Dissertation committee:
Αριστείδης Παρμακέλης, Αναπληρωτής Καθηγητής, Ε.Κ.Π.Α.
Κωνσταντίνος Τριάντης, Αναπληρωτής Καθηγητής, Ε.Κ.Π.Α.
Μαρία Θ. Στουμπούδη, Διευθύντρια Ερευνών, Ινστιτούτο Θαλάσσιων Βιολογικών Πόρων & Εσωτερικών Υδάτων, ΕΛ.ΚΕ.Θ.Ε.
Brant C. Faircloth, Moreland Family Professor, Louisiana State University
Ιωάννης Καραούζας, Ερευνητής, Ελληνικό Κέντρο Θαλασσίων Ερευνών
Σάββας Μ. Γεννίτσαρης, Επίκουρος Καθηγητής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Παναγιώτης Παφίλης, Καθηγητής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
The present doctoral thesis is the first comprehensive study of freshwater gastropods in the under-studied aquatic systems of the Aegean Sea. Freshwater gastropods are characterized as habitat specialists with low dispersal abilities that have successfully colonized all types of freshwater bodies. The Aegean archipelago with a complex geological and palaeogeographical history represents an ideal system for biodiversity and evolutionary studies. The insular aquatic systems of the Aegean archipelago are characterized by small area size and, nearly half of them, ephemeral presence of water due to semi-arid conditions. These features result in them being the most vulnerable ecosystems in the Aegean Archipelago. The aims of the doctoral thesis are to investigate the patterns of biodiversity and ecology of freshwater gastropods throughout the study area, as well as to reconstruct the phylogenetic relationships of selected gastropod taxa.
In Chapter 1, freshwater gastropod fauna from the aquatic systems of 11 Aegean islands was documented. Overall, 40 native and 4 invasive freshwater gastropod species belonging to 9 and 4 families, respectively, were identified by employing morphological, anatomical, and molecular data analyses. Our field work showed that most taxa mostly occur in karst springs and small headwater streams/brooks fed by the springs rather than other aquatic systems. We also identified diversity hotspots within this group, characterized by a high number of native and endemic species. Canonical Correspondence Analysis (CCA) was performed to examine if there is correlation between species presence and environmental variables. CCA succeeded in explaining 81% of the variance of the data. Lastly, we investigated the presence of any hierarchical community structure in our data using network analyses looking for patterns according to the delineated freshwater ecoregions of Greece and the paleogeography of the Aegean. Our analyses did not reveal any strong modular structure when either of the two algorithms was implemented. However, when Louvain method was applied in all native species, eastern Aegean islands were grouped together and were separated from the remaining ones. This pattern is an indication of the defining impact of the Mid-Aegean (MAT) creation on the existing biotic affinities.
In Chapter 2, the two gastropod genera, namely Bythinella and Pseudamnicola that stood out for their large number of species present in Greece and the high levels of endemism, were selected, to reconstruct their phylogenetic relationships using mtDNA molecular markers and investigate the evolutionary processes responsible for shaping their current distribution on the island of Crete. For Bythinella, our findings strongly support the presence of at least five species in Crete, out of which four correspond to already described species and one was recovered for the first time in the present study. The analyses revealed a relatively old timeframe of diversification, dating back to the middle Miocene (ca.14 Mya), with allopatric speciation events being the main drivers of the evolution of the genus within the island. For Pseudamnicola, there is strong evidence for the presence of at least two species in Crete and a relatively recent time of diversification that started in the late Miocene. Dispersion processes followed by isolation of the populations and/or recent speciation, seem to be the underlying process for the current distribution of Pseudamnicola lineages.
