Genetic tracing of the illegal trade of the white-bellied pangolin (Phataginus tricuspis) in western Central Africa.

The white-bellied pangolin is subject to intense trafficking, feeding both local and international trade networks. In order to assess its population genetics and trace its domestic trade, we genotyped 562 pangolins from local to large bushmeat markets in western central Africa. We show that the two lineages described from the study region (WCA and Gab) were overlapping in ranges, with limited introgression in southern Cameroon. There was a lack of genetic differentiation across WCA and a significant signature of isolation-by-distance possibly due to unsuspected dispersal capacities involving a Wahlund effect. We detected a c. 74.1-82.5% decline in the effective population size of WCA during the Middle Holocene. Private allele frequency tracing approach indicated up to 600 km sourcing distance by large urban markets from Cameroon, including Equatorial Guinea. The 20 species-specific microsatellite loci provided individual-level genotyping resolution and should be considered as valuable resources for future forensic applications. Because admixture was detected between lineages, we recommend a multi-locus approach for tracing the pangolin trade. The Yaoundé market was the main hub of the trade in the region, and thus should receive specific monitoring to mitigate pangolins' domestic trafficking. Our study also highlighted the weak implementation of CITES regulations at European borders.

Phylogenomics of African radiation of Praomyini (Muridae: Murinae) rodents: First fully resolved phylogeny, evolutionary history and delimitation of extant genera.

The tribe Praomyini is a diversified group including 64 species and eight extant rodent genera. They live in a broad spectrum of habitats across whole sub-Saharan Africa. Members of this tribe are often very abundant, they have a key ecological role in ecosystems, they are hosts of many potentially pathogenic microorganisms and comprise numerous agricultural pests. Although this tribe is well supported by both molecular and morphological data, its intergeneric relationships and the species contents of several genera are not yet fully resolved. Recent molecular data suggest that at least three genera in current sense are paraphyletic. However, in these studies the species sampling was sparse and the resolution of relationships among genera was poor, probably due to a fast radiation of the tribe dated to the Miocene and insufficient amount of genetic data. Here we used genomic scale data (395 nuclear loci = 610,965 bp long alignment and mitogenomes = 14,745 bp) and produced the first fully resolved species tree containing most major lineages of the Praomyini tribe (i.e. all but one currently delimited genera and major intrageneric clades). Results of a fossil-based divergence dating analysis suggest that the radiation started during the Messinian stage (ca. 7 Ma) and was likely linked to a fragmentation of the pan-African Miocene forest. Some lineages remained in the rain forests, while many others adapted to a broad spectrum of new open lowland and montane habitats that appeared at the beginning of Pliocene. Our analyses clearly confirmed the presence of three polyphyletic genera (Praomys, Myomyscus and Mastomys). We review current knowledge of these three genera and suggest corresponding taxonomic changes. To keep genera monophyletic, we propose taxonomic re-arrangements and delimit four new genera. Furthermore, we discovered a new highly divergent genetic lineage of Praomyini in southwestern Ethiopia, which is described as a new species and genus.

Description of Heteracris lecoqi Yetchom-Fondjo amp; Kekeunou sp. nov. and Heteracris hannai Wandji amp; Kekeunou sp. nov. and redescription of Heteracris guineensis (Krauss, 1890) (Orthoptera: Acrididae) with comments on its ecology in the Southern part of Cameroon.

The specimens studied were collected with sweep net and pitfall in the forests, agro-forests, herbaceous fallows, and crop fields of 14 localities in the southern part of Cameroon, from August 2015 to February 2018. The results show that Heteracris hannai sp. nov. and Heteracris lecoqi sp. nov. differ from previously known species of the same genus by the pattern of coloration and the details or shape of the phallic complex. H. lecoqi sp. nov. is characterized by light brownish body; male cercus with flattened, downcurved and obtuse apex; lophus strongly curved; interlophal space with V shape; apodeme of cingulum bow, convergent, with V-shape; valve of cingulum in lateral view longer than apical valve of penis; ramus in ventral view joined. H. hannai sp. nov. is distinct to other species by brown to grey body; male cercus with apex rounded, curved inside; lophus curved; interlophal space with U-shape; apodeme of cingulum thick, slightly parallel, with U shape; valve of cingulum in lateral view hardly longer than apical valve of penis; ramus in ventral view opened. The characteristics of H. guineensis are closer to H. hannai sp. nov. than to H. lecoqi sp. nov. H. lecoqi sp. nov. was collected only in the fallows while H. hannai sp. nov. was collected in the forests, agro-forests, fallows, and crop fields. Compared to H. guineensis, both new species are scarce in the natural vegetation and their distribution area is limited to two and four localities respectively for H. lecoqi sp. nov. and H. hannai sp. nov. All these three grasshopper's species were recorded as accidental species in all types of vegetation.

Development and characterization of 20 polymorphic microsatellite markers for the white-bellied pangolin Phataginus tricuspis (Mammalia, Pholidota).

