Phylogenetic analysis of "Breviceps adspersus" documents B. passmorei Minter et al., 2017 in Limpopo Province, South Africa - Biotaxa
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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,
USA; and Department of Biological Sciences, Marquette
character (Minter et al., 2004). Several new mossambicus
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: heinicke@umich.edu 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 as B. 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 range400 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 Eswatini 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
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Accepted by Hinrich KaiserAppendix 1. 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:
African Amphibian Conservation Research Group (AACRG), Museum of Comparative Zoology (MCZ), Museum of Vertebrate Zoology (MVZ), Port Elizabeth Museum (PEM), South African Institute
of Aquatic Biodiversity (SAIAB). Other sample IDs acronyms reflect identifiers used in the original sources.
Genbank Accession Number
Species Sample ID Locality Latitude Longitude
12S 16S RAG1 SLC8a3
Breviceps acutirostris PEM A11119 South Africa: Western Cape, Grootwatersbos -33.98476 20.81055 MH340328 MH340406 MH340099 MH340249
Breviceps adspersus AMB 7972 Namiba: Kavango-East, vicinity of Shamruva Camp -18.03550 20.97153 MH340294 MH340372 MH340065 MH340217
Breviceps adspersus B01 no locality NC_023379 NC_023379
Breviceps adspersus MCZ A-137796 South Africa: Limpopo, Farm Celine -22.70806 29.52833 MT944255 MT944282 MT944317
Breviceps adspersus MCZ A-148557 Namibia: Zambezi, Katima Mulilo -17.62356 24.19958 MH340293 MH340371 MH340064 MH340216
Breviceps adspersus MCZ A-148563 Namiba: Kavango-East, vicinity of Shamruva Camp -18.03550 20.97153 MH340295 MH340373 MH340066 MH340218
Breviceps adspersus MCZ A-148595 Zimbabwe: Matabeland North, road to Miombo Safari Camp -18.62150 26.86494 MT944256 MT944283 MT944318
Breviceps adspersus MCZ A-148599 Zimbabwe: Matabeland North, road to Miombo Safari Camp -18.67792 26.94397 MT944257 MT944284 MT944319
Breviceps adspersus MCZ A-148600 Zimbabwe: Matabeland North, road to Miombo Safari Camp -18.67792 26.94397 MT944258 MT944285
Breviceps adspersus MCZ A-148601 Zimbabwe: Matabeland North, Hwange Road -18.67550 26.94594 MT944259 MT944286
Breviceps adspersus MCZ A-148603 Zimbabwe: Matabeland North, Hwange Road -18.67097 26.95347 MT944238 MT944249 MT944222
Breviceps adspersus MCZ A-148608 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.64778 26.89142 MT944260 MT944287
Breviceps adspersus MCZ A-148610 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.67056 26.92144 MT944261 MT944288 MT944320
Breviceps adspersus MCZ A-148615 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.67044 26.92117 MT944262 MT944289 MT944321
Breviceps adspersus MCZ A-148620 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.62422 26.86842 MT944263 MT944290 MT944322
Breviceps adspersus MCZ A-148622 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.58933 26.88119 MT944264 MT944291
New Records of Breviceps passmorei in Limpopo Province, South Africa
Breviceps adspersus MCZ A-148623 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.58933 26.88119 MT944292 MT944323 MT944342
Breviceps adspersus MCZ A-148625 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.65697 26.90300 MT944293 MT944324
Breviceps adspersus MCZ A-148629 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.63208 26.87642 MT944294 MT944325
Breviceps adspersus MCZ A-148630 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.63208 26.87642 MT944265 MT944295 MT944326
Breviceps adspersus MCZ A-148636 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.64578 26.88894 MT944266 MT944296 MT944327
Breviceps adspersus MCZ A-148637 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.64578 26.88894 MT944297
Breviceps adspersus MCZ A-148641 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.64578 26.88894 MT944267 MT944298 MT944328
Breviceps adspersus MCZ A-148642 Zimbabwe: Matabeland North, vic. of Miombo Safari Camp -18.64578 26.88894 MT944268 MT944299 MT944329
Breviceps adspersus MCZ A-148650 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944269 MT944300
Breviceps adspersus MCZ A-148651 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944270 MT944301 MT944330
Breviceps adspersus MCZ A-148652 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944271 MT944302 MT944331
403Appendix 1. (cont.)
