Sandi Willows-Munro

DNA-led rediscovery of the giant sable antelope in Angola

The giant sable antelope (Hippotragus niger variani), unique to Angola, was feared extinct after almost three decades of civil war. Comparisons of mitochondrial DNA sequences derived from dung samples recently collected in the field and from old museum specimens of certain provenance provide the first documented evidence to date that this enigmatic antelope has survived. Its DNA-led rediscovery in the former combat zone was subsequently confirmed by photographic evidence. The Angolan isolate constitutes a distinct monophyletic group that shows a dramatic population decline from historic levels. It represents a diagnosable conservation unit which is characterised by unique cranial morphological features (Blaine 1922 1922:317–339), a highly restricted range, and the presence of fixed genetic differences in all of its common relatives.

Exploring the diversity and molecular evolution of shrews (family Soricidae) using mtDNA cytochrome b data

The taxonomy of the shrews (family Soricidae) is primarily underpinned by the analysis of morphology characters. These morphological characters are, however, often plesiomorphic and difficult to interpret in an evolutionarily meaningful way. Recent molecular studies have provided some clarity at lower-taxonomic levels, but studies giving resolution to deeper levels of the soricid phylogeny are lacking. By constructing the most taxonomically inclusive molecular phylogeny for this cosmopolitan family, this study aims to test the utility of the routinely used mitochondrial cytochrome b gene to resolve the higher-level relationships within the Soricidae. Given the life history characteristics of shrews (high metabolic rate in Soricinae, small body size, fast generation time and generally short life span), the metabolic theory predicts accelerated nucleotide turnover in this family. By examining the molecular evolution of the cytochrome b molecule, this study identifies both lineage- and codon-specific mutational rate differences. We suggest that codon-specific mutational rate patterns are best explained by unique selective regimes operating on the different codon positions, while lineage-specific rate variation among genera belonging to Soricidae may reflect differences in the mode and speed of speciation events which generated species.

Molecular Systematics of the Cape Parrot (Poicephalus robustus): Implications for Taxonomy and Conservation

The taxonomic position of the Cape Parrot (Poicephalus robustus robustus) has been the focus of much debate. A number of authors suggest that the Cape Parrot should be viewed as a distinct species separate from the other two P. robustus subspecies (P. r. fuscicollis and P. r. suahelicus). These recommendations were based on morphological, ecological, and behavioural assessments. In this study we investigated the validity of these recommendations using multilocus DNA analyses. We genotyped 138 specimens from five Poicephalus species (P. cryptoxanthus, P. gulielmi, P. meyeri, P. robustus, and P. rueppellii) using 11 microsatellite loci. Additionally, two mitochondrial (cytochrome oxidase I gene and 16S ribosomal RNA) and one nuclear intron (intron 7 of the β-fibrinogen gene) markers were amplified and sequenced. Bayesian clustering analysis and pairwise FST analysis of microsatellite data identified P. r. robustus as genetically distinct from the other P. robustus subspecies. Phylogenetic and molecular clock analyses on sequence data also supported the microsatellite analyses, placing P. r. robustus in a distinct clade separate from the other P. robustus subspecies. Molecular clock analysis places the most recent common ancestor between P. r. robustus and P. r. fuscicollis / P. r. suahelicus at 2.13 to 2.67 million years ago. Our results all support previous recommendations to elevate the Cape Parrot to species level. This will facilitate better planning and implementation of international and local conservation management strategies for the Cape Parrot.

A Review of Over a Decade of DNA Barcoding in South Africa: A Faunal Perspective

For over a decade, molecular short standardised DNA fragments, termed DNA barcodes, have been developed for species discrimination around the world. As of 2010, the vast majority of barcoding research was biased toward particular taxonomic groups and geographic regions largely because researchers in developed countries were the ones with the resources and capacity to carry out such work. To rectify this, the International Barcode of Life Project was launched with the intent to extend the geographic and taxonomic coverage of the barcode reference library. South Africa committed to this mission in an attempt to catalogue all of its known biodiversity and, possibly, help identify new species. To date, approximately 48 000 South African faunal barcodes are housed in the Barcode of Life Data System (BOLD), which represent only 2.3% of all known South African animal species. Although insects are the best represented in absolute terms, with over 37 000 samples recorded, they are still grossly lacking with just over 1% representation. Much like the global trend, there is a general taxonomic bias, with fish, birds and mammals showing the greatest representation. Moreover, geographic bias is also present, with the Free State province particularly under-represented on BOLD, likely owing to limited human capacity. Although few studies have been published with respect to barcoding, the majority reveal that the cytochrome c oxidase 1 (CO1) gene, used in isolation or in conjunction with other molecular markers, can greatly benefit South African biodiversity research. Several limitations of DNA barcoding are discussed and recommendations specific to South Africa provided.