Pangolins, or scaly anteaters, have recently been flagshiped as one of the most illegally traded mammals, and as a corollary, as potential intermediate hosts at the origin of the COVID-19 pandemic. In order to improve the traceability of their trade, we developed 20 polymorphic microsatellite loci for the white-bellied pangolin (Phataginus tricuspis), the species most frequently found on African bushmeat markets. We genotyped 24 white-bellied pangolins from the Douala market, Cameroon, originating from the Ebo forest c. 75 km north-east of Douala. The number of alleles per locus ranged from 4 to 12 (mean = 6.95), and mean observed and expected heterozygosities were 0.592 (0.208-0.875) and 0.671 (0.469-0.836), respectively. Genetic diversity was higher than that cross-estimated from microsatellite loci developed for other species of pangolins. Two loci deviated from Hardy-Weinberg equilibrium and two loci showed linkage disequilibrium. Genetic variance (PCoA) was increased with the addition of 13 pangolins of unknown origin, possibly suggesting that the Douala market is fed from differentiated source populations of white-bellied pangolins. Each of the 37 individuals had a unique multilocus genotype. The unbiased probability of identity (uPI) and the probability of identity among siblings (PIsibs) were both very low (uPI = 8.443 e-21; PIsibs = 1.011 e-07). Only five microsatellite loci were needed to reach the conservative value of PIsibs < 0.01, overall indicating a powerful discriminating power of our combined loci. These 20 newly developed microsatellite loci might prove useful in tracing the local-to-global trade of the white-bellied pangolin, and will hopefully contribute to the DNA-assisted implementation of future conservation strategies at reasonable costs.

The phylogeny of the African wood mice (Muridae, Hylomyscus) based on complete mitochondrial genomes and five nuclear genes reveals their evolutionary history and undescribed diversity.

Wood mice of the genus Hylomyscus, are small-sized rodents widely distributed in lowland and montane rainforests in tropical Africa, where they can be locally abundant. Recent morphological and molecular studies have increased the number of recognized species from 8 to 18 during the last 15 years. We used complete mitochondrial genomes and five nuclear genes to infer the number of candidate species within this genus and depict its evolutionary history. In terms of gene sampling and geographical and taxonomic coverage, this is the most comprehensive review of the genus Hylomyscus to date. The six species groups (aeta, alleni, anselli, baeri, denniae and parvus) defined on morphological grounds are monophyletic. Species delimitation analyses highlight undescribed diversity within this genus: perhaps up to 10 taxa need description or elevation from synonymy, pending review of type specimens. Our divergence dating and biogeographical analyses show that diversification of the genus occurred after the end of the Miocene and is closely linked to the history of the African forest. The formation of the Rift Valley combined with the declining global temperatures during the Late Miocene caused the fragmentation of the forests and explains the first split between the denniae group and remaining lineages. Subsequently, periods of increased climatic instability during Plio-Pleistocene probably resulted in elevated diversification in both lowland and montane forest taxa.

A Checklist of Short-horned Grasshopper Species (Orthoptera: Caelifera) from Littoral Region of Cameroon with description of a new species of the genus Hemierianthus Saussure, 1903 (Orthoptera: Chorotypidae).

The Littoral Region of Cameroon is one of the most highly disturbed regions in Cameroon that remain poorly studied and where no investigation regarding the grasshopper fauna has been conducted. Hence a survey was conducted from August 2015 to November 2018 in 11 localities of this region. In each locality, grasshoppers were sampled in three vegetation types (forests, fallows and cultivated farms) using sweep nets sampling method. We collected in total 51 short-horned grasshopper species belonging to the families Acrididae, Chorotypidae, Euschmidtiidae, Pyrgomorphidae and Thericleidae. Among these species, 43 belong to Acrididae, 5 to Pyrgomorphidae and only one belongs to the Chorotypidae, Euschmidtiidae and Thericleidae respectively. The Chorotypidae species Hemierianthus mbongueensis Yetchom Xu sp.nov. is also described in this study. Twelve grasshopper species were widely distributed as they occurred in all study sites and 11 were recorded exclusively from one study site. This study showed that there is high species richness of short-horned grasshoppers in the Littoral Region of Cameroon. However, more sampling effort in different ecosystems of this region and other regions of Cameroon will certainly improve our understanding on grasshopper taxonomy, ecology and biogeography.

Bushmeat genetics: setting up a reference framework for the DNA typing of African forest bushmeat.

The bushmeat trade in tropical Africa represents illegal, unsustainable off-takes of millions of tons of wild game - mostly mammals - per year. We sequenced four mitochondrial gene fragments (cyt b, COI, 12S, 16S) in >300 bushmeat items representing nine mammalian orders and 59 morphological species from five western and central African countries (Guinea, Ghana, Nigeria, Cameroon and Equatorial Guinea). Our objectives were to assess the efficiency of cross-species PCR amplification and to evaluate the usefulness of our multilocus approach for reliable bushmeat species identification. We provide a straightforward amplification protocol using a single 'universal' primer pair per gene that generally yielded >90% PCR success rates across orders and was robust to different types of meat preprocessing and DNA extraction protocols. For taxonomic identification, we set up a decision pipeline combining similarity- and tree-based approaches with an assessment of taxonomic expertise and coverage of the GENBANK database. Our multilocus approach permitted us to: (i) adjust for existing taxonomic gaps in GENBANK databases, (ii) assign to the species level 67% of the morphological species hypotheses and (iii) successfully identify samples with uncertain taxonomic attribution (preprocessed carcasses and cryptic lineages). High levels of genetic polymorphism across genes and taxa, together with the excellent resolution observed among species-level clusters (neighbour-joining trees and Klee diagrams) advocate the usefulness of our markers for bushmeat DNA typing. We formalize our DNA typing decision pipeline through an expert-curated query database - DNA BUSHMEAT - that shall permit the automated identification of African forest bushmeat items.