404
Genbank Accession Number
Species Sample ID Locality Latitude Longitude
12S 16S RAG1 SLC8a3
Breviceps adspersus MCZ A-148653 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944239 MT944250 MT944223
Breviceps adspersus MCZ A-148654 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944272 MT944303 MT944332
Breviceps adspersus MCZ A-148655 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944304 MT944333
Breviceps adspersus MCZ A-148656 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944305 MT944334
Breviceps adspersus MCZ A-148657 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944273 MT944306 MT944335
Breviceps adspersus MCZ A-148658 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944307
Breviceps adspersus MCZ A-148659 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944274 MT944308 MT944336
Breviceps adspersus MCZ A-148660 Zimbabwe: Matabeland North, Miombo Safari Camp -18.62883 26.87206 MT944309
Breviceps adspersus MCZ A-149504 Namibia: Otjozondjupa, Farm Ohange -19.52850 17.56417 MT944240 MT944251 MT952163
Breviceps adspersus SVN 766 South Africa: Limpopo, Lephalale (Ellisras) -23.73193 27.57980 MH340291 MH340369 MH340062 MH340214
Breviceps adspersus SVN 768 South Africa: Limpopo, Lephalale (Ellisras) -23.73193 27.57980 MH340292 MH340370 MH340063 MH340215
Breviceps cf. bagginsi PEM A10566 South Africa: Eastern Cape, Silaka NR -31.65949 29.49955 MH340297 MH340375 MH340068 MH340220
Breviceps cf. bagginsi PEM A10565 South Africa: Eastern Cape, Silaka NR -31.65531 29.50457 MH340298 MH340376 MH340069 MH340221
Breviceps cf. bagginsi PEM A10568 South Africa: Eastern Cape, Silaka NR -31.65895 29.50165 MH340299 MH340377 MH340070 MH340222
Breviceps cf. bagginsi PEM A10567 South Africa: Eastern Cape, Silaka NR -31.65531 29.50457 MH340300 MH340378 MH340071 MH340223
Breviceps cf. bagginsi PEM A10569 South Africa: Eastern Cape, Silaka NR -31.65895 29.50165 MH340301 MH340379 MH340072 MH340224
Breviceps cf. bagginsi PEM A11122 South Africa: Eastern Cape, Mkambati NR -31.20223 29.83080 MH340302 MH340380 MH340073 MH340225
Breviceps branchi ZMB 77781 South Africa: Northern Cape, Holgat -28.93289 16.76914 JQ965934
Breviceps carruthersi n/a no locality AF215366
Breviceps carruthersi PEM A11996 South Africa: KwaZulu-Natal -28.02278 32.27306 MF288975
Breviceps carruthersi PEM A11997 South Africa: KwaZulu-Natal MF288977
Breviceps carruthersi PEM A11998 South Africa: KwaZulu-Natal MF288978
Breviceps carruthersi PEM A11999 South Africa: KwaZulu-Natal -28.02111 32.27167 MF288983
Breviceps carruthersi PEM A12000 South Africa: KwaZulu-Natal MF288984
Breviceps carruthersi SAIAB 204591 South Africa: KwaZulu-Natal -28.02278 32.27306 MF288976
Breviceps carruthersi SAIAB 204592 South Africa: KwaZulu-Natal MF288979
Breviceps carruthersi SAIAB 204593 South Africa: KwaZulu-Natal -28.02278 32.27306 MF288980
Breviceps carruthersi SAIAB 204594 South Africa: KwaZulu-Natal MF288981
Breviceps carruthersi SAIAB 204595 South Africa: KwaZulu-Natal MF288982
Breviceps carruthersi SVN 822 South Africa: KwaZulu-Natal, Pietermaritzburg -29.64000 30.39000 MH340320 MH340398 MH340091 MH340241
Matthew P. Heinicke et al.
Breviceps carruthersi SVN 823 South Africa: KwaZulu-Natal, Pietermaritzburg -29.64000 30.39000 MH340321 MH340399 MH340092 MH340242Appendix 1. (cont.)
