Biological Sciences

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DNA barcoding of KwaZulu-Natal afromontane forest parmelia (parmeliaceae) species: a molecular approach to accurate specimen identification and sensitivity to climate change.
(2018) Ndhlovu, Nqobile Truelove.; Willows-Munro, Sandi.; Beckett, Richard Peter.
Accurate species identification is challenging, especially in groups with subtle taxonomically diagnostic characters such as lichens. Molecular-based techniques have shown to be a valuable tool for accurate specimen identification in fungi, in particular the use of DNA barcoding has become popular. Specifically, the internal transcriber spacer (ITS) region has been shown to successfully discriminate a broad range of fungal species. In this study, the utility of the ITS DNA barcode for use as a species diagnostic tool in the cosmopolitan lichen-forming fungus, Parmelia (Parmeliaceae) was investigated. Sixty-eight ITS sequences were generated from specimens collected from five sites around the province of KwaZulu-Natal and analysed. Phylogenetic analysis indicated that unlike European Parmelia species that form strong monophyletic clades, what appeared to be morphologically very similar Parmelia species in KwaZulu-Natal are paraphyletic or polyphyletic. No barcode gap was detected between the intra and interspecific distances. This suggests that the taxonomy of Parmelia lichens in South Africa needs to be thoroughly revised. The molecular data presented in this study provides evidence of previously hidden species-level diversity in Parmelia and as such contributes to the knowledge and understanding of the biodiversity of lichenized fungi in South Africa. The thermotolerance of Parmelia collected from different sites along an altitudinal gradient around Kwa-Zulu Natal was invesigated Chlorophyll fluorescence was used to assess the performance of lichen photobionts following stress, while ion leakage that of the mycobiont. For heat tolerance, results suggested that tolerance was correlated with the climatic conditions in which the lichens grow. Material from the coastal site of Hawaan were more heat tolerant than that from the three Midlands sites. Counter to our expectations, the coastal collections were more cold tolerant than those from the other sites. However, the genus clearly contains genetic variation with respect to stress tolerance, suggesting that it may have the potential to adapt to climate change.
Effects of high light intensity and desiccation stress on moss species.
(2021) Mbatha, Sikhethile.; Beckett, Richard Peter.
Bryophytes are desiccant tolerant non-vascular plants, capable of growing and surviving in extreme conditions. They are divided into three groups: liverworts, hornworts, and mosses. Most mosses grow in shady and moist environments, although some formpart of arid soil crusts where they protect soil from erosion. The moss flora of the Afromontane vegetation around Pietermaritzburg is dominated by two acrocarpous mosses, Atrichum androgynum, and Dicranella subsubulata. A. androgynum tends to grow in wetter, more shaded habitats and is a rather delicate species, while D. subsubulata grow in open, drier habitats and is more robust. Rarely, the species grow together, for example at the transition of a shaded indigenous woodland to plantations. We hypothesized that the more robust species has higher stress tolerance and has largely constitutive stress tolerance mechanisms. By contrast, we hypothesized that the more delicate species is less tolerant and may have inducible tolerance mechanisms. In the present study, desiccation tolerance and tolerance to high light stress were investigated in A. androgynum and D. subsubulata. Results confirmed that D. subsubulata was more tolerant of high light stress than A. androgynum. Exposure to moderate light intensities did not increase tolerance to subsequent high light stress in either species. Similarly, D. subsubulata was more desiccation tolerant than A. androgynum. Not consistent with our original hypothesis, mild desiccation, and treatment ABA-induced tolerance to desiccation in both species. Furthermore, detailed studies of the antioxidant enzyme peroxidase showed that enzyme activity was induced during slow drying in both D. subsubulata than A. androgynum. It appears that inducible tolerance mechanisms are present in both species. The work presented here represents a contribution to the autecology of two important mosses in the KwaZulu-Natal midlands Afromontane vegetation.
