DOI: 10.5061/dryad.wdbrv15m8
title: Data from: Ultraconserved yet informative for species delimitation: UCEs resolve long-standing systematic enigma in Central European bees

description: Accurate and testable species delimitation hypotheses are essential for
 measuring, surveying and managing biodiversity. Today, taxonomists often
 rely on mitochondrial DNA barcoding to complement morphological species
 delimitations. Although COI barcoding has largely proven successful in
 assisting identifications for most animal taxa, there are nevertheless
 numerous cases where mitochondrial barcodes do not necessarily reflect the
 species history. For instance, what is regarded as one single species can
 be associated with two distinct DNA barcodes, which can point either to
 cryptic diversity or to deep within-species mitochondrial divergences with
 no reproductive isolation. In contrast, two or more species can share
 barcodes, for instance due to mitochondrial introgression. These intrinsic
 limitations of mitochondrial DNA barcoding can only be addressed with
 nuclear genomic markers, which are expensive, labour intensive, poorly
 repeatable, and often require high-quality DNA. To overcome these
 limitations, we examined the use of ultraconserved nuclear genetic
 elements (UCEs) as a quick and robust genomic approach to address such
 problematic cases of species delimitation. This genomic method was
 assessed using six different bee species complexes suspected to harbour
 cryptic diversity, mitochondrial introgression, or mitochondrial
 paraphyly. The sequencing of UCEs recovered between 686 and 1860
 homologous nuclear loci and provided explicit species delimitation
 hypotheses in all investigated species complexes. These results provide
 strong evidence for the suitability of UCEs as a fast method for species
 delimitation even in recently diverged lineages. Furthermore, this study
 provided the first conclusive evidence for both mitochondrial
 introgression among distinct species, and mitochondrial paraphyly within a
 single bee species.
description: UCE library
 preparation Whole body DNA extractions were
 performed overnight in a proteinase K buffer at 56°C and purified using a
 Qiagen Biosprint 96 extraction robot following the manufacturer’s
 protocol. Extracts were quantified using Qubit v3 (Thermofisher
 Scientific) and 50 ng DNA per specimen were sonicated to 500 bp fragment
 length using a Bioruptor ultrasonicator (Diagenode). Two independent
 dual-indexed libraries each containing 96 specimens were constructed using
 a Kapa Hyper prep kit (Roche) using one fourth of the manufacturer’s
 recommended volumes (as described in Branstetter, Longino, Ward, &
 Faircloth, 2017). PCR amplifications were performed in the recommended
 volumes. PCR products were quantified using a Qubit v3 and each row of a
 96-well PCR plate were pooled equimolarly (i.e. for total of 8 pools).
 Libraries were UCE enriched using the Hymenopteran v2 hybridization kit
 (UCE Hymenoptera 2.5Kv2 Principal/Full, myBaits, Arborbiosci). Each
 enrichment was performed on a single pool of 12 specimens using 500 ng.
 The enrichment protocol followed the manufacturer’s recommendations with a
 hybridization step of 24 h at 65°C, followed by a PCR amplification with
 14 cycles. Pools were sequenced on a Miseq using the Illumina v3 kits (2 x
 300 bp; Illumina). Bioinformatic
 processing of UCE data Fastq reads were demultiplexed on
 the Miseq and data from all runs were merged and processed mainly using
 PHYLUCE tools (Faircloth, 2016). Raw data were cleaned with illumprocessor
 (Faircloth, 2016), a tool wrapped around trimmomatic (Bolger, Lohse,
 & Usadel, 2014). Clean reads were assembled with SPAdes<i>
 </i>v3.12.0 (Nurk et al., 2013) using the single-cell flag (“--sc”),
 careful option (“--careful”) and a coverage cutoff value of five
 (“--cov-cutoff”). Obtained contigs were mapped against the corresponding
 UCE reference file using Lastz (Harris, 2007) and matching reads were
 extracted and aligned by species complex using MAFFT (Katoh &
 Standley, 2013). Alignments were edge-trimmed using the PHYLUCE “seq-cap”
 program; a strategy recommended for closely related species (&lt;
 30-50 MYA) (Faircloth, 2016). Loci shared by less than 75% of the maximum
 number of specimens were filtered out. Remaining alignments were
 concatenated and saved in fasta format. An additional filtering step was
 applied to remove specimens with more than 90% missing data.