Gene flow persists millions of years after speciation in Heliconiusbutterflies
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Hybridization, or the interbreeding of two species, is now recognized as an important process in the evolution of many organisms. However, the extent to which hybridization results in the transfer of genetic material across the species boundary (introgression) remains unknown in many systems, as does the length of time after initial divergence that the species boundary remains porous to such gene flow.
Here I use genome-wide genotypic and DNA sequence data to show that there is introgression and admixture between the melpomene/cydno and silvaniform clades of the butterfly genus Heliconius, groups that separated from one another as many as 30 million generations ago. Estimates of historical migration based on 523 DNA sequences from 14 genes suggest unidirectional gene flow from the melpomene/cydno clade into the silvaniform clade. Furthermore, genetic clustering based on 520 amplified fragment length polymorphisms (AFLPs) identified multiple individuals of mixed ancestry showing that introgression is on-going.
These results demonstrate that genomes can remain porous to gene flow very long after initial divergence. This, in turn, greatly expands the evolutionary potential afforded by introgression. Phenotypic and species diversity in a wide variety of organisms, including Heliconius, have likely arisen from introgressive hybridization. Evidence for continuous gene flow over millions of years points to introgression as a potentially important source of genetic variation to fuel the evolution of novel forms.
KeywordsGene Flow Triose Phosphate Isomerase Introgressive Hybridization Mixed Ancestry AFLP Locus
Hybridization has long been recognized as an important mechanism of diversification in plants [1, 2], and the exchange of genetic material via horizontal gene transfer has played a significant role in the evolution of many prokaryotic genomes . In animals however, hybridization has historically been viewed as rare and evolutionarily inconsequential . Despite this bias in opinion, we now know that hybridization is relatively wide-spread among animal species , and in some instances, it has likely had important evolutionary ramifications, such as in the origin of new species [6, 7, 8, 9, 10]. Surveys of hybridization in animals show that it occurs predominantly between closely-related sister species , and well-characterized examples of interspecific gene flow generally involve species that diverged very recently [10, 11, 12, 13]. These observations are consistent with theory and data which show that genetic incompatibilities that result in hybrid sterility and inviability accumulate as species diverge . Hybrid sterility and inviability, in turn, reduce or eliminate the opportunity for gene exchange. Despite these general trends, there are occasional examples of hybridization between distantly-related non-sister species in various animal groups [15, 16, 17, 18, 19, 20]. Do these cases result in the long-term sharing of genetic material or are they simply evolutionary dead-ends?
While the existence of rare hybrids between the melpomene/cydno and silvaniform clades provides a potential avenue for gene flow between them, it is unknown whether introgression occurs over these large phylogenetic distances. To determine whether these distantly related groups continue to exchange genes, I used two complementary population genetic datasets to measure the extent of historical gene flow and contemporary admixture between sympatric populations of the two clades.
Results and Discussion
Historical migration inferred from DNA sequence data
It is important to note that the evolutionary history of the DNA sequences studied here violates one of the underlying assumptions of the Isolation with Migration model. The IM model assumes that the two populations being examined are sister-taxa, each being more closely related to the other than either is to any other population. That is clearly not the situation with these inter-clade comparisons. However, if the sampled species provide an unbiased approximation of the genetic divergence between the two groups under study (the silvaniform and melpomene/cydno clades in this case), then it seems reasonable to model the system under the Isolation with Migration framework. The fact that analyses based on independent data (AFLPs, see below) are consistent with the IM results lends support to both the approach and the results.
Gene flow has a variable influence across the genome
AFLP based estimates of mixed ancestry. Admixture proportions (with 95% confidence intervals) and ancestry probabilities for the four individuals that exhibited evidence of between-clade mixed ancestry.
Proportion of genome derived from:
Probability of pure ancestry
Probability of mixed ancestry with the opposite clade
Gene flow has persisted for millions of years after speciation
Together, these population genetic data are consistent with a history of divergence with gene flow between the melpomene/cydno and silvaniform clades. Average pairwise mtDNA divergence between these two groups is 5.7% (SE = 0.5%). Using the estimate of 1.1 – 1.2% divergence per lineage per million years , this equates to approximately 2.5 million years of divergence. With a minimal Heliconius generation time of one month, this represents as many as 30 million generations of evolution along each lineage since the speciation event that precipitated cladogenesis.
These data suggest that the process of divergence that ultimately results in reproductively isolated species can be prolonged. The fact that genomes can remain open to gene flow very long after the speciation process is initiated greatly expands the evolutionary novelty that can be generated from introgression. Some portion of the phenotypic and species diversity in Heliconius has very likely arisen from introgressive hybridization [6, 26]. The results presented here suggest that the melpomene/cydno and silvaniform clades of Heliconius have experienced continuous gene flow over millions of years. Thus, introgression has had the potential to provide a ready source of genetic variation to fuel this expansive adaptive radiation. For many organisms, even rare hybridization with distantly related species may allow for the continued exchange of genetic material which may serve as a long-term source of variation for adaptive change.
