Amphibians of Northern KwaZulu-Natal : a phylogenetic study

Abstract

Northern KwaZulu-Natal (KZN) is a biodiversity hotspot containing the Maputaland centre of endemism and boasts the highest diversity of frog species in South Africa. The tropical to subtropical climate with relatively high rainfall coupled with its unique geography have resulted in a mosaic of habitats and proliferation of frog species in this region. KwaZulu-Natal has become a priority area for conservation due to increasing environmental pressures from population growth and development. Effective conservation and management of biodiversity depend largely on our knowledge of taxonomy. Evolutionary patterns of convergence and parallelism, occurring among relatively closely related taxa, present a great challenge to amphibian taxonomy and diversity research. This not only leads to challenges in identification resulting from similarities between species, but can also completely mask cryptic species. DNA barcoding is a relatively modern tool that uses a short standard gene fragment to identify specimens. Additionally, it can also lead to the discovery of new or cryptic species and provides standardisation for species description. This study aimed to contribute to a better understanding of frog diversity by expanding the DNA barcoding reference library for the frogs of northern KZN and screening for possible cryptic species. Due to a lack in reference sequences of the mitochondrial cytochrome c oxidase subunit I marker, the complementary 16S rDNA marker was also included in this study for phylogenetic analysis. A total of 350 individual frog and tadpole voucher specimens were collected. All voucher specimens, tissue samples, and photographs were processed and accessioned. A subset of 141 specimens was chosen for molecular analysis. The successfully obtained sequences, 224 in total, represent 35 species and two unidentified Breviceps sp. specimens, accounting for 69% of the known frog species occurring within the study area. Only the 16S marker was obtained for thee of these species. All sequences and data were uploaded and added to the Biodiversity of Life Database under project code LDPJR. The Barcode Index Number (BIN) system was used to corroborate identifications of sequenced specimens. Identifications were also supported by the acquisition of additional reference sequences (COI and 16S) from BOLD and GenBank and their subsequent analysis through the construction of Neighbour Joining (NJ) trees. Screening for the presence of possible cryptic species was performed through the employment of the Automatic Gap Discovery algorithm, the construction of NJ trees, and the evaluation of genetic divergence between the elucidated groups. The possible presence of cryptic species was found in ten genera. Many other taxa were shown to need further investigation to strengthen morphological characterisation, establish species genetic boundaries, correct distribution estimates and correct identification of reference sequence records. While DNA barcoding alone does not provide enough information to identify and describe new species, it does flag taxa in need of further investigation and provides a rapid identification system, thus aiding in the prioritisation of research efforts and efficiency