The present study aimed to obtain a better understanding of the genotypic and phenotypic variation in the astaxanthin producing green algae Haematococcus pluvialis. More specifically, we wanted to find out 1) whether H. pluvialis is a single species or comprises multiple species by analyzing variation patterns in molecular markers, morphological characteristics and ecophysiological preferences, 2) to quantify the genetically based component of phenotypic variation at the inter- and intraspecific level in Haematococcus with a focus on traits related to astaxanthin productivity, 3) to obtain a first view on how responses of different strains belonging to different genotypes may diverge with environmental changes, specifically with varying irradiance 4) to better understand the host specificity of P. sedebokerense, a common pathogen of H. pluvialis.
The first part of this thesis was designed to generate a phylogenetic framework for further comparative studies.
In Chapter 3, genetic, morphological and ecophysiological patterns were studied in European Haematococcus strains isolated from 15 locations across Europe to assess potential species level variation. For comparison, the European strains collected were complemented with South American isolates and public culture collection strains. Genetic differentiation between strains was investigated using the complete ITS1 rDNA region (ITS1-5.8S-ITS2) and for a subset of strains the rbcL2 chloroplast gene. Also the presence of CBCs3 in ITS1 and ITS2 secondary structures was evaluated. Six lineages could be resolved in the ITS rDNA phylogeny - supported by both GMYC4 and statistical parsimony network analysis5 - of which three contained all the newly collected European strains and most of the culture collection strains. The rbcL alignment showed less variation than the ITS, yet resolving two out of the three ITS lineages to which our European strains belonged. Up to three CBC’s were found between each of the three European lineages in the ITS1 secondary structure, while one CBC was found between lineages in the ITS2 secondary structure. Combined, these results allowed the identification of three species of Haematococcus: an epitype for H. pluvialis was proposed and two new species were described: H. rubicundus and H. rubens. DGGE6 was performed as a tool to distinguish the three species from each other, without the need to sequence. The presence of three additional lineages within Haematococcus suggest that further cryptic species diversity exists and remains to be explored.
For a subset of strains belonging to the three described species, the further congruence with morphology and temperature preferences was determined.
Although showing a high degree of intraspecific variation and considerable overlap, significant interlineage differences in morphology and growth rate were found, supporting our species boundaries. H. pluvialis and H. rubens had on average more elongated cells, more noticeable cytoplasmic strands and pear shaped protoplasts. Optimal temperatures for growth were similar for H. pluvialis, H. rubicundus and H. rubens, varying between 17 and 23 °C, yet H. pluvialis had on average a lower maximal growth rate than H. rubicundus and H. rubens.
The second part of this thesis aimed at exploring the phenotypic diversity in Haematococcus in relation to its capacity to produce biomass and astaxanthin. This was done through the study of differences in physiological responses and the intraspecific variation between strains.
In Chapter 4, the genetically based component of phenotypic variation was quantified in a common garden experiment, where comparisons of phenotypic traits of genetically distinct strains were made under strict identical environmental conditions. Specifically, using this approach under growth conditions (pre-stress) and under astaxanthin inducing conditions (post-stress), we examined genetically based intra- and interspecific (co-) variation in six traits related to astaxanthin productivity using a total of 30 strains belonging to H. pluvialis and H. rubicundus. Besides including 24 newly isolated strains, six H. pluvialis strains from culture collections were examined. A significant reservoir of intraspecific variation was found for all six traits both pre- and post-stress, characterized by high broad sense heritability estimates7, reinforcing the genetic determinism of this variation. Next, overlap between species was found in the six traits. Yet despite the overlap, species differences were found in five out of six traits. As much as fifteen fold variation in astaxanthin productivity was found between the poorest and the best performing strain. Strikingly, strains from culture collection had a lower astaxanthin productivity compared to natural isolates of H. pluvialis, possibly reflecting loss of photo protective capacity, during their long term maintenance. The variation in total astaxanthin productivity largely co-varied with post stress traits, rather than pre-stress traits, focus for future astaxanthin productivity improvement
In Chapter 5, we focused on the ecophysiological responses of different strains to irradiance during the growth phase. The responses of six different Haematococcus strains, belonging to two species (H. pluvialis and H. rubicundus) were assessed when exposed to a light gradient comprised of nine different irradiances (2, 7, 15, 30, 52, 79, 114, 154 and 222 μmol photons m-2 s-1). Systematic comparisons of batch grown stationary cultures of the six strains were made in terms of cell type, cell size, mortality, growth rate, cell density, photosynthetic activity as well as quantitative and qualitative pigment composition. Significant strain differences were found for all traits with few exceptions, namely growth rate and cell density. Significant irradiance effects were found for all traits except cell biovolume. Moreover, irradiance x strain, or G x E interaction8 effects were found for cell mortality, Fv/Fm9 ratio, cell density as well as for pigment composition implying that is was not possible to denote a single optimum irradiance resulting in increased growth and biomass accumulation valid for all Haematococcus.
The final part of this thesis aimed at utilizing phenotypic variation in the search for resistant strains amongst Haematococcus.
Chapter 6, focused on the relation between the highly threatening pathogen P. sedebokerense and Haematococcus, through quantitative phenotyping to identify resistant Haematococcus strains. Specifically, the host specificity of P. sedebokerense (strain PS1) was examined on 44 Haematococcus strains in a laboratory controlled infectivity assay, where growth and photosynthetic activity was measured in presence and absence of PS1. Moreover, at the end of the trial, the presence of PS1 in infected cultures was quantified through a novel method developed for this purpose, by measuring the fluorescence intensity after staining with fluorescein labeled WGA10, specific to P. sedebokerense. Altogether the measurements resulted in three infectivity proxies allowing comparative studies across strains. All 44 Haematococcus strains differed significantly in susceptibility to infection for all three infectivity proxies. Differences were was not related to phylogenetic background nor strain sampling origin. Half of the strains exhibiting potential low susceptibility to PS1, possessed cells in flagellated state, unlike the remaining which were palmelloid and aplanospore dominated. Correspondingly, we showed that vulnerability to PS1 of a highly susceptible H. pluvialis strain was decreased in a long term selection experiment through the dominance of flagellated phenotypes over several generations of infection. Our results demonstrated that strain resistance may be genetically determined and that the morphological flagellated state among others, may provide protection.
In conclusion, the results of this thesis contribute to the understanding of genetic, morphological and physiological diversity in Haematococcus strains both at the intraspecific as at the interspecific level. This work contributes to three of the main pillars for microalgae development (genetic diversity, environmental adaptation, phenotypic characterization) embodied within the applied pipeline, where the ultimate goal is to obtain high yielding strains with wide adaptability and high resilience to pathogens