Herpetology Notes, volume 14: 397-406 (2021) (published online on 22 February 2021)

Phylogenetic analysis of “ adspersus” documents B. passmorei Minter et al., 2017 in Limpopo Province,

Matthew P. Heinicke1,*, Mohamad H. Beidoun1, Stuart V. Nielsen1,2, and Aaron M. Bauer3

Abstract. Recent systematic work has shown the Breviceps mossambicus 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 and . 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 .

Keywords. , cryptic species, Microhylidae, rain , systematics, Transvaal

Introduction , 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 distributed across eastern northeastern South Africa], has a southern distribution, and (AmphibiaWeb, 2020; Frost, 2020). ranging from Namibia across much of , The genus includes two major clades: the gibbosus Zimbabwe, and South Africa to western . 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, ) 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 , 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 to include only regions for which sequence data from KwaZulu-Natal range from 0–2%. for Limpopo specimens were available. The resulting aligned regions were 400 bp (12S), 456 bp (16S), 559 bp Discussion (RAG1), and 875 bp (SLC8a3), respectively, for a total Records of Breviceps identified as B.“ adspersus” are aligned data set of 2290 bp. A greedy search algorithm documented across most of northeastern South Africa, was implemented in PartitionFinder 2.1.1 (Lanfear et al., including nearly every quarter degree square in Limpopo 2017) to identify a best-fitting partitioning scheme and Province (Minter et al., 2004). The vast majority of models of evolution. Each of the four genes and each these records, including two B. adspersus syntypes from codon position of the protein-coding genes RAG1 and northeastern South Africa, have no associated genetic SLC8a3 were considered potentially unique partitions or acoustic data that can reliably distinguish true B. for this analysis. The best-fitting partitioning scheme and adspersus from other morphologically similar species. models were used for phylogenetic analyses. A maximum However, the Farm Celine specimen can be added to likelihood (ML) phylogenetic analysis was performed previously sequenced samples from Lephalale (Ellisras) using the full concatenated data set in IQ-TREE 1.5 as representing true B. adspersus, confirming that the (Nguyen et al., 2015), with 1000 bootstrap replicates species occurs across at least the northwestern portions to assess branch support. In order to ensure there is no of Limpopo Province. In contrast, it is clear from the significant discordance among loci, we also performed genetic data that the other Limpopo Province specimens separate ML analyses for 12S/16S, RAG1, and SLC8a3, we sequenced in this study are actually B. passmorei. using the same models for these subsets as were used in At the time of its description, B. passmorei was known the analysis of the full data set. to occur only in far northeastern KwaZulu-Natal Province (Minter et al., 2017). Subsequent work involving this species did not appreciably extend its known range 400 Matthew P. Heinicke et al.

Figure 2. Multi-gene maximum likelihood phylogeny of the Breviceps mossambicus group. Designations of undescribed taxa follow Nielsen et al. (2018). Outgroups (B. gibbosus group species and Callulina kreffti) are not shown. Samples sequenced in this study are bold. Bootstrap support values >50% are indicated at nodes.

(Nielsen et al., 2018; Phaka et al., 2019; Pretorius, 2019; Reeder, 2019). The new Limpopo Province localities are from localities within 70 km of one another in the far southeastern portion of the province, 315–360 km north of the type locality of B. passmorei in KwaZulu-Natal Province. The nearest genetically verified B. adspersus is the sample sequenced from Farm Celine in this study, another 200–250 km to the north. Given the presence of B. passmorei in multiple localities in both Limpopo and KwaZulu-Natal provinces, it is likely that the species also occurs in areas with suitable habitat across intervening regions of Mpumalanga Province, South Africa, as well as in adjacent and Mozambique. Indeed, specimens from near Swadini are less than 4 km from the Mpumalanga Province border. Figure 3. Breviceps passmorei from Swadini-Hoedspruit Road, Limpopo Province, South Africa. Photo by Aaron M. Bauer. New Records of Breviceps passmorei in Limpopo Province, South Africa 401

