Integrative taxonomic analysis of new collections from the central Angolan highlands resolves the taxonomy of African pipistrelloid bats on a continental scale

SUPPORTING INFORMATION : TABLE S1. Details of specimens included in molecular, morphological (craniodental or bacular) and/or morphometric analysis of pipistrelloid bats from Angola, Eswatini, South Africa, Mozambique and Botswana. Abbreviation of Museums: DM (Durban Natural Science Museum); TM (Ditsong National Museum of Natural History); UNESWA (University of Eswatini). Availability of skull or baculum specimens indicated by ‘x’. GenBank numbers are included for specimens having DNA sequences for the three mitochondrial genes, cytochrome b (cytb), cytochrome oxidase subunit 1 (CO1) and 12S ribosomal RNS (12S). Specimens marked with asterisks under ‘Skull’ indicate those having skulls available but not measured in final morphometric analyses.TABLE S2. Uncorrected P-distances (number of base differences per site from averaging over all sequence pairs) from the CO1 mitochondrial gene are shown, obtained between (bottom left) and within (on diagonals in bold face; ‘n/c’ indicates sample sizes too small for calculation) groups of pipistrelloid bats from Angola, Eswatini and South Africa in relation to GenBank sequences of comparable African species for the CO1 mitochondrial gene. See Supporting Information, Table S1 for details of specimens used in the study. This analysis involved 118 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 478 positions in the final dataset. Analyses were conducted in MEGA X (Kumar et al., 2018). Abbreviations for groups are as follows: Anan = Afronycteris nana, Lang = Laephotis angolensis, Lbot = L. botswanae, Lcap(Ang) = L. capensis (Angola), Lcap(AngSA) = L. capensis (South Africa and Angola), Nanc = Neoromicia anchietae, Nsom = N. somalica, Nzul = N. zuluensis, Phesp = Pipistrellus hesperidus, Prust(Ang) = P. rusticus (Angola), Prust(SA) = P. rusticus (southern Africa), Outgroup = outgroup (Miniopterus spp.).TABLE S3. Uncorrected P-distances (number of base differences per site from averaging over all sequence pairs) from the 12S RNA mitochondrial gene are shown, obtained between (bottom left) and within (on diagonals in bold face; ‘n/c’ indicates sample sizes too small for calculation) groups of pipistrelloid bats from Angola, Eswatini and South Africa in relation to GenBank sequences of comparable African species for the 12S RNA mitochondrial gene. See Supporting Information, Table S1 for details of specimens used in the study. This analysis involved 72 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 783 positions in the final dataset. Analyses were conducted in MEGA X (Kumar et al., 2018). Abbreviations for groups are as follows: Anan(Ang) = Afronycteris nana (Angola), Anan(EA) = A. nana (East Africa), Anan(WA) = A. nana (West Africa), Lang = Laephotis angolensis/botswanae (Angola, South Africa, Mozambique), Lcap(EA) = L. capensis (East Africa), Lcap(SA) = L. capensis (southern Africa), Lnam = L. namibensis, Nanc = Neoromicia anchietae, Nbem = N. bemainty (Madagascar), Nsom = N. somalica (East Africa), Nsp_nov = N. sp. nov. (= N. hlandzeni from South Africa and Eswatini), Nzul(Ang) = N. zuluensis (Angola), Nzul(SA) = N. zuluensis (South Africa, Eswatini), Phesp(SA) = Pipistrellus hesperidus (South Africa, Eswatini), Pcf.rust(SWA) = P. cf. rusticus (West Africa, Eswatini), Prust = P. rusticus (Angola), Outgroup = outgroup (Miniopterus spp).FIGURE S1. Bayesian phylogeny of mitochondrial cytochrome oxidase one sequences (478 nucleotides) of Vespertilionidae genera Afronycteris, Neoromicia, Laephotis, Pipistrellus sampled from Angola, South Africa and Eswatini and the outgroup, Miniopterus spp. (Miniopteridae). Bayesian phylogenetic analysis was performed using the Hasegawa– Kishino–Yano model incorporating invariant sites and a gamma distribution (HKY+I+G). Posterior probabilities of > 0.7 are indicated at internal nodes. Samples obtained in this study are indicated in bold. Branch colours indicate posterior probabilities at each node. FIGURE S2. Bayesian phylogeny of mitochondrial 12S sequences (478 nucleotides) of pipistrelle-like Vespertilionidae genera Afronycteris, Neoromicia, Laephotis and Pipistrellus sampled from Angola, South Africa and Eswatini and outgroup Miniopterus spp. (Miniopteridae). Bayesian phylogenetic analysis was performed using the Hasegawa– Kishino–Yano model incorporating invariant sites and a gamma distribution (HKY+I+G). Posterior probabilities of > 0.7 are indicated at internal nodes. Samples obtained in this study are indicated in bold. Branch colours indicate posterior probabilities at each node.Ten years ago, the genus-level and species-level taxonomy of African pipistrelloid bats was in a state of flux. In spite of advances in the past decade, gaps in collecting from species-rich regions like Angola have hampered efforts to revise this group. We report on new collections of pipistrelle-like bats from the poorly sampled central highlands of Angola (1000–1500 m a.s.l.) as well as comparative material from lower-lying areas of Eswatini and South Africa. Specimens identified as Neoromicia anchietae, collected 400–700 km east of the holotype locality in the western highlands of Angola, were genetically and morphologically distinctive from N. anchietae s.l. from South Africa and Eswatini. We describe herein this latter lineage as a distinct species from low-lying areas of south-eastern Africa, distinct from N. anchietae s.s., which is therefore restricted to the central and western Angolan highlands. We also identified shallow to deep genetic divergence between different African regions in other recognized pipistrelloid species, such as conspecificity between the long-eared species Laephotis angolensis from Angola and Laephotis botswanae from northern Botswana, northern Namibia and south-western Zambia. Our phylogeny supports a recently proposed generic classification of African pipistrelloid bats.The University of Venda, the National Research Foundation and the Department of Science and Innovation under the South African Research Chair Initiative (SARChI) on ‘Biodiversity Value and Change within the Vhembe Biosphere Reserve’ hosted at the University of Venda and co-hosted by the Centre for Invasion Biology at the University of Stellenbosch and the South African Research Chair at the University of Pretoria on Infectious Diseases of Animals (Zoonoses)https://academic.oup.com/zoolinneanhj2023Mammal Research InstituteMedical VirologyZoology and Entomolog