Shade tree diversity, cocoa pest damage, yield compensating inputs and farmers' net returns in West Africa.

Cocoa agroforests can significantly support biodiversity, yet intensification of farming practices is degrading agroforestry habitats and compromising ecosystem services such as biological pest control. Effective conservation strategies depend on the type of relationship between agricultural matrix, biodiversity and ecosystem services, but to date the shape of this relationship is unknown. We linked shade index calculated from eight vegetation variables, with insect pests and beneficial insects (ants, wasps and spiders) in 20 cocoa agroforests differing in woody and herbaceous vegetation diversity. We measured herbivory and predatory rates, and quantified resulting increases in cocoa yield and net returns. We found that number of spider webs and wasp nests significantly decreased with increasing density of exotic shade tree species. Greater species richness of native shade tree species was associated with a higher number of wasp nests and spider webs while species richness of understory plants did not have a strong impact on these beneficial species. Species richness of ants, wasp nests and spider webs peaked at higher levels of plant species richness. The number of herbivore species (mirid bugs and cocoa pod borers) and the rate of herbivory on cocoa pods decreased with increasing shade index. Shade index was negatively related to yield, with yield significantly higher at shade and herb covers<50%. However, higher inputs in the cocoa farms do not necessarily result in a higher net return. In conclusion, our study shows the importance of a diverse shade canopy in reducing damage caused by cocoa pests. It also highlights the importance of conservation initiatives in tropical agroforestry landscapes.

Nonlinear projection methods for visualizing Barcode data and application on two data sets.

Developing tools for visualizing DNA sequences is an important issue in the Barcoding context. Visualizing Barcode data can be put in a purely statistical context, unsupervised learning. Clustering methods combined with projection methods have two closely linked objectives, visualizing and finding structure in the data. Multidimensional scaling (MDS) and Self-organizing maps (SOM) are unsupervised statistical tools for data visualization. Both algorithms map data onto a lower dimensional manifold: MDS looks for a projection that best preserves pairwise distances while SOM preserves the topology of the data. Both algorithms were initially developed for Euclidean data and the conditions necessary to their good implementation were not satisfied for Barcode data. We developed a workflow consisting in four steps: collapse data into distinct sequences; compute a dissimilarity matrix; run a modified version of SOM for dissimilarity matrices to structure the data and reduce dimensionality; project the results using MDS. This methodology was applied to Astraptes fulgerator and Hylomyscus, an African rodent with debated taxonomy. We obtained very good results for both data sets. The results were robust against unbalanced species. All the species in Astraptes were well displayed in very distinct groups in the various visualizations, except for LOHAMP and FABOV that were mixed up. For Hylomyscus, our findings were consistent with known species, confirmed the existence of four unnamed taxa and suggested the existence of potentially new species.

Assessment of three mitochondrial genes (16S, Cytb, CO1) for identifying species in the Praomyini tribe (Rodentia: Muridae).

The Praomyini tribe is one of the most diverse and abundant groups of Old World rodents. Several species are known to be involved in crop damage and in the epidemiology of several human and cattle diseases. Due to the existence of sibling species their identification is often problematic. Thus an easy, fast and accurate species identification tool is needed for non-systematicians to correctly identify Praomyini species. In this study we compare the usefulness of three genes (16S, Cytb, CO1) for identifying species of this tribe. A total of 426 specimens representing 40 species (sampled across their geographical range) were sequenced for the three genes. Nearly all of the species included in our study are monophyletic in the neighbour joining trees. The degree of intra-specific variability tends to be lower than the divergence between species, but no barcoding gap is detected. The success rate of the statistical methods of species identification is excellent (up to 99% or 100% for statistical supervised classification methods as the k-Nearest Neighbour or Random Forest). The 16S gene is 2.5 less variable than the Cytb and CO1 genes. As a result its discriminatory power is smaller. To sum up, our results suggest that using DNA markers for identifying species in the Praomyini tribe is a largely valid approach, and that the CO1 and Cytb genes are better DNA markers than the 16S gene. Our results confirm the usefulness of statistical methods such as the Random Forest and the 1-NN methods to assign a sequence to a species, even when the number of species is relatively large. Based on our NJ trees and the distribution of all intraspecific and interspecific pairwise nucleotide distances, we highlight the presence of several potentially new species within the Praomyini tribe that should be subject to corroboration assessments.