The Cape Dwarf Chameleon (Bradypodion pumilum) is a medium sized chameleon (adults averaging 50–70 mm body size) that has a restricted distribution within the winter rainfall region in southwestern South Africa (Fig. 1). All members of this genus are viviparous, and the Cape Dwarf averages one to several clutches of 10–15 offspring per year (Jackson 2007; Tolley and Burger 2007). Although undocumented, mortality of neonates is probably high given the intense reproduction schedule and high fecundity. Adult short-term survival has been estimated to be low, on the order of 0.9 per 10 day period, projecting to a rough annual estimate of <3% survival (Raw 2009; Tolley et al. in press). Although this is similar to that of some Anolis lizards (e.g. Andrews and Nichols 1990), survival appears to be lower than in other lizards of similar body size (see review by Shine and Charnov 1992).

Fig. 1
figure 1

Map showing approximate distribution of Bradypodion pumilum (within dotted line), the two sampling areas (black dots), and the extent of habitat transformation (lighter shades indicate greater transformation)

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Bradypodion pumilum is under extreme anthropogenic pressure given that the majority of its distribution has suffered from intense urbanization and agricultural transformation (Driver et al. 2005) which has severely fragmented the majority of its habitat (Fig. 1). In addition, the trend for urbanization is continuing, with approximately 6.5 km2 per year of undeveloped land being lost to transformation in the Cape Town municipal area, where this species is primarily distributed (Rebelo et al. In review). This habitat loss is particularly alarming in the face of rapid predicted climatic changes, and climatic niche models suggest this species could suffer additional losses (Houniet et al. 2009). In urban areas, this chameleon is presently limited to planted vegetation (frequently non-indigenous), or isolated patches of highly disturbed habitat. Specifically, it is often found living in the overgrowth of exotic vegetation on road verges, riverine thicket, abandoned urban ground, or in residential areas (Tolley and Burger 2007). B. pumilum undergoes substantial demographic fluctuations in these small remnant populations, and is currently being monitored for the mode and pattern of these changes (Tolley et al. in press; Raw 2009).

Fortunately, part of its distribution lies in montane habitats, much of which is protected by provincial and national parks, or on private reserves. However, these protected areas amount to only about 40% of its total area of occupancy. In addition, this chameleon is not without pressure in the protected areas, as the dominant montane vegetation is a naturally fire-prone shrubland (fynbos), and frequent natural fires are known to affect dwarf chameleon populations (KAT pers. obs.). Because of the intense pressure on this species in urban areas, and the potential for extreme fluctuations in the number of individuals in both natural and transformed areas, it is currently being evaluated as a candidate for the IUCN Redlist (KAT, In review). In order to provide comprehensive conservation assessments, and to provide information that can be used toward a framework for conservation planning, an understanding of its population structure, genetic diversity and mating system, with respect to the effects of habitat fragmentation is necessary. Here, we describe polymorphic microsatellite markers that were developed in order to conduct studies aimed at understanding the conservation status of this species.

Microsatellite markers were developed using an enrichment protocol developed by Glenn and Schable (2005). Approximately 4 mg of genomic DNA (gDNA) from one individual was digested with RsaI and XmnI, and SuperSNX24 linkers were ligated onto the ends of gDNA fragments. Linkers act as priming sites for polymerase chain reactions (PCR) in subsequent steps. Five tetranucleotide [(AAAT)8, (AACT)8, (AAGT)8, (ACAT)8, (AGAT)8] and five trinucleotide [(ACT)12, (AAT)12, (AAG)8, (ATC)8, (AAC)6] biotinylated probes were hybridized to gDNA in two separate reactions (one for each repeat type). Each biotinylated probe-gDNA complex was added to streptavidin-coated magnetic beads (Dynabeads® M-280 Invitrogen, Carlsbad, California). This mixture was washed twice with 2 × SSC, 0.1% SDS and four times with 2 × SSC, 0.1% SDS at 52°C. Between washes, a magnetic particle collecting unit was used to capture the magnetic beads which are bound to the biotin-gDNA complex. This allows us to capture gDNA containing repeats while other fragments (i.e. those not containing repeats) are washed away. Enriched fragments were removed from the biotinylated probes by denaturing at 95°C and precipitated with 95% ethanol and 3 M sodium acetate. To increase the amount of enriched fragments, a “recovery” PCR was performed in a 25 μl reaction containing 1× PCR buffer (10 mM Tris–HCl, 50 mM KCl, pH 8.3), 1.5 mM MgCl2, 0.16 mM of each dNTP, 10× BSA, 0.52 μM of the SuperSNX24 forward primer, 1U Taq DNA polymerase, and approximately 25 ng enriched gDNA fragments. Thermal cycling, performed in an MJ Research DYAD, was as follows: 95°C for 2 min followed by 25 cycles of 95°C for 20 s, 60°C for 20 s, and 72°C for 90 s, and a final elongation step of 72°C for 30 min. Subsequent PCR fragments were cloned using the TOPO-TA Cloning® kit following the manufacturer’s protocol (Invitrogen). Bacterial colonies containing a vector with gDNA (i.e. white colonies) were used as a template for subsequent PCR. These PCR products were cleaned using MultiScreen-PCR Filter Plates following the manufacturer’s protocol (Millipore, Billerica, Massachusetts). DNA sequencing was performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, California). Sequencing reactions were precipitated with ethanol and 125 mM EDTA and run on an ABI 3730 DNA Analyzer. Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) was used to develop microsatellite PCR primers.

