Although genome-scale data generation is becoming more tractable for phylogenetics, there are large quantities of single gene fragment data in public repositories and such data are still being generated. We therefore investigated whether single mitochondrial genes are suitable proxies for phylogenetic reconstruction as compared to the application of full mitogenomes. With near complete taxon sampling for the southern African dwarf chameleons (Bradypodion), we estimated and compared phylogenies for the complete mitogenome with topologies generated from individual mitochondrial genes and various combinations of these genes. Our results show that the topologies produced by single genes (ND2, ND4, ND5, COI, and COIII) were analogous to the complete mitogenome, suggesting that these genes may be reliable markers for generating mitochondrial phylogenies in lieu of generating entire mitogenomes. In contrast, the short fragment of 16S commonly used in herpetological systematics, produced a topology quite dissimilar to the complete mitogenome and its concatenation with ND2 weakened the resolution of ND2. We therefore recommend the avoidance of this 16S fragment in future phylogenetic work.

Arboreal species tend to have specific adaptations allowing them to effectively negotiate the complexity of their habitats. For example, chameleons have a prehensile tail and limbs adapted to grasping branches. However, the impact of branch diameter and orientation on their movement has been poorly studied. Using ecological, morphological, and pull force data from two species of Bradypodion from different habitats, we examined if chameleons use different substrates and if perch orientation and diameter affect pull strength by quantifying their grip forces on different sized dowels. We found that the habitat of Bradypodion ventrale is homogeneous, and dominated by sparse patches of narrow, vertical perches. For this species, perch diameter significantly affected pull strength in both horizontal and vertical pull directions, with chameleons performing best on a vertical 1.5-mm dowel and a horizontal 3-mm dowel. In contrast, Bradypodion pumilum typically occurs in more variable vegetation, ranging from low shrubs to wooded habitat with high canopy. Our results show that the habitat has perches that are on average wider than for B. ventrale with a greater size range. The performance of B. pumilum was less impacted by perch diameter and pull direction suggesting that B. pumilum is able to use perches of different diameters in their more heterogeneous habitat.

We conducted a study on interpopulation variation of colour patterns in two congeneric chameleon species, which have an analogous life history. Both species are able to rapidly change colour pattern, and their context-dependent colour patterns often vary across a wide geographical range. Specifically, we tested four hypotheses that can explain the observed interpopulation variation of colour patterns by a series of behavioural field trials where the colour patterns of individuals were recorded and later analysed by a deep neural network algorithm. We used redundancy analysis to relate genetic, spectral and behavioural predictors to interpopulation colour pattern distance. Our results showed that both isolation by distance (IBD) and alternative mating tactics were significant predictors for interpopulation colour pattern variation in Chamaeleo chamaeleon males. By contrast, in Chamaeleo dilepis, the interpopulation colour pattern variation was largely explained by IBD, and evidence for alternative mating tactics was absent. In both chameleon species, the environmental colours showed no evidence of influencing chameleon interpopulation colour pattern variation, regardless of sex or behavioural context. This contrasting finding suggests that interpopulation context-dependent colour pattern variations in each species are maintained under a different set of selective pressures or circumstances.

In biodiversity research, the retrieval of genetic material from organisms is a common and essential component for assessing genetic diversity. The welfare of the organism, however, needs to be balanced against the overall goal of the intended research. One sampling technique often applied to retrieve DNA material from small reptiles is the removal of a small portion of the distal end of the tail. While most squamate reptiles have tail autotomy, some species (e.g. many iguanid lizards and snakes) do not regenerate tail tissue. We therefore explored the efficacy of a minimally disruptive technique, buccal swabbing, as an alternative to tissue sampling via tail clipping, particularly for species without tail autotomy, using dwarf chameleons (Bradypodion spp.) as a case study. The two sampling techniques were compared to assess the efficacy of DNA retrieval. We also evaluated the financial implications of each technique. The results indicate that buccal swabs paired with a specialised DNA extraction kit offer a feasible (although expensive), once-off alternative to tissue sampling, but with no material left for biobanking. Deviations in swab type used and the DNA extraction process (i.e. using more affordable extraction procedures) resulted in poor DNA retrieval and unreadable sequences. This finding suggests that buccal swabbing can be a suitable alternative when finances are not constrained, an expensive extraction kit is available, and biobanking is not a concern. For researchers from low- to middle-income economies, this expensive alternative may hamper research progress by placing a financial obstacle in the way, and therefore the next best option is tissue sampling. Significance: This study provides guidance on the efficacy of buccal swabs as a viable alternative to tissue samples collected via tail clipping for DNA retrieval from small reptiles. The results indicate that swabs may be a feasible alternative to tissue samples when finances are not constrained. Deviations in buccal swabbing method (i.e. using more cost-effective alternatives) performed poorly in DNA retrieval and do not offer competitive alternatives to tissue samples. Although buccal swabs were shown to offer an alternative to tissue samples, the financial implications to research in low- to middle-income economies may hinder research goals unnecessarily.

