FBIP: Amphibian biodiversity and eco-tourism

Study Extent

KwaZulu Natal, Kosi Bay, Tembe, St Lucia, Ndumo, Sodwana, Bonamanzi, Munguzi, Richards Bay, Kosi Bay, Pongola

Sampling

We propose three transects: An east-west transect from the Swaziland border to Kosi Bay and two North-South transects. One along the 32o longitude to Richards Bay and the second along the coast to Richards Bay. Individual species recordings will be made using a digital recorder fitted with a high-end rifle mic. In order to enable long-term recordings and monitoring of as many frog species as possible a passive acoustic monitoring (PAM) recorder/song meter equipped with an ambient temperature sensor, solar panel and a weather station will be set up in key localities (protected areas already identified) within the study area. Active sampling: involves homing in on calling frogs and forming human transect lines and walking in a line through the selected site. Frogs will be collected by hand and mainly at night. Baited traps will be used to collect aquatic frog species such as Xenopus. Traps will be baited with chicken livers and checked daily. Any by catch will be released unharmed. Dip netting will be used to collect different tadpoles species at all collection sites. Upon collection frogs will be placed in plastic bags or containers that are marked according to the collection site, and transported back to a field laboratory, where they will be assigned unique field numbers. For all frog specimens photos will be taken and blood extracted for DNA barcoding and blood parasites biodiversity via standard procedure from the femoral artery or vein as per the method of Netherlands et al. (2014). A drop of blood will be used to prepare thin blood smears, which will be air-dried, fixed using absolute methanol, and stained using a modified Giemsa stain; the remaining blood will fixed in 70% ethanol for future molecular analysis. Only two voucher specimens per species per locality will be euthanized using a 3 % ethyl-4-aminobenzoate (MS 222) solution. A muscle tissue sample from one hind leg will be preserved in 70% molecular grade ethanol and the carcass fixed in a natural position in 10% neutral buffered formalin (for future locality and genetic records). DNA will be extracted from the samples using the standard protocol for human or animal tissue and cultured cells as detailed in the NucleoSpin®Tissue Genomic DNA Tissue Kit.

Method steps

1. TRANSECTS We propose three transects: An east-west transect from the Swaziland border to Kosi Bay and two North-South transects. One along the 32o longitude to Richards Bay and the second along the coast to Richards Bay. METHODS & APPROACH: All the proposed methods have been tried and tested in a pilot study in the area during 2014. This study dealt with species diversity, habitat utilization and blood parasites of amphibians in and around Ndumo Game Reserve. VOICE RECORDINGS: Individual species recordings will be made using a digital recorder fitted with a high-end rifle mic. In order to enable long-term recordings and monitoring of as many frog species as possible a passive acoustic monitoring (PAM) recorder/song meter equipped with an ambient temperature sensor, solar panel and a weather station will be set up in key localities (protected areas already identified) within the study area. Calls will be analysed using Song Scope analysis software. COLLECTION OF HOST SPECIES: Active sampling: involves homing in on calling frogs and forming human transect lines and walking in a line through the selected site. Frogs will be collected by hand and mainly at night. Baited traps will be used to collect aquatic frog species such as Xenopus. Traps will be baited with chicken livers and checked daily. Any by catch will be released unharmed. Dip netting will be used to collect different tadpoles species at all collection sites. HANDLING OF SPECIMENS: Upon collection frogs will be placed in plastic bags or containers that are marked according to the collection site, and transported back to a field laboratory, where they will be assigned unique field numbers. Additional data such as the date of collection, locality coordinates, photographs, type of species, weight, length and sex will also be recorded. Specimens will be kept cool and moist in individual breathable plastic containers with a small volume of water (to maintain moisture). Tadpoles will be placed in plastic sorting trays at the site of collection and at least five representatives of each species will be placed in formalin/ethanol for further identification back at the field laboratory. NON-INVASIVE COLLECTION OF SAMPLES: For all frog specimens photos will be taken and blood extracted for DNA barcoding and blood parasites biodiversity via standard procedure from the femoral artery or vein as per the method of Netherlands et al. (2014). All frogs will be processed the morning after collection and will be released where collected within 24hr of capture. FIXING OF PARASITES: A drop of blood will be used to prepare thin blood smears, which will be air-dried, fixed using absolute methanol, and stained using a modified Giemsa stain; the remaining blood will fixed in 70% ethanol for future molecular analysis. EUTHANASIA AND DISSECTION OF FROGS: Only two voucher specimens per species per locality will be euthanized using a 3 % ethyl-4-aminobenzoate (MS 222) solution. A muscle tissue sample from one hind leg will be preserved in 70% molecular grade ethanol and the carcass fixed in a natural position in 10% neutral buffered formalin (for future locality and genetic records). MOLECULAR WORK: DNA will be extracted from the samples using the standard protocol for human or animal tissue and cultured cells as detailed in the NucleoSpin®Tissue Genomic DNA Tissue Kit. For frogs: To identify potential species, sequence fragments of two mitochondrial genes (12S, 16S) will be used. The nuclear gene Tyrosinase exon 1 (Tyr), will also be used to separated populations into the most likely haplotype phases. Phylogenetic analyses will be completed through comparisons of the generated sequences in this study, to each other and to similar and available sequences downloaded from GenBank. Both model-based (Maximum likelihood) and character-based (Maximum parsimony) phylogenetic analyses will be used, along with haplotype networks through TCS 1.21 (Clement et al. 2000) that implements statistical parsimony to estimate gene genealogies (Templeton et al. 1992). ECO-TOURISM & LOCAL COMMUNITY INVOLVEMENT - A NEW INITIATIVE: Eco-tourism strategies have already been discussed with Ezemvelo officials and the regional ecologist and they are keen to get involved. Members from the local communities will be recruited, trained and educated as frog tour guides. The Frog APP for tablets and cell phones (See attachment B) as developed by the research group, will be used as technology platform to support the strategy As amphibian eco-tourism becomes sustainable and well managed the initiative can be introduced to other areas in South Africa. The Ndumo Game Reserve (NGR) is the perfect reserve to pilot an amphibian eco-tourism project. It does not have dangerous game such as lions or elephants, and has an significant diversity of frog species and excellent habitat for guided tours. The NGR has already initiated birding tours by knowledgeable locals from the surrounding community over the past few years, making us confident that we can be the first to initiate the first managed frogging tours. Through the APP data collected by the guides and tourists will be uploaded to the FrogMAP database at the Animal Demography Unit at UCT.

All specimen identified to Genus and a most to Species level

1. Anura
   common name: Frogs rank: order

KwaZulu Natal, Kosi Bay, Tembe, St Lucia, Ndumo, Sodwana, Bonamanzi, Munguzi, Richards Bay, Kosi Bay, Pongola