Final Report Summary - ALOE-DIVERSITY (Phylogeny and ethnobotany: Testing predictive approaches to plant drug discovery in the genus Aloe L. (Asphodelaceae))
Project context
Recent advances in the study of evolution have led to a deeper understanding of how the related plant species arranged on a phylogenetic tree share certain characteristics. These new insights hold considerable promise in the prioritisation of plant species for useful natural products from the plant kingdom and targets for conservation. Aloe vera supports one of the world's major natural products industries, and is just one of over 500 species in the genus Aloe, one of the largest and most recognisable genera of succulent plants, native to Africa, Arabia and Madagascar. Aloes are valued by people wherever they grow, particularly for folk medicine. So far, there is no explanation for why so few Aloe species are used commercially besides A. vera, nor how best to identify species for new markets being established in Africa. Sustainability is an important consideration, since many aloes are rare and threatened. Another consideration is the properties of the leaves. The major constituents of Aloe leaves are polysaccharides, associated with anti-inflammatory and wound-healing activity, and anthraquinone compounds, linked to laxative effects and antimicrobial activity. Until now, it has been difficult to identify the most promising and the most threatened species of Aloe due to the complicated taxonomy and lack of insights into their evolution. Too few species have been studied to even to forecast expected properties in closely related species. These limitations were overcome using a predictive phylogenetic approach that has considerably developed the evolutionary hypothesis for Aloe, and tested species-level patterns in their biochemical properties. The findings will have bearing on the selection of Aloe species for expanding natural products industries in Africa, and on targeting conservation efforts towards the most threatened species.
Project work and results
The main objective of ALOE-DIVERSITY was to build a comprehensive picture of evolution in Aloe and test a phylogenetic framework to identify promising utility species, based on surveys of useful biochemical features across the genus, and species of high conservation concern. The objectives were met with minor modifications to the work plan and methodology; time owing primarily to the research group moving to a new Department.
1.Evolution in Aloe: this work required field- and laboratory-based work. Olwen Grace attended the Molecular Evolution training course (Cesky Krumlov, Czech Republic, 23 January-04 February 2011) and led a collecting trip (November 2011) in Ethiopia with collaborator Sebsebe Demissew of the National Herbarium. Two collaborators from African universities working on Aloe visited Olwen Grace to be trained in molecular techniques. Plant material and DNA extracts were obtained from the field and living collections through a network of collaborators at the Royal Botanic Gardens, Kew and Royal Botanic Garden Edinburgh (UK), Natural History Museum of Oslo (Norway), University of Addis Ababa (Ethiopia), South African National Biodiversity Institute (South Africa) and University of Dar es Salaam (Tanzania). Three genomic regions in >180 species of Aloe (about a third of the genus) and relatives were sequenced. This is a five-fold increase in the number of taxa used in previous studies and places ca. 150 species in clades (some of which are new) in the phylogeny for the first time and amounts to 697 new sequences for Genbank. The data matrix is complete and final analyses to produce the dated phylogeny and a biogeographical history of Aloe are under way. The earliest lineages within Xanthorrhoeaceae subfamily Asphodeloideae have been clarified and dated for the first time using secondary calibration points derived from the fossil record. The generic circumscription of Aloe and closely related genera has troubled taxonomists for decades. The findings will underpin a new working classification which overcomes the problem of paraphyly among alooids without compromising the usefulness of the classification in practical terms. This is the first such study since the 1970s and the first to use a phylogeny to add insights into where and how the diversity of Aloe species in succulent hot spots was established. The work was presented at three international conferences. Four manuscripts are in preparation to fulfil this objective and will be submitted in the coming months, along with three popular articles.