Genbank Accession Number
Species Sample ID Locality Latitude Longitude
12S 16S RAG1 SLC8a3
Breviceps carruthersi SVN 822 South Africa: KwaZulu-Natal, Pietermaritzburg -29.64000 30.39000 MH340320 MH340398 MH340091 MH340241
Breviceps carruthersi SVN 823 South Africa: KwaZulu-Natal, Pietermaritzburg -29.64000 30.39000 MH340321 MH340399 MH340092 MH340242
Breviceps fichus MW 1826 Tanzania AY531859 AY531836
Breviceps fichus MW 1848 Tanzania -7.75000 35.67000 AY531860 AY531837
Breviceps fuscus PEM A11108 South Africa: Western Cape, Knysna -33.94400 23.14822 MH340330 MH340408 MH340101 MH340251
Breviceps gibbosus PEM A11124 South Africa: Western Cape, Piketberg -32.88579 18.72963 MH340331 MH340409 MH340102 MH340252
Breviceps macrops isolate 43 South Africa: Northern Cape, Port Nolloth -29.28619 16.88222 MH340333 MH340411 MH340104 MH340254
Breviceps montanus isolate 46 South Africa: Western Cape, Silvermine NR -34.09976 18.43351 MH340336 MH340414 MH340107 MH340257
Breviceps mossambicus 1031BreMos n/a EF017947 EF018056 EF107383
Breviceps mossambicus MCZ A-137055 Malawi: Southern Region, Mulanje -15.93333 35.51667 MH340305 MH340383 MH340076 MH340228
Breviceps mossambicus MCZ A-148815 Tanzania: Rondo Forest Reserve -10.11806 39.17778 KY177082
Breviceps mossambicus MVZ 265910 Mozambique: Zambezia, Gurue -15.46394 36.97785 MH340304 MH340382 MH340075 MH340227
Breviceps mossambicus PEM A14008 Mozambique: Niassa, Lichinga -13.30806 35.24411 MH340311 MH340389 MH340082 MH340234
Breviceps mossambicus SAIAB 88176.1 Mozambique: Cabo Delgado, Pemba -12.96361 40.52944 MH340308 MH340386 MH340079 MH340231
Breviceps mossambicus SAIAB 88176.2 Mozambique: Cabo Delgado, Pemba -12.96361 40.52944 MH340309 MH340387 MH340080 MH340232
Breviceps mossambicus SAIAB 88161.1 Mozambique: Nampula, Ile de Mozambique -15.03094 40.74094 MH340306 MH340384 MH340077 MH340229
Breviceps mossambicus SAIAB 88161.2 Mozambique: Nampula, Ile de Mozambique -15.03094 40.74094 MH340307 MH340385 MH340078 MH340230
Breviceps mossambicus SAIAB 88586.1 Mozambique: Nampula -15.03072 40.74122 MH340310 MH340388 MH340081 MH340233
New Records of Breviceps passmorei in Limpopo Province, South Africa
Breviceps mossambicus RdS_903 no locality JF836942 JF836996
Breviceps mossambicus PEM A11021 Mozambique: Cabo Delgado, Balama -13.28867 38.68153 MH340312 MH340390 MH340083 MH340235
Breviceps mossambicus T1931 Tanzania -10.11806 39.17778 KF954743 KF954750
Breviceps namaquensis isolate 51 South Africa: Northern Cape, Port Nolloth -29.28619 16.88222 MH340341 MH340419 MH340112 MH340262
Breviceps passmorei AACRG 3207 South Africa: KwaZulu-Natal MH115695
Breviceps passmorei AACRG 3208 South Africa: KwaZulu-Natal MH115696
Breviceps passmorei MCZ A-137743 South Africa: Limpopo, Swadini-Hoedspruit Road -24.48028 30.87361 MT944252 MT944277 MT944312 MT944339
Breviceps passmorei MCZ A-137751 South Africa: Limpopo, Swadini-Hoedspruit Road -24.49056 30.85222 MT944278 MT944313 MT944340
Breviceps passmorei MCZ A-137754 South Africa: Limpopo, Swadini-Hoedspruit Road -24.48222 30.85750 MT944279 MT944314
Breviceps passmorei MCZ A-137777 South Africa: Limpopo, Swadini-Hoedspruit Road -24.49028 30.85306 MT944253 MT944280 MT944315 MT944341
Breviceps passmorei MCZ A-137782 South Africa: Limpopo, Swadini-Hoedspruit Road -24.48528 30.85972 MT944254 MT944281 MT944316 MT944342
405Appendix 1. (cont.)
