The CSIRO Collection of Living Microalgae: An Australian Perspective on Microalgal Biodiversity and Applications S.I. Blackburn, I.D. Jameson, D. Frampton, M. Brown, M. Mansour, A. Negri, N.S. Parker, S. Robert, C.J. Bolch, P.D. Nichols and J.K. Volkman CSIRO Marine Research, Hobart, Tasmania, Australia [email protected] [email protected] ABSTRACT The CSIRO Collection of Living Microalgae maintains over 800 strains from 140 genera representing the majority of marine and some freshwater microalgal classes. The Collection is incorpoarted within the CSIRO Microalgal Research Centre (CMARC) which provides a supply service of microalgal strains to industry, government and university organizations. Research within CMARC and in partnership with collaborators spans a wide base within the three themes of Environment, Aquaculture and Biotechnology. Some of the research projects undertaken include physiological studies of different life-history stages of microalgae, toxin production in harmful algal bloom (HAB) species, phylogenetic studies of different populations of HAB species, optimizing the nutritional benefit of microalgal diets in larval and broodstock aquaculture species, including important nutrients such as vitamins and polyunstaurated fatty acids. Research is also being undertaken into optimizing high biomass production systems through the use of photobioreactors. 1. THE CSIRO COLLECTION OF LIVING MICROALGAE The CSIRO Collection of Living Microalgae maintains over 800 strains from 140 genera representing the majority of marine and some freshwater microalgal classes (see Fig.1 for summary, list of strains available from the authors or downloadable from http://www.marine.csiro.au). There are also some selected micro-heterotrophic strains. This collection is the largest and most diverse microalgal culture collection in Australia and, with NIES-Collection (National Institute for Environmental Studies, Environment Agency) in Japan, ranks as a major microalgal collection in the the Asia-Pacific region. 1-1. Development of the CSIRO Collection of Living Microalgae The collection originated in the 1960s from fundamental research into the pigment composition of marine microalgae, a theme which has continued through to the present and which has culminated in the incorporation of microalgal pigments as essential oceanographic reference standards (Jeffrey et al.1997). The aquisition of microalgal strains has focused on isolates from Australian waters - over 80% of strains have been isolated from diverse localities and climatic zones, from tropical northern Australia to the Australian Antarctic Territory, from oceanic, inshore coastal, estuarine, intertidal and freshwater environments (Fig. 2). Additionally, emphasis has also been placed on representation of 39 different populations of a single species, usually by more than one strain. This is particularly the case for harmful bloom-forming species where there are up to 20 strains of a single species from a single locality. 250 40 strains N= 803 35 200 genera N=139 30 150 25 20 100 15 10 50 5 number of strains per Class number of genera per Class 0 0 Dinophyceae Cyanophyceae Cryptophyceae Chlorophyceae Rhodophyceae Chrysophyceae Xanthophyceae Prasinophyceae Euglenophyceae Raphidophyceae Bacillariophyceae Dictyochophyceae Prymnesiophyceae Eustigmatophyceae Fig. 1. Number of strains and genera in different algal classes held in the CSIRO collection of living microalgae. 250 Australian Overseas 200 150 100 Number of strains 50 0 Other Dinophyceae Cyanophyceae Cryptophyceae Rhodophyceae Chlorophyceae Chrysophyceae Prasinophyceae Euglenophyceae Raphidophyceae Bacillariophyceae Dictyochophyceae Prymnesiophyceae Eustigmatophyceae Fig. 2. Number of strains of Australian and non-Australian origin in different classes held in the CSIRO collection of living microalage. 40 All strains in the culture collection are unialgal and the majority are clonal. A subset of strains, including particular strains used by the aquaculture industry and some used for genetic and toxin profiles, are axenic. Since the mid 1980s research has focused on the rapidly developing aquaculture industry in Australia and key environmental issues such as harmful and toxic algal blooms. Since 1995 the potential for biotechnological development of bioactive and novel compounds, particularly lipids, from microalgae has been explored. In parallel, the issue of efficient, high production of quality-controlled microalgae using photobioreactors has been a research focus. Such systems will allow the full potential of microalgae for biotechnology and aquaculture applications to be realized. 