Chameleons were sampled from two general localities near Cape Town: the Stellenbosch area (N = 19) and the Hottentots-Holland Mountains approximately 40 km SE of Stellenbosch (N = 24). Tissue was taken from 1 to 2 mm tail clips and stored in 96% ethanol until subsequent DNA extraction. Extractions were performed using QIAGEN DNeasy extraction kit. Eight microsatellite loci yielded consistent unambiguous peaks. PCR reactions for six loci were carried out in a 10 μl reaction volume containing 10 ng of DNA template, 20–50 pM of each primer, 0.2 mM dNTPs, 0.75–1.5 mM MgCl2 (Table 1), 1× PCR buffer (7.5 mM MCl2, Ph 8.5) and 0.08 μl (5 U/μl) of Promega Taq polymerase. Thermal cycling parameters were as follows: 94°C for 1 min, followed by 35 cycles at 94°C for 45 s, Ta for 30 s (Table 1), 72°C for 45 s and a final extension at 72°C for 90 s. For loci Bpu26 and Bpu571 optimization was not successful using Promega reagents. As a result, we used the QIAGEN Multiplex PCR Kit to amplify these loci in a final reaction volume of 7 μl containing approximately 5 ng of DNA template, 3.5 μl of the QIAGEN PCR Master Mix, 6.0 mM MgCl2 (Table 1), and 17.5 pM of each primer using the following thermal cycling conditions: an initial activation step at 95°C for 15 min, followed by 34 cycles of 94°C for 30 s, Ta (Table 1) for 90 s, 72°C for 90 s, and a final extension at 60°C for 30 min. PCR products were combined either in a monoplex (1.2 μl) or multiplex format (0.6 μl) with a mixture containing Rox-350 size standard, formamide and loading dye, and run on a 377 ABI Prism automated sequencer. Sizing of alleles was performed using GENOTYPER version 2.5 (Applied Biosystems Inc.).

Table 1 Characteristics of microsatellite loci and primers developed for Bradypodion pumilum
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All loci were polymorphic, with the number of alleles ranging from five to 26 alleles and high levels of heterozygosity (Table 1). Tests for linkage disequilibrium between loci and Hardy Weinberg equilibrium were performed using GENEPOP ON THE WEB (Raymond and Rousset 1995). Loci Bpu28, Bpu94 and Bpu115 deviated significantly from Hardy-Weinberg equilibrium. No significant linkage disequilibrium was detected for any of the loci after applying a Bonferroni correction for multiple comparisons (Rice 1989).

Deviations from HW expectations may be the result of extreme demographic fluctuations that have been observed in this species (KAT, pers. obs.). Both anecdotal information and ongoing monitoring of populations in urban fragments suggest that B. pumilum may be prone to population crashes with quick recoveries. In natural settings, this species also occurs in the fire prone “fynbos” vegetation (a Mediterranean type heathland), where tens to hundreds of hectares of habitat can be burnt in a single fire, during which populations can be severely reduced (M. Burger and A. A. Turner, personal communication). Thus, populations of this species that have undergone these extreme fluctuations could be prone to repeated founder events, altering expectations of HW frequencies.

The use of these microsatellite loci in studies on the conservation genetics of this species will be extremely valuable for understanding the effects of habitat fragmentation on population structure and gene flow, temporal changes in allele frequencies with regards to demographic fluctuations, population bottlenecks and inbreeding, as well as the type of mating system. This information will be particularly useful for providing a framework that contributes to a comprehensive evaluation of the conservation status, and also for making recommendations to Cape Town and other municipalities regarding the use of urban corridors (Rebelo et al. In review) for threatened wildlife.