Ten years of barcoding at the African Centre for DNA Barcoding.

The African Centre for DNA Barcoding (ACDB) was established in 2005 as part of a global initiative to accurately and rapidly survey biodiversity using short DNA sequences. The mitochondrial cytochrome c oxidase 1 gene (CO1) was rapidly adopted as the de facto barcode for animals. Following the evaluation of several candidate loci for plants, the Plant Working Group of the Consortium for the Barcoding of Life in 2009 recommended that two plastid genes, rbcLa and matK, be adopted as core DNA barcodes for terrestrial plants. To date, numerous studies continue to test the discriminatory power of these markers across various plant lineages. Over the past decade, we at the ACDB have used these core DNA barcodes to generate a barcode library for southern Africa. To date, the ACDB has contributed more than 21 000 plant barcodes and over 3000 CO1 barcodes for animals to the Barcode of Life Database (BOLD). Building upon this effort, we at the ACDB have addressed questions related to community assembly, biogeography, phylogenetic diversification, and invasion biology. Collectively, our work demonstrates the diverse applications of DNA barcoding in ecology, systematics, evolutionary biology, and conservation.

Exposing the illegal trade in cycad species (Cycadophyta: Encephalartos) at two traditional medicine markets in South Africa using DNA barcoding.

Species in the cycad genus Encephalartos are listed in CITES Appendix I and as Threatened or Protected Species in terms of South Africa's National Environmental Management: Biodiversity Act (NEM:BA) of 2004. Despite regulations, illegal plant harvesting for medicinal trade has continued in South Africa and resulted in declines in cycad populations and even complete loss of sub-populations. Encephalartos is traded at traditional medicine markets in South Africa in the form of bark strips and stem sections; thus, determining the species traded presents a major challenge due to a lack of characteristic plant parts. Here, a case study is presented on the use of DNA barcoding to identify cycads sold at the Faraday and Warwick traditional medicine markets in Johannesburg and Durban, respectively. Market samples were sequenced for the core DNA barcodes (rbcLa and matK) as well as two additional regions: nrITS and trnH-psbA. The barcoding database for cycads at the University of Johannesburg was utilized to assign query samples to known species. Three approaches were followed: tree-based, similarity-based, and character-based (BRONX) methods. Market samples identified were Encephalartos ferox (Near Threatened), Encephalartos lebomboensis (Endangered), Encephalartos natalensis (Near Threatened), Encephalartos senticosus (Vulnerable), and Encephalartos villosus (Least Concern). Results from this study are crucial for making appropriate assessments and decisions on how to manage these markets.

Isozyme and allozyme markers distinguishing two morphologically similar, medically important Mastomys species (Rodentia: Muridae).

BACKGROUND: Two common southern African mice species, Mastomys coucha and M. natalensis, are widely distributed throughout the subregion and overlap in many areas. They also share a high degree of morphological similarity, making them impossible to distinguish in the field at present. These multimammate mice are documented carriers of serious disease vectors causing Lassa fever, plague and encephalomyocarditis, which coupled to their cohabitation with humans in many areas, could pose a significant health risk. A preliminary study reported the presence of isozyme markers at three loci (GPI-2, PT-2, -3) in one population each of M. coucha and M. natalensis. Two additional populations (from the Vaal Dam and Richards Bay) were sampled to determine the reliability of these markers, and to seek additional genetic markers. RESULTS: Fifteen proteins or enzymes provided interpretable results at a total of 39 loci. Additional fixed allele differences between the species were detected at AAT-1, ADH, EST-1, PGD-1, Hb-1 and -2. Average heterozygosities for M. coucha and M. natalensis were calculated as 0.018 and 0.032 respectively, with a mean genetic distance between the species of 0.26. CONCLUSIONS: The confirmation of the isozyme and the detection of the additional allozyme markers are important contributions to the identification of these two medical and agricultural pest species.

Persistent organochlorine pesticides detected in blood and tissue samples of vultures from different localities in South Africa.

Gas chromatography was used to establish the presence of quantifiable residues of 14 persistent chlorinated hydrocarbon pollutants in whole blood, clotted blood, heart, kidney, liver and muscle samples obtained from individual African whitebacked (Pseudogyps africanus), Cape griffon (Gyps coprotheres) and Lappetfaced (Torgos tracheliotos) vultures from different localities in South Africa. The levels of pesticides measured in whole blood samples of live specimens were compared between nestlings from two natural breeding colonies, adults from a wildlife area and birds held in captivity. Statistically significant (P<0.05) differences between populations were detected in geometric means calculated for gamma-BHC (lindane), alpha(cis)-chlordane and alpha-endosulfan. Five of the organochlorine contaminants displayed significant variations between concentrations detected in the clotted blood, organs and muscles excised from vulture carcasses. This includes residues of gamma-BHC, alpha-chlordane, dieldrin, beta-endosulfan and heptachlor epoxide. Values of the respective biocides measured in vulture samples were generally low in comparison to results documented for a number of avian species. Although no threat is posed by any of the organochloride pesticides, continual monitoring of especially breeding colonies is recommended. Furthermore, the suitability of African whitebacked vulture nestlings as basic bioindicators is highly advocated.

A review of the use of allozyme electrophoresis in plant systematics.

