Thesis (M.S.)--Wake Forest University. Dept. of Biology, 2003. / Vita. Includes bibliographical references (leaves 20-22).

The genus Erica is the most species rich in the Cape Floristic Kingdom, yet there are limited data on the various flower-pollinator interactions and breeding systems for the majority of these species. Until recently research has focussed on predictive floral traits, including flower shape, length of corolla and corolla opening to identify likely pollinators in this genus. Field observations provide an empirical test of such predictions. This study investigated three species of Erica and compared their pollination and breeding systems and floral biology. Research, including field experiments and pollinator observations was undertaken in the Vogelgat Private Nature Reserve, Maanschynkop Nature Reserve and Boskloof farm in the vicinity of Hermanus in the Western Cape Province of South Africa. Field observations were conducted to determine what flower- visitor interactions occurred, while nectar volumes and sugar concentrations were measured to determine the value of the reward to the different visitors. Selective exclusion and breeding system experiments were carried out to determine whether these Erica species were capable of autonomous self-fertilization or whether they are pollinator dependent for breeding success. The unusual morphology of Erica lanuginosa suggested rodent-pollination. Field observations, including photographs of visits to plants in the field, the presence of pollen in scat sample and selective exclusion and breeding system experiments identified rodents, primarily Acomys subspinosus to be the primary pollinator. Long-proboscid flies of the family Nemestrinidae were found, on the basis of field observations to be responsible for pollination of the endemic Erica aristata. This was supported by with nectar volume and sugar concentration samples which are consistent with other long-proboscid fly-pollinated plant species. Selective exclusion and breeding system experiments undertaken confirmed that Erica aristata required a pollinator to set seed. Observations and breeding trials revealed bird-pollination in Erica sessiliflora. Nectar volume and sugar concentrations in Erica sessiliflora were in line with other sunbird-pollinated plant species, providing the necessary rewards for sunbirds visiting this species. Specialised pollination by single pollinators was found in all three species and results from breeding system experiments show that out-crossing is important. Further research into pollinator-flower interactions in the genus Erica is necessary, not least to understand more fully the conservation importance of specific pollinators.

South Africa has the highest species diversity of ericaceous plants belonging to the Erica genus. There are over 850 identified species in the Cape Floral Region. The Albany Centre of Endemism (ACOE) is located within this region and is a hotspot of diversity consisting of various plant genera. The success of Erica plants is ubiquitously attributed to mycorrhizal relationships they engage in with a diverse group of fungi. This symbiosis is known as the ericoid mycorrhizal (ERM) association. The overall aim of this study was to establish the diversity of root fungi associated with Erica plants using morphological, molecular and 454 pyrosequencing techniques. Six Erica species were identified using leaf and flower morphology according to taxonomic keys. The identified plants were Erica cerinthoides, Erica demissa, Erica chamissonis, Erica glumiflora, Erica caffra and Erica nemorosa. Roots from sampled plants were stained and examined microscopically to determine their mycorrhizal status. Ericoid mycorrhizal associations together with dark septate endophyte (DSE) structures and hyphae that did not form any specific structure were observed in all the roots. In addition arbuscular mycorrhizal (AM) structures in the form of vesicles were detected in E. glumiflora and E. cerinthoides. In order to identify the culturable fungi associated with the respective hosts, sterilised roots were placed on various culture media for cultivation. Thereafter isolated fungi were morphologically classified into 67 morphotypes. These were mostly sterile and darkly pigmented. Non-sporulating mycelia of variable colouration such as white, cream-yellowish, beige, green and brown were also observed. Further identification was carried out using molecular techniques. DNA was extracted separately from pure cultures and amplified using ITS1 and ITS4 primers in a polymerase chain reaction (PCR). Thereafter sequencing and Basic Local Alignment Search Tool (BLAST) were used to identify the isolates to generic level. The fungi were taxonomically classified into 54 operational taxonomic units and 94 percent were Ascomycetes and Helotiales was the dominant order. Unclassified Helotiales with affinities to fungi currently identified as Epacrid root fungus was common in all hosts. Other isolates that were identified included Oidiodendron, Meliniomyces, Phialocephala, Cadophora, Lachnum, Leohumicola Cryptosporiopsis, Chaetomium, Acremonium and Epicoccum species. Basidiomycetes were represented by two OTUs belonging to the genus Mycena. Four OTUs comprised fungi that had no significant alignments in the reference databases. Direct root DNA extraction together with 454 pyrosequencing was used to detect the diversity of culturable and unculturable fungi associated with the identified hosts. The ITS2 region was targeted for sequencing. Although Ascomycetes remained the dominant phyla, Basidiomycetes were also detected in all host plants. Glomeromycota was present in E. caffra and E. cerinthoides. Helotiales was dominant in all Erica plants with the exception of E. cerinthoides and E. chamissonis which were dominated by the order Chaetothyriales. The OTUs identified to genus level included Epacris pulchella root fungus, Oidiodendron cf. maius, Acremonium implicatum, Leohumicola, Lachnum, Capronia and Mycena species. Culture-based techniques and pyrosequencing detected similar fungal composition comprising Ascomycetes, while, pyrosequencing was able to detect Glomeromycetes and Basidiomycetes.