Phylogenetic analysis of the polyprotein coding region of an infectious South African bursal disease virus (IBDV) strain.

Infectious bursal disease virus (IBDV) causes Gumboro disease, which is highly contagious and immunosuppressive in young chickens. A virulent form of IBDV reached South Africa in 1989 and to date there has been little molecular information available for this strain. In this study, the polyprotein coding region of the South African strain SA-KZN95 was sequenced and analysed along with 52 representative sequences of other serotype I and II strains. We explored the relative impact of recombination on phylogenetic reconstruction using a multidimensional scaling approach. Phylogenetic analyses consistently placed the South African isolate within the very virulent IBDV clade. Selection analyses were also conducted to identify evolutionarily relevant amino acid residues. Previously, 19 residues in the polyprotein were shown to be potentially diagnostic for the different IBDV pathotypes. This study identified an additional two unique residues in the polyprotein which may be used as genetic signatures in future viral identifications. Better strain identification would aid in the development and application of vaccines.

Influence of killing method on Lepidoptera DNA barcode recovery.

The global DNA barcoding initiative has revolutionized the field of biodiversity research. Such large‐scale sequencing projects require the collection of large numbers of specimens, which need to be killed and preserved in a way that is both DNA‐friendly and which will keep voucher specimens in good condition for later study. Factors such as time since collection, correct storage (exposure to free water and heat) and DNA extraction protocol are known to play a role in the success of downstream molecular applications. Limited data are available on the most efficient, DNA‐friendly protocol for killing. In this study, we evaluate the quality of DNA barcode (cytochrome oxidase I) sequences amplified from DNA extracted from specimens collected using three different killing methods (ethyl acetate, cyanide and freezing). Previous studies have suggested that chemicals, such as ethyl acetate and formaldehyde, degraded DNA and as such may not be appropriate for the collection of insects for DNA‐based research. All Lepidoptera collected produced DNA barcodes of good quality, and our study found no clear difference in nucleotide signal strength, probability of incorrect base calling and phylogenetic utility among the three different treatment groups. Our findings suggest that ethyl acetate, cyanide and freezing can all be used to collect specimens for DNA analysis.

Testing of microsatellite multiplexes for individual identification of Cape Parrots (Poicephalus robustus): paternity testing and monitoring trade

BACKGROUND Illegal trade in rare wildlife species is a major threat to many parrot species around the world. Wildlife forensics plays an important role in the preservation of endangered or threatened wildlife species. Identification of illegally harvested or traded animals through DNA techniques is one of the many methods used during forensic investigations. Natural populations of the South African endemic Cape Parrot (Poicephalus robustus) are negatively affected by the removal of eggs and chicks for the pet trade. METHODS In this study, 16 microsatellite markers specifically designed for the South African endemic Cape Parrot (P. robustus) are assessed for their utility in forensic casework. Using these 16 loci, the genetic diversity of a subset of the captive Cape Parrot population was also assessed and compared to three wild Cape Parrot populations. RESULTS It was determined that the full 16 locus panel has sufficient discriminatory power to be used in parentage analyses and can be used to determine if a bird has been bred in captivity and so can be legally traded or if it has been illegally removed from the wild. In cases where birds have been removed from the wild, this study suggests that a reduced 12 locus microsatellite panel has sufficient power to assign confiscated birds to geographic population of origin. DISCUSSION The level of genetic diversity observed within the captive Cape Parrot population was similar to that observed in the wild populations, which suggests that the captive population is not suffering from decreased levels of genetic diversity. The captive Cape Parrots did however have double the number of private alleles compared to that observed in the most genetically diverse wild population. This is probably due to the presence of rare alleles present in the founder population, which has not been lost due to genetic drift, as many of the individuals tested in this study are F1-F3 wild descendants. The results from this study provide a suit of markers that can be used to aid conservation and law enforcement authorities to better control legal and illegal trade of this South African endemic.

A molecular phylogeny of the spiny lobster Panulirus homarus highlights a separately evolving lineage from the Southwest Indian Ocean

Accurate species description in the marine environment is critical for estimating biodiversity and identifying genetically distinct stocks. Analysis of molecular data can potentially improve species delimitations because they are easily generated and independent, and yield consistent results with high statistical power. We used classical phylogenetic (maximum likelihood and Bayesian inference) and coalescent-based methods (divergence dating with fossil calibrations and coalescent-based species delimitation) to resolve the phylogeny of the spiny lobster Panulirus homarus subspecies complex in the Indo-West Pacific. Analyses of mitochondrial data and combined nuclear and mitochondrial data recovered Panulirus homarus homarus and Panulirus homarus rubellus as separately evolving lineages, while the nuclear data trees were unresolved. Divergence dating analysis also identified Panulirus homarus homarus and Panulirus homarus rubellus as two distinct clades which diverged from a common ancestor during the Oligocene, approximately 26 million years ago. Species delimitation using coalescent-based methods corroborated these findings. A long pelagic larval life stage and the influence of ocean currents on post-larval settlement patterns suggest that a parapatric mode of speciation drives evolution in this subspecies complex. In combination, the results indicate that Panulirus homarus rubellus from the Southwest Indian Ocean is a separately evolving lineage and possibly a separate species.

