Herpetology Notes, volume 14: 397-406 (2021) (published online on 22 February 2021) Phylogenetic analysis of “Breviceps adspersus” documents B. passmorei Minter et al., 2017 in Limpopo Province, South Africa Matthew P. Heinicke1,*, Mohamad H. Beidoun1, Stuart V. Nielsen1,2, and Aaron M. Bauer3 Abstract. Recent systematic work has shown the Breviceps mossambicus species group to be more species-rich than previously documented and has brought into question the identity of many populations, especially in northeastern South Africa. We obtained genetic data for eight specimens originally identified as B. adspersus from Limpopo Province, South Africa, as well as numerous specimens from the core range of B. adspersus in Namibia and Zimbabwe. Phylogenetic analysis shows that there is little genetic variation across the range of B. adspersus. However, most of our Limpopo specimens are not B. adspersus but rather B. passmorei, a species previously known only from the immediate vicinity of its type locality in KwaZulu-Natal. These new records extend the known range of B. passmorei by 360 km to the north. Our results emphasize the need to obtain fine- scale range-wide genetic data for Breviceps to better delimit the diversity and biogeography of the genus. Keywords. Brevicipitidae, cryptic species, Microhylidae, rain frog, systematics, Transvaal Introduction Breviceps adspersus, with a lectotype locality listed as “Damaraland” [= north-central Namibia], and other The genus Breviceps Merrem, 1820 includes 18 or 19 syntypes from both Damaraland and “Transvaal” [= described species of rain frogs distributed across eastern northeastern South Africa], has a southern distribution, and southern Africa (AmphibiaWeb, 2020; Frost, 2020). ranging from Namibia across much of Botswana, The genus includes two major clades: the gibbosus Zimbabwe, and South Africa to western Mozambique. group and the mossambicus group (Nielsen et al., 2018). Breviceps mossambicus (type locality: Island of Although there is some geographic overlap between the Mozambique, Nampula Province, Mozambique) has an ranges of these groups, most gibbosus group species eastern distribution from Tanzania through Mozambique occur in the South African Cape region, while most to eastern South Africa. Breviceps poweri (type locality: species in the mossambicus group occur in tropical and Kabwe, Zambia) has a northern distribution centred subtropical savannas. For most of the 20th century, three on the Zambezi River basin (Fig. 1; Du Preez and species were considered to be part of the mossambicus Carruthers, 2018; Channing and Rödel, 2019). group. These species, B. adspersus Peters, 1882, B. Even though this group and these species have been mossambicus Peters, 1854, and B. poweri Parker, 1934, recognized for many decades, it has long been recognized are distinguished from one another by differences in that the systematics of these Breviceps populations is more colour pattern, digit length, and number and shape of complex. Studies of external morphology have identified subarticular tubercles. They also occur in different areas. populations with individuals of intermediate phenotypes for putatively diagnostic characters where the ranges of these species are thought to overlap in parts of Angola, 1 Department of Natural Sciences, University of Michigan at Mozambique, South Africa, and Zimbabwe (Poynton, Dearborn, 4901 Evergreen Road, Dearborn, Michigan 48128, 1982; Poynton and Broadley, 1985). The situation in USA. northeastern South Africa is particularly complex, where 2 Department of Natural History, Florida Museum of Natural advertisement call has been identified as the most reliable History, University of Florida, Gainesville, Florida 32611, character (Minter et al., 2004). Several new mossambicus USA; and Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233, USA. group species have been described from this general area 3 Department of Biology and Center for Biodiversity and (KwaZulu-Natal, South Africa), and these are genetically Ecosystem Stewardship, Villanova University, 800 Lancaster distinct and identifiable based on advertisement call but Avenue, Villanova, Pennsylvania 19085, USA. morphologically indistinguishable from B. mossambicus * Corresponding author. E-mail: [email protected] or B. adspersus: B. bagginsi Minter, 2003, B. sopranus © 2021 by Herpetology Notes. Open Access by CC BY-NC-ND 4.0. Minter, 2003, B. carruthersi Du Preez et al. in Minter et 398 Matthew P. Heinicke et al. Figure 1. Sample localities of southern African Breviceps mossambicus group specimens included in this study. New localities for B. passmorei are indicated by diamond symbols with enclosed dots. Approximate ranges of the widespread species B. adspersus, B. mossambicus, and