406
Genbank Accession Number
Species Sample ID Locality Latitude Longitude
12S 16S RAG1 SLC8a3
Breviceps passmorei PEM A-12003 South Africa: KwaZulu-Natal -27.04333 32.29056 MF288985
Breviceps passmorei PEM A-12252 South Africa: Limpopo, Cleveland Nature Reserve -24.02100 31.19075 MT944275 MT944310 MT944337
Breviceps passmorei PEM D0001 South Africa: KwaZulu-Natal, Pinda GR -27.84757 32.33324 MH340319 MH340397 MH340090 MH340240
Breviceps passmorei PEM A-12256 South Africa: Limpopo, Witriver Road -24.55350 31.30186 MT944276 MT944311 MT944338 MT944344
Breviceps passmorei SAIAB 204596 South Africa: KwaZulu-Natal -27.04319 32.28836 MF288986
Breviceps passmorei SAIAB 204597 South Africa: KwaZulu-Natal MF288987
Breviceps passmorei SAIAB 204598 South Africa: KwaZulu-Natal MF288988
Breviceps passmorei SAIAB 204617 South Africa: KwaZulu-Natal MF288989
Breviceps pentheri RB12-Mis01 South Africa: Eastern Cape, Grahamstown -33.30000 26.53000 MH340296 MH340374 MH340067 MH340219
Breviceps poweri ELI 325 Dem. Rep. of Congo: Tanganyika, Manono -7.27770 27.38980 MH340313 MH340391 MH340084 MH340236
Breviceps poweri SAIAB 98182.1 Zambia: North-Western, Kalumbila -12.23778 25.34194 MH340314 MH340392 MH340085 MH340237
Breviceps poweri SAIAB 98788.1 Zambia: Lusaka -15.51028 28.26053 MH340315 MH340393 MH340086 MH340238
Breviceps poweri SAIAB 98788.2 Zambia: Lusaka -15.51028 28.26053 MH340316 MH340394 MH340087 MH340239
Breviceps rosei WC-988 South Africa: Western Cape, Cape Town -34.07470 18.53806 MH340348 MH340426 MH340119 MH340269
Breviceps sopranus PEM A11126 South Africa: KwaZulu-Natal, Hluhluwe -28.24754 31.99219 MH340303 MH340381 MH340074 MH340226
Breviceps sp. 1 PEM A10739 South Africa: Limpopo, Lajuma -23.03586 29.42783 MH340317 MH340395 MH340088 —
Breviceps sp. 4 MVZ 265908 Mozambique: Inhambane, Vilanculos -21.99272 35.29633 MH340322 MH340400 MH340093 MH340243
Breviceps sp. 5 WC-1214 South Africa: Mpumalanga, Lydenburg -25.14911 30.61938 MH340324 MH340402 MH340095 MH340245
Breviceps sp. 5 WC-1267 South Africa: Mpumalanga, Lydenburg -25.14911 30.61938 MH340325 MH340403 MH340096 MH340246
Breviceps sp. 5 PEM A11460 South Africa: Mpumalanga, Belfast -25.71843 30.02079 MH340323 MH340401 MH340094 MH340244
Breviceps sp. 6 PEM A11464 South Africa: Mpumalanga, Wakkerstroom -27.33747 30.12379 MH340326 MH340404 MH340097 MH340247
Breviceps sp. 7 MVZ 265909 Mozambique: Maputo, Ponta do Ouro -26.84061 32.88620 MH340327 MH340405 MH340098 MH340248
Breviceps sylvestris SVN 812 South Africa: Limpopo, Woodbush Forest Reserve -23.81000 29.99000 MH340349 MH340427 MH340120 MH340270
Breviceps verrucosus PEM A11121 South Africa: KwaZulu-Natal, Kokstad -30.48907 29.40611 MH340352 MH340430 MH340122 MH340273
Callulina kreffti MVZ 234046 Tanzania -5.08333 38.66667 DQ347056 DQ347339 DQ347281 EF107395
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