It is unclear if there is any overlap between the References geographic ranges of B. adspersus and B. passmorei, AmphibiaWeb (2020): AmphibiaWeb. Available at: https:// and whether key biological or physical features define amphibiaweb.org. Accessed on 1 June 2020. the range limits of either species in northeastern South Beck, H.E., Zimmermann, N.E., McVicar, T.R., Vergopolan, N., Berg, Africa. The B. adspersus and B. passmorei localities A., Wood, E.F. (2018): Present and future Köppen-Geiger climate with confirmed genetic data in Limpopo Province are classification maps at 1-km resolution. Scientific Data 5: 180214. in regions dominated by similar bushveld vegetation, as Channing, A., Minter, L. (2004): A new rain frog from Tanzania (Microhylidae: Breviceps). African Journal of Herpetology 53: is the type locality of B. passmorei in KwaZulu-Natal 147–154. Province (Mucina et al., 2006). These localities are also Channing, A., Rödel, M.-O. (2019): Field Guide to the Frogs & Other climatically similar and sit in the same Köppen-Geiger of Africa. Cape Town, South Africa, Struik. climate zone (Beck et al., 2018). Since there are no major Du Preez, L.M., Carruthers, V. (2017): Frogs of Southern Africa: a climate or vegetation differences between these regions, Complete Guide. Cape Town, South Africa, Struik. it is possible that physical features play a stronger role Frost, D.R. (2020): Species of the World: an Online in defining the range limits of B. adspersus and B. Reference. Version 6.1. Available at: https://amphibiansoftheworld. amnh.org/index.php. Accessed on 1 June 2020. passmorei. The overall range of B. adspersus broadly Kirchhof, S., Krämer, M., Linden, J., Richter, K. (2010): The reptile coincides with the distribution of Kalahari sands, which species assemblage of the Soutpansberg (Limpopo Province, terminate in Botswana adjacent to northwestern Limpopo South Africa) and its characteristics. Salamandra 46: 147–166. Province (Thomas and Shaw, 1990). The herpetofauna of Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. (2018): MEGA northwestern Limpopo Province as a whole has been noted X: molecular evolutionary genetics analysis across computing to have a strong Kalahari element (e.g., Schmidt, 2002; platforms. Molecular Biology and Evolution 35: 1547–1549. Schmidt et al., 2005). It is separated from southeastern Lanfear, R., Frandsen, P.B., Wright, A.M., Senfeld, T., Calcott, B. (2017): PartitionFinder 2: new methods for selecting partitioned Limpopo Province by the intervening high elevation models of evolution for molecular and morphological phylogenetic Waterberg and Soutpansberg mountains (Partridge et analyses. Molecular Biology and Evolution 34: 772–773. al., 2010). These highlands have a significantly different Larkin, M.A., Blackshields, G., Brown, N.P., Chenna, R., herpetofauna containing Afromontane elements (e.g., McGettigan, P.A., McWilliam, H., et al. (2007): Clustal W and Kirchhof et al., 2010), including at least one undescribed Clustal X, version 2.0. Bioinformatics 23: 2947–2948. Breviceps population (“sp. 1” of Nielsen et al., 2018). Merrem, B. (1820): Versuch eines Systems der Amphibien. Marburg, These highlands may therefore act as a barrier between Germany, Johann Christian Krieger. Minter, L.R. (2003): Two new cryptic species of Breviceps B. adspersus in the northwest and B. passmorei in the (Anura: Microhylidae) from southern Africa. African Journal of southeast, but more genetic and/or acoustic data from Herpetology 52: 9–21. Breviceps populations across Limpopo Province are Minter, L.R., Burger, M., Harrison, J.A., Braack, H.H., Bishop, P.J., needed to investigate this possibility. Indeed, given Kloepfer, D., Eds. (2004): Atlas and Red Data Book of the Frogs the phylogenetic complexity of the B. mossambicus of South Africa, Lesotho and Swaziland. Washington, D.C., USA, group, such data are needed from across the range of the Smithsonian Institution. species group as a whole, especially in the physically Minter, L.R., Netherlands, E.C., Du Preez, L.H. (2017): Uncovering a hidden diversity: two new species of Breviceps (Anura: heterogeneous regions of northeastern South Africa, Brevicipitidae) from northern KwaZulu-Natal, South Africa. Eastern Zimbabwe, and western Mozambique. Zootaxa 4300: 195–216. Mucina, L., Rutherford, M.C., Powrie, L.W. (2006): Vegetation atlas Acknowledgments. We thank Marius Burger for providing one of of South Africa, Lesotho and Swaziland. In: The Vegetation of the samples used in this study. Permits for field work were issued South Africa, Lesotho and Swaziland, p. 748–789. Mucina, L., to A.M. Bauer, W.R. Branch, V. Egan, and D.G. Broadley over the Rutherford, M.C., Eds., Pretoria, South Africa, SANBI. period 2004–2011 by the Department of Economic Development, Nguyen, L.T., Schmidt, H.A., Haeseler, A. von, Minh, B.Q. (2015): Environment and Tourism, Limpopo Province, the Ministry of IQ-TREE: a fast and effective stochastic algorithm for estimating Environment and Tourism, Republic of Namibia, and the Parks maximum-likelihood phylogenies. Molecular Biology and and Wildlife Management Authority of Zimbabwe. Funding was Evolution 32: 268–274. provided by the US National Science Foundation (grants DEB- Nielsen, S.V., Daniels, S.R., Conradie, W., Heinicke, M.P., Noonan, 1556255, 1556559 and 1556585) and the University of Michigan. B.P. (2018): Multilocus phylogenetics in a widespread African anuran lineage (Brevicipitidae: Breviceps) reveals patterns of diversity reflecting geoclimatic change. Journal of Biogeography 45: 2067–2079. 402 Matthew P. Heinicke et al.