Lichen photobiology in relation to climate change : protection in peltigeralean lichens against excess ultraviolet (UV) radiation using induced melanins and the effects of UV on melanin synthesizing enzymes.
(2015) Matee, Lusanda Patrick Herbert.; Beckett, Richard Peter.
Depletion of the stratospheric ozone has allowed more solar ultraviolet (UV) radiation (100-400 nm) to reach the lower atmosphere. Despite the success of the continued implementation of Montreal protocol of 1987 and its amendments aimed at the protection of the ozone layer, it is possible that UV will remain elevated in some regions due to decreasing removal by clouds and aerosols. Strong interactions between ozone depletion and changes in climate induced by increasing greenhouse gases could result in increased UV radiation levels reaching the surface. Increased exposure to UV-B can cause significant damage to organisms including protein damage, inhibition of photosynthesis, formation of high energy reactive oxygen species (ROS), disruption of nitrogen fixation and induction of cell mutation and inhibition of growth. Lichens are composite organisms that arise from a symbiotic association between fungi and photoautotrophic algal partners, which may be microalgae and/or cyanobacteria. They have developed several protective strategies to protect themselves from excessive radiation. Lichens such as Cladonia rangiferina, Lobaria pulmonaria and Umbilicaria rossica are known to synthesize melanins in response to elevated levels of UV-B radiation. Synthesis of melanins is generally catalysed by enzymes that are involved in phenol coupling including P450 monooxygenases, ascorbate oxidases, peroxidases, laccases and tyrosinases (Nezbedová et al. 2001). The work presented in this thesis investigated the enzymes involved in melanin biosynthesis in lichens from suborder Peltigerineae. We present a detailed characterization of redox enzymes from Lobaria pulmonaria and Pseudocyphellaria aurata with a special focus on tyrosinases. Furthermore, an evaluation of the response of L. pulmonaria to UV radiation was done. The role of the redox enzymes tyrosinases, laccases and peroxidases in melanin synthesis is elucidated by exposing L. pulmonaria thalli to various combinations of UV and PAR. Laboratory experiments clearly indicated that L. pulmonaria and P. aurata have redox enzymes that are of similar nature to those of their free-living symbionts and other lichens. Tyrosinase from L. pulmonaria oxidized monophenolic compounds and was able to metabolize L-DOPA, thus showing characteristics of a typical fungal tyrosinase. L-DOPA can also be metabolized by peroxidases and laccase suggesting that peroxidases and laccases may participate in melanisation reactions. A field experiment showed that exposing shade-adapted L. pulmonaria to solar radiation in the field induces DOPA melanin synthesis. The synthesis of the brown cortical compounds occurred when lichen thalli were exposed to direct sunlight or placed under a wavelength neutral filter that marginally reduced overall light. Melanin synthesis was accompanied by an increase in laccase activity in lichens that were exposed to natural sunlight. By contrast, no changes in enzyme activity occurred in lichen thalli placed under the wavelength neutral filter. Placing lichen thalli under filters that removed both UV-A and UV-B prevent melanisation and was accompanied by an increase in tyrosinase activity. Filtering out UV-B had no effect on enzyme activity but slight browning occurred. Results showed that laccases could be involved in melanin biosynthesis under some conditions, but provided no evidence for a role of tyrosinases in melanisation. However, it could be that melanisation requires tyrosinase, but rates of melanisation are controlled by the levels of melanin precursors such as tyrosine. No differences in maximal PSII efficiency and chlorophyll contents occurred between the radiation treatments indicating that no deterioration of photosynthetically apparatus occurred and the photobionts remained relatively healthy even when exposed to UV. Taken together these results suggest that further research in needed into determining the levels of melanin precursors, the roles of lichen redox enzymes in melanin synthesis and whether a transcriptional activation of these redox enzymes by UV radiation in lichens exists or not. Furthermore, additional roles for tyrosinase need to be investigated, for example defence against pathogens.