In Chapter 3, the phylogenetic pattern of the genus Pseudamnicola throughout its Mediterranean range was also investigated using molecular data available and newly produced. This is the first time that molecular data from Greek taxa are incorporated in a phylogenetic analysis for Pseudamnicola of this spatial scale. Our findings are in favor of Pseudamnicola having diversified quite recently during the late Miocene. The ancestral distributional area recovered was the Italian Peninsula & Islands together with the Ionian Drainages. The main speciation events of the genus are estimated to have taken place during the Pleistocene. Two different dispersal routes are suggested for the gradual spread of Pseudamnicola within the Mediterranean. One of the routes involved a westward dispersion towards the Iberian Peninsula and then to NW Africa. The second dispersal route involves a westward dispersion towards Italy and Spain and an eastward one towards the Aegean Archipelago and the Eastern Mediterranean.
During this study, it became obvious that the molecular markers commonly used in phylogeographic or phylogenetic studies of freshwater gastropods, are rarely successive in fully resolving the phylogenetic relationships of the taxa under question. This was also the case in part of the phylogenetic analyses performed in this study. Within the frame of this study, sequence data originating from three different mtDNA genes (cytochrome c oxidase subunit I: COI, 16S rDNA and cytochrome b) were systematically checked for their ability to unambiguously resolve the phylogenetic relationships of the taxa investigated. Among the three mtDNA genes, COI was always the one that provided the highest level of phylogenetic resolution, whereas 16S rDNA and cytochrome b were systematically failing to provide additional phylogenetic information for resolving the relationships of the taxa involved. In some cases, the phylogenetic analyses relying on COI sequence data, were also suffering from low resolution levels. Therefore, it has become obvious that in order to resolve the phylogenetic relationships of freshwater gastropods, a new set of molecular markers must be identified.
In Chapter 4, hence, within the frame of this study, we aimed to evaluate the effectiveness of the Ultra-Conserved Elements (UCEs) in resolving freshwater gastropods relationships. UCEs are highly conserved regions of genomic DNA that can function as regulators and/or enhancers of gene expression and have been discovered across a variety of both vertebrate and invertebrate taxa.
Therefore, after the generation of four (4) low depth gastropod genomes and the inclusion of six (6) already available gastropod genomes, a caenogastropod dataset of 24 taxa was constructed that qualified for the identification (via specialized bioinformatic analyses) of UCEs that were shared among gastropods. Following the identification of the UCEs, a set of baits targeting the conserved regions shared by gastropods was designed. Finally, the utility of these baits for collecting UCE sequence data across gastropod lineages, with a particular emphasis on enriching UCE loci from freshwater caenogastropod taxa, was evaluated. Based on the results of the bioinformatic analyses, a set of 12092 probes were identified, and these were commercially synthesized as an RNA target capture array detecting 1234 loci, in total, out of which 1224 UCE loci were shared across the alignments of the 24 caenogastropd species participating in the study. Using the sequence data generated through high throughput sequence, a phylogenetic analysis involving the 24 gastropod taxa was performed. The resulting phylogeny demonstrated the utility of the gastropod-specific UCE loci in resolving the relationships among and within gastropod clades and supported the monophyly of the three major gastropod clades represented by multiple taxa: Caenogastropoda, Neritimorpha and Heterobranchia. Furthermore, our analysis strongly supports Neritimorpha as a sister group to Caenogastropoda and recovers Heterobranchia as a sister group to Caenogastropoda and Neritimorpha.
In previous studies, UCE loci have been found to be very useful for recovering both deep-level phylogenetic relationships and shallow-level genus and population relationships. Therefore, within the frame of this study, in Chapter 5, the variability and utility of UCEs in reconstructing species-level relationships within the genus Bythinella was also investigated. European and Greek taxa were employed to investigate the performance of UCEs in resolving phylogenetic relationships and compare their efficiency against commonly used mtDNA markers, such as COI. The phylogenetic tree resulting from the UCEs sequence data was fully resolved and outperformed the tree generated using COI data alone, laying the groundwork for the efficient use of UCEs for inferring phylogenies and delineating species at lower taxonomic levels.