B. poweri are indicated. al., 2017, and B. passmorei Minter et al. in Minter et al., During previous general faunal surveys, we and our 2017. Two other mossambicus group species, B. fichus colleagues collected a small sample of eight Breviceps Channing & Minter, 2004 and B. ombelanonga Nielsen specimens at four localities in Limpopo Province, South et al., 2020, occur in Tanzania and Angola, respectively. Africa. All were identified at the time of collection as Additional genetic lineages with divergences as great as B. adspersus. We have also collected other Breviceps or greater than those between named Breviceps species samples from the core range of B. adspersus in have been documented in northeastern South Africa and Namibia and Zimbabwe, outside the region containing Mozambique (Nielsen et al., 2018). populations of questionable taxonomic affinity and west Thus, instead of a few widespread forms, the of the putative hybrid zone identified by Poynton (1982). mossambicus group consists of numerous species We herein use multi-gene DNA sequence data obtained that are poorly defined by traditional morphological from these samples, in combination with published characters. The presence of cryptic species, individuals sequences, to evaluate three possible scenarios for the of intermediate phenotype, and undescribed lineages identity of each of our Limpopo “Breviceps adspersus” means that the true identities of many mossambicus samples: (1) each is correctly identified asB. adspersus, group populations are difficult to ascertain. This is (2) each is referable to one of the other named species especially the case in eastern South Africa and western or genetic lineages of Breviceps, or (3) each represent an Mozambique, where the ranges of putative B. adspersus additional undescribed taxon. and B. mossambicus overlap and most documented genetic lineages occur. Molecular data provide needed insight in these cases. New Records of Breviceps passmorei in Limpopo Province, South Africa 399 Material and Methods Results We generated a data set consisting of fragments of The combined phylogenetic analysis (Fig. 2) recovered the mitochondrial 12S and 16S and nuclear RAG1 and a monophyletic B. mossambicus group, with relationships SLC8a3 genes. For samples sequenced in this study, among species identical to those inferred by Nielsen et al. DNA was extracted from ethanol-preserved tissues (2018). As expected, all new samples from Namibia and using a Qiagen DNeasy blood and tissue kit under the Zimbabwe cluster with B. adspersus with strong support, manufacturer’s protocol. Targeted gene regions were as does newly sequenced specimen MCZ A-137796 from amplified in 25 µL PCR reactions using primer pairs Farm Celine, Limpopo Province, South Africa (bootstrap listed in Nielsen et al. (2018). Successfully amplified PCR support = 100). There is no significant discordance among products were purified using Axygen AxyPrep magnetic loci (i.e., there is no evidence of hybridization with B. beads and sequenced at the University of Michigan mossambicus or any other species). The B. adspersus Advanced Genomics core. Sequence inspection and samples display minimal genetic variation across their assembly was performed using MEGA X (Kumar et al., geographic range. The mitochondrial (combined 12S and 2018). All specimens newly sequenced in this study have 16S) pairwise sequence divergence is 0.8% between the been deposited at the Museum of Comparative Zoology, sample from Farm Celine and a near-topotypic central Harvard University, Massachusetts, USA (MCZ) or the Namibian specimen (MCZ A-149504) even though Port Elizabeth Museum, Eastern Cape Province, South their collection localities are 1300 km apart. The other Africa (PEM). New sequences are deposited in Genbank seven newly sequenced Limpopo Province samples, under accession numbers MT944238–MT944344. from Cleveland Nature Reserve, Swadini-Hoedspruit Additional 12S, 16S, RAG1, and SLC8a3 sequences of Road (Fig. 3), and Witriver Road, do not cluster with B. mossambicus group taxa were obtained from Genbank B. adspersus. Instead, they are phylogenetically nested to represent all major lineages identified by Nielsen et with topotypic B. passmorei from KwaZulu-Natal, South al. (2018); exemplars of each B. gibbosus group species Africa, forming a strongly supported monophyletic group were included as outgroups. The complete data set is (bootstrap = 100). These samples are also recovered as listed in Appendix 1. most closely related to B. passmorei in all single locus Sequences of each gene were aligned using ClustalW analyses. Mitochondrial pairwise sequence divergences (Larkin et al., 2007) implemented in MEGA X and between these new samples and topotypic B. passmorei trimmed