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The polysaccharide hydration phenomenon is nowadays the subject of intense research. The interaction of native and modified polysaccharides and polysaccharides-based bioconjugates with water has an important influence on their functional behaviour. Notwithstanding that the hydration phenomenon has been studied for decades, there is still a lack of awareness about the influence of hydration water on the polysaccharide´s structure and consequences for industrial or medicinal applications. The hydration of polysaccharides is often described by the existence of water layers differing in their physical properties depending on the distance from the polysaccharide. Using the differential scanning calorimetry (DSC) such water layers were categorized according their properties upon cooling in hyaluronan (HYA, sodium salt of ß-1,4-linked units of ß-1,3-linked D-glucuronic acid and N-acetyl-D-glucosamine), a model polysaccharide in the present work. The amount of non-freezing water, i.e. water in close proximity of HYA chain which does not freeze et all, was determined around 0.74gH2O/gHYA for HYA with molecular weight from 100 to 740kDa and 0.84gH2O/gHYA for molecular weight of 1390kDa. The amount of freezing-bound water, the water pool which is affected by presence of HYA but freezes, was determined in the range from 0.74 to 2gH2O/gHYA. Above this value only non-freezing and bulk water are present since melting enthalpy measured above this concentration reached the same value as for pure water. Since this approach suffers from several experimental problems, a new approach, based on the evaporation enthalpy determination, was suggested. The analysis of the evaporation enthalpies revealed an additional process associated with apparent energy release taking part below the water content of 0.34gH2O/gHYA. Existence of this phenomenon was observed also for protonated form of HYA. The existence of energy compensating process was confirmed with the Kissinger-Akahira-Sunose method which allowed determination of actual water evaporation/desorption enthalpies in all stages of the evaporation process. In fact, the apparent evaporation enthalpy value increased until water content of 0.34gH2O/gHYA, and then dropped down to lower values which were, still higher than the value of the pure water evaporation enthalpy. By the use of time domain nuclear magnetic resonance (TD-NMR) technique it was revealed that this phenomenon is the plasticisation of HYA.
Further, it was revealed that the non-freezing water determined by the use of DSC consists of two water fractions, i.e. 15% of water structurally integrated, interacting directly with polar sites, and 85% of water structurally restricted, embedded in-between the HYA chains. The occurrence of plasticisation concentration close to equilibrium moisture content provided the possibility to influence the HYA physical structure during the drying. In this way three samples of native HYA, dried under various conditions were prepared and their physical properties were analyzed. The samples differed in kinetics of rehydration, plasticisation concentration, glass transitions, and morphology. The properties of water pool were studied in solutions of 10"25mg HYA/mL as well. The fast filed cycling (FFC) NMR relaxometry showed the existence of three water fractions which correlation times spanned from 10"6 to 10"10 seconds, progressively decreasing in dependency on its distance from HYA chain. The formation of a weak and transient intramolecular water bridge between HYA chains was observed. It was shown that, unlike the inorganic electrolytes, polyelectrolytes hydration is a dynamic process which reflects not only the technique used for the analysis, experimental conditions but also the conformation of the polysaccharide and its "thermal" and "hydration" history.
It was demonstrated that some native polysaccharide structures can be easily modified by manipulation of preparation conditions, giving fractions with specific physicochemical properties without necessity of any chemical modification.
In this thesis, I present the results of my studies on taxonomy, systematics, and biogeography of Impatiens (Balsaminaceae) in Madagascar and the Comoro islands.
In Chapter 1 I reviewed the literature on taxonomy and classification of Balsaminaceae, on habitat, world distribution, morphology, molecular phylogenetics and infrageneric classification of the genus Impatiens. In Chapters 2-15 (Fischer & Rahelivololona 2002, 2003, 2004, 2007, 2015a, 2015b, 2015c, 2016, Fischer et al. 2004. 2017, 2018a, b submitted, Rahelivololona et al. 2003) I presented the first results of a revision of Balsaminaceae of Madagascar and the Comoro islands including the description of 78 new species. In Chapter 16 (Yuan et al. 2004) we worked on the phylogeny and biogeography of Balsaminaceae inferred from ITS sequences using combined results from molecular phylogenetic and morphological analyses. In Chapter 17 (Rahelivololona et al. 2018) we conducted a phylogeny and assessment of the infrageneric classification of species in the Malagasy Impatiens (Balsaminaceae) with a particular emphasis on taxa collected from Marojejy.
Below I summarise the most important findings of each chapter and provide an outlook for future studies.
How many species of Impatiens occur in Madagascar and the Comoro islands?