2: Biochemical assessment of Aloe: this work involved laboratory-based bioassays and analytical methods and was based at the Faculty of Pharmaceutical Science at the University of Copenhagen. Together with Prof. Anna Jäger, Olwen Grace co-supervised a Masters student whose project provided a preliminary phylogenetic assessment of anthraquinones and antibacterial activity of leaf extracts from 42 species of Aloe. Plant material was obtained from the University of Copenhagen's botanic garden. Extracts of the (green) outer leaf mesophyll were separated using TLC and HPLC methods and compared to standardised references. Differences in the composition of anthraquinones were seen among species, although levels of the active principles were low. An antimicrobial screen against four bacteria (gram negative and gram positive) did not identify any extracts with a minimum inhibitory concentration below the acceptable 1mg/ml threshold, and further bioassays on these extracts were therefore halted. In a separate study, the inner (clear) leaf mesophyll of 31 Aloe species was partially hydrolysed and the products detected by GC-MS. Principal components analysis showed that monosaccharide composition is conservative and has a strong phylogenetic signal. Preliminary work showed that considerable method development would be necessary to run the planned bioassay to measure immunomodulatory activity of the inner leaf mesophyll, and this could not be taken further due to time constraints. Monosaccharide composition in Aloe could be particularly useful in the selection and authentication of species for new natural products markets, with further assessment of structure-activity functions and monosaccharide composition as a putative proxy for polysaccharide structure. The work resulted in one Masters dissertation and a manuscript has been submitted to Phytochemistry.
3 and 4: Phylogenetic and ethnoguided approaches to species prioritisation in Aloe and recommendations: this work involved an assessment of phylogenetic signals in ethnobotanical utility data, biochemical characteristics and conservation threat data across the genus Aloe, involving bioinformatic approaches to mapping and optimising characters in a phylogenetic framework and statistical correlation tests. Analyses are currently under way to complete this work. The robust hypothesis of evolution in Aloe (Objective 1) provided a framework for the evaluation of phylogenetic signal in various descriptive and comparative datasets gathered for representative samplings of species across the full geographical and morphological diversity of the genus, including leaf mesophyll monosaccharides (Objective 2), endemism, and conservation threat status data (IUCN and CITES) and a substantial unpublished utility dataset from Olwen Grace's PhD study, standardised to TDWG Levels 1 and 2. In a speciose, ethnobotanically important plant group such as this, the ethno-guided approach holds promise for identifying species that may be suitable for new product development. The predictive phylogenetic method, however, could offer deeper insights into the distribution of various properties and characteristics across the entire phylogeny of Aloe, allowing more targeted approach to future utility as well as conservation interventions. An invited review of the economic botany of Aloe by Olwen Grace was published in the South African Journal of Botany, and a manuscript describing the results of the analyses above is in preparation.
Recent advances in the study of evolution have led to a deeper understanding of how the related plant species arranged on a phylogenetic tree share certain characteristics. These new insights hold considerable promise in the prioritisation of plant species for useful natural products from the plant kingdom and targets for conservation. Aloe vera supports one of the world's major natural products industries, and is just one of over 500 species in the genus Aloe, one of the largest and most recognisable genera of succulent plants, native to Africa, Arabia and Madagascar. Aloes are valued by people wherever they grow, particularly for folk medicine. So far, there is no explanation for why so few Aloe species are used commercially besides A. vera, nor how best to identify species for new markets being established in Africa. Sustainability is an important consideration, since many aloes are rare and threatened. Another consideration is the properties of the leaves. The major constituents of Aloe leaves are polysaccharides, associated with anti-inflammatory and wound-healing activity, and anthraquinone compounds, linked to laxative effects and antimicrobial activity. Until now, it has been difficult to identify the most promising and the most threatened species of Aloe due to the complicated taxonomy and lack of insights into their evolution. Too few species have been studied to even to forecast expected properties in closely related species. These limitations were overcome using a predictive phylogenetic approach that has considerably developed the evolutionary hypothesis for Aloe, and tested species-level patterns in their biochemical properties. The findings will have bearing on the selection of Aloe species for expanding natural products industries in Africa, and on targeting conservation efforts towards the most threatened species.
Project work and results
The main objective of ALOE-DIVERSITY was to build a comprehensive picture of evolution in Aloe and test a phylogenetic framework to identify promising utility species, based on surveys of useful biochemical features across the genus, and species of high conservation concern. The objectives were met with minor modifications to the work plan and methodology; time owing primarily to the research group moving to a new Department.
