Vespertilionini

Torrent, Laura, Juste, Javier, Garin, Inazio, Aihartza, Joxerra, Dalton, Desiré L., Mamba, Mnqobi, Tanshi, Iroro, Powell, Luke L., Padidar, Sara, Mudarra, Juan Luis Garcia, Richards, Leigh & Monadjem, Ara, 2025, Taxonomic revision of African pipistrelle-like bats with a new species from the West Congolean rainforest, Zoological Journal of the Linnean Society 204 (2) : -

Tribe Vespertilionini

Phylogenetic analysis of Cytb and COI identified several wellsupported clades, although basal nodes were generally poorly supported. Our phylogeny of Vespertilionini clearly distinguished the seven genera (Afonycteris, Afopipistrellus , Hypsugo , Laephotis , Neoromicia , Nycticeinops , and Pseudoromicia ) that are currently recognized in Africa. Te members of the genera Laephotis and Neoromicia form monophyletic clades that are sister to each other based on both ML and BI analyses ( Fig. 2). Similarly, Afonycteris and Pseudoromicia genera form monophyletic clades supported by both ML and BI analyses ( Fig. 2). However, in the COI phylogenies, only monophyly of the genera Laephotis , Neoromicia , and Afonycteris were supported (Supporting Information, Fig. S1). In the ML phylogeny, the members of the genus Afopipistrellus , except for Af. eisentrauti , formed a monophyletic clade sister to the genus Hypsugo ‘ sensu stricto ’ (from North Africa and elsewhere in the Palaearctic and Indo-Malayan regions) ( Fig. 2). Interestingly, in the BI phylogeny, Ny. schlieffenii and Ny. cf. schlieffenii were supported as sister to the Afopipistrellus clade, whereas Af. eisentrauti was not. Moreover, Af. crassulus together with a sample of Af. cf. crassulus from Tanzania were placed in our topology with high bootstrap support as a sister species of Af. bellieri . Furthermore, the new sequences from ‘ Hypsugo musciculus ’ from Equatorial Guinea were also placed in the topology within Afopipistrellus , showing sister-relationships to Af. happoldorum and to Af. grandidieri . A final group of samples from Equatorial Guinea corresponded to the species Af. happoldorum ( Fig. 2).

Within the genus Pseudoromicia , both ML and BI analyses recovered a clade formed by Ps. brunnea , Ps. isabella , Ps. kityoi , Ps. mbamminkom , Ps. roseveari , and Ps. cf. tenuipinnis ( Fig. 2). Te three new sequences from Equatorial Guinea were obtained from individuals of white-winged bats, identified morphologically as Ps. tenuipinnis in the field and confirmed based only on the genetic material. Two of these sequences grouped with a Ps. cf. tenuipinnis sample from Tanzania in a group formed by Ps. mbamminkom , Ps. kityoi , Ps. roseveari , and Ps. sp. from Tanzania. However, one sequence was placed within the species Ps. nyanza ( Fig. 2). Te remaining samples from Equatorial Guinea belonging to the tribe were included within the clades that defined the species Afonycteris nanus , Ps. mbamminkom , and Ps. roseveari for both ML and BI analyses ( Fig. 2).

K2P genetic divergence between Afopipistrellus species ranged from 7.4% between Af. happoldorum and Af. grandidieri , to 28.3% between Af. crassulus and Af. eisentrauti . Divergence of Af. musciculus from Hypsugo species ranged from 16.5% to 19.7% (Supporting Information, Table S3). Te divergence between Nycticeinops and Afopipistrellus species ranged from 12.6% between Ny. cf. schlieffenii and Af. bellieri , to 27.8% between Ny. schlieffenii and Af. crassulus . Te genetic divergence between Ps. nyanza and Ps. cf. nyanza was 10.5%, and between these two taxa and Ps. cf. tenuipinnis was 21.3% and 21.5%, respectively. Moreover, the divergence between Ps. brunnea and Ps. mbamminkom , and Ps. roseveari was 8.1% and 6.4% respectively (Supporting Information, Table S3).

Te PCA ordination of craniodental variables for African Afopipistrellus and Nycticeinops species accounted for 85% of the variation, within the two first axes. Te first principal component (PC1) represented a size gradient with positive loadings for all measurements ( Table 1). Te ordination allocated Af. musciculus in a distinct region of the morphospace in which the remaining species were distributed along the size axis with some degree of overlap among them ( Fig. 3). Afopipistrellus musciculus appears on the lef of the graph, thus being the smallest Afopipistrellus of the species studied. Te second principal component (PC2) had a distinct high positive loading (0.927) for the variable GSH, indicating that the main difference in shape among the studied samples was the skull height ( Table 1). Te samples of Af. musciculus presented the most inflated skull of all Afopipistrellus and Nycticeinops studied ( Fig. 3). Te complete set of body and craniodental measurements of all Afopipistrellus specimens from Equatorial Guinea used in this study is reported in Supporting Information Tables S4–S 6.

