Genomic Mining of Phylogenetically Informative Nuclear Markers in Bark and Ambrosia Beetles

Abstract

Deep level insect relationships are generally difficult to resolve, especially within taxa of the most diverse and species rich holometabolous orders. In beetles, the major diversity occurs in the Phytophaga, including charismatic groups such as leaf beetles, longhorn beetles and weevils. Bark and ambrosia beetles are wood boring weevils that contribute 12 percent of the diversity encountered in Curculionidae, one of the largest families of beetles with more than 50000 described species. Phylogenetic resolution in groups of Cretaceous age has proven particularly difficult and requires large quantity of data. In this study, we investigated 100 nuclear genes in order to select a number of markers with low evolutionary rates and high phylogenetic signal. A PCR screening using degenerate primers was applied to 26 different weevil species. We obtained sequences from 57 of the 100 targeted genes. Sequences from each nuclear marker were aligned and examined for detecting multiple copies, pseudogenes and introns. Phylogenetic informativeness (PI) and the capacity for reconstruction of previously established phylogenetic relationships were used as proxies for selecting a subset of the 57 amplified genes. Finally, we selected 16 markers suitable for large-scale phylogenetics of Scolytinae and related weevil taxa.

Molecular phylogeny of bark and ambrosia beetles (Curculionidae: Scolytinae) based on 18 molecular markers

“…1). As many genera as possible were represented, except for the previously well sampled HDAC Rpd3_for1 GARTAYAAYAARCARATGC and HDAC Rpd3_for2 CRGARATYTGYATHAAYTGGGG -to use with previously published reverse primers (Pistone et al, 2016). The optimal annealing temperature for these primers was 52 ∘ C for FEN1 and 62 ∘ C for HDAC Rpd3.…”

“…Nucleotide sequences were blasted in GenBank for gene target verification, accepting a minimum E value threshold of 1E-4. All genes considered in this study were previously examined for paralogy and/or multiple copies in the OrthoDB database (Waterhouse et al, 2013) and the majority of them (with the exclusion of Arr2, Iap2 and SOD1) were frequently present in single copy in arthropod genomes (Pistone et al, 2016). Careful phylogenetic analyses of each of the three genes with potential multiple copies did not indicate signs of anomalous relationships between sequences (Pistone et al, 2016).…”

“…All genes considered in this study were previously examined for paralogy and/or multiple copies in the OrthoDB database (Waterhouse et al, 2013) and the majority of them (with the exclusion of Arr2, Iap2 and SOD1) were frequently present in single copy in arthropod genomes (Pistone et al, 2016). Careful phylogenetic analyses of each of the three genes with potential multiple copies did not indicate signs of anomalous relationships between sequences (Pistone et al, 2016). Sequences with simple intron-exon patterns were aligned using bioedit v. 7.2.5 (Hall, 1999) with manual adjustments, locating intron borders based on previously published alignments (Pistone et al, 2016).…”

“…Paper I: Pistone D, Mugu S and Jordal BH (2016) Deep level insect relationships are generally difficult to resolve, especially within taxa of the most diverse and species rich holometabolous orders. In beetles, the major diversity occurs in the Phytophaga, including charismatic groups such as leaf beetles, longhorn beetles and weevils.…”

“…As a means of improving phylogenetic resolution in bark and ambrosia beetles, we add 13 new markers (Pistone et al, 2016) to the previous five standard markers used for weevil systematics . Our main hypothesis implies that morphologically defined tribes as proposed by are monophyletic, except for recently resolved groups including Micracidini LeConte, Ipini Bedel and Dryocoetini Lindemann .…”