Integrative taxonomic analysis of new collections from the central Angolan highlands resolves the taxonomy of African pipistrelloid bats on a continental scale Author Taylor, Peter John Author Strydom, Erika Author Richards, Leigh Author Markotter, Wanda Author Toussaint, Dawn Cory Author Kearney, Teresa Author D., F. P. Author Woody Author Cotterill Author Howard, Alexandra Author Weier, Sina Monika Author Keith, Mark Author Neef, Gӧtz Author Mamba, Mnqobi L. Author Magagula, Siphesihle Author Monadjem, Ara text Zoological Journal of the Linnean Society 2022 2022-10-07 196 4 1570 1590 http://dx.doi.org/10.1093/zoolinnean/zlac071 journal article 199801 10.1093/zoolinnean/zlac071 9bd5667a-f485-4324-a1c3-340f61909fb8 0024-4082 7390987 14158A89-807B-4157-94E9-CB6870644CE3 NEOROMICIA HLANDZENI SP. NOV. LOWVELD SEROTINE Pipistrellus (Hypsugo) anchietae Hill & Harrison (1987) (In part): Zambia ? Pipistrellus (Hypsugo) anchietae Koopman (1993) (In part): South Africa . Hypsugo anchietae Cotterill (1996) : Zimbabwe . Hypsugo anchietae Kearney et al. (2002) : KwaZuluNatal Province, South Africa . Hypsugoanchietae Kearney (2005): Zimbabwe , Limpopo and KwaZulu-Natal provinces, South Africa . Figure 5. Photographs and drawings of bacula of N. hlandzeni sp. nov. from Eswatini (DM 8423, 8422), KwaZulu-Natal Province, South Africa (DM 2269, 8787, 5353, 5358), together with N. anchietae from Botswana (TM 48489), Angola (FWC 7521), and Zambia (NHM 70.2632; from Hill & Harrison, 1987 ). In the top row, D=dorsal, L=lateral and V=ventral view. Images in the lower row are of the ventral view, except for the far right image which is of the lateral view. Abbreviations of museums as follows: DM = Durban Natural Science Museum; TM = Ditsong National Museum of Natural History; NHM = The Natural History Museum, London. FWC indicates the field number of Fenton (Woody) Cotterill. The vertical scale bar on the bottom right indicates 1 mm. Hypsugo anchietae Simmons (2005) (In part): Zimbabwe , South Africa . Hypsugo anchietae Monadjem et al. (2010) (In part): Zimbabwe , Mozambique , Eswatini , South Africa . Neoromicia anchietae Monadjem et al. (2020) (In part): Zimbabwe , Mozambique , Eswatini , South Africa . Laephotis anchietae Simmons & Cirranello (2022) (In part): Zimbabwe , Mozambique , Eswatini , South Africa . Holotype : Durban Natural Science Museum ( DM ) No. 8423. The adult male specimen collected by Ara Monadjem (field number AM 2066 ) on 5 September 2005 , has muscle tissue in 90% ethanol and the body preserved in ethanol, with skull ( Fig. 6 ) and baculum ( Fig. 5 ) extracted and cleaned. cytb sequences are available from GenBank (No. KM 886057 ). Type locality: Eswatini (formerly Swaziland ), Mlawula Nature Reserve , near the Siweni train siding in the north-eastern lowveld savanna region of the country (latitude 26.17998°S ; longitude 32.04871°E ). The specimen was netted in riparian vegetation over a small side channel of the Mbuluzi River at an elevation of 110 m a.s.l. Paratypes : DM No. 8422; Ditsong National Museum of Natural History ( TM ) No. 47718. Both adult males, with bacula, from Mlawula Nature Reserve , Eswatini . Etymology: eHlandzeni means the ‘lowveld’ or wilderness in the SiSwati language indicating the specific lowveld (low-lying savanna) habitat of the new species which differentiates it from N. anchietae known from higher-lying (> 1000 m a.s.l.) elevations. With SiSwati names, the prefix is typically dropped when a noun is turned into a name. Diagnosis: The species is distinguished genetically (11% divergence in cytb sequences) from its sister species, N. anchietae from Angola . When compared with craniometric data of positively identified (from either mtDNA sequences or baculum) N. hlandzeni from South Africa and Eswatini ( N = 17; Table 2 ; Fig. 3 ), specimens of N. anchietae s.s. from Angola ( N = 20) have distinctly smaller-sized skulls ( Fig. 3 ; Table 2 ). Greatest skull length varies from 11.2 to 12.9 mm (mean 12.1 mm ) in Angolan N. anchietae and from 12.7 to 13.5 mm (mean 13.1 mm ) in N. hlandzeni . Based on material examined in this study, there is a definite and clear dental feature that distinguishes both N. anchietae and N. hlandzeni from P. hesperidus . Unless the teeth are worn, the inner, anterior (larger of two) upper incisor is clearly bifid in N. anchietae and N. hlandzeni , although sometimes this is only manifested as a ‘step’ in the tooth. Neither the step, nor the bifid condition was found in any P. hesperidus examined in this study. This condition of the inner upper incisor is confirmed byVan Cakenberghe & Happold (2013) . The original description by Seabra (1900) refers to the inner upper incisor as ‘tricuspid’. The baculum is> 1.30 mm long and bears a bilobed tip and base, with one of the basal lobes more enlarged than the other. Eswatini and South African individuals of N. hlandzeni can be distinguished from Angolan and Zambia samples of N. anchietae (also> 1.30 mm long) by their lack of well-defined lateral projections of the tip lobes ( Fig. 5 ). Table 5. Bacular measurements of 10 Neoromicia anchietae s. l . individuals from southern Africa. Museum abbreviations: DM = Durban Natural Science Museum; TM = Ditsong National Museum of Natural History