Melanisation of lichens : the composition of melanin and the role of ultraviolet light (uv) in peltigeralean and non-peltigeralean lichens.
(2017) Mafole, Tshepiso.; Beckett, Richard Peter.
Lichens are unique organisms widely known for their ability to tolerate extreme environmental conditions due to the symbiotic relationship between a fungus and algae or cyanobacteria. This includes the ability to synthesise melanins to protect themselves from ultraviolet radiation and high light. Melanins are found in different forms, the eumelanins (DOPA), often synthesised by Peltigeralean lichens while the non-Peltigeralean produce dark pigments that appear not to be DOPA melanins. Increased levels of UV and high light affect the physiology of many organisms, as a result this study investigates the effects of the photoprotective pigment, melanin on the photosynthetic apparatus of both chlorophycean and cyanobacterial bionts. The first aim of this thesis was to study the effect of using different light regimes to induce melanins. The second aim was to compare the properties of melanin between different lichens with those from free-living fungi. Lastly, the effect of melanisation on the photosynthesis was investigated. Results presented here suggest that melanins are insoluble in organic solvents, except DMSO and strongly absorb in the UVB and UVA wavelengths. The induction of melanin was slow and was better induced beneath the screens that transmitted UV. Transplanting nonmelanised Lobaria pulmonaria, to an open site for four weeks induced melanic pigments. Melanised thalli had normal chlorophyll contents and normal maximum rates of photosynthesis. Chlorophyll fluorescence analysis showed that the maximum quantum yield and relative electron transfer rates were similar to those of nonmelanised thalli. However, at light levels lower than 100 μmol photons m-2 s-1 melanisation of the upper cortex of the lichen reduced rates of CO2 fixation by more than 40%. Melanic thalli also had a higher chlorophyll a/b ratio and more xanthophyll cycle pigments. In Lobaria retigera, the +UV screen decreased the photosynthetic rate more than other light treatments. Photoinhibition of wet thalli was rapid compared to dry, though complete recovery was reached after a day. From these results, it can be confirmed that melanisation has a protective action against high light as melanised thalli were more tolerant to excess light. While protecting photobionts from high light, melanisation reduced photosynthetic efficiency and protects lichens from photoinhibition in both the wet and dry states.
Production of hydroxyl radicals by lichens via extracellular hydroquinone-driven redox cycling.
(2016) Moyo, Calvin Eddington.; Beckett, Richard Peter.
Abstract available in PDF file.
The role of secondary metabolites in protecting lichens from climate change induced stress.
(2022) Ndhlovu, Nqobile Truelove.; Beckett, Richard Peter.
Lichen secondary metabolites play a great diversity of roles in lichen biology by acting as UV screens, antimicrobials, herbivore deterrents or allelopathic compounds. The main aim of the work described in this thesis was to test the potential roles of secondary metabolites in the tolerance of lichens (twelve species) to a variety of abiotic stresses. The first stress considered was that of tolerance to high levels of photosynthetically active radiation (PAR). Photosynthetic organisms possess a great diversity of mechanisms to protect themselves from the potentially stressful effects of high PAR. In addition to non-photochemical quenching and antioxidant mechanisms, it has been suggested that lichens use secondary metabolites for photoprotection. A few studies have shown that even faintly pigmented or unpigmented lichen substances can reduce photoinhibition. Here, the acetone rinsing technique was used to harmlessly remove secondary metabolites from all lichen thalli and then the chlorophyll fluorescence technique was used to measure their tolerance to photoinhibition in desiccated and hydrated states. Results showed that colorless lichen substances can increase the tolerance of lichen photobionts to photoinhibition when thalli are hydrated, apparently by increasing reflectance. Interestingly, substances can also photoprotect lichens in the dry state, while having no effect on reflectance. The acetone rinsing technique was also used to compare the relative importance of lichen substances in photoprotection in sun and shade collections of four species of Afromontane lichens. Results showed that lichens