Thesis (MSc)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: Erica is a genus of about 860 species world wide, with 700 of these found in South Africa’s southwestern and southern Cape, making it by far the most speciose genus in the Cape Floristic Region. This poses a particular challenge in the construction of a molecular phylogeny of the genus. The choice of suitably variable gene regions is a crucial decision on which the successful phylogenetic reconstruction of this important genus is critically dependent. The aim of this project was therefore to determine which DNA regions, both chloroplast and nuclear, would be sufficiently variable to give adequate informative characters that may be useful at the species level phylogenetic reconstruction. A subset of 30 species, representing the range of morphological diversity and pollinator preference within Erica, was selected for study. For each of these species the variability in eight chloroplast regions (trnL-F, matK, trnS-G, rps12- rpl20, psbAtrnH, trnC-D, rps4-trnT and trnT-L) and the nuclear ITS region was investigated. The psbA-trnH, trnC-D, rps4-trnT and trnT-L chloroplast regions were found to be problematic to amplify and to possess too few Parsimony Informative Characters to be of use in phylogenetic reconstruction. Four of the chloroplast regions, trnS-G, trnL-F, matK and rpS12-rpL20 and the nuclear ITS region could be amplified and sequenced with success. The ITS region was found to be reasonably variable, with the chloroplast genes showing less variability. The DNA extraction method employed showed itself to be of critical importance in the success of the study. Two DNA extraction protocols, both modified from the original Doyle and Doyle (1987) method, were tested. The one included double the amount of β-mercaptoethanol and Polyvinylpyrrolidone (PVP) and the other included an extended phenol: chloroform: isoamylalcohol step. These variables, together with the effectiveness of these methods on fresh vs. silica dried plant samples, were investigated to determine which of the two would yield high quantities and qualities of DNA and result in the best method for the extraction of DNA from Erica species. / AFRIKAANSE OPSOMMING: Erica is ‘n genus van omtrent 860 spesies wêreldwyd, met 700 van hierdie spesies aanwesig in die suidwes en suid Kaap van Suid Afrika, wat dit by verre die mees spesieryke genus in die Kaapse Floristiese Streek maak. Dit stel ’n besondere uitdaging in die konstruksie van ’n molekulêre filogenie van die genus. Die keuse van geskikte variërende geen-areas is ‘n belangrike besluit waarvan die suksesvolle filogenetiese rekonstruksie van hierdie belangrike genus krities afhanklik sal wees. Die doel van hierdie projek was dus om te bepaal watter DNS areas, buide chloroplas en kern, genoegsaam varieer om voldoende informatiewe kenmerke te lewer om bruikbaar te wees in ’n spesie-vlak molekulêre rekonstruksie. ’n Subgroep van 30 spesies, wat die reeks van morfologiese diversiteit en bestuiwer voorkeure in Erica verteenwoordig, is dus vir die studie geselekteer. Vir elk van hierdie spesies is die variasie in agt chloroplast areas (trnL-F, matK, trnS-G, rps12- rpl20, psbA-trnH, trnC-D, rps4-trnT en trnT-L) en die kern ITS area ondersoek. Dit was problematies om die psbA-trnH, trnC-D, rps4-trnT en trnT-L chloroplast areas te amplifiseer, en daar is gevind dat hulle te min Parsimonie Informatiewe Kenmerke besig om bruikbaar te wees in filogenetiese rekonstruksie. Vier van die chloroplas areas, trnS-G, trnL-F, matK en rpS12-rpL20 en die kern ITS kon suksesvol geamplifiseer word en die basisvolgordes kon suksesvol bepaal word. Daar is gevind dat die ITS area redelik variërend is, terwyl chloroplas areas minder variasie getoon het. Die DNS ekstraksie metode wat gebruik is het die kritiese belang van die ekstraksie metode in die sukses van die studie bewys. Twee DNS protokolle, beide gemodifiseer van die oorspronklike Doyle en Doyle (1987) metode, is getoets. Die een het dubbel die hoeveelheid β-mercaptoetanol en Polyvinylpyrrolidone (PVP) bevat, en die het ’n uitgebruide fenol: chloroform: isoamylalkohol stap ingesluit. Hierdie veranderlikes, saam met die effektiwiteit van hierdie metodes op vars teenoor silika-gedroogde plant monsters, is ondersoek om vas te stel watter een van die twee die hoogste kwaliteit en kwantiteit DNS sou lewer en dus sal lei tot die beste DNS ekstraksie metode vir Erica spesies.