As sequencing technologies become more affordable, it is now realistic to propose studying the evolutionary history of virtually any organism on a genomic scale. However, when dealing with non-model organisms it is not always easy to choose the best approach given a specific biological question, a limited budget, and challenging sample material. Furthermore, although recent advances in technology offer unprecedented opportunities for research in non-model organisms, they also demand unprecedented awareness from the researcher regarding the assumptions and limitations of each method. In this review we present an overview of the current sequencing technologies and the methods used in typical high-throughput data analysis pipelines. Subsequently, we contextualize high-throughput DNA sequencing technologies within their applications in non-model organism biology. We include tips regarding managing unconventional sample material, comparative and population genetic approaches that do not require fully assembled genomes, and advice on how to deal with low depth sequencing data.

The European honeybee (Apis mellifera) is a key pollinator and has in the last decades suffered significant population decline. A combination of factors, including decrease in genetic diversity and introduction of Varroa mites, have been suggested to be responsible for these losses, but no definitive cause has yet been appointed. In Europe not only have wild colonies been severely affected, but managed hives have had a massive decline in numbers. To test the hypothesis that honeybees’ genetic diversity has decreased in the recent past, we used reduced representation genome sequencing of 40 historical honeybee specimens collected in Natural History collections across Europe and compared them to genomic data from 40 individuals from extant populations (collected post 2006). Our results are consistent with the existence of five evolutionary lineages as previously described, and show a decrease in genetic diversity between historical and extant individuals of the same lineage, as well as high levels of admixture in historical specimens. Our data confirm that a loss of genetic diversity has occurred during the last century, potentially increasing honeybees’ vulnerability to contemporary ecological and anthropogenic stressors.

Newts of the genus Triturus (Amphibia, Caudata, Salamandridae) are distributed across Europe and adjacent Asia. In spite of its prominence as a model system for evolutionary research, the phylogeny of Triturus has remained incompletely solved. Our aim was to rectify this situation, to which we employed nuclear encoded proteins (40 loci) and mitochondrial DNA-sequence data (mtDNA, 642 bp of the ND4 gene). We sampled up to four populations per species covering large parts of their ranges. Allozyme and mtDNA data were analyzed separately with parsimony, distance, likelihood and Bayesian methods of phylogenetic inference. Existing knowledge on taxonomic relationships was confirmed, including the monophyly of the genus and the groups of crested newts (four species) and marbled newts (two species). The genetic coherence of species and subspecies was also confirmed, but not always with high statistical support (depending on taxon, characters under consideration, and method of inference). In spite of our efforts we did not obtain sufficient phylogenetic signal to prefer one out of twelve potential topologies representing crested newts relationships. We hypothesize that the lack of phylogenetic resolution reflects a hard polytomy and represents the (near)-simultaneous origin of crested newt species. Using a calibration point of 24 Ma (million years before present) for the most recent common ancestor of Triturus-species, the crested newt radiation event is dated at 7-6 Ma (scenario 1) or at 11-10 Ma (scenario 2), depending on the application of an allozyme versus mtDNA molecular clock. The first biogeographical scenario involves the spread of crested newts from the central Balkans into four compass directions. This scenario cannot be brought into line with potential vicariance events for south-eastern Europe. The second scenario involves the more or less simultaneous origin of four species of crested newts through large-scale vicariance events and is supported by the paleogeographical reconstruction for the region at the end of the Middle Miocene. The subspecies Triturus carnifex macedonicus carries in one large area the mtDNA that is typical for the neighbouring species T. karelinii, which is attributed to introgression and a recent range shift. It is nevertheless a long distinct evolutionary lineage and we propose to elevate its taxonomic status to that of a species, i.e., from Triturus c. macedonicus (Karaman, 1922) to Triturus macedonicus (Karaman, 1922).

Ancestry inference for an individual can only be as good as the reference populations with allele frequency data on the SNPs being used. If the most relevant ancestral population(s) does not have data available for the SNPs studied, then analyses based on DNA evidence may indicate a quite distantly related population, albeit one among the more closely related of the existing reference populations. We have added reference population allele frequencies for 14 additional population samples (with >1100 individuals studied) to the 125 population samples previously published for the Kidd Lab 55 AISNP panel. Allele frequencies are now publicly available for all 55 SNPs in ALFRED and FROG-kb for a total of 139 population samples. This Kidd Lab panel of 55 ancestry informative SNPs has been incorporated in commercial kits by both ThermoFisher Scientific and Illumina for massively parallel sequencing. Researchers employing those kits will find the enhanced set of reference populations useful.