Nielsen, S.V., Conradie, W., Ceríaco, L.M.P., Bauer, A.M., Heinicke, Poynton, J.C. (1982): On species pairs among southern African M.P., Stanley, E.L., Blackburn, D.C. (2020): A new species of rain amphibians. African Zoology 17: 67–74. frog (Brevicipitidae, Breviceps) endemic to Angola. ZooKeys Poynton, J.C., Broadley, D.G. (1985): Amphibia Zambesiaca 979: 133–160. 1. Scolecomorphidae, Pipidae, Microhylidae, Hemisidae, Parker, H.W. (1934): A Monograph of the Frogs of the Family Arthroleptidae. Annals of the Natal Museum 26: 503–553. Microhylidae. London, United Kingdom, Trustees of the British Pretorius, W.W. (2019): Ecology and calling behaviour of the anurans Museum. of northern Zululand, South Africa. Unpublished MS thesis, Partridge, T.C., Dollar, E.S.J., Moolman, J., Dollar, L.H. (2010): The North-West University, Potchefstroom, South Africa. 112 pp. geomorphic provinces of South Africa, Lesotho and Swaziland: a Reeder, J.E. (2019): Amphibians of northern KwaZulu-Natal: physiographic subdivision for earth and environmental scientists. a phylogenetic study. Unpublished MS thesis, North-West Transactions of the Royal Society of South Africa 65: 1–47. University, Potchefstroom, South Africa. 102 pp. Peters, W.C.H. (1854): Diagnosen neuer Batrachier, welche Schmidt, A.D. (2002): The herpetofauna of the Langjan Nature zusammen mit der früher (24. Juli und 18. August) gegebenen Reserve (Limpopo Province, Republic of South Africa). Übersicht der Schlangen und Eidechsen mitgetheilt werden. Herpetozoa 15: 121­–135. Bericht über die zur Bekanntmachung geeigneten Verhandlungen Schmidt, A.D., Snyman, P.H., Gruschwitz, M. (2005): Diversity of der Königlich Preußischen Akademie der Wissenschaften zu reptiles in the bushveld of the Blouberg Nature Reserve (Limpopo Berlin 1854: 614–628. Province, South Africa). Herpetozoa 18: 35–53. Peters, W.C.H. (1882): Naturwissenschaftliche Reise nach Thomas, D.S.G., Shaw, P.A. (1990): The deposition and development Mossambique auf Befehl Seiner Majestät des Königs Friedrich of the Kalahari Group sediments, Central Southern Africa. Journal Wilhelm IV in den Jahren 1842 bis 1848 ausgeführt. Zoologie 3 of African Earth Sciences (and the Middle East) 10: 187–197. (Amphibien). Berlin, Germany, G. Reimer. Phaka, F.M., Netherlands, E.C., Kruger, D.J.D., Du Preez, L.H. (2019): Folk and indigenous names for frogs in Zululand, South Africa. Journal of Ethnobiology and Ethnomedicine 15(17): 1–8.