Catalogue number	Locality	Total	Basal	Basal	Basal notch	Tip	Tip	Tip notch	Greatest
length	width	height	height	width	length	depth	constriction
DM 8423 (Holotype)	Eswatini : Mlawula Nature Reserve	1.91	0.79	0.63	0.25	0.64	0.49	0.13	0.20
DM 8422 (Paratype )	Eswatini : Mlawula Nature Reserve	1.73	0.44	0.40	0.25	0.60	0.45	0.10	0.17
TM 47718 ( Paratype )	Eswatini: Mlawula Nature Reserve	1.74	0.62	-	-	0.55	0.41	0.10	0.13
DM 8512	Eswatini : Mbuluzi Nature Reserve	1.70	0.60	0.41	0.19	0.56	0.58	0.07	0.17
DM 2269	South Africa : KwaZulu-Natal , iSimangaliso	1.70	0.80	0.52	0.29	0.67	0.35	0.11	0.18
Wetland Park
DM 8787	South Africa : KwaZulu-Natal , Phinda	1.76	0.62	0.58	-	0.62	0.47	0.10	0.20
Private Game Reserve
DM 5353	South Africa : KwaZulu-Natal , Harold	1.62	0.58	0.36	-	0.61	0.42	0.10	0.12
Johnson Nature Reserve
DM 5358	South Africa : KwaZulu-Natal , Umkomaas,	1.34	0.49	0.37	0.14	0.41	0.32	0.08	0.11
Empisini Nature Reserve
DM 16054	Angola	1.63	0.47	0.55	0.24	0.72	0.44	0.14	0.14
TM 48489	Botswana	1.09	0.43	0.29	0.12	0.54	0.34	0.09	0.16

Description: In pelage coloration and general body size and appearance, the new species is indistinguishable from N. anchietae (see description of Seabra, 1900 in Van Cakenberghe & Seamark, 2020 ; Van Cakenberghe & Happold, 2013 ). The new bat represents a relatively small-sized vespertilionid species with relatively large ears. The thumb is relatively long and has a distinct white marking at the base, on the thumb pad ( Fig. 7 ). The tragus is about half the length of the ear, with a rounded and convex posterior surface and basal lobe. The antitragus is clearly visible and triangular in shape. The pelage is longish (up to 6 mm in length), bicoloured (hairs darker below), dark brown to yellowish brown dorsally and light brown, cream or white ventrally, darker in the pelvic area. The skull is relatively small and fragile for an African vespertilionid with a ‘notably depressed head’, i.e. concave lateral profile ( Seabra, 1900 ; Fig. 6 ). There are five upper cheek teeth (including a minute anterior premolar), five lower cheek teeth and one canine and two upper and three lower incisors on each side. The anterior (inner) upper incisor is typically bifid or retains a ‘step’ in individuals with worn teeth. The posterior (outer) incisor is much smaller, about half the length of the anterior incisor or less. In cranial shape, the new species, together with N. anchietae , have an upturned end to the skull in the region of the incisors and canines relative to P. rusticus and P. hesperidus giving a concave lateral profile, whereas the others have a straighter profile. Furthermore, the zygomatic arch is not straight but has a wavy profile in N. anchietae and N. hlandzeni , whereas in P. rusticus and P. hesperidus it is straight. Figure 6. Dorsal, ventral, lateral cranial and lateral mandibular views of DM 8423 (holotype) and DM 8422 (paratype) of N. hlandzeni sp. nov. Scale bar represents 1 mm. Figure 7. Lateral photograph of the alcohol-preserved head and anterior portion of the forearm and thumb of the holotype of N. hlandzeni , DM 8423 (left) and close-up photographs of the thumb and proximal forearm of the holotype, DM 8423 (centre) and paratype, DM 8422 (right) showing a distinct white marking at the base to the thumb. The thumb (encircled) is slightly longer than that of a typical pipistrelloid bat. Based on eight genotyped and released individuals in the Eastern Cape Province of South Africa ( Moir et al. , 2020 ), of which mtDNA sequences of five were included in our molecular study ( Fig. 2 ), the echolocation call parameters of this species are almost indistinguishable from those of P. hesperidus : duration = 2.73 ± 0.32 ms; maximum frequency = 87.09 ± 8.74 kHz; minimum f r e q u e n c y = 4 6.0 6 ± 1.2 6 k H z; c h a r a c t e r i s t i c f r e q u e n c y = 4 6. 6 3 ± 1. 2 9 k H z; f r e q u e n c y a t knee = 50.5 ± 2.08 kHz. Biology: The species seems to be associated with coastal and scarp forests in the Eastern Cape and KwaZuluNatal provinces of South Africa ( Moir et al. , 2020 ), the lowveld savanna of Eswatini and well wooded riparian areas in Zimbabwe and Mozambique ( Monadjem et al. , 2020 ). These habitats contrast somewhat with the higher-elevation habitat of N. anchietae based on the type specimen from Cahata in western Angola ( c . 1300 m a.s.l.) and the localities in central Angolan represented in the current study ( 1000–1300 m a.s.l.). Further molecular sampling is required to determine whether previous records from Zambia , Botswana and the Democratic Republic of Congo ( DRC ) represent N. anchietae or N. hlandzeni . Preliminary bacular analyses and comparison with published records indicate that Zambian samples represent N . anchietae s.s.