Native to Southeastern Asia, the ambrosia beetle Xylosandrus crassiusculus is invasive worldwide. Its invasion is favoured by its cryptic lifestyle, symbiosis with a fungus that facilitates a broad range of host plants, and predominant sib-mating reproduction.

We used COI and RAD sequencing to i) characterise the worldwide genetic structure of the species, ii) disentangle the origin(s) of the non-native populations on the three invaded continents and (iii) analyse the genetic diversity and pathways within each invaded region.

The DNA amount extracted from each individual of Xylosandrus crassiusculus was too small to allow the direct construction of RAD libraries, therefore we carried out whole genome amplification. We used the Genomiphi kit V3 following the manufacturer's procedure. We constructed the Individual RAD libraries as follows. DNA was digested using 250 ng of DNA in 22 µL per sample and 0.5 µL of the PstI-HF enzyme for a total volume of 25 µL. The digested fragments from each specimen were tagged with a unique 5- or 6- bp barcode and a P1 adapter using 1.5 µL of P1 adapter (100 nM) and 0.5 µL of T4 Ligase (2.000.000 U/ml) for a total volume of 30.5 µL. Specimens were then pooled 32 by 32 to create seven libraries. Libraries were sonicated on a Covaris S220 (duty cycle 10%, intensity 5, 200 cycles/burst, duration 75 s) to obtain 300-600 bp fragments. Each library was then tagged with a 5- or 6- nucleotide barcode and a P2 adapter using 1 µL of P2 adapter (10 nM) and 0.5 µL of Quick Ligase (2,000,000 U/ml). The sizing and purification steps were realized using AMPure XP beads (Agencourt). We performed 5 PCR enrichment with 15 cycles (30 ng DNA input, NEB Phusion High-Fidelity PCR Master Mix) for each library to increase fragment diversity. After quality control using the Agilent 2100 Bioanalyzer, the libraries were pooled altogether at an equimolar ratio and sent to MGX-Montpellier GenomiX for sequencing. The library was verified on a Fragment Analyser (Agilent, HS NGS fragment Kit), quantified by qPCR (Kapa Library quantification kit) and sequenced on a SP lane in paired-end 2 x 150 nt mode on a Novaseq6000 (Illumina) according to the manufacturer's instructions. We used the RADIS pipeline (Cruaud et al. 2016) to i) demultiplex individuals using process_radtags (Catchen et al. 2013), ii) homogenise read length and remove a few low-quality bases by trimming reads to 139 bp and iii) remove PCR duplicates using clone_filter (Catchen et al. 2013). Using the BWA-MEM algorithm (Li and Durbin 2009), we mapped the loci obtained on Ambrosiella xylebori's genome (Vanderpool et al. 2018) (accession number: ASM277803v1) to create a blacklist. We did the same with the reference genome GRCh38.p13 of Homo sapiens (accession number: GCA_000001405.28)to create a second blacklist.