Phylogeny and biogeography of Campanula L. section Quinqueloculares (Boiss.) Phitos (Campanuloideae, Campanulaceae)

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Λιβέρη, Ελένη

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Campanula (Campanuloideae, Campanulaceae) is a one of the most speciose and variable genera including ca. 400 species, which are distributed across the circumboreal region. It is characterized by a long and complex taxonomic history. Traditional classifications were based on morphological characters that often resulted in artificial grouping. The last two decades, however, extensive phylogenetic studies of the broader Campanuloideae utilizing molecular data and modern analytical methods have been presented. These studies have revealed many interesting patterns and insights into the evolution of Campanuloideae and have provided compelling evidence for the polyphyly of Campanula. Mediterranean hotspots for the genus such as the Middle East, Greece, and the Balkan Peninsula are particularly species-rich and have attracted much attention from botanists. Campanula section Quinqueloculares, as traditionally defined, belongs to C. subgenus Campanula and consists of ca. 39 species, which are distribibuted mainly in Greece and W Turkey with high number of endemics. One species is distributed in Italy and France, while two species have been recorded to Caucasus and Middle East. The section is considered one of the most morphologically variable groups in Campanula comprising biennial (monocarpic) or, more rarely, perennial chasmophytes, characterized by a 5-locular ovary, a style with five stigmas, appendages more or less covering the ovary, and a capsule opening with five pores. The main common taxonomic feature was the 5-locular ovary; even though a small group of species with trilocular ovary was recorded. The few early evolutionary hypotheses for C. sect.Quinqueloculares were based on morphological, karyological and biogeographical data resulting to a correlation with paleogeographical events in the Aegean area. From phylogenetic point of view, almost the half species of C. sect.Quinqueloculares, which were included in broader Campanula frameworks, were recoverd into a large but completely unresolved clade of Campanula and related genera (CAM17) mostly growing in Greece, Turkey and Caucasus. However, the comb-like structure of CAM17 to a large extent does not allow generating clear hypotheses on the evolution of C. sect. Quinqueloculares. Main obstacles for the phylogenetic studies were the incomplete taxon sampling and the insufficient molecular markers for this section. Furthermore, the absence of a phylogenetic study focused on C. sect. Quinqueloculares in addition to the few available evolutionary hypotheses formulated several decades ago, render C. sect. Quinqueloculares as a challenging case within CAM17, and presumably a key group for the interpretation of the evolutionary processes in the whole clade. The current PhD Thesis is focused on the study of C. sect. Quinqueloculares employing different methods and evaluating various perspectives. A complete taxon sampling is achived through field work throughout Greece and collaborations with Universities and Herbaria of other countries. The main goals of the study include testing the monophyly and reconstructing the phylogeny of C. sect. Quinqueloculares. In this framework, we aim to infer the historical biogeographic patterns and to reveal the significant biological and geological processes that may have shaped the evolution of this group. In order to accomplish these goals, molecular data for every taxon are obtained whereas the taxonomic contribution of morphological, palynological, and karyological features of most taxa are considered. The photosynthetic traits of C. sect. Quinqueloculares are also evaluated. The comparative morphological study of C. sect. Quinqueloculares and the recording of their main morphological features are based on collected material and herbarium specimens of all described until now taxa. The morphological diversity of the section is very high, especially for some species-complexes, and our results reinforce the already expressed opinions for “extreme polymorphism”. However, some species such as C. laciniata and C. merxmuelleri are particularly distinct. The complexes of C. lyrata, C. rupestris and C. topaliana are found the most variable morphologically; thus, requiring further taxonomic reconsiderations. The re-establishment of C. nisyria as distinct species and not synonym with any species of C. lyrata complex, is well-evident from its morphological traits corroborated by molecular data. Regarding C. topaliana complex, the subsp. topaliana and subsp. cordifolia probably should be merged based on morphological and molecular data. The study of C. rupestris complex, characterized by a consistent 3-locular ovary, revealed high variation on number of ovary locules (from 3 to 5) rendering the number of ovary locules as a diagnostic feature questionable. Seed and pollen morphology of C. sect. Quinqueloculares is examined using Scanning Electron Microscopy and their taxonomic significance is evaluated. The study of seed morphology provides data on size, shape, color and seed coat of 16 Greek taxa. The seed morphology is found to be relatively uniform except the seed coat. The seeds are characterized of small size, oblong to elliptic/obelliptic shape and light to dark brown color. However, two different patterns are described based on the nearness of striations on the seed surface: a) regularly striate and b) narrowly striate. All the data for seed morphology are presented for first time here. The pollen morphology of 12 taxa from C. sect. Quinqueloculares is studied revealing spheroidal, radially symmetrical and isopolar pollen grains with three pores for all taxa. The size of pollen grains is small with the exception of C. laciniata, which has medium-sized grains. The pollen ornamentation shows considerable variation resulting to two different types: a) striato-reticulate and microechinate in most studied taxa and b) rugulate and echinate pollen grains observed in C. laciniata and C. pelviformis. The palynological