collected from sunny microhabitats have higher tolerance to photoinhibition than those from shaded locations. Furthermore, removal of lichen substances increases sensitivity to photoinhibition much more in sun than shade collections. Results further emphasized the importance of lichen secondary metabolites in photoprotection. This study also considered whether melanins, a pigmented secondary metabolite can cause problems with using fluorimetry techniques for e.g., to measure NPQ. A dissecting technique was used to remove the lower cortices and medullas of two lichen species so that NPQ could be measured from the underside of the thallus with an imaging PAM. Results confirmed that NPQ can be satisfactorily assessed with a standard fluorimeter by taking measurement from above using intact thalli. However, interestingly, photobionts from the bottom of the photobiont layer tended to have slightly lower rates of photosynthetic activity and lower NPQ than those at the top, i.e., display mild “shade” characteristics. The results presented in the final chapter looked at the protective role of secondary metabolites against desiccation-induced stress. It was hypothesized that secondary metabolites may act as antioxidants that protect lichens from desiccation-induced stress. Unfortunately, the findings of this part of the work did not present a consistent story, but rather indicated that according to species, the secondary metabolites can act as antioxidants or prooxidants. Nevertheless, taken together, the work presented in this thesis clearly shows that lichen secondary metabolites, whether pigmented or unpigmented, play important roles in photobiont photoprotection.
Roles of ROS scavenging enzymes and ABA in desiccation tolerance in ferns.
(2018) Mkhize, Kwanele Goodman Wandile.; Beckett, Richard Peter.
The fern species Loxogramme abyssinica and Crepidomanes inopinatum occur widely in KwaZulu Natal Afromontane forests. Although both species are suspected to be desiccation tolerant (DT), little is known about their mechanisms of tolerance. The present study was undertaken to characterize DT in both ferns, specifically the roles of the reactive oxygen species (ROS) scavenging enzymes superoxide dismutase (SOD) and peroxidase (POX), sugar afccumulation, and abscisic acid. Broadly speaking, DT mechanisms are either constitutive (always present) or induced in response to stress. C. inopinatum dries rapidly because it is a “filmy” fern, lacking a cuticle; in contrast L. abyssinica possesses a cuticle, and therefore dries slowly. It was predicted that the fast-drying C. inopinatum would rely mainly on constitutive mechanisms, while the slow-drying L. abyssinica would depend on inducible mechanisms. Plant were collected from the field, transported to the laboratory and then subjected to desiccation. Two methods of desiccation were used. The first one was a relatively mild, slow and short term (48 h) desiccation over calcium acetate, and the second was a harsher, rapid and long term (1 week) desiccation over silica gel. Measurement of chlorophyll fluorescence parameters showed that both species displayed rapid recovery during rehydration after slow or fast desiccation, confirming that both species are genuinely poikilohydric. POX activity remained constant in both species during slow desiccation and subsequent rehydration, suggesting that ROS scavenging by POX is a constitutive DT mechanism. However, the absolute POX activity of C. inopinatum was much higher than in L. abyssinica. Rapid long term desiccation reduced POX activity in both species, but the activity recovered during rehydration. In both species, slow desiccation increased SOD activity, and activity declined to original values during rehydration. Slow and fast drying increased the concentrations of soluble sugars in both species, and concentrations rapidly declined to initial values during rehydration. ABA pretreatment had little effect on DT in either species, although tolerance was slightly increased in L. abyssinica. Results of this study suggested that both species depend largely on inducible DT mechanisms. Counter to the original hypothesis, inducible mechanisms occur even in filmy ferns that desiccate rapidly. The results from this project will contribute to our understanding of how ferns can survive in stressful environments in South Africa, and potentially could help improve abiotic stress tolerance in crop plants.

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Browsing Biological Sciences by Author "Beckett, Richard Peter."