Studying Earthworms (Annelida: Oligochaeta) in South Africa

ABSTRACT Earthworms are an important component of southern African invertebrate diversity, due both to their influential roles in soil ecosystems, and the relatively large number of species. As of 2010, there were 282 indigenous earthworm species (most endemic) known to South Africa belonging to three families: Microchaetidae, Tritogeniidae and Acanthodrilidae. In addition, 44 introduced species from six families had been recorded. However, earthworms are rarely included in environmental monitoring or conservation programmes—partly because sampling and species identification are difficult and many sampling methods are destructive and/or toxic. In this paper we review the earthworm sampling techniques most commonly used by screening data from a digitised literature collection on South African earthworms and on-line global searches. By examining a case study sampling of three vegetation types, this paper highlights taxonomic challenges and the effort required to properly curate specimens. The study provides recommendations for future sampling and highlights some key priorities for future work on the group. From the literature review in early 2012, it is clear that collection techniques are often insufficiently recorded in published work. A total of 10 938 publications from the period 1950 to 2012 were found from the literature search and digitised collection and from these only 32 papers recorded the sampling methodology (mainly hand sorting) for South African research, pointing to the need to adopt standard sampling and reporting protocols. We also tested two of the most popular methodologies in the field. Sampling was conducted in January and February 2012 at four sites, with 24 plots at each site (12 digging and 12 using mustard extraction). A total of 2 094 earthworms collected could be assigned a species name, with introduced species predominating at both disturbed and natural sites. It took a team of three to five people digging and hand collecting all earthworm specimens encountered in a plot of 50 cm × 50 cm × 20 cm deep around 45 to 60 minutes. However, much more time was spent curating and identifying samples. While we recommend following the ISO (ISO11268-3, ISO23611-1) protocol for collecting introduced taxa, to get a complete inventory of South African earthworms a range of sampling techniques will be required; in particular, a large 1 m × 1 m × 20 cm plot is required for many large bodied native taxa, and the collection of giant earthworms will require different approaches. The identification of specimens requires skills that are scarce in the country and so there is an urgent need for training and funding for fundamental work on earthworm taxonomy. An atlasing project could serve as a focal point for future research. In providing some general recommendations based on the long and fruitful history of research on earthworms in South Africa, we are optimistic that a better understanding of the group will help us to both improve our usage of natural resources and provide insights into this vitally important edaphic group.

Plant-pollinator interactions over time: Pollen metabarcoding from bees in a historic collection

Pollination is a key component in agricultural food production and ecosystem maintenance, with plant–pollinator interactions an important research theme in ecological and evolutionary studies. Natural history collections provide unique access to samples collected at different spatial and temporal scales. Identification of the plant origins of pollen trapped on the bodies of pollinators in these collections provides insight into historic plant communities and pollinators’ preferred floral taxa. In this study, pollen was sampled from Megachile venusta Smith bees from the National Collection of Insects, South Africa, spanning 93 years. Three barcode regions, the internal transcribed spacer 1 and 2 (ITS1 and ITS2) and ribulose‐1,5‐biphosphate carboxylase (rbcL), were sequenced from mixed pollen samples using a next‐generation sequencing approach (MiSeq, Illumina). Sequenced reads were compared to sequence reference databases that were generated by extracting sequence and taxonomic data from GenBank. ITS1 and ITS2 were amplified successfully across all (or most) samples, while rbcL performed inconsistently. Age of sample had no impact on sequencing success. Plant classification was more informative using ITS2 than ITS1 barcode data. This study also highlights the need for comprehensive reference databases as limited local plant sequence representation in reference databases resulted in higher‐level taxon classifications being more confidently interpreted. The results showed that small, insect‐carried pollen samples from historic bee specimens collected from as early as 1914 can be used to obtain pollen metabarcodes. DNA metabarcoding of mixed origin pollen samples provided a faster, more accurate method of determining pollen provenance, without the need for expert palynologists. The use of historic collections to sample pollen directly from pollinators provided additional value to these collections. Sampling pollen from historic collections can potentially provide the spatial and temporal scales for investigations into changes in plant community structure or pollinator floral choice in the face of global climate change.