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

cies were sequenced. Our study produced 72 sequences and included all species mentioned in checklist, with eight described species receiving a DNA barcode for the first time and five addi- tional are of species likely new to science. Continuous efforts in Cameroon, especially in north- ern areas in savannah biomes will undoubtably bring country’s checklist on par with neighboring Gabon, as well as help obtain more unique DNA barcodes. Our research supports the comments of Moulin et al. [23] on genus Plistospilota, taxonomi- cally complicated genus from the tribe Polispilotini, with original descriptions of the species within this genus are mostly too brief, which makes morphological species identification diffi- cult. At the same time, barcoding seems to clearly separate and group samples into distinct clusters in both Moulin et al. and our own analysis. Molecular phylogeny can become a valu- able tool in aiding traditional taxonomical methods in revising this taxon, which it desperately needs. Several collected specimens of Mantodea were not identified to species level. Three males and ten females of the genus Negromantis were found to be morphologically closely related to Negromantis modesta Giglio-Tos, 1915 but they differed from the original description of the species in number of posteroventral spines on the forefemora. Barcodes obtained from these specimens did not match with any entry in GenBank or BOLD. Considerable differences in barcodes should be evaluated by examining the type material of the genus.

During identification of a specimen of genus Galepsus, it was found to have unique genital morphology. While the structure of head vertex fits the diagnosis of the nominative subgenus, none of the species with described copulatory organs possess the same genitalia. Based on external morphology and coloration, this specimen is related to Galepsus (Galepsus) laticeps Werner, 1907 but has distinct genitalia. Available sequences of G. (Galepsus) laticeps and G. (Syngalepsus) bucheti Moulin, 2018 differ significantly from this sample, with distance being 54.5% and 63.9% respectively. This specimen likely represents a species new to science, how- ever, examination of type material of the species of this subgenus is required. Several collected specimens of genus Sphodromantis resembled quite closely a described taxon, Sphodromantis lineola pinguis La Greca, 1967. They have same body shape, spination of the forelegs and very similar structure of genitalia in males. At the same time, these specimens differed in larger body size and most noticeably, pigmented spots on forefemora. Such PLOS ONE Diversity of praying mantises in Cameroon PLOS ONE | https://doi.org/10.1371/journal.pone.0304163 May 23, 2024 19 / 23

combination of morphological characters was not matched with any described species. DNA barcode of the sample has the distance of 14% with a sequence of typical S. lineola, while on phylogenetic tree the unidentified species formed a separate branch with same species. It might be suggested that femoral spots are an early developing character resulted from predator pressure, as the spots are a part of threat display of the mantis. Larger body size might also facilitate in repelling the offenders. One of the advantages of using DNA barcodes is identification of samples, otherwise impossible to determine due to life stage or sex. This is illustrated on obtained tree in Steno- pyga extera. One of the samples (VGPC23383) we obtained the sequence from was immature female mantis, belonging to genus Stenopyga. However, identification to species-level based on morphological characters was not possible, as the diagnostic characters for the genus include wing coloration (not developed at nymphal stage) and structure of male genitalia (obviously absent in a female) [20]. While building phylogenetic tree, the barcode of immature Stenopyga clustered with a sequence from same locality of an adult male Stenopyga extera, which was pos- sible to identify based on morphological characters. The distance between two samples is <0.2%, which confirms these two specimens are conspecific. DNA barcoding proved to be useful in identification of specimens with abnormal morpho- logical traits. One female of Polyspilota aeruginosa (VGPC23764) was found to possess black-

ened prosternum, atypical for this species. Given that P. aeruginosa is a taxon with high intraspecific variation [24], it is likely an individual aberration. Molecular analysis supports this hypothesis, as the maximum likelihood tree places this specimen close to sequences from CAR, moreover, the pairwise distance between a typical looking sample and the one with blackened prosternum both collected in Ebogo II is about 0.7%. Comparison of the dissected genitalia of this female with a typically looking female revealed no noticeable differences in copulatory organs. Cameroon is an often-disregarded region when it comes to biodiversity assessments, partic- ularly regarding Mantodea. Our research represents the first comprehensive study on praying mantises of Cameroon, enriching the catalogue of species and enhancing it through the use of DNA barcodes. The use of genetic information allowed us to compare existing phylogeny of the order and to complement existing efforts from neighboring countries. Phylogenetic tree built using sequences of Mantodea of Central African subregion is adequately congruent with current taxonomical consensus. However, several collected specimens likely belong to previ- ously unrecognized taxa, highlighting the importance of expanding the study area and increas- ing the frequency of sampling to reveal even more undescribed species. The tree, complemented by pairwise distances, demonstrate unresolved taxonomy of several genera, such as Negromantis, Plistospilota. To address these knowledge gaps, it is crucial to employ a

combination of morphological and molecular approaches to fill in the missing data on the biology and ecology of praying mantises in the Central African subregion and, by extension, the entire continent. Supporting information S1 Table. Primer pairs used for amplification of the samples and their molecular voucher and accession codes. (DOCX) S2 Table. Pairwise distances. (XLSX) PLOS ONE Diversity of praying mantises in Cameroon PLOS ONE | https://doi.org/10.1371/journal.pone.0304163 May 23, 2024 20 / 23

Acknowledgments We would like to thank Nicolas Moulin (Muse´um national d’Histoire naturelle, Paris, France) for providing unpublished barcode sequences of Mantodea from Cameroon, CAR and Gabon. Fieldwork in Cameroon would not have been possible without Anna Maňourova´ (Swedish University of Agricultural Sciences, Alnap, Sweden), who helped organizing the fieldwork. We wish to thank Tchana family for the warmest welcome and hospitality in Yaounde´, and all the inhabitants of Ebogo II village who made material collection possible. We are grateful to Sergei Bimbirekov (Moscow, Russia) for taking magnificent pictures of praying mantises and Chris- tian Schwarz (Ruhr University Bochum, Bochum, Germany) for providing rare literature. Andreas Schmidt-Rhaesa (Museum der Natur Hamburg, Hamburg, Germany) has lent us his expert opinion on the identity of horsehair worms. Author Contributions Conceptualization: Valeriy Govorov. Data curation: Valeriy Govorov, Evgeny Shcherbakov, Barbora Černa´ Bolfikova´. Funding acquisition: Petr Jansˇta, Barbora Černa´ Bolfikova´. Investigation: Valeriy Govorov. Methodology: Valeriy Govorov, Evgeny Shcherbakov, Barbora Černa´ Bolfikova´. Project administration: Valeriy Govorov, Barbora Černa´ Bolfikova´. Resources: Barbora Černa´ Bolfikova´. Supervision: Evgeny Shcherbakov, Petr Jansˇta, Barbora Černa´ Bolfikova´. Validation: Valeriy Govorov. Visualization: Valeriy Govorov. Writing – original draft: Valeriy Govorov. Writing – review & editing: Valeriy Govorov, Evgeny Shcherbakov, Petr Jansˇta, Barbora Černa´ Bolfikova´. References

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