NEW YORK (GenomeWeb News) – In a study appearing online today in Nature, a Swedish team describes the genome sequencing approach that it used to study the genetics of speciation in two closely related bird species.
Researchers from Uppsala University started by generating a flycatcher reference genome using DNA from a collared flycatcher, Ficedula albicollis. They then resequenced the genomes of 10 unrelated collared flycatchers and 10 pied flycatchers from the species F. hypoleuca, believed to have diverged from collared flycatchers within the past two million years.
Together with transcriptome data from several bird tissues, these genome sequences highlighted the extensive genetic similarities that exist between birds from each species. But it also uncovered several dozen sites in the flycatcher genome where sequence divergence was especially high and the proportion of shared polymorphisms was very low for the two bird species.
These regions tended to turn up in parts of the genome suspected of housing centromeres and other repeat-rich sequences, rather than parts of the flycatcher genome that directly code for proteins.
"We have hypothesized that what we are seeing is signals from centromeric regions," the study's first author Hans Ellegren, an evolutionary biology researcher at Uppsala University, told GenomeWeb Daily News.
"That would argue that there is something related to how chromosomes separate or segregate during meiosis," he explained, "since the centromeres are the sort of anchor points for spindle fibers which bind chromosomes and pull them apart during segregation."
Given the level of conservation described for centromeres-related processes, Ellegren added, such meiotic effects might theoretically represent "a very general and common mechanism for explaining speciation."
Collared flycatchers and pied flycatchers are known to breed with one place where their ranges overlap, including parts of the Czech Republic and Bulgaria and islands in the Baltic Sea, Ellegren explained. But hybrid offspring from these matings tend to have poor fitness and fertility, pointing to some level of reproductive incompatibility between the species.
Aspects of divergence between the two bird species have been explored in the past, particularly since the flycatchers' relationship to one another — and their propensity for breeding in easy-to-access nest boxes — make them attractive research models. Nevertheless, little to no research has been done on the genetic and genomic events underlying this species divergence.
"When it comes to the genetic basis [of flycatcher speciation], essentially nothing was known," Ellegren said.
To begin exploring this, he and his colleagues used Illumina technology to tackle the flycatcher genome. From paired-end and mate-pair reads generated for a wild male collared flycatcher, they assembled a 1.1 billion base flycatcher reference genome de novo. This sequence was physically mapped to individual chromosomes with the help of a low-density linkage map.
The team also did transcriptome sequencing on around 10 flycatcher tissues, including tissues collected during embryonic development, as well as samples representing adult somatic and gonadal tissues. In addition, the zebra finch genome, published in 2010, proved useful not only for helping to define flycatcher coding genes, but also for some of the chromosome-level analyses done for the new study.
In an effort to identify genetic players in flycatcher speciation, the team resequenced 10 male collared flycatcher genomes and as many male pied flycatcher genomes, each to a mean depth of around 5.7-fold coverage.
When they compared these sequences to one another and to the newly sequenced flycatcher reference genome, researchers saw that collared flycatchers and pied flycatchers were even more similar genetically than expected, Ellegren explained. For instance, roughly half of the nearly 10 million SNPs identified in the birds' genome sequences were shared between the two species, he said, pointing to incomplete lineage sorting.
Even so, around 50 regions in the genome showed particularly high levels of divergence in the bird species. These areas, dubbed "divergence islands," contained far fewer shared SNPs and higher levels of linkage disequilibrium than the rest of the genome, the researchers reported. The nucleotide diversity and allele frequency patterns were also distinct from those generally observed in the genome.
"In contrast to the low background or baseline level of divergence, we found some localized regions that had up to 50 times higher divergence than the average background level," Ellegren said.
While these divergence islands turned up on all of the autosomal chromosomes, their dispersal doesn't appear to be random, he noted, since each chromosome had between one to three divergence islands regardless of whether it was a very large chromosome or one of the birds' minute microchromosomes.
Moreover, many of these sites appeared in parts of the flycatcher reference genome that failed to assemble properly or else at the ends of chromosomes where centromeric sequences have been described in other birds. Still other divergent areas seemed to overlap with telomeres.
Together, the study's authors explained, these features hint that repeat structures in the flycatcher genome may have had a role in the selective processes that happened in parallel in F. hypoleuca and F. albicollis as each became a distinct species.
On the other hand, when they focused on the "Z" sex chromosome, investigators did not see the sorts of divergence peaks present across the autosomal chromosomes. Instead, baseline divergence across the sex chromosome was generally much higher than it was in other parts of the genome — on par with the level of separation found in the autosomal divergence islands.
"That suggests that there is something about the sex chromosomes that is particularly difficult when it comes to gene flow," Ellegren said, noting that sex chromosomes have long been hypothesized to be hotspots for speciation.
The researchers are currently doing similar genomic analyses on the hybrid offspring of collared flycatchers and pied flycatchers to see how genetic features in those birds compare to the parental species.
They have also developed a SNP chip based on the polymorphisms present in the flycatcher genome sequences that they plan to use for genotyping studies on flycatchers within defined pedigrees. Those studies should make it possible to develop a finer scale linkage map for the birds and to trace recombination patterns along the flycatcher chromosomes, Ellegren explained.