Molecular analysis

DNA Sequencing

Tissue samples were taken from thigh muscle or toe clippings of 30 adults of the Afrixalus spinifrons complex from 15 localities in KwaZulu-Natal, the Eastern Cape and Western Cape and stored in 95-100% ethanol. Whole genomic DNA was extracted from a small amount of tissue using the Qiagen Animal Tissue spin-column protocol. The following gene fragments were selected for sequencing: a partial sequence of 545 base pairs (bp) of the mitochondrial gene fragment 16S ribosomal RNA and two partial nuclear protein-coding genes; 300 bp recombination activating gene (RAG1) and 450 bp proopiomelanocortin (POMC). These markers were selected based on their suitability to deep and shallow phylogenetic inference (Vieites et al. 2007). Double-stranded PCR was performed in 25μl reactions with 0.110μM of primer to amplify the target gene fragments using the primers and PCR conditions shown in Table 5.2. PCR product was sent to Inqaba Biotech, Pretoria for sequencing.

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Table 5.2 Primer sequences and PCR conditions used for molecular analysis in this study. Sources: 1. Palumbi et al. 1991; 2. Chiari et al. 2004; 3. Vieites et al. 2007.

Gene Primer

name Sequence (5‟  3‟) Source PCR conditions

16S 16S AL CGCCTGTTTATCAAAAACAT 1 94(90), [94(45), 55(45), 72(90) x 33], 72(300)

16S 16S BH CCGGTCTGAACTCAGATCACGT 1

Rag1 AmpF1 ACAGGATATGATGARAAGCTTGT 2 94(300), [94(30), ↑52-57 (40), 68(180)

GCAA 3 95(120),[95(60),58(60),72(90) x 35],

72(600) POMC DRV R1 GGCRTTYTTGAAWAGAGTCATTA

GWGG 3

Data Analysis

Ultimately, only partial sequences of the 16S gene were analysed due to low PCR yields of the RAG1 and POMC fragments. 16S sequences were aligned using the MUSCLE algorithm implemented in MEGA 5 (Tamura et al. 2011). The Maximum Parsimony (MP) analysis was performed with MEGA 5 following a branch-and-bound search on 43 equally-weighted informative characters, with gaps considered as missing data. Bootstrap percentage support values were calculated following heuristic search and stepwise addition with 1,000 replicates using the close-neighbour-interchange method. A T92 + I + G model was selected by modeltest 3.06 (Posada & Crandall 1998) and used for Maximum Likelihood (ML) and Bayesian analysis. Bayesian analysis was conducted using the software MrBayes 3.04b (Huelsenbeck & Ronquist 2001; 2004), with four chains running for a million generations, sampling each 100 cycles. Bayesian posterior probabilities were computed after removing the first 1,000 trees as the burn-in phase. Bayesian Inferences (BI) was run three times independently to assess for convergence, using the Tracer software available at http://tree.bio.ed.ac.uk/software.

173 5.3.5 Bioacoustic analysis

Recordings of male A. s. spinifrons and A. s. intermedius advertisement calls were made in the field from October 2011 to January 2012 at various localities (Table 5.3) using a NAGRA ARES-ML recorder (Software Version 3.24 2009). Air temperature was recorded using an ExTech Instruments waterproof thermometer since temporal call features such as call duration and pulse rate and duration can be affected by ambient temperature (Gerhardt & Bee 2007). Calls were edited using WaveSurfer version 1.8.8p4 (Sjölander & Beskow 2000). Call components were separated, and only the trill component was used for analysis as this has been shown by phonotaxis experimentation on A. delicatus to be the component that functions in female attraction (Backwell 1988). Sound analysis was performed with Sound Ruler Acoustic Analysis (Version 0.9.6.0) using default settings (Gridi-Papp 2007). The trills of each call bout were analysed separately and the mean values of key temporal factors, namely pulse rate (number of pulses per second), pulse duration and the spectral factors, dominant and fundamental frequency were measured for each call. The average of each factor was used for statistical analysis.

Table 5.3: Localities at which call recordings were made for Afrixalus spinifrons intermedius (n = 19) and Afrixalus spinifrons spinifrons (n= 11).

