at which H. heurippa shows a stronger association with H. c. cordula. Fixed sites are indicated by dotted lines showing a likelihood value of 244 for a complete association of H. heurippa with H. m. melpomene and −244 for that with H. c. cordula. Colors represent different coding regions. The majority of unlinked SNPs (634) show shared polymorphism among the three species (240> ΔLnL >−240). At unlinked loci, H. heurippa and H. c. cordula shared fixed polymorphism at only 8 SNPs whereas H. heurippa and H. m. melpomene did not share any fixed polymorphism. For the HmB locus, sequenced BAC clones are indicated above the gene annotation .

Figure 2. Distribution of average likelihood values at SNPs in unlinked and HmB linked loci.

The average of likelihood values for each unlinked marker and for 1,000 bp windows in the HmB region was calculated. This histogram shows the distribution of these values. Dotted lines represent the 95% two-tailed interval for unlinked genes. The asterisk over the bars indicates those 1,000 bp windows showing average values that lie outside the unlinked genes distribution (p<0,005). Positive values outside that distribution correspond to 3′ kinesin whereas negative values are those in kin_2.

Figure 3. HmB linkage disequilibrium analysis.

Pairwise estimates of linkage disequilibrium (r²) among 199 SNPs in the HmB locus (those with a rare allele frequency less than 10% were excluded) for combined H. c. cordula and H. heurippa population samples. Physical distance between sites is shown in the adjacent map.

Figure 4. Gene genealogies for 3′ kinesin and its flanking regions.

(A) sorting nexin (sdp); (B) 3′ kinesin; and (C) 5′ kinesin partial sequence (kin_2). Filled color circles represent alleles of each species. The 3′ kinesin tree is rooted with H. numata as outgroup (black). Numbers above and below the branches are bootstrap support values for likelihood and parsimony analyses respectively. (B) shows H. heurippa most closely related to H. m. melpomene, while (A,C), the genomic regions surrounding 3′ kinesin, show H. heurippa alleles more closely related to H. c. cordula. A similar tree topology was obtained from an amino acid alignment (data not shown).

Figure 5. Expression pattern of kinesin in forewings.

Of (A) H. m. cythera and (B) H. cydno. A similar expression pattern to that present in (A) is also observed in H. m. rosina forewings (Figure S3), consistent with the red band phenotype of these two races. The lack of any localized kinesin expression in H. cydno forewings is consistent with the absence of a red band in this species. (C) Model of how kinesin expression (K, solid line), might interact with an unknown gene (X, dotted line) to regulate forewing red band expression.