results are newly generated for 10 of the 12 studied taxa. Although, data for more taxa are needed, the two types of pollen ornamentation seem to support phylogentic relationships in some cases. Overall, seed and pollen morphology are found to be an overlooked feature in Greek Campanula taxa. However, this study sets the framework for their research and additionally supports their complementary role in Campanula taxonomy and systematic mainly at specific rank. The determination of chromosome number, ploidy level and karyomorphometric analysis is presented for 12 taxa of Campanula section Quinqueloculares employing the classical squash technique and combination of stains. The chromosome number 2n = 2x = 34 predominates in all examined taxa with the exception of C. laciniata, for which a new chromosome number (2n = 4x = 68) was found in a population from W Crete. The karyotypes in all examined taxa are symmetrical comprising of mostly metacentric and submetacentric chromosomes, small in size, but they differ in the presence and the size of satellites. The chromosome count (2n = 34) and karyotype morphology of C. topaliana subsp. delphica is given for first time. New populations of C. cymaea, C. kamariana, C. pelviformis, and C. topaliana subsp. cordifolia were karyologically investigated confirming the previous references. The karyotype morphology of C. anchusiflora, C. andrewsii subsp. hirsutula, C. kamariana, C. lavrensis, C. merxmuelleri, C. nisyria and C. rupestris is given for first time. Additionally, microphotographs are firstly provided hereby for all investigated taxa. The taxonomic value of chromosome data for C. sect. Quinqueloculares is limited due to the almost constant chromosome number (2n = 34), the similar chromosome morphology and the small chromosome size, which renders the karyomorphometric analysis extremely difficult. However, the karyological study of more populations is necessary since it may reveal important evolutionary processes, e.g. polyploidy, as in the case of C. laciniata. The molecular study of Campanula section Quinqueloculares utilizes individual and combined data matrices consisting of six plastid (NADHS-2, rpoC1-1, rpoC2-1, rpoC2-2, rpoC2-3, trnT-trnL) and four nuclear (2017561, ITS, PPR11, PPR70) markers for 121 taxa in total. The results indicate that C. sect. Quinqueloculares, as traditionally circumscribed, is polyphyletic. The topology of the phylogenetic trees of chloroplast markers, ITS and 2017561, as well as of combined data is similar. The nuclear markers of PPR family genes show slightly different topology in their phylogenetic trees, but the species of sect. Quinqueloculares are found again scattered in different branches. Species are largely clustered into two well-supported clades, except for three taxa (C. crispa, C. medium, C. pelia) excluded from these groups. Additionally, a few taxa (C. isaurica, C. koyuncui, C. sartorii, C. teucrioides) belonging to other sections are confidently nested within the two Quinqueloculares clades. The first clade (Greek clade) includes one isophylloid species (C. sartorii) nested with 24 Greek endemics belonging to C. sect. Quinqueloculares, which are distributed in mainland Greece, Evia, Northen Sporades and Kiklades islands. Only two species of the clade, ie. C. merxmuelleri and C. saxatilis subsp. saxatilis, extend their distribution to NE Aegean islands and Crete, respectively. C. sartorii, endemic to Kiklades (Andros, Tinos), was not considered member of the traditional circumscribed sect. Quinqueloculares, but our results indicate it always nested within this clade. The second clade (Southeastern Aegean-Turkish clade) comprises 20 taxa of C. sect. Quinqueloculares and 3 species of C. sect. Rupestres, all distributed in E Aegean islands, Crete, Karpathos-Saria and Turkey. The Turkish endemics C. isaurica, C. koyuncui and C. teucrioides were traditionally classified under sect. Rupestres, but in our phylogenetic analyses are resolved within this clade together with Quinqueloculares taxa. Divergence time estimates suggest that these clades originated in the Late Miocene. Temporal and geographic patterns are consistent with a vicariant scenario driven by geological events during the Miocene, such as the formation of the Mid-Aegean trench and the Messinian salinity crisis. The response of various indices of the photosynthetic machinery efficiency to temperature change was examined in species of sect. Quinqueloculares both experimentally in the laboratory (15°C, 24°C, 35°C) and seasonally in the field (winter, spring, summer). The methods of chlorophyll fluorescence and leaf reflectance spectrometry were used. According to the results, the photosynthetic machinery of the studied taxa of the sect. Quinqueloculares is generally more sensitive to high temperatures in the laboratory and respectively during summer in the field. The effect is most pronounced in C. pelviformis and C. merxmuelleri. The low winter temperature which is generally considered as a second stressful parameter for Mediterranean plants does not seem to be particularly stressful for the studied species. Concluding, after decades of attempts to clarify the evolutionary history of Campanula at broad and narrow scales, it seems that we are still far from a comprehensive understanding of this enigmatic group. In order to resolve the taxonomic complexity and elucidate the historical evolution of Campanula in the Mediterranean region and beyond, more focused studies with increased genomic data and complete taxon sampling are necessary. The current PhD represents the first phylogenetic study focused on C. sect. Quinqueloculares and based on complete taxon sampling, 10 molecular markers as well as data on morphology, palynology, karyology and ecophysiology in a taxonomic context. The results reveal the polyphyletic origin and intriguing evolutionary aspects of C. sect. Quinqueloculares, which comprise an important piece of Campanula evolutionary history and also set the background for a new classification of the section in a phylogenetic context.



Campanula section Quinqueloculares, Evolution, Phylogenetic analyses, Divergence time estimation, Taxonomy, Karyology, Palynology