Systematics and evolution of predatory flower flies (Diptera Syrphidae) based on exon-capture sequencing

Flower flies (Diptera: Syrphidae) are one of the most species-rich dipteran families and provide important ecosystem services such as pollination, biological control of pests, recycling of organic matter and redistributions of essential nutrients. Flower fly adults generally feed on pollen and nectar, but their larval feeding habits are strikingly diverse. In the present study, high-throughput sequencing was used to capture and enrich phylogenetically and evolutionary informative exonic regions. With the help of the baitfisher software, we developed a new bait kit (SYRPHIDAE1.0) to target 1945 CDS regions belonging to 1312 orthologous genes. This new bait kit was successfully used to exon capture the targeted loci in 121 flower fly species across the different subfamilies of Syrphidae. We analysed different amino acid and nucleotide data sets (1302 loci and 154 loci) with maximum likelihood and multispecies coalescent models. Our analyses yielded highly supported similar topologies, although the degree of the SRH (global stationarity, reversibility and homogeneity) conditions varied greatly between amino acid and nucleotide data sets. The sisterhood of subfamilies Pipizinae and Syrphinae is supported in all our analyses, confirming a common origin of taxa feeding on soft-bodied arthropods. Based on our results, we define Syrphini stat.rev. to include the genera Toxomerus and Paragus. Our divergence estimate analyses with beast inferred the origin of the Syrphidae in the Lower Cretaceous (125.5-98.5 Ma) and the diversification of predatory flower flies around the K-Pg boundary (70.61-54.4 Ma), coinciding with the rise and diversification of their prey.Peer reviewe

Data from: Oligonucleotide primers for targeted amplification of single-copy nuclear genes in apocritan Hymenoptera

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

Data from: Oligonucleotide primers for targeted amplification of single-copy nuclear genes in apocritan Hymenoptera

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

Type material of Lepidoptera and Trichoptera in the ZFMK collection, Bonn

Volume: 58Start Page: 169End Page: 21

Amplicons_NT.tar

Supplementary 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"

Rating of obtained polymerase chain reaction (PCR) products.

<p>Rating of the PCR products obtained from using the degenerate oligonucleotide primers shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039826#pone-0039826-t003" target="_blank">Table 3</a> to amplify ten target genes in six apocritan Hymenoptera. ++  =  target PCR product in excess. +  =  target PCR product sufficient for direct sequencing. +/−  =  target PCR product insufficient for direct sequencing. –  =  no target PCR product. (?)  =  unclear whether or not PCR products include amplicon of target gene.</p>*<p>Secondary PCR amplification product likely hampering direct sequencing.</p

Hypothesized phylogenetic relationships of apocritan Hymenoptera studied in this investigation [4], [12].

<p>Taxa with sequenced genomes are highlighted in green; their genome sequences were analyzed to identify single-copy genes and to design degenerate oligonucleotide PCR primers. DNA of non-highlighted species was used to assess the functionality of the inferred PCR and sequencing primers.</p

Oligonucleotide sequencing primer pairs.

<p>The nucleotide sequences of the sequencing primers were attached as a binding site to the 5′ end of the degenerate oligonucleotide polymerase chain reaction (PCR) primers. Each of the oligonucleotide primers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039826#pone.0039826.s002" target="_blank">Table S1</a> is compatible with at least one of the sequencing primers added to the 5′ end of the PCR primer. <i>T</i>_m  =  approximate melting temperature [°C].</p

Empirically evaluated degenerate oligonucleotide PCR primer pairs.

<p>The ten degenerate oligonucleotide primers were tested with the respective binding sites for sequencing primer HOG-Seq-A (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039826#pone-0039826-t002" target="_blank">Table 2</a>) attached to the 5′ end and used to amplify ten target genes in six apocritan Hymenoptera. <i>d</i>  =  degree of degeneration. <i>T</i>_m  =  approximate melting temperature [°C].</p