I collected 56 H. cydno, 44 H. pachinus, 27 H. melpomene, and 44 H. hecale individuals from various locations throughout Costa Rica. None of the sampled individuals exhibited phenotypic evidence of introgression from the opposite clade. All specimens were collected as adults in the summer of 2000 and 2002. Tissue was preserved in 95% ethanol and DNA was extracted with a DNeasy Tissue Kit (Qiagen) or using a phenol/chloroform extraction protocol.
DNA sequencing and analysis
I sequenced multiple haplotypes for one mitochondrial locus and 13 nuclear loci from the four species using primers and methods described previously [24, 33]. The loci were apterous (ap), cubitus interruptus (ci), cytochrome oxidase I &II (CO, mtDNA), Distal-less (Dll), elongation factor 1α (ef1α), engrailed (en), invected (unv), Mannose phosphate isomerase (Mpi), patched (ptc), scalloped (sd), scarlet (st), Triose phosphate isomerase (Tpi), white (w), and wingless (wg). I also sequenced portions of the genes cinnabar  and decapentaplegic  but these loci were excluded from the analyses because only one H. hecale sequence was obtained for each. All sequences have been deposited in GenBank under accession numbers AY744577–AY744672, AY745254–AY745278, AY745315–AY745335, AY745356–AY745490, DQ448305–DQ448516 and EF041105–EF041122. For analysis, the datasets for CO,ef1α,Mpi, and Tpi were supplemented with published sequences from GenBank .
I used the comparative DNA sequence data and the Isolation with Migration model implemented in IM  to infer historical rates of between-species gene flow. IM cannot handle alignment gaps or evidence of recombination in DNA sequence data. Therefore, I removed gaps and searched for evidence of recombination using the four-gamete test in DnaSP 3.5 . For those loci that exhibited evidence of recombination, the sequence alignment was divided into portions that showed no evidence of recombination and only one portion was included in the analysis. In an effort to preserve as much genealogical information as possible, the portion with the most polymorphisms was chosen. The size of these non-recombining portions ranged from 60 bp for st in the H. cydno/H. hecale comparison to 1111 bp for wg in the H. melpomene/H. hecale comparison.
For each comparison between H. hecale and one of the three melpomene/cydno clade species, I ran IM in two different ways. First, I used the data for all loci to estimate a single pair of bidirectional population migration rates (Figure 2a–c). Then, using the same dataset, I allowed each locus to have a separate pair of population migration rates (Figure 3). For each analysis, IM was run with 10 Metropolis-coupled chains for 300,000 burn-in steps followed by 2 × 107steps of data collection. Following other implementations of IM , I used the HKY model for the mitochondrial locus and the infinite-sites model for the nuclear loci.
I used published DNA sequences from six genes  and MrBayes  to estimate a phylogeny of the silvaniform and melpomene/cydno clades of Heliconius. Four Metropolis-coupled Markov chains were run for 250,000 burn-in generations followed by 1.75 × 106 generations of data collection. The age of the melpomene/cydno and silvaniform split was estimated based on 1606 base pairs of mtDNA from ref. 36, assuming an evolutionary rate of 1.15% per lineage per million years . Gene genealogies for ci and w were also estimated using MrBayes, based on the GTR+I+Γ model of molecular evolution.
AFLP genotyping and analysis
Using the ABI Plant Mapping Kit (PE Applied Biosystems), I genotyped each individual with four selective AFLP primer combinations; EcoRI-ACT/MseI-CAT, EcoRI-ACT/MseI-CTG, EcoRI-ACA/MseI-CAT, and EcoRI-ACA/MseI-CTG. AFLP fragments were separated using an ABI 3100 automated genotyper and then scored using ABI GENEMAPPER software version 3.7. In total, each individual was typed for the presence or absence of a fragment at 925 AFLP loci. In an effort to focus on the most informative and reliable markers, only the 520 AFLPs that had a minor allele frequency ≥ 0.05 were used in further analyses.
I used the AFLP data and the Bayesian-based genetic clustering program STRUCTURE 2.2 [30, 31] to define populations and estimate admixture among them. The data were analyzed in two ways. First, I performed naïve admixture clustering assuming four populations. As part of this analysis, I estimated the 95% posterior probability interval around each individual admixture proportion. Second, I performed an additional round of admixture clustering after first indicating the population of origin for each individual and setting the prior probability of pure ancestry to 0.95. As part of this analysis, I estimated the posterior probability that each individual was misclassified or had an ancestor from each of the other species within the last three generations. For both analyses, STRUCTURE was run for 10,000 burn-in generations followed by 106 generations of data collection.
I thank Larry Gilbert, Ulrich Mueller, Joan Strassmann, Dave Queller, Lauren Blume, Laura Young, Andres Vega, and Kenny Kronforst for facilitating this research. I also thank reviewers for comments on the manuscript. This work was funded by National Science Foundation Grants DEB 0415718 & DEB 0640512.
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