1.Evolution in Aloe: this work required field- and laboratory-based work. Olwen Grace attended the Molecular Evolution training course (Cesky Krumlov, Czech Republic, 23 January-04 February 2011) and led a collecting trip (November 2011) in Ethiopia with collaborator Sebsebe Demissew of the National Herbarium. Two collaborators from African universities working on Aloe visited Olwen Grace to be trained in molecular techniques. Plant material and DNA extracts were obtained from the field and living collections through a network of collaborators at the Royal Botanic Gardens, Kew and Royal Botanic Garden Edinburgh (UK), Natural History Museum of Oslo (Norway), University of Addis Ababa (Ethiopia), South African National Biodiversity Institute (South Africa) and University of Dar es Salaam (Tanzania). Three genomic regions in >180 species of Aloe (about a third of the genus) and relatives were sequenced. This is a five-fold increase in the number of taxa used in previous studies and places ca. 150 species in clades (some of which are new) in the phylogeny for the first time and amounts to 697 new sequences for Genbank. The data matrix is complete and final analyses to produce the dated phylogeny and a biogeographical history of Aloe are under way. The earliest lineages within Xanthorrhoeaceae subfamily Asphodeloideae have been clarified and dated for the first time using secondary calibration points derived from the fossil record. The generic circumscription of Aloe and closely related genera has troubled taxonomists for decades. The findings will underpin a new working classification which overcomes the problem of paraphyly among alooids without compromising the usefulness of the classification in practical terms. This is the first such study since the 1970s and the first to use a phylogeny to add insights into where and how the diversity of Aloe species in succulent hot spots was established. The work was presented at three international conferences. Four manuscripts are in preparation to fulfil this objective and will be submitted in the coming months, along with three popular articles.
2: Biochemical assessment of Aloe: this work involved laboratory-based bioassays and analytical methods and was based at the Faculty of Pharmaceutical Science at the University of Copenhagen. Together with Prof. Anna Jäger, Olwen Grace co-supervised a Masters student whose project provided a preliminary phylogenetic assessment of anthraquinones and antibacterial activity of leaf extracts from 42 species of Aloe. Plant material was obtained from the University of Copenhagen's botanic garden. Extracts of the (green) outer leaf mesophyll were separated using TLC and HPLC methods and compared to standardised references. Differences in the composition of anthraquinones were seen among species, although levels of the active principles were low. An antimicrobial screen against four bacteria (gram negative and gram positive) did not identify any extracts with a minimum inhibitory concentration below the acceptable 1mg/ml threshold, and further bioassays on these extracts were therefore halted. In a separate study, the inner (clear) leaf mesophyll of 31 Aloe species was partially hydrolysed and the products detected by GC-MS. Principal components analysis showed that monosaccharide composition is conservative and has a strong phylogenetic signal. Preliminary work showed that considerable method development would be necessary to run the planned bioassay to measure immunomodulatory activity of the inner leaf mesophyll, and this could not be taken further due to time constraints. Monosaccharide composition in Aloe could be particularly useful in the selection and authentication of species for new natural products markets, with further assessment of structure-activity functions and monosaccharide composition as a putative proxy for polysaccharide structure. The work resulted in one Masters dissertation and a manuscript has been submitted to Phytochemistry.
3 and 4: Phylogenetic and ethnoguided approaches to species prioritisation in Aloe and recommendations: this work involved an assessment of phylogenetic signals in ethnobotanical utility data, biochemical characteristics and conservation threat data across the genus Aloe, involving bioinformatic approaches to mapping and optimising characters in a phylogenetic framework and statistical correlation tests. Analyses are currently under way to complete this work. The robust hypothesis of evolution in Aloe (Objective 1) provided a framework for the evaluation of phylogenetic signal in various descriptive and comparative datasets gathered for representative samplings of species across the full geographical and morphological diversity of the genus, including leaf mesophyll monosaccharides (Objective 2), endemism, and conservation threat status data (IUCN and CITES) and a substantial unpublished utility dataset from Olwen Grace's PhD study, standardised to TDWG Levels 1 and 2. In a speciose, ethnobotanically important plant group such as this, the ethno-guided approach holds promise for identifying species that may be suitable for new product development. The predictive phylogenetic method, however, could offer deeper insights into the distribution of various properties and characteristics across the entire phylogeny of Aloe, allowing more targeted approach to future utility as well as conservation interventions. An invited review of the economic botany of Aloe by Olwen Grace was published in the South African Journal of Botany, and a manuscript describing the results of the analyses above is in preparation.