A longstanding question in evolutionary biology is how natural selection and environmental pressures shape the mitochondrial genomic architectures of organisms. Mitochondria play a pivotal role in cellular respiration and aerobic metabolism, making their genomes functionally highly constrained. Evaluating selective pressures on mitochondrial genes can provide functional and ecological insights into the evolution of organisms. Collembola (springtails) are an ancient hexapod group that includes the oldest terrestrial arthropods in the fossil record, and that are closely associated with soil environments. Of interest is the diversity of habitat stratification preferences (life forms) exhibited by different species within the group. To understand whether signals of positive selection are linked to the evolution of life forms, we analysed 32 published Collembola mitogenomes in a phylomitogenomic framework. We found no evidence that signatures of selection are correlated with the evolution of novel life forms, but rather that mutations have accumulated as a function of time. Our results highlight the importance of nuclear-mitochondrial interactions in the evolution of collembolan life forms and that mitochondrial genomic data should be interpreted with caution, as complex selection signals may complicate evolutionary inferences.

Molecular phylogenetics and the application of species delimitation methods have proven useful in addressing limitations associated with morphology based taxonomy and have highlighted the inconsistencies in the current taxonomy for many groups. For example, the genus Chamaeleo, which comprises 14 species with large distributions across mainland Africa and parts of Eurasia, exhibits relatively minor phenotypic differentiation between species, leading to speculation regarding the presence of cryptic diversity in the genus. Therefore, the aims of the present study were to construct a robust and comprehensive phylogeny of the genus and highlight potential species-level cryptic diversity. Additionally, we sought to ascertain the most likely biogeographic origin of the genus and understand its spatio-temporal diversification. Accordingly, we made use of species delimitation methods (Bayesian and divergence based) to investigate the extent of cryptic diversity in Chamaeleo, and applied an ancestral area reconstruction to examine the biogeographic origin of the group. Our phylogenetic analyses suggested the presence of at least 18 taxa within Chamaeleo. Notably, three taxa could be recognised within C. dilepis, none of which are equivalent in context with any of the currently described subspecies. There were also three taxa within C. gracilis and two within C. anchietae. The single available tissue specimen identified as C. necasi was embedded within the C. gracilis clade. Our ancestral area reconstruction points to a southern African/Zambezian origin for Chamaeleo, with diversification beginning during the cooling and aridification of Africa that characterised the Oligocene Epoch, ca. 34-23 Mya (Million years ago). Species-level diversification began in the Miocene Epoch (ca. 23-5 Mya), possibly tracking the aridification that triggered the shift from forest to more open, mesic savanna for most clades, but with tectonic events influencing speciation in a Palearctic clade. These findings lay the foundation for a future integrative taxonomic re-evaluation of Chamaeleo, which will be supported with additional lines of evidence before implementing any taxonomic changes.

Large-scale monitoring of wild populations in remote areas using traditional live-capturing methods is logistically and financially challenging. Devices that can be used to obtain biological material remotely and store it for an extended period have considerable potential to monitor population densities and health status, but their applicability remains largely unexplored. The present study describes a device that collects trace amounts of DNA from the saliva of small mammals that is deposited on the surface of a collection medium (WaxTags®). The device’s performance was evaluated on Australian brushtail possums ( Trichosurus vulpecula ), an invasive pest species and the most significant vector of bovine tuberculosis infective agent ( Mycobacterium bovis ), under field conditions in Canterbury, New Zealand. The retrieved DNA was used to amplify eight possum-specific microsatellite markers and bacterial 16S rRNA. The design is mechanically robust, and the quality of the recovered DNA was adequate for microsatellite-based identification of individual possums, estimation of population density, and partial reconstruction of their oral microbiomes as a potential indicator of health. Several medically important bacteria, including strains of environmental Mycobacterium sp., were detected. The design can be refined to monitor other animals’ populations proactively and provide different levels of information necessary to manage wild populations.

During austral winter, the southern and eastern coastlines of South Africa witness one of the largest animal migrations on the planet, the KwaZulu-Natal sardine run. Hundreds of millions of temperate sardines, Sardinops sagax, form large shoals that migrate north-east towards the subtropical Indian Ocean. Recent studies have highlighted the role that genetic and environmental factors play in sardine run formation. In the present study, we used massively parallel sequencing to assemble and annotate the first reference transcriptome from the liver cells of South African sardines, and to investigate the functional content and transcriptomic diversity. A total of 1,310,530 transcripts with an N50 of 1578 bp were assembled de novo. Several genes and core biochemical pathways that modulate energy production, energy storage, digestion, secretory processes, immune responses, signaling, regulatory processes, and detoxification were identified. The functional content of the liver transcriptome from six individuals that participated in the 2019 sardine run demonstrated heterogeneous levels of variation. Data presented in the current study provide new insights into the complex function of the liver transcriptome in South African sardines.

