How population size and pollinator availability mould breeding system and population genetics in plants? The case of Jacob's Ladder (Polemonium caeruleum, Polemoniaceae), a pollen-limited species of the "Polish Plant Red-Book"

Mutualistic plant-pollinator interactions are crucial for the functioning of many terrestrial ecosystems and global food supply. However, global diversity loss, which has intensified in recent decades, disrupts the pollination process, making it a significant subject of the study. Populations of rare species and those at the edges of their range are particularly vulnerable to extinction. While pollinators play a pivotal role in shaping plant reproductive success, there are also other factors that influence it. Studies on plant reproductive success typically focus on individual factors, instead of considering them simultaneously. Additionally, most studies on plant pollinator interactions tend to focus on specialist plant species, while overlooking generalist species. In my study, I investigated the ecological and evolutionary consequences of population fragmentation of Polemonium caeruleum, a red-listed, boreal plant from the Polemoniaceae family and the only representative of the genus Polemonium in the Polish flora. In Poland, the species reaches its southern range limit and its populations are in decline. P. caeruleum is usually a self-compatible, insect-pollinated plant, characterised by a generalist pollination system, that reproduces only by seeds. By using multidimensional methods, I was able to verify the following hypotheses: 1. The decrease in population size of P. caeruleum affects the reproductive success of plants due to lower pollinator visitation frequency. 2. The decrease in population size leads to changes in the breeding system (transition from self-incompatibility to self-compatibility), resulting in decreased intra-population genetic diversity as a result of breeding among closely related individuals. 3. The chemistry of P. caeruleum nectar varies among populations and is shaped by availability of nutrients in the habitat, hence influencing the insect assemblages visiting flowers. 4. Bacterial communities inhabiting the nectar of P. caeruleum differ spatially. In order to verify the above hypotheses, I conducted research in 15 populations of P. caeruleum located in Poland, distributed across the country and differing in habitat conditions and number of individuals. I performed experiments on randomly selected flowers, testing reproductive success and pollen limitation level in the years 2014-2018. I also recorded insect activity visiting the flowers in the years 2015-2018. I conducted the experiments on the breeding system in the years 2016-2018. In 2016, I collected leaf samples and genotyped them using AFLP (Amplified Fragment Length Polymorphism) markers to assess genetic variation in populations. l also performed flower measurements and collected nectar samples that I analysed using HPLC (High Pressure Liquid Chromatography) in 2018. I conducted analyses of nectar inhabiting bacteria assemblages using culture-independent 16S rRNA gene amplicon sequencing (MiSeq, Illumina) in 2017. The decrease in population size of P. caeruleum resulted in decreased reproductive success of plants and increased pollen limitation level. However, my results did not confirm the presence of a positive effect of population size on insect visit frequency. On the other hand, increased insect visits had a negative impact on pollen limitation level (hypothesis 1). Furthermore, my analyses showed that honeybees visiting flowers of P. caeruleum played a contradictory role: while these insects were the most frequent flower visitors they also decreased the pollen limitation level, their activity also reduced plant reproductive success. The studied populations demonstrated varying degrees of self-compatibility, with most of them characterised by a mixed-mating system, albeit to a different degree. No correlation was found between the breeding system (level of self-compatibility) and population size. However, my analyses revealed that population size negatively influenced seed production via self-pollination. The increase in seed production through self-pollination in small populations suggests the development of a reproductive assurance mechanism. This process was facilitated by changes in flower morphology, which indicate the development of a selfing syndrome. Furthermore, the analysis showed that flower morphology is shaped by pollinator mediated selection. My study revealed that populations of P. caeruleum in Poland exhibit relatively low inter population genetic diversity and moderate among-population genetic diversity, coupled with and a high degree of inbreeding. Intra-population genetic variation was not correlated with the size of population or the degree of self-compatibility. This lack of association may be due to changes in population size and in the level of self-compatibility (breeding system) over the years. Moreover, such lack of relationship may result from the breeding system of studied populations, because a positive relationship between population size and its genetic variability is usually stronger in self-incompatible than in self-compatible populations. Additionally, low genetic variability and high inbreeding in populations may hinder the ability to adapt to changing environmental conditions, putting even large populations at risk of extinction. The prevalence of mixed-mating among studied populations ensures reproduction in an unpredictable pollination environment. This strategy may also provide benefits in case of fluctuations in population size (hypothesis 2). The investigation of nectar chemistry revealed variations both among regions and among populations within Poland. The presence of selected nutrients in the habitat may influence nectar characteristics, but the observed nectar traits had little effect on insect visitation (hypothesis 3). Moreover, my study confirmed that there are spatial differences in the bacterial communities inhabiting nectar (hypothesis 4). My study expands our knowledge of the biology of P. caeruleum and provides a comprehensive view of the factors contributing to the decline of its populations in Poland. Such long-term, across-population studies enhance our understanding of the pressures driving plant reproductive success and form the scientific foundation for an effective conservation strategy. Although the findings pertain to a single species, they can potentially be extended to other species characterised by a similar biology, rare species or groups representing related taxa.