The role of electrophoretic data is discussed as it applies to plant taxonomy and systematic studies. Nei's (Am. Nat. 106 (1972) 283-292; Genetics 89 (1978) 583-590) genetic distances calculated for a large number of populations, species and genera were taken from published data. The relation between Nei's genetic identity measures and taxonomic rank (populations, species and genera) are shown graphically. The graphs obtained in this way (from 3021 pairs of plant taxa) differ substantially from previous graphs published by Thorpe (Ann. Rev. Ecol. Syst. 13 (1982) 139-168; in: G.S. Oxford, D. Rollinson (Eds.), Protein Polymorphism: Adaptive and Taxonomic Significance, Academic Press, London, 1983, pp. 131-152) and Thorpe and Solé-Cava (Zool. Scripta 23 (1994) 3-18). These authors suggested that the divergence between the different taxonomic ranks is roughly similar across a wide range of taxa. The latter was based on values for 2664 (Thorpe, 1982) and 8060 (Thorpe, 1983) pairs of animal and plant taxa, but the plant data contributed little to the total. For any given taxonomic rank, we found that plants are genetically more closely related than animals (possibly with the exception of birds). This result is important because the empirical relationships of genetic distance measures, to different levels of taxonomic separation, is often used for distinguishing and identifying cryptic or sibling species where conventional methods are unable to resolve systematic problems.

Allozyme variation in four populations of African whitebacked vultures (Gyps africanus) and phylogenetic relationships between four vulture species from southern Africa.

Genetic variation detected by protein electrophoresis at 41 presumptive gene loci was assayed in four populations of Gyps africanus and compared to values previously obtained for Gyps coprotheres. Values calculated for percentage of polymorphic loci (P=34.15%, 0.99 criterion) and average heterozygosity (&Hmacr;=0.108, +/-0.032) in G. africanus, confirm low levels of genetic variation as reported for G. coprotheres. Allele frequency data, assessed at 19 loci, were obtained to evaluate genetic differentiation among four vulture species. Six (31.58%) of the 19 shared loci were polymorphic. Values of 1.26 (+/-0.1), 26.32% and 0.076 (+/-0.047) for G. africanus, 1.21 (+/-0.1), 21.05% and 0.097 (+/-0.045) for Torgos tracheliotus, 1.11 (+/-0.7), 21.05% and 0.053 (+/-0.053) for Neophron percnopterus and 1.05 (+/-0.5), 5.26% and 0.044 (+/-0.047) for G. coprotheres were obtained for the mean number of alleles per locus, P and &Hmacr;, respectively. An average between-population fixation index (F(ST)) value of 0.322 was obtained, which is indicative of significant (P<0.01) differentiation between the four accipitrid species studied. Considerable concordance was obtained between dendograms produced from different analyses, pointing to the distinctiveness of N. percnopterus, which has evolved along a separate lineage as G. africanus, G. coprotheres and T. tracheliotus. Along the latter lineage G. africanus is clustered together with G. coprotheres which is consistent with the morphological similarities of these species.

Allozyme and DNA sequence comparisons of nine species of Encephalartos (Zamiaceae).

Phylogenetic relationships between Encephalartos altensteinii Lehmann, E. arenarius R.A. Dyer, E. horridus (Jacquin) Lehmann, E. latifrons Lehmann, E. lehmannii Lehmann, E. longifolius (Jacquin) Lehmann, E. princeps R.A. Dyer and E. trispinosus (Hooker) R.A. Dyer were studied, using E. ferox Bertoloni f. as outgroup. Three continuous and one discontinuous buffer systems were used and gene products of 14 enzyme coding loci were examined by horizontal starch gel-electrophoresis. Genetic variation was studied in a cultivated population of E. lehmannii and the average heterozygosity value for this population is 13.5%, which falls within the range reported for other cycad species. Fixed allele differences between the species studied was not found at any of the loci studied, which suggest that these species are closely related. DNA sequence analysis of rbcL and ITS 1 & 2 genes (1428 and 895 basepairs, respectively) confirmed the close genetic relationships between these taxa. According to ITS and rbcL sequences E. altensteinii and E. princeps are sibling taxa which form a sister group to E. arenarius, E. horridus, E. latifrons, E. lehmannii, E. longifolius, and E. trispinosus. The genetic distances between both groups were 0.12-0.47% for ITS and 0.08-0.16% for rbcL DNA. The results indicate recent (probably pleistocenic) speciation for this group of cycads, and the relationships are discussed with reference to affinities based on morphology and distribution.

Biochemical genetic markers to identify two morphologically similar South African Mastomys species (Rodentia: Muridae).

The two common southern African mice species (Mastomys coucha and M. natalensis) are morphologically almost identical, making field identification impossible at present. Specimens from two localities were collected and tissue and blood samples taken. The habitat type of each locality was studied, and a distribution map compiled. A definite correlation between biome-type and species range was found to be present. Three isozyme markers were identified: glucose phosphate isomerase in liver, and two general (non-specific) protein coding loci in muscle. In addition, we also identified species characteristic haemoglobin components in both species. This is the first study to report genetic variation within, and differentiation between these species. Our results are of medical importance because Mastomys coucha carries bubonic plague and M. natalensis carries Lassa Fever.