Species Location Date Air Temp

(°C)

# of males recorded A. s. intermedius Fort Nottingham (Kingussie Dam)

Hilton Wetland

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For comparison of trill components of the two subspecies, statistical analysis with STATISTICA (Ver. 10; Statsoft, Inc. 2011) was performed to determine whether there was any significant difference between the call parameters, pulse rate, fundamental frequency, dominant frequency and call duration. Tests for normality were performed for each variable to determine whether parametric or non-parametric testing was necessary for the data. T-tests to compare the means of each parameter were performed for each parameter. Only „call duration‟ was not normally distributed, so the non-parametric equivalent to the t-test for that variable (the Mann-Whitney U-test) was run for this variable. Standard deviation and coefficient of variation was calculated for each variable.

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5.4 Results

Since the morphology of the Afrixalus spinifrons complex has already been assessed comprehensively (Pickersgill 1984, 1996, 2007), it was not deemed necessary to re-examine all type material and other museum specimens for the purposes of this study. A subset of specimens were selected from the most important collections in South Africa, and along with specimens collected during the study period for examination. In particular, attention was paid to specimens from the Eastern Cape, which were referred to A. s. spinifrons based on morphological examination (Pickersgill 1996). Paratypes were loaned from the Durban Natural Science Museum and Natal Museum. Unfortunately, those from the former were in an unexaminable state (completely dried out). Collection of 30 new specimens (KZN Wildlife permit 5080/2011) also provided a good opportunity to examine colouration in life, since colouration in preserved specimens can differ markedly from live specimens (McDiarmid 1994).

Size, Colouration and Sexual Dimorphism

Snout-Vent measurement data shows that A. knysnae specimens were significantly larger than A. s. spinifrons and A. s. intermedius (15.7% and 14.4% respectively). Sexual dimorphism traits appear to be similar between spinifrons and intermedius. Female SVL was on average (21.42 mm, n = 15) 12.7% larger than males (19.01 mm, n = 35). For the average measurements for each taxon, both males and females were included (A. s. intermedius females = 10, males = 27; A. s. spinifrons females = 13, males = 43; A. knysnae males = 3). In general, females were paler in colour than males and had fewer, less dense asperities. Where asperities were apparent, they were usually uniform in distribution. Females frequently exhibited pale (bluish) dots on the lower surfaces of the limbs and lateral surface. This characteristic was observed for females of both A. s. spinifrons and A. s. intermedius from throughout the range (Fig 5.7). Males are characterised by having a brightly coloured yellow gular sac (du Preez & Carruthers 2009). Asperities on males were distributed over the entire dorsum, including the limbs and were more highly concentrated, particularly on the snout (although there is significant variation within these patterns).

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Figure 5.7: (A) a male Afrixalus spinifrons spinifrons from Tala Nature Reserve showing defined dorsal colouration and pronounced asperities) and (B) a female Afrixalus spinifrons intermedius from the Type locality at Rosetta, showing pale colouration and few asperities.

Males were generally darker in colour and had more clearly defined dorsal and lateral banding, however colour is also highly variable both between individuals of the different taxa and for the same individual depending on exposure to light (Figure 5.8).

Figure 5.8: Colour variation in a single A. s. intermedius male from Himeville following (A) exposure to dark conditions, (B) exposure to light.

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Overall, colouration in A. s. spinifrons was darker with more defined patterning than in A. s.

intermedius. Snout characteristics and degree of spinosity were found to be highly variable between and within taxa, with blunt, tapering and rounded snout shapes being represented in both A. s. spinifrons and A. s. intermedius. On average, snout shape was found to be tapering in specimens from the Eastern Cape (Figure 5.9) and midlands and more rounded and swollen (bulbous) in coastal specimens (Fig 5.10). Colouration was usually paler and more uniform in A. s. intermedius, while asperities were less pronounced and more evenly distributed across the dorsum. The pattern of spinosity was also inconsistent, ranging from being concentrated on the snout to evenly distributed over the entire dorsum, with no clear pattern discernable for specimens from the different regions. Similarly, gular shape was also found to be highly variable. These results are summarised in Table 5.4.

Figure 5.9: Male Afrixalus spinifrons spinifrons from Coffee Bay area, Transkei, Eastern Cape showing pale lateral bands, evenly distributed asperities and tapering snout. Photograph, Donnavan Kruger.

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Figure 5.10: Male Afrixalus spinifrons spinifrons from Prospecton, Durban showing bulbous snout and dense asperities.

Table 5.4: Comparison of characteristics of A. s. spinifrons from the KwaZulu-Natal coast (ⁿ = 33) and Eastern Cape coast (n = 21); A. s. intermedius from the KwaZulu-Natal

Snout shape Short and bulbous Tapering, not

swollen Relatively long,