1-2. Genetic Diversity Microalgae are known for their cosmopolitanism at the morphological species level, with very low endemicity being shown (Norton et al.1996). However this morphological cosmopolitanism can hide a plethora of diversity at the intra-specific level. There have been a number of studies of genetic diversity on different microalgae using various approaches such as interbreeding, isozymes, growth rates and a range of molecular genetic techniques. The diversity identified by these studies ranges from large regional and global scales (Chinain et al.,1997) to between and within populations at small scales (Gallagher, 1980; Medlin et al.,1996; Bolch et al.,1999a,b). Variation at the intra-specific level, between morphologically indistinguishable microalgae, can usually only be identified using strains isolated from the natural environment and cultured in the laboratory. For selected microalgae we have demonstrated the distinctness of Australian populations compared with populations from outside Australia. The dinoflagellate Gymnodinium catenatum has formed recurrent toxic blooms in south-eastern Tasmanian waters since 1986. Cargo vessel ballast water from Japan or Spain has been proposed as one likely vector of introduction. Using both interbreeding and molecular approaches we have shown considerable within-population variation in G. catenatum as well as a complex multi-group mating system. The interbreeding data showed a slightly closer relationship between Tasmanian and Spanish populations than between Tasmanian and Japanese populations (Blackburn et al.2001), whereas multidimensional scaling analysis (MDS) of Randomly Amplified Polymorphic DNA (RAPD) indicated that Australian strains were almost equally related to both the Spanish/Portuguese population and the Japanese population (Bolch et al.1999b). Analysis of molecular variance (AMOVA) found that genetic variation was partitioned mainly within the Tasmanian populations (87%) compared to the variation between the regions (8%) and between populations within regions (5%). The potential source population for Tasmania’s introduced G. catenatum remains equivocal; however, strains from the more recently discovered mainland Australian population (Port Lincoln, South Australia, 1996) clustered with Tasmanian strains, supporting the notion of a secondary relocation of Tasmanian G. catenatum populations to the mainland via a shipping vector. Geographic and temporal clustering of strains was evident among the Tasmanian strains, indicating that genetic exchange between neighboring estuaries is limited and that Tasmanian G. catenatum blooms are composed of localized, estuary-bound subpopulations (Fig. 3). Another phylogenetic study of the toxic cyanobacterial bloom-forming species Nodularia 41 spumigena highlighted some similar findings. Australian populations of N. spumigena were distinctly different from Nodularia strains from Canada and Europe including the proposed type strain for the species. Within Nodularia strains, genetic variation could be resolved at several levels, from intra-generic through to intra-population levels, clearly defining generic, species and population limits of genetic variation in these clonal organisms (Bolch et al.1999a). Unlike G. catenatum, within population heterogeneity was not high in Australian N. spumigena. This may reflect a difference in genetic complexity and life history between these eutcaryotic and prokaryotic microalgae. Culture collections maintain living and characterised biodiversity for research and comparison with new strain acquisitions as well as with natural biodiversity. It is essential to have identifiable and stable genotypes. While there are recorded instances of change or loss of particular characteristics in long term culture (Coleman, 1977), in general, culturing guarantees genetic continuity and stability of a particular strain. We are now developing cryopreservation strategies to limit the potential for genetic drift, beginning with our cyanobacterial subset of strains. Cryopreservation is also attractive because it decreases the demanding task of regular subculturing. 1 HU JP TRA DE '93 ) 3 0 ( PT M I D DE '87 SP PTL02 PTL01-4 -1 -2 -1012 DIM (1) Fig. 3. Three-dimensional MDS analysis of RAPD data from G. catenatum strains, not including the two outgroup species. Kruskal stress = 0.193. (a) Plot of first and second dimension of the three-dimensional MDS analysis of G. catenatum strains. Region/population clusters (bounded by solid line): Huon Estuary and Hastings Bay (HU/HA), Derwent Estuary 1987 (DE”87) and 1993 (DE’93), Triabunna (TRA), and Japan, Spain and Portugal (JP/SP/PT). Port Lincoln, South Australia strains (PTL) marked by strain number. (b) Plot of the first and third dimension of the three-dimensional MDS analysis of G. catenatum strains. G. catenatum clusters bounded by solid or shaded lines: Huon Estuary