Supplementary File 1 : Molecular Protocols for Each Laboratory

Supplementary file1. Molecular protocols in each laboratory.

Centre de Biologie pour la Gestion des Populations (CBGP) in Montpellier, France – Gaël Kergoat

DNA was extractedfrom the anterior part of each larva to limit the amount of fatty tissues present.This was performed using the QiagenDNeasyTissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s recommendations.

Typical PCR reactions were prepared in 25-µl volumes using 0.2 µl of Taq-polymerase (5units/µl), 2.5 µl of 10× PCR buffer, 18.3 µl of purified water, 1 µl of 25 mMMgCl2, 0.25 µl of each 100 µM primer, 0.5 µl of 2.5 mMdeoxynucleotides (dNTPs) and 2 µl of genomic DNA.

For the 12S, 16S and Cytbfragments, the PCR cycling conditions were an initial denaturing step at 94°C for 3 min, followed by 35 amplification cycles (denaturing at 92°C for 1 min, annealing at the appropriate temperature for the fragment for 1 min, extension at 72°C for 1 min), and a final extension step at 72°C for 10 min.For the COI fragments, the protocol was the same only the initial denaturing step occurred at 95°C for 3 min. For the 28S D4–D5 fragment, the PCR cycling conditions were an initial denaturing step at 94°C, followed by 40 amplification cycles (denaturing at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 90 s), and a final extension step at 72°C for 10 min.For the 18S fragments (three overlapping fragments), the PCR cycling conditions were an initial denaturing step at 94°C for 3 min, followed by 35 amplification cycles (denaturing at 94°C for 30 s, annealing at 56–58°C for 30 s, extension at 72°C for 90 s), and a final extension step at 72°C for 10 min.

The PCR products wereprocessed by the French sequencing centre Genoscope usinga BigDye® 3.1 Sequencing Kit and Applied 3730xl sequencers.The resulting sequences of complementary strands werefurther edited and reconciled using Geneious 5.1 (available at

(For additional details, see: Kergoat G, Soldati L, Claemens AL et al. 2014. Higher level molecular phylogeny of darkling beetles (Coleoptera: Tenebrionidae). Systematic Entomology 39: 486-499.)

InstitutAgronomiquenéo-Calédonien(IAC) in Nouméa, New Caledonia – Kelly Letellier

DNA was extracted fromthe legs and thorax fragments using the QiagenDNeasyTissue Kit (Qiagen, Hilden, Germany), following the manufacturer’s recommendations.

Amplifications were performed in a 20-µL reaction volume with 16.75 µL of H2O, 3.8 µL of buffer, 1.5 µl of MgCl2, 0.25 µl of dNTP, 0.75 µl of each 10 ng/µl primer, 0.2 µl of Taq polymerase and 1 µlof DNA at 10ng/µl. The PCR consisted of an initial denaturing step at 94°C for 4 min, followed by 35 amplification cycles (denaturing at 94°C for 30 s, annealing at 52°C for 1 min, extension at 72°C for 90 s), and a final step at 72°C for 7 min.

The PCR products were checked on agarose gels. DNA fragments were then diluted to 10ng/μl and the sequencing reaction was performed with the reverse primer using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA). Amplification was performed in a 20-µL reaction volume. The PCR consisted of an initial step of denaturationat 96°C for 1 min, followed by90 cycles (denaturing at 96°C for 10 s, annealing at 52°C for 5 s, elongation at 72°C for 4 min), and a final phase at 15°C for 7 min. The reaction volume was adjusted to 30 µl, followed by gel filtration purification on Sephadex® G50 columns (Sigma-Aldrich, Saint Louis, USA). Sequences were obtained with an automated capillary sequencer (DNA sequencer 3130XL Sequencer 16 Capillaries-Applied Biosystems®) and cleaned using BioEdit 7.2.5 software (available at

Museum national d'Histoire naturelle (MNHN) in Paris, France – Romain Nattier

DNA was extracted from the whole individual using the QIAamp DNA MicroKit (QIAGEN, Courtaboeuf, France), following the manufacturer’s instructions.

Amplifications were performed in a 30-µl reaction volume with 1 µl of each 10 pMprimer, 20 µl of H20, 3 µl of buffer, 0.6 µl of dNTP, 0.4 µl of Taq polymerase and 4µl of DNA. The PCR consisted of an initial denaturing step at 95°C for 5 min, 35 amplification cycles (denaturing at 95°C for 1 min, annealing at 48°C for 1 min, extension at 72°C for 90 s), and a final step at 72°C for 10 min.

The PCR products were checked on agarose gels and sequenced in both directions with the same primers at Eurofins (MWG Operon, Ebersberg, Germany). The sequences were then cleaned and coding sequences were translated using the invertebrate mitochondrial genetic code to check for the absence of stop codons using Sequencher v. 4.8 (GeneCodes Corporation, Ann Arbor, MI, USA).

Australian Museum Research Institute (AMRI) in Sydney, Australia – Andrew Mitchell

DNA was extracted fromthe legs and thorax fragments using the GenEluteTM Mammalian Genomic DNA Miniprep Kit(Sigma-Aldrich, Sydney, Australia), following the manufacturer’s recommendedprotocol but with DNA elution volumes adjusted to 100 µl.

PCRs were prepared using an Invitrogen Platinum Taq PCR kit (Life Technologies, Mulgrave, Australia). Each reaction volume contained 1.5 µl of 10X PCR buffer, 0.84 µl of50 mM MgCl2, 0.3 µl of 10 mMdNTP mix, 3.10-6µl of each of the 10 µM forward and reverse primers, 4 µL of genomic DNA extraction,0.075 µL (0.375 U) of Platinum Taq DNA polymerase, and was completed to 15µL with H20. Thermal cycling was performed using an Eppendorf Mastercycler ep gradient S PCR machine, and consisted of an initial step at 94°C for 2 min, followed by 40 cycles, or 35 cycles for reamplification reactions (denaturing at 94°C for 30 s, annealing at 50°C for 40 s, extension at 72°C for 60 s), and a final extension at 72°C for 7 min, followed by storage at 10°C.

The PCR products were electrophoresed on a 1.5% agarose gel and visualized on a UV transilluminator. Successful PCRs were only sequenced if the sequence of that gene region had not previously been obtained for that DNA extraction.

DNA sequencing was performed on an ABI3730xl, and sequence trace files were checked for accuracy and assembled into contigs using GENEIOUS v. 6.5 (Kearse et al. 2012).

(For additional details, see: Mitchell A 2015. Collecting in collections: a PCR strategy and primer set for DNA barcoding of decades-old dried museum specimens. MolecularEntomologyResources15: 1102-1111.)