m o d e r n t e c h niques in plant taxo nomy

Mesembs and Molecules

Discovering the Delosperma family tree via molecules
by M.H. Buys, Compton Herbarium, South African National Biodiversity Institute, Kirstenbosch and Dept. of Botany and Zoology, University of Stellenbosch
Delosperma gautengense with broader petals than found in D. herbeum. Photo: M.H. Buys.

he genetic fingerprinting of criminals no longer belongs to the realm of science fiction. Similar molecular data has become a common tool at the disposal of plant taxonomists, contributing tremendously to the better understanding of how plant families are related to each other. However, when descending from the rank of family down the hierarchy of life, ranks below, i.e. groups of genera, individual genera or species, have not all yet been reliably identified by molecular means. This holds especially true for the vygie family (Aizoaceae/Mesembryanthemaceae) where researchers have failed to get meaningful groupings of genera or species after analysing a number of genes. Yet, species remain the most fundamental rank in our line of work. Clear and reliable species boundaries are required not only for taxonomic purposes, but also for conservation endeavours. Delosperma herbeum and D. davyi are a case in point. D. davyi is known from a few populations in Gauteng and is listed as a Red Data species. Its conservation is thus currently actively mediated by nature conservation authorities. However, D. davyi could be a mere variety or ecotype of the widespread D. herbeum, in which case it is not in need of conservation. Differences between the two species
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lie in growth form, with D. davyi developing long, trailing or climbing, persisting vegetative branches as opposed to the predominantly erect annual flowering branches in D. herbeum. From an ecological perspective, D. davyi occurs in shady riverine woods and D. herbeum occurs in open grassland. In the view of the highlighted inability of gene sequencing to provide meaningful data, a different molecular technique known as amplified fragment length polymorphism (AFLP) was considered. Instead of determining the order of all the building blocks in a particular gene, as is done in gene sequencing, the entire DNA content of the plant is chopped up into fragments beginning and ending with predetermined short sequences. Plants that share the same complement of fragments are perceived to be related. The AFLP technique has been used with great success in studying taxonomic issues below the rank of species, i.e. at the population level, and is therefore ideally suited to address the D. davyi/D. herbeum issue mentioned above. But, could this technique perchance also distinguish between vygie species, keeping in mind that many fynbos families and genera (vygies included) appear to be recently evolved, and perhaps, from a molecular point of view, still act like populations?

In response to the D. davyi/D. herbeum affair, a number of Delosperma herbeum populations from the North West, Gauteng, Free State and Limpopo provinces of South Africa were analysed as well as a single population of D. davyi (see accompanying map). To test the AFLP technique to distinguish between vygie species, populations of D. knox-daviesii, D. gautengense, D. purpureum, D. leendertziae and D. vogtsii (all occurring in Gauteng) were also included in the study. After scoring a total of 158 fragments for 99 individuals representing a total of 30 populations, a cluster analysis, where individuals are grouped/clustered on the basis of shared similarities, was embarked upon. The majority of individuals formed locality-specific groupings. In addition, some broad correlations appear to exist between the clusters obtained in D. herbeum and their distribution. The Sun City and Groot Marico populations (labelled 21 and 22 on the map) form a cluster to the north-west of the study area. There also appears to be a connection between the north-eastern Pretoria and Naboomspruit populations (labelled 19 and 20 on the map). The geographic signal in the Vredefort Dome (populations 5-7) is less clear. Population 5 clusters with samples from Potchefstroom and Klerksdorp, whereas popu-

MAP: The localities of the 22 populations of Delosperma herbeum analysed. Shaded polygons encircling populations refer to main clusters in the Neighbor-Joining tree. TOP: A new species of Delosperma? Resembling Delosperma herbeum in habit, but a separate entity based on AFLP data and possessing exclusively mauve flowers. ABOVE: Delosperma herbeum with petals tipped with mauve. Photos: M.H. Buys. RIGHT: Neighbor-Joining tree of AFLP data obtained from Delosperma. Using a molecular technique known as amplified fragment length polymorphism (AFLP), the DNA content of the plant is chopped up into fragments beginning and ending with predetermined short sequences. Plants that share the same complement of fragments are perceived to be related. This tree is the result of the cluster analysis, using data from a number of Delosperma populations from the North West, Gauteng, Free State and Limpopo provinces of South Africa. Circled numbers refer to population numbers on the map.

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lation 6 has ties to the north, clustering with the population from Fochville, and population 7, from south of the Vaal River, has apparent similarities with the populations to the northeast ranging from Sasolburg to Johannesburg. D. davyi and D. herbeum from North Riding form a cluster separate from the remaining populations of D. herbeum. While the above results are somewhat ambiguous regarding the status of D. davyi, further statistical analyses not reported here, as well as morphological considerations, suggest D. davyi to be a superfluous name. The AFLP data can readily distinguish between species of Delosperma. The yellow flowered D. vogtsii groups with the white flowered D. gautengense. The mauve coloured D. knox-daviesii attaches itself, in turn, to this group. The yellow flowered D. leendertziae groups with the mauve flowered D. purpureum. The yet unidentified and possibly new mauve flowered species from Ditholo clusters with the mainly white flowered D. herbeum. Although flower colour is known to be an unreliable taxonomic character, it was tempting to consider species with similarly coloured flowers and of equal morphology, e.g. D. leendertziae and D. vogtsii, to be one and the same entity. Our results suggest this not to be the case. Where-as DNA sequencing approaches have failed to resolve even generic relationships - let alone species - in the vygie family, the AFLP technique holds promise for vygie systematics at lower ranks.
*Extracted and modified in part from Buys et al. 2008. Applying AFLPs in Aizoaceae: The Delosperma herbeum complex as a case study. Biochemical Systematics and Ecology 36, 92-100, with permission from Elsevier.

TOP: Delosperma davyi with the typical longer leaves and internodes which are probably due to the shady habitat in which it occurs. ABOVE : Delosperma herbeum, the most widespread Delosperma species in Gauteng and North West provinces. Flowers here pure white. ABOVE RIGHT: Delosperma vogtsii, resembling D. leendertziae, but on average possessing more petals than the latter and distinguishable based on AFLP data. BELOW: Delosperma leendertziae, resembling D. vogtsii, but on average possessing fewer petals than the latter, and distinguishable based on AFLP data. BELOW RIGHT: Transfer of knowledge. Heidi Hartmann pointing out diagnostic characters of Delosperma herbeum to Wim Buys. LEFT: Delosperma knox-daviesii with petals more numerous than in D. purpureum. BOTTOM LEFT: Delosperma purpureum with broad petals similar to D. gautengense. Note mauve filaments. Photos: M.H. Buys.

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