South Africa harbours remarkable biological diversity with three of the 34 recognised global biodiversity hotspots placed within its borders. One of these is the Succulent Karoo, which together with the Nama-Karoo, forms the Greater Karoo region. Notwithstanding a paucity of studies from this region, it would appear that, although mammal diversity is low, endemism is high. Here, as part of the Karoo BioGaps project, we use a molecular approach to assess small mammal diversity and endemism in the Karoo. We focus on rock rats (Micaelamys) and elephant shrews (Elephantulus and Macroscelides). Using a DNA-informed identification approach, we reveal two, well supported, monophyletic clades of Micaelamys; one that corresponds to M. granti. Our study is the first to publish sequence data for this species. Furthermore, when unverified records are excluded, the range of M. granti is far smaller (∼99 000 km2) than that given by the IUCN red list assessments (236 027 km2), which lends support to the species being a Karoo endemic. Our macrosceledid samples grouped into four well supported clades of the genera Elephantulus and Macroscelides. Very high intraspecific diversity was present within E. pilicaudus compared with other species in our study and this newly described species may harbour cryptic diversity. Our geographic analyses confirm that the range of this species, previously considered to be a Nama-Karoo endemic, extends beyond this region. This study adds more information to the nominal data currently available for the species, Elephantulus pilicaudus.

1075 Chamaeleo is a pan-African genus of chameleons that also extends into southern Europe, 1076 southern Asia, and the Middle East, with much geographic overlap and confusing phenotypic 1077 variation between its taxa. The large, overlapping ranges of members of Chamaeleo present an 1078 interesting but complex case for biogeographic ancestral area reconstruction. A previous study 1079 (Chapter 2) found strong evidence for the presence of cryptic species within the Chamaeleo 1080 dilepis complex. The aims of the present study were twofold: firstly, to reconstruct the ancestral 1081 biogeographic history of the genus Chamaeleo and secondly, to investigate whether there is 1082 evidence for niche partitioning between the three cryptic species within the C. dilepis complex. 1083 A time-calibrated phylogenetic analysis was run on a dataset encompassing all known species 1084 of Chamaeleo. Dispersal-Extinction-Cladogenesis (DEC) was used on this time-calibrated 1085 phylogeny to reconstruct the ancestral biogeography of Chamaeleo. Additionally, Maxent was 1086 used to carry out ecological niche modelling on the three clades within C. dilepis. The common 1087 ancestor of Chamaeleo emerged during the Eocene, most likely in the Zambezian region. 1088 Chamaeleo probably exploited mesic corridors that opened up during forest contractions of the 1089 Oligocene and Miocene to move into North Africa and Eurasia. Niche partitioning is evident 1090 between C. dilepis clades and only the suitable niche of C. dilepis 2 appears relatively stable 1091 since the late Pleistocene. These results provide a glimpse at the evolutionary biogeographic 1092 history of Chamaeleo and could aid future revisions of distribution maps pertaining to the C. 1093 dilepis complex. 1094

The spatial genetic structure of a species, and whether distinct genetic lineages are present, is strongly influenced by their biology and habitat requirements. Given habitat specificity and low vagility, many herpetofaunal species are reservoirs for high levels of cryptic diversity; chameleons are a case in hand. The common flap-necked chameleon Chamaeleo dilepis has a large range that spans much of sub-Saharan Africa. Within South Africa, the species is largely confined to the north-eastern and central areas of the country, and occurs from the coastal forests in southern KwaZulu-Natal westwards into Namibia. Their large range. together with anecdotal evidence that there is considerable morphological and phenotypic diversity across the range, suggests a questionable taxonomy with possible cryptic lineages. The aims of the present study were to investigate whether C. dilepis is genetically structured across parts of its South African range, and whether this species (as it currently stands) might include cryptic lineages. To this end, 72 C. dilepis individuals sampled from four localities across South Africa (Gauteng, n = 2; KwaZulu-Natal, n = 2) were sequenced for two mitochondrial markers (ND4 and 16S). The phylogenetic results suggest that C. dilepis is indeed spatially structured. In addition, the large sequence divergence values between groups strongly suggests the presence of cryptic lineages and, pending the inclusion of more data from a larger geographic range, the group may be in need of a taxonomic revision.