|Zusammenfassung||The field of molecular phylogenetics has benefited greatly from the recent advances of modern sequencing approaches that allow for the generation of large genomics data sets Nonetheless a lack of suitable genetic markers and incomplete taxon sampling remain common problems in studies of evolutionary relatedness. Most phylogenetic studies are based on mitochondrial DNA (mtDNA) because information about the nuclear genome and strategies to develop new genetic markers are often not available. The use of appropriate genetic markers and the inclusion of both a geographically and phylogenetically comprehensive taxon sampling are required for adequately reconstructing evolutionary histories among different taxa. This is particularly true for studies of recent diversification.
Mayflies (Ephemeroptera) are ancient freshwater insects, dating back more than 300 million years, but at the same time have been reported to successfully colonize and diversify on recently formed Atlantic oceanic islands. This combination of ancient origin and recent diversification makes them a fascinating study system for molecular phylogenetics. In the first part of my thesis, I investigated the recent diversification and colonization history of mayflies on 13 Atlantic oceanic islands of the Azores, Madeira, and the Canary Islands. The island fauna provides an ideal setting to understand how speciation and dispersal shape present-day freshwater biodiversity.
A first step in the research was an assessment of the species richness of the island fauna, because current taxonomic estimates are uncertain. Earlier research on mayflies in Europe, Africa, Madagascar, and North America has repeatedly uncovered otherwise cryptic diversity based on analysis of mtDNA. This suggests that past morphological estimates may underestimate species richness, and that a comprehensive understanding of island biodiversity and its evolution requires molecular-based taxonomy. In order to assess the biodiversity and date the origin of the island fauna, I used phylogenetic analyses based on universal mtDNA markers combined with a generalized mixed Yule- coalescent (gmyc) approach. In total, I found twelve island-endemic species within three species groups (Baetis canariensis s.l., B. pseudorhodani s.l., and Cloeon dipterum s.l.) that have diversified within the last 15 million years in parallel throughout the island archipelagos. While intriguing, the results also pointed out the limitations of mtDNA
markers for the study of recent diversification events. The study clearly demonstrated a need for the development of new genetic markers that provide increased phylogenetic signal in order to resolve the relationships of closely related species groups.
To investigate relationships among newly diverged species, many polymorphisms are needed, and these should ideally be derived from multiple unlinked markers. Since mayflies are a non-model organism i.e. no reference genome is available, I generated a whole genome draft and used these data to design 59 nuclear DNA (nDNA) markers to establish a basis for inferring the evolutionary history of the C. dipterum s.l. species group. Prior to my work, there were only two suitably variable nuclear markers available, namely 28S ribosomal RNA (rRNA) and PEPCK. I applied species tree reconstruction methods using the multispecies coalescent approach, a phylogenetic framework developed within the last five years and suitable for large nDNA data sets. This model was used to overcome both the lack of phylogenetic signal and the potentially conflicting signal derived from gene tree incongruences. Using this approach, I delineated six different Cloeon species, three on the islands and three on the European mainland. The phylogeny resolved complex colonization routes on a large geographic scale (Macaronesian islands, the European mainland and North America). The three Macaronesian Cloeon species appear to have originated from European source populations and different species co-occur in the same freshwater habitats. The diversification within the C. dipterum s.l. species group was mainly promoted by allopatric speciation, whereby strong natural selection on ecological traits i.e. freshwater habitat adaptations and shifts in life history traits are presumed to play a key role. Future research identifying specific ecological, morphological, or behavioral traits, as well as genes that are under natural selection will be needed to understand the mechanistic basis of speciation.
The second part of my thesis focused on evolution over much longer temporal scales, namely ancient origins of the extant winged insects. It remains one of the open questions in the field of insect evolution and systematics, and is thought to act as foundation to understand the evolution of flight as one of the most fascinating evolutionary processes, leading to the development of the most diverse and successful animal group. All winged insects (Pterygota) are placed into one of two groups, based on wing function. The inability to fold back the wings, as seen in the Ephemeroptera and Odonata (dragonflies and damselflies), is considered to be an ancestral condition and these orders are therefore referred to as the Palaeoptera (old wings). In contrast, all other orders are able to fold their wings and as such referred to as Neoptera (new wings). The phylogenetic position of the Palaeoptera within the winged insects is one of the unresolved problems in insect systematics and is thus referred to as the ‘Palaeoptera problem’. Morphological and molecular data have provided support for three competing hypotheses: (1) the Palaeoptera hypothesis, stating the Ephemeroptera + Odonata as sister group to the Neoptera, (2) the basal Ephemeroptera hypothesis (Ephemeroptera + (Odonata + Neoptera)), and (3) the basal Odonata hypothesis (Odonata + (Ephemeroptera + Neoptera)). To date molecular phylogenetic reconstructions have been inferred with a limited number of genes, mostly mitochondrial and ribosomal genes, or a limited number of mayfly taxa (i.e. phylogenomic studies).
To resolve the ‘Palaeoptera problem’, I increased the taxon sampling to a total of 93 insect taxa, including 19 mayflies and I used as marker the protein-coding regions of the mitochondrial genomes (mitogenomes) in order to overcome the highly sensitive sequence alignment step. I applied two different phylogenetic tree reconstruction methods, namely Bayesian inference and maximum-likelihood. I identified taxa with unstable topological positions under the different statistical models, and tested the effects of excluding these taxa on the overall phylogenetic accuracy. First, I sequenced and annotated the mitogenomes of the three mayfly species Baetis rutilocylindratus, Cloeon dipterum, and Habrophlebiodes zijinensis. A comparison among mayfly mitogenomes showed that the gene content and gene orientation was conserved, including 37 protein- coding genes and low AT content. I found that the pruning of identified problematic taxa greatly improved the node support values of the tree reconstruction. Interestingly, also the chosen outgroup was identified as being a problematic taxon. The Bayesian inferences provided support for the basal Ephemeroptera hypothesis, whereas the maximum- likelihood phylogeny supported the basal Odondata hypothesis. The increased number of taxa, the exclusion of problematic taxa and the use of mitogenomes proved to be well suited to reconstruct ancient relationships. The contradicting results of the two phylogenetic methods support the growing evidences that phylogenetic methods based on Bayesian inference might be more appropriate for reconstructing ancient relationships. Thus, the relationships of the Palaeoptera remained unresolved but the results point out the need to investigate the suitability of currently used phylogenetic methods for resolving ancient splits.
Taken together, my thesis presents one of the first genetically comprehensive studies on aquatic insects, combining molecular phylogenetic approaches based on a large set of nDNA markers and mitogenomes. I found that the increase of nDNA markers and the development of bioinformatics approaches for recently evolved species groups and the use of mitogenomes for ancient taxa are extremely important for understanding evolution because of their capacity to reconstruct well supported phylogenetic trees.