Computational protocol: Co1 DNA supports conspecificity of Geomyphiluspierai and G.barrerai (Coleoptera, Scarabaeidae, Aphodiinae) and is a good marker for their phylogeographic investigation in Mexican mountains

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Protocol publication

[…] To collect Geomyphilus beetles, the gopher nests were excavated (only those that seemed recently used). The galleries were followed up to find the nests and latrines where the dung beetles might be found. This sampling technique is very effort-consuming because the nests can be located very deep and they are not always found. Once the nests and latrines were reached, the organic matter was removed as well as the soil within 20 cm from it, and then all the soil was carefully inspected for scarabs. The depth of the nest and latrines varies depending on the type of soil, slope and surrounding vegetation. They were found from 30 cm to 1.5 m deep in different biotopes (scrublands and grasslands) in the studied mountains (Fig. ). All the individuals were immediately transferred to 90% ethanol and conserved for further morphological identification and molecular analyses. All specimens examined were identified by Giovanni and Marco Dellacasa.The DNA was extracted from ethanol-preserved specimens. Either of two different techniques were used, both designed to obtain molecular information while keeping voucher cuticle of each individual. For the first one, after complete evaporation of ethanol, the cuticle of each specimen was cut at two points on the prothoracic-mesothoracic joint soft integument in order to facilitate the lysis of internal tissues. This was done with a sterile syringe needle on a glass slide before insertion of individual in a vial for proteinase K digestion. For the second technique, one leg was removed from the beetle, then put in an Eppendorf vial and crushed with a pestle. For both techniques, the entire cut individual or the crushed leg were immersed in 100 ml Buffer ATL + 15 ml Proteinase K and incubated for 19–30 h at 70 °C, before proceeding to the DNA purification using Qiagen’s DNeasy Blood & Tissue Kit following the manufacturer’s protocol. The specimens’ cuticles (tech. 1) were conserved in glycerol and the body remaining (tech. 2) in ethanol as vouchers.Checking congruence between phylogenetic topologies from genes with different transmission patterns such as mt DNA (maternal transmission) and nuclear DNA (biparental transmission) is useful to confirm cryptic species (see ). For this purpose, Co1 and IT2 appeared to be good candidates, since they were shown to be variable both at the inter- and intraspecific levels in some scarab beetle genera (). Two fragments from one mitochondrial protein-coding gene (Co1) and one nuclear ribosomal internal transcribed spacer (ITS2) gene were amplified. Primer pairs used for amplification of Co1 were LCO_1490 and HCO_2198 () or LEP-F1 and LEP-R1 (); and for the nuclear region ITS2 we used ITS2f () and RhITSR ().Polymerase chain reaction (PCR) was performed using the Qiagen’s Taq PCR Core kit. The following reagent concentrations were used for a final volume of 25 µL per tube: 1×PCR buffer, 0.036U/µl Qiagen Taq Polymerase, 300 µM dNTPs and 0.6 µM of each Co1 primer or 0.43 µM of each ITS2 primer, and 2.5 mM MgCl2 for CO1 or 3.0 mM MgCl2 for ITS2. For the Co1, thermal cycling parameters comprised an initial denaturation step of 10 minutes at 94 °C, followed by 5 cycles of 94 °C for 40 s, 49 °C for 60 s and 62 °C for 60 s, and 35 cycles of 94 °C for 40 s, 52 °C for 60 s and 62 °C for 60 s, with a final elongation at 62 °C for 10 min. For the ITS region, thermal cycling parameters comprised an initial denaturation of 10 minutes at 94 °C, followed by 5 cycles of 94 °C for 40 s, 56 °C for 60 s and 72 °C for 60 s, and 35 cycles of 94 °C for 52 s, 52 °C for 60 s and 72 °C for 60 s, with a final elongation at 72 °C for 10 min. Purification and sequencing was realized by Genoscreen (France, Lille) using a 96-capillary sequencer ABI3730XL.Obtained DNA sequences were aligned using Muscle () in Seaview4.1 (). The absence of any stop codon was first checked in the Co1 sequence alignment. Maximum likelihood phylogenetic analyses were performed using PhyML () and nucleotidic diversities were compared between samples and between taxa using DNAsp 5.10.01 (; ). A median-joining network (Bandelt et al. 1999) of DNA fragments was constructed using NETWORK v4.5.1.6 (fluxus-engineering.com) to visualize the frequency and geographical distribution of Co1 haplotypes. […]

Pipeline specifications

Software tools MUSCLE, SeaView, PhyML, DnaSP
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Canis lupus familiaris
Diseases Rodent Diseases