Accepted by Hinrich Kaiser New Records of Breviceps passmorei in Limpopo Province, South Africa 403 16

H3402 SLC8a3 MT944342 MH340217 MH340249 MH340218

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8.64578 8.64578 8.62883 18.03550 18.62883 18.63208 18.65697 33.98476 17.62356 22.70806 18.67792 18.67550 18.67097 18.64778 18.62422 18.58933 18.63208 18.67056 18.67044 18.58933 18.64578 18.64578 18.62883 18 .03550 18.62150 18.67792 2 Latitude ------amp - i Camp

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s adspersu iceps eviceps adspersus eviceps adspersuseviceps Breviceps adspersus Brevicepsadspersus Brevicepsadspersus Species Brevicepsacutir adspersusBreviceps Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Br Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus adspersusBreviceps Brev Brevicepsadspersus Brevicepsersus adsp Breviceps adspersus Brevicepsersus adsp Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Br Breviceps adspersus Genbank accession numbers and collection localities of specimens used in this study. Sample IDs use collection acronyms when available, including for all newly sequenced specimens: including acronyms when available, IDs use collection Sample used in this study. of specimens localities numbers and collection Appendix 1. Genbank accession African Institute Museum (PEM), South Zoology (MVZ), Port Elizabeth Vertebrate Zoology (MCZ), Museum of Group (AACRG), Amphibian Conservation Research Museum of Comparative African Aquatic Biodiversity (SAIAB). Other sample IDs acronyms reflect identifiers used in the original sources. of

404 Matthew P. Heinicke et al.

SLC8a3 MT952163 MH340225 MH340224 MH340223 MH340222 MH340220 MH340221 MH340215 MH340214 MH340241 MH340242

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12S MT944239 MT944272 MT944273 MT944274 MT944240 MH340302 MH340301 MH340300 MH340299 MH340297 MH340298 MH340292 MH340291 MH340320 MH340321

16.76914 29.83080 29.50165 29.50457 29.50165 29.49955 29.50457 27.57980 27.57980 26.87206 26.87206 26.87206 26.87206 26.87206 26.87206 26.87206 26.87206 17.56417 32.27306 32.27167 32.27306 32.27306 30.39000 30.39000 Longitude

28.93289 31.20223 31.65895 31.65531 31.65895 31.65949 31.65531 23.73193 23.73193 18.62883 18.62883 18.62883 18.62883 18.62883 18.62883 18.62883 18.62883 19.52850 28.02278 28.02111 28.02278 28.02278 29.64000 29.64000 Latitude ------

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South Africa: Northern Cape, Holgat South Africa: Eastern Cape, Mkambati NR South Africa: Eastern Cape, Silaka NR South Africa: Eastern Cape, Silaka NR South Africa: Eastern Cape, Silaka NR South Africa: Eastern Cape, Silaka NR South Africa: Eastern Cape, Silaka NR South Africa: Limpopo, Lephalale (Ellisras) South Africa: Limpopo, Lephalale(Ellisras) Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Zimbabwe:Matabeland North, Miombo Safari Camp Ohange Farm Otjozondjupa, Namibia: Locality nolocality South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu

148653 148654 148655 148656 148657 148658 148659 148660 149504 ------A10568

ZMB77781 PEMA11122 PEMA10569 PEMA10567 PEM PEMA10566 PEMA10565 SVN768 MCZ A MCZ A MCZ A MCZ A MCZ A MCZ A MCZ A MCZ A MCZ A SVN766 Sample ID n/a PEMA11996 PEMA11997 PEMA11998 PEMA11999 PEMA12000 SAIAB204591 SAIAB204592 SAIAB204593 SAIAB204594 SAIAB204595 SVN822 SVN823

(cont.)

Brevicepsbranchi Breviceps cf. bagginsi Breviceps cf. bagginsi Breviceps cf. bagginsi Breviceps cf. bagginsi Breviceps cf. bagginsi Breviceps cf. bagginsi Brevicepsadspersus Species Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepsadspersus Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepscarruthersi Brevicepsadspersus Brevicepscarruthersi Appendix 1.

