First assessment of the biodiversity of praying mantises (Insecta: Mantodea) in Cameroon with DNA barcoding

comprehensive monographs and dedicated taxon revisions (see Taxonomic checklist in Results). Species that had no recent literature references were identified by studying their orig- inal descriptions. When required for species identification, male and female genitalia were sep- arated from the specimens, macerated in 10% KOH solution for 4–36 h and rinsed with water and ethanol afterwards. Prepared genitalia are stored in microvials with glycerol together with corresponding specimens. External morphology and genitalia were studied using Leica M205 C stereomicroscope (Wetzlar, Germany). Photographs of the live specimens were made with Sony Alpha a6000 digital camera (Tokyo, Japan) and Sony SEL30M35 30mm f/3.5 macro lens. DNA barcoding was performed for a representative number of specimens (n = 72), selected using diversity and material quality criteria. DNA was extracted from tissue (meta- or meso- thoracic leg) using DNeasy Blood & Tissue kit (Qiagen) and the isolates were checked for qual- ity and quantity on NanoDrop spectrophotometer. Samples with high DNA concentration (>20–25 ng/μL) were diluted with water until optimal concentration. For 56 samples PCR was done using COH6 (5’- TAD ACT TCD GGR TGD CCA AAR AAY CA -3’) and COL6b (5’- ACA AAT CAT AAA GAT ATY GG -3’) primers [34], the thermal cycle included 1 cycle of initial denaturation at 94˚C for 5 min, followed by 30 cycles of denaturation at 94˚C for 1 min, annealing at 55˚C for 1 min and extension 72˚C for 1 min, finishing with 1 cycle of

final extension at 72˚C for 10 min. For other 16 samples, where amplification with COH6/ COL6b primers failed, another primer pair was used, LCO1490 (5’- GGT CAA CAA ATC ATA AAG ATA TTG G -3’) and HCO2198 (5’- TAA ACT TCA GGG TGA CCA AAA AAT CA -3’) [16], the thermal cycle included initial denaturation at 94˚C for 1 min, 5 cycles of 94˚C for 40 s, 45˚C for 40 s, 72˚C for 1 min, followed by 35 cycles of 94˚C for 40 s, 51˚C for 40 s, 72˚C for 1 min and a final extension of 72˚C for 5 min. For details on samples amplifica- tion primers, see S1 Table. The amplified products were visualized in 1% agarose gel, purified using Gel / PCR DNA fragment extraction kit (Geneaid) and sequenced with forward primer at Biology Section, Faculty of Science, Charles University using BigDye1 Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) on POP-7 polymer (Applied Biosystems). Raw sequences were trimmed and edited in Geneious v10.2.6 (https://www.geneious.com) software and aligned by ClustalW approach [35] as implemented in Geneious. These sequence data have been submitted to the NCBI databases under accession numbers OR820652—OR820723 (see S1 Table). Available DNA barcodes from samples collected in CAR (n = 73 [23]) were obtained from BOLD database. In addition, several unpublished sequences from Cameroon (n = 10) and Gabon (n = 10) were provided by Nicolas Moulin (Muse´um national d’Histoire naturelle, Paris, France). Raw chromatograms were curated, converted to the sequences and aligned together with our sequences using same software tools as described above.

Pairwise genetic distances and maximum likelihood trees were calculated in IQ-TREE 1.6.12 [36]. Sequences were initially partitioned according to 1st, 2nd and 3rd codon position. ModelFinder [37] was used to determine the best partition scheme and nucleotide substitution model according to Bayesian information criterion. The best partition scheme was keeping the original three partitions, and the best models were TPM3+F+I+G4, TIM3e+I+G4 and GTR+F +G4, respectively. Support for the clades found on the ML tree was calculated using SH-aLRT, aBayes and ultrafast bootstrap [38]. A map of collecting sites (Fig 1) was created with open-source desktop geographic informa- tion system application QGIS 3.36 (QGIS Development Team) using Shuttle Radar Topogra- phy Mission (SRTM) digital elevation model [39]. Country boundaries and populated places data were obtained from https://www.naturalearthdata.com. Abbreviations of institutions of type material deposition: ANSP: Academy of Natural Sciences, Philadelphia, USA. PLOS ONE Diversity of praying mantises in Cameroon PLOS ONE | https://doi.org/10.1371/journal.pone.0304163 May 23, 2024 4 / 23