To provide additional information on the taxonomic revision of Impatiens in Madagascar and the Comoro islands, the identification of already described species as well as the description of new species was conducted. Based on herbarium specimens from BR, G, K, NEU, P, TAN and on living plants collected during several field trips, 78 new species and 6 nomina nova have been published and another 70 new taxa are already identified. Actually more than 260 species occur in Madagascar and the Comoro islands and all of them are endemic. For each species, a description of the morphology, phenology, ecology and known distribution range was provided. Apart from new taxa, the delimitation of already described species like Impatiens firmula Baker and Impatiens hildebrandtii Baill. could be clarified by studying the types and by observing the variability in the field.
Are the groups of Impatiens in Madagascar monophyletic, and what is the systematic position of Trimorphopetalum?
Yuan & al. (2004) conducted a molecular phylogenetic study to examine the morphological and karyological evolution, and the historical biogeography of the Balsaminaceae family by using nucleotide sequence data of internal transcribed spacer regions of nuclear ribosomal DNA. The results support the monophyly of the Malagasy endemic section Trimorphopetalum and show that the cleistogamous Impatiens inaperta should be included in the sect. Trimorphopetalum which is the most derived within Impatiens. Therefore, the section Preimpatiens proposed by Perrier de la Bâthie (1934) is paraphyletic.
Rahelivololona & al. (2018) provided a phylogenetic study focused on three subdivisions (based on macromorphological characters) proposed by Perrier de la Bâthie (1934). The analysis was done using two nuclear AP3/DEF homologues (ImpDEF1 and ImpDEF2) and the plastid atpB-rbcL spacer to reassess or assess the monophyly of the Malagasy Impatiens, of the sections Preimpatiens (Humblotianae and Vulgare groups) and Trimorphopetalum. A focus was on the species of Impatiens from the Marojejy National Park and of the morphologically variable species I. elatostemmoides, I. “hammarbyoides”, I. inaperta and I. manaharensis, using monophyly as the primary criterion.
As results the Malagasy Impatiens are paraphyletic and the section Preimpatiens sensu Perrier de la Bâthie (1934) (= subgen. Impatiens sensu Fischer & Rahelivololona 2002) was not resolved as a monophyletic group. The section Trimorphopetalum sensu Perrier de la Bâthie (1934) (= subgen. Trimorphopetalum sensu Fischer & Rahelivololona 2002), however, was strongly confirmed as a monophyletic lineage (BS: 92; BPP: 1). Neither the Humblotianae group nor the Vulgare group was supported as monophyletic. None of the morphologically variable species appeared to be monophyletic and the sampled species of Impatiens from the Marojejy National Park do also not form a monophyletic group.
What are the biogeographical position and the distribution patterns of Impatiens in Madagascar and the Comoro islands?
Investigation of the geographical affinities and species distribution of section Impatiens (including Humblotianae group and Vulgare group) and section Trimorphopetalum were conducted and the origin and evolution as well as species richness and endemism were discussed.
The isolation, the climate and the complex topography of Madagascar have generated the microhabitats and ecological niches favourable to the diversification of Impatiens species. Impatiens of Madagascar with 260 endemic species is actually the largest genus in Madagascar. Therefore, Madagascar and the Comoro islands are among the most species-rich regions in the world for Impatiens.
Future studies
In Impatiens on Madagascar, there remain numerous unresolved questions that need to be adressed:
• A further study based on a much larger molecular data set and sampling from the entire geographic ranges of Impatiens in Madagascar is needed to retest the monophyly of the different subgenera and sections, as well as a molecular dating of the Malagasy Impatiens.
• The study of pollinators as a key for understanding the radiation and species richness is required: Within Impatiens the different shapes of spur are related to pollinators (bees, birds, butterflies and moths). Therefore pollinator observation of specific species need to be done to understand the radiation of species by adaptation and coevolution with these pollinators. A pollination study with a large number of species within section Trimorphopetalum will help to understand the mechanism of complete disappearance of the spur, the shift of pollinators and the evolution of species richness.
• The destruction of the natural habitats of Impatiens and the subsequent reduction of humidity in logged area constitute a severe threat for the survival of many species. The conservation and reforestation of vulnerable areas such as Ankaratra, Daraina, Mandraka and Col des Tapia near Antsirabe is required.
• In terms of conservation and to mitigate the threat on the genus, a study on the ex-situ-conservation of Malagasy Impatiens species is very important as long as some species are suitable for horticultural purposes (e.g. Impatiens mayae-valeriae, Impatiens emiliae and species with broad red spur).
• Finally, the publication of the revision of Impatiens of Madagascar and the Comoro islands will help other botanists to identify the species and will thus increase our knowledge on the group.