New Records of Breviceps passmorei in Limpopo Province, South Africa 405

SLC8a3 EF107383 MT944339 MT944341 MT944342 MT944340 MH340230 MH340233 MH340229 MH340232 MH340252 MH340254 MH340234 MH340231 MH340251 MH340235 MH340227 MH340257 MH340262 MH340241 MH340242 MH340228

RAG1 EF018056 MT944312 MT944315 MT944316 MT944313 MT944314 MH340078 MH340081 MH340077 MH340080 MH340102 MH340104 MH340082 MH340079 MH340101 MH340083 MH340075 MH340107 MH340112 MH340091 MH340092 MH340076

16S JF836996 EF017947 KF954750 KY177082 AY531836 AY531837 MT944277 MT944280 MT944281 MT944278 MT944279 MH340385 MH340388 MH340384 MH340387 MH340409 MH340411 MH340389 MH340386 MH340408 MH340390 MH340382 MH340414 MH340419 MH115695 MH115696 MH340398 MH340399 MH340383 Genbank Accession NumberGenbank Accession

12S JF836942 KF954743 AY531859 AY531860 MT944252 MT944253 MT944254 MH340307 MH340310 MH340306 MH340309 MH340331 MH340333 MH340311 MH340308 MH340330 MH340312 MH340304 MH340336 MH340341 MH340320 MH340321 MH340305

40.74094 40.74122 40.74094 40.52944 39.17778 18.72963 16.88222 35.24411 40.52944 23.14822 38.68153 36.97785 18.43351 16.88222 30.87361 30.85306 30.85222 30.85972 30.85750 30.39000 30.39000 35.67000 35.51667 39.17778 Longitude

7.75000 15.03094 15.03072 15.03094 12.96361 10.11806 32.88579 29.28619 13.30806 12.96361 33.94400 13.28867 15.46394 34.09976 29.28619 24.48028 24.49028 24.49056 24.48528 24.48222 29.64000 29.64000 15.93333 10.11806 - Latitude ------

Hoedspruit Road Hoedspruit Road Hoedspruit Hoedspruit Road Hoedspruit Road Hoedspruit Hoedspruit Road Hoedspruit

Natal Natal Natal, Pietermaritzburg Natal, Pietermaritzburg Natal,

- - - - Zambezia,Gurue

Mozambique: Nampula Mozambique: nolocality Mozambique: Nampula, Ile de Mozambique de Ile Nampula, Mozambique: Mozambique: Nampula, Ile de Mozambique de Ile Nampula, Mozambique: Mozambique: Cabo Delgado, Pemba CaboDelgado, Mozambique: Mozambique: Cabo Delgado, Pemba Delgado, Cabo Mozambique: SouthAfrica:Western Cape,Piketberg Tanzania Mozambique: Niassa, Lichinga South Africa: Northern Cape, Port Nolloth n/a SouthAfrica:Western Cape,Knysna Mozambique: Mozambique: Balama Delgado, Cabo Mozambique: South Africa: KwaZulu South Africa: KwaZulu South Africa: Western Cape, Silvermine NR South Africa: Northern Cape, Port Nolloth South Africa: Limpopo, Swadini - South Africa: Limpopo, Swadini - Locality Tanzania South Africa: Limpopo, Swadini - South Africa: Limpopo, Swadini - South Africa: Limpopo, Swadini - South Africa: KwaZulu South Africa: KwaZulu Tanzania Malawi: Southern Region, Mulanje Tanzania:Rondo ReserveForest

137743 137777 137751 137782 137754 137055 148815 ------A- AB 88586.1 AB I SA RdS_903 SAIAB 88161.2 SAIAB 88161.1 isolate43 SAIAB 88176.1 SAIAB 88176.2 PEMA11124 T1931 PEMA14008 isolate46 1031BreMos PEMA11108 isolate51 3208 AACRG MCZ A MCZ A MW1848 MVZ265910 PEMA11021 AACRG 3207 AACRG MCZ A MCZ A MCZ MCZ A Sample ID SVN822 SVN823 MW1826 MCZ A

Brevicepsmossambicus Brevicepsmossambicus Brevicepsmossambicus Brevicepsmossambicus Brevicepsmacrops Brevicepsmontanus Brevicepsmossambicus Brevicepsmossambicus Brevicepsgibbosus Brevicepsmossambicus Brevicepsmossambicus Brevicepsmossambicus Species Brevicepscarruthersi Brevicepscarruthersi Brevicepsfichus Brevicepsfuscus Brevicepsmossambicus Brevicepsmossambicus Brevicepsnamaquensis Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepsmossambicus Brevicepspassmorei Brevicepsfichus Brevicepsmossambicus Appendix 1. (cont.)

