The ribosomal small subunit (SSU) rRNA gene has emerged as an important genetic marker for taxonomic identification in environmental sequencing datasets. In addition to being present in the nucleus of eukaryotes and the core genome of prokaryotes, the gene is also found in the mitochondria of eukaryotes and in the chloroplasts of photosynthetic eukaryotes. These three sets of genes are conceptually paralogous and should in most situations not be aligned and analyzed jointly. To identify the origin of SSU sequences in complex sequence datasets has hitherto been a time-consuming and largely manual undertaking. However, the present study introduces Metaxa ( http://microbiology.se/software/metaxa/ ), an automated software tool to extract full-length and partial SSU sequences from larger sequence datasets and assign them to an archaeal, bacterial, nuclear eukaryote, mitochondrial, or chloroplast origin. Using data from reference databases and from full-length organelle and organism genomes, we show that Metaxa detects and scores SSU sequences for origin with very low proportions of false positives and negatives. We believe that this tool will be useful in microbial and evolutionary ecology as well as in metagenomics.

Background: Gene trees that arise in the context of reconstructing the evolutionary history of polyploid species are often multiply-labeled, that is, the same leaf label can occur several times in a single tree. This property considerably complicates the task of forming a consensus of a collection of such trees compared to usual phylogenetic trees.

Phylogenetic relationships within Silene sect. Physolychnis are investigated using DNA sequences from two low‐copy nuclear regions and three plastid regions. We expand the circumscription of Physolychnis to include also Siberian and Eurasian taxa, with S. zawadzkii and S. uralensis as delimiters of the section. Two main clades are identified within Physolychnis, the Siberian S. ajanensis group and an Asian/American clade. Several taxa, previously classified to sect. Odontopetalae, sect. Occidentales, and sect. Morrisonmontanae, are shown to belong to the Asian/American clade, which otherwise corresponds to Bocquet's (1969) circumscription of sect. Physolychnis. Silene sect. Physolychnis also includes several additional, mainly Asian taxa (e.g., S. davidii, S. seoulensis, S. keiskei, S. viscosa, S. quadriloba, and homoeologues from S. songarica, S. firma, and S. tolmatchevii). Several putative allopolyploids are identified, in several cases stemming from crosses between the two main clades. The two allotetraploids S. involucrata and S. sachalinensis, although morphologically and geographically distinct, have indistinguishable ancestries.

Hybridization has played a major role during the evolution of angiosperms, mediating both gene flow between already distinct species and the formation of new species. Newly formed hybrids between distantly related taxa are often sterile. For this reason, interspecific crosses resulting in fertile hybrids have rarely been described to take place after more than a few million years after divergence. We describe here the traces of a reproductively successful hybrid between two ancestral species of Silene, diverged for about six million years prior to hybridization. No extant hybrids between the two parental lineages are currently known, but introgression of the RNA polymerase gene NRPA2 provides clear evidence of a temporary and fertile hybrid. Parsimony reconciliation between gene trees and the species tree, as well as consideration of clade ages, help exclude gene paralogy and lineage sorting as alternative hypotheses. This may represent one of the most extreme cases of divergence between species prior to introgressive hybridization discovered yet, notably at a homoploid level. Although species boundaries are generally believed to be stable after millions of years of divergence, we believe that this finding may indicate that gene flow between distantly related species is merely largely undetected at present.

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.

Structural rearrangements have been shown to be important in local adaptation and speciation, but have been difficult to reliably identify and characterize in non-model species. Here we combine long reads, linked reads and optical mapping to characterize three divergent chromosome regions in the willow warbler Phylloscopus trochilus, of which two are associated with differences in migration and one with an environmental gradient. We show that there are inversions (0.4–13 Mb) in each of the regions and that the divergence times between inverted and non-inverted haplotypes are similar across the regions (~1.2 Myrs), which is compatible with a scenario where inversions arose in either of two allopatric populations that subsequently hybridized. The improved genomes allow us to detect additional functional differences in the divergent regions, providing candidate genes for migration and adaptations to environmental gradients.

Chromoanagenesis is a genomic event responsible for the formation of complex structural chromosomal rearrangements (CCRs). Germline chromoanagenesis is rare and the majority of reported cases are associated with an affected phenotype. Here, we report a healthy female carrying two de novo CCRs involving chromosomes 4, 19, 21 and X and chromosomes 7 and 11, respectively, with a total of 137 breakpoint junctions (BPJs). We characterized the CCRs using a hybrid-sequencing approach, combining short-read sequencing, nanopore sequencing, and optical mapping. The results were validated using multiple cytogenetic methods, including fluorescence in situ hybridization, spectral karyotyping, and Sanger sequencing. We identified 137 BPJs, which to our knowledge is the highest number of reported breakpoint junctions in germline chromoanagenesis. We also performed a statistical assessment of the positioning of the breakpoints, revealing a significant enrichment of BPJ-affecting genes (96 intragenic BPJs, 26 genes, p < 0.0001), indicating that the CCRs formed during active transcription of these genes. In addition, we find that the DNA fragments are unevenly and non-randomly distributed across the derivative chromosomes indicating a multistep process of scattering and re-joining of DNA fragments. In summary, we report a new maximum number of BPJs (137) in germline chromoanagenesis. We also show that a hybrid sequencing approach is necessary for the correct characterization of complex CCRs. Through in-depth statistical assessment, it was found that the CCRs most likely was formed through an event resembling chromoplexy—a catastrophic event caused by erroneous transcription factor binding.

Long-distance migration requires adaptations in a suite of behavioral, physiological and morphological traits. However, almost nothing is known about the genetic basis of these adaptations. The willow warbler Phylloscopus trochilus occurs in Europe with a northern and southern subspecies that show drastically different migration routes and wintering areas. Previous studies have demonstrated that the subspecies are genetically extremely similar except for three divergent chromosome regions, of which two are associated with the differences in migratory phenotypes and one is associated with an environmental gradient. Here we use a combination of long-read sequencing, linked-read sequencing and optical mapping to construct more complete and contiguous assemblies for both of the subspecies. We find evidence for inversions in each of the three divergent regions, which range from 0.4 to 13 Mb in size, and that breakpoints are associated with tandem repeat arrays or segmental duplications. The divergence times between inverted and non-inverted haplotypes are similar across the regions (~1.2 Myrs), which is compatible with a scenario where the inversions arose in either of two allopatric populations that subsequently hybridized. The improved genome assemblies and annotation also allowed us to detect additional functional differences in the divergent regions that provide candidate genes for migration and local adaptations to environmental gradients.