BACKGROUND: Published nucleotide sequence data from the mega-diverse insect order Hymenoptera (sawflies, bees, wasps, and ants) are taxonomically scattered and still inadequate for reconstructing a well-supported phylogenetic tree for the order. The analysis of comprehensive multiple gene data sets obtained via targeted PCR could provide a cost-effective solution to this problem. However, oligonucleotide primers for PCR amplification of nuclear genes across a wide range of hymenopteran species are still scarce. FINDINGS: Here we present a suite of degenerate oligonucleotide primer pairs for PCR amplification of 154 single-copy nuclear protein-coding genes from Hymenoptera. These primers were inferred from genome sequence data from nine Hymenoptera (seven species of ants, the honeybee, and the parasitoid wasp Nasonia vitripennis). We empirically tested a randomly chosen subset of these primer pairs for amplifying target genes from six Hymenoptera, representing the families Chrysididae, Crabronidae, Gasteruptiidae, Leucospidae, Pompilidae, and Stephanidae. Based on our results, we estimate that these primers are suitable for studying a large number of nuclear genes across a wide range of apocritan Hymenoptera (i.e., all hymenopterans with a wasp-waist) and of aculeate Hymenoptera in particular (i.e., apocritan wasps with stingers). CONCLUSIONS: The amplified nucleotide sequences are (a) with high probability from single-copy genes, (b) easily generated at low financial costs, especially when compared to phylogenomic approaches, (c) easily sequenced by means of an additionally provided set of sequencing primers, and (d) suitable to address a wide range of phylogenetic questions and to aid rapid species identification via barcoding, as many amplicons contain both exonic and fast-evolving intronic nucleotides.

Amplicons_NT.tarSupplementary File Archive: "Amplicons_NT.tar.gz." Multiple nucleotide sequence alignments in FASTA format of expected amplicons when applying the PCR oligonucleotide primer pairs inferred in our study. Each multiple nucleotide sequence alignment contains the nucleotide sequence of each of the nine species of Hymenoptera whose genomes we analyzed plus the position and nucleotide sequence (with IUPAC ambiguity code; complement sequence of reverse primer) of the inferred forward and reverse PCR oligonucleotide primer. Anatomy of the file names: [Identifier of ortholog group]_[Identifier of oligonucleotide primer pair specific to ortholog group]_[Identifier of recommended pair of sequencing primers].NT.fas For example, the file name "HOG2939_01_B.NT.fas" contains the nucleotide sequences expected to be amplified from the genes belonging to ortholog group "HOG2939" when using the first oligonucleotide primer pair that has been inferred for this ortholog group and the sequencing oligonucleotide primer pair B. HOG stands for "Hymenoptera Ortholog Group".