406 Matthew P. Heinicke et al.

SLC8a3 EF107395 MT944344 MH340239 MH340238 MH340269 MH340226 MH340243 MH340237 MH340248 MH340270 MH340273 MH340240 MH340219 MH340236 MH340247 MH340244 MH340245 MH340246

RAG1 DQ347281 MT944337 MT944338 MH340087 MH340086 MH340119 MH340074 MH340088 MH340093 — MH340085 MH340098 MH340120 MH340122 MH340090 MH340067 MH340084 MH340097 MH340094 MH340095 MH340096

16S DQ347339 MF288985 MF288986 MF288987 MF288988 MF288989 MT944310 MT944311 MH340394 MH340393 MH340426 MH340381 MH340395 MH340400 MH340392 MH340405 MH340427 MH340430 MH340397 MH340374 MH340391 MH340404 MH340401 MH340402 MH340403 Genbank Accession NumberGenbank Accession

12S DQ347056 MT944275 MT944276 MH340316 MH340315 MH340348 MH340303 MH340317 MH340322 MH340314 MH340327 MH340349 MH340352 MH340319 MH340296 MH340313 MH340326 MH340323 MH340324 MH340325

28.26053 28.26053 18.53806 31.99219 29.42783 35.29633 25.34194 32.88620 29.99000 29.40611 38.66667 32.33324 26.53000 27.38980 30.12379 31.19075 31.30186 30.02079 32.28836 30.61938 30.61938 32.29056 Longitude

5.08333 7.27770 15.51028 15.51028 34.07470 28.24754 23.03586 21.99272 12.23778 26.84061 23.81000 30.48907 27.84757 33.30000 27.33747 24.02100 24.55350 25.71843 27.04319 25.14911 25.14911 27.04333 - - Latitude ------

Natal,Hluhluwe Natal,Kokstad Natal,Pinda GR Natal Natal Natal Natal Natal ------

Kalumbila Western, - KwaZulu

Zambia:Lusaka SouthAfrica:Western Cape,Cape Town South Africa: Limpopo, Lajuma Vilanculos Inhambane, Mozambique: Zambia:Lusaka Tanzania South Africa: KwaZulu Dem. Rep. of Congo: Tanganyika, Manono Tanganyika, Congo: of Rep. Dem. Mozambique: Maputo, Ponta do Ouro do Ponta Maputo, Mozambique: Zambia:North South Africa: Limpopo, Woodbush Forest Reserve WoodbushForest Limpopo, Africa: South South Africa: KwaZulu South Africa: KwaZulu South Africa: KwaZulu South Africa: South Africa: KwaZulu SouthAfrica:EasternCape, Grahamstown South Africa: Limpopo, Witriver Road South Africa: KwaZulu SouthAfrica:Mpumalanga, Wakkerstroom South Africa: Mpumalanga, Lydenburg Mpumalanga, Africa: South Locality South Africa: KwaZulu SouthAfrica:Limpopo, Cleveland Nature Reserve Lydenburg Mpumalanga, Africa: South SouthAfrica:Mpumalanga, Belfast

12256 12003 12252 - - Mis01 A- 988 1267 1214 325 - - - WC SAIAB 98788.2 PEMA10739 MVZ265908 SAIAB 98788.1 SVN812 MVZ234046 PEMA11126 PEM ELI ELI MVZ265909 SAIAB 98182.1 PEMA11121 PEM A PEM A PEM PEMD0001 SAIAB204597 SAIAB204598 SAIAB204617 RB12 - WC SAIAB204596 PEMA11464 WC Sample ID PEMA11460

(cont.) sp. 1 sp. 4 sp. 7 sp. 5 sp. 5 sp. 5

Brevicepsrosei Brevicepspoweri Brevicepssopranus Breviceps Breviceps Breviceps Brevicepspoweri Brevicepssylvestris Brevicepsverrucosus Callulina kreffti Brevicepspassmorei Brevicepspoweri Brevicepspoweri Breviceps Species Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspassmorei Brevicepspentheri Breviceps Brevicepspassmorei Breviceps sp. 6 Breviceps Appendix 1.