Te bacula of Afopipistrellus showed remarkable variation in shape among species. Te baculum of Af. eisentrauti was small and lacked the long, slender, and tapered shaf of other species within the genus (Supporting Information, Figs S2, S 3, Table S7). Unlike the Af. eisentrauti baculum drawing presented in Hill and Harrison (1987: 69), the Af. eisentrauti specimen (EBD 19104M) had a slightly pointed tip (Supporting Information, Fig. S2). Te bacula of Af. musciculus and Af. happoldorum were long and slender with a narrow tip and two basal lobes, Af. musciculus was c. 1 mm shorter in length, the base conspicuously bifurcated and overall thinner than Af. happoldorum (Supporting

(I) model. Filled red circles on nodes denote bootstrap (BS)

values ≥ 70% and Bayesian posterior probabilities (PP) ≥ 0.90. Open circles outlined in black indicate BS ≥ 70% and PP <0.90, and open circles outlined in red indicate BS <70% and PP> 0.90. DRC refers to the Democratic Republic of the Congo. Labels include species name, GenBank accession number or specimen/sample number, and country of collection (Supporting Information, Table S2). Branch colours indicate individual species/clade membership.

Information, Fig. S2, Table S7). Moreover, the baculum of Af. happoldorum had the thickest shaf of the five Afopipistrellus species. Te baculum of Af. crassulus was the thinnest of all Afopipistrellus species, only comparable to Af. bellieri , with an overall long shape and a narrow tip, and short but conspicuous basal lobes (Supporting Information, Figs S2, S 3, Table S7).

Te mean echolocation call parameters of Af. crassulus (FME 46.65, StartF 90.94, and EndF 40.15 kHz) were higher than for Af. happoldorum (FME40.55, StartF 78.87, and EndF 35.20kH z) ( Table 2). Te echolocation calls of Ps. cf. mbamminkom , Ps. roseveari , Ps. cf. nyanza , and Ps. cf. tenuipinnis from Equatorial Guinea overlapped in the three call parameters measured with a FME between 39.21 and 40.40 kHz, StartF between 60.33 and 65.98 kHz, and EndF between 30.37 and 35.15 kHz ( Table 2).

Tribe Pipistrellini

For the tribe Pipistrellini , our reconstructions, under both ML and BI criteria, recovered the three genera currently recognized in Africa ( Pipistrellus , Scotoecus , and Vansonia ) ( Fig. 4). In the Cytb phylogeny, within the genus Pipistrellus , a strongly supported clade comprising specimens captured on Bioko Island stood out as a distinct group, sister to Pi. hesperidus and Pi. simandouensis but clearly differentiated from these two species ( Fig. 4). Likewise, a COI phylogenetic tree incorporating sequences from both tribes also reaffirms the monophyly of the group. However, the relationship between the three species must be interpreted carefully because the topology from the COI phylogeny did not support the Cytb hypothesis ( Fig. 4; Supporting Information, Fig. S1). Moreover, both ML and BI analyses placed Pi. cf. hesperidus from Senegal apart from the other sequences identified as Pi. hesperidus from Kenya and showing sister-relationships with a separate clade representing Pi. rusticus from Namibia ( Fig. 4).

K2P genetic divergence between the undescribed lineage from Bioko and Pi. hesperidus and Pi. simandouensis were 6.4% and 11.1%, respectively. Genetic divergence between this new lineage and the other Pipistrellus varied from 10.3% to 26.3% ( Table 3).

Te ordination of the samples for the two main axes of a PCA multivariate analysis on the craniodental measurements of the African Pipistrellus species shows that most taxa are distributed along the morphospace’s first axis with litle overlap ( Fig. 5). Te first two principal axes accounted for 87% of the total variation, and PC1 represented a size variation with negative loadings for all craniodental measurements ( Table 4). Hence, the smallest species ( Pi. nanulus ) appeared on the right side of PC1. In contrast, the new lineage from Bioko is located on the lef side of the gradient ( Fig. 5), indicating that this group represents the largest of the series studied. PC2, which can be interpreted as summarizing the variation in shape, had two high negative loadings; the most significant negative value (–0.781) corresponded to skull height (GSH), and the second most significant negative value (–0.428) corresponded to postorbital width (POB) ( Table 4). Species with higher projections on PC2 had narrower interorbital regions and flater skulls. Pipistrellus simandouensis from West Africa scarcely overlaps with the lineage found on Bioko Island ( Fig. 5).

Te penises and bacula of the Pipistrellus taxa from Bioko, as well as Pi. simandouensis , Pi. hesperidus , Pi. rusticus , and Pi. nanulus , are presented in Figures 6 and 7 and Table 5. Overall, the shape of the penises was similar, with a swollen glans and long whitish hairs, except for Pi. nanulus that presented the same thickness from the base to the glans of the penis ( Fig. 6). Te bacula of the five taxa had elongated shafs, bifurcated tips, wide bases, and are of similar size, except for Pi. nanulus , which shows a distinctly larger and sturdier baculum than the other species ( Fig. 7).

Based on the molecular and morphological differentiation presented above, we conclude that the new lineage from Bioko Island, Equatorial Guinea, represents an unknown Pipistrellus species. We first reassess the taxonomy of Af. crassulus and Af. musciculus , and then describe the new species of Pipistrellus .

Taxonomy