The project is largely based on collections gathered during earlier projects. A supplementary material will be gathered during in the first year of the project. The analyzed material will come from 50 localities, completed for all taxa. The fieldwork will be organized in co-operation between the teams from Montenegro and from Poland. It needs to be stressed that the museum collections generally lack material fixed properly for molecular investigation. A limited number of reference taxa, other than GenBank/BOLD (Barcode of Life Database) data, will be obtained from own collections or from other specialists. The whole study will be organized within three main key activities.
Key Activity 1: Assessment of the taxa diversity at the morphological and molecular level (taxonomic identification, COI barcoding, selection of MOTUs for phylogeny reconstruction, analysis of population molecular structure, descriptions of new species). This task will be based upon taxonomic identification of the gathered collections. Species definition at the morphological level will be tested versus Molecular Operational Taxonomic Units (MOTUs) newly defined with the standard barcoding fragment of cytochrome oxidase subunit I (COI) based on the genetic distance and phylogeny-based methods. MOTUs present in more than one locality will be analyzed for their molecular population structure with the COI, supplemented with the 16S rDNA marker (see below), if needed. The proposed task allows for a first reliable assessment of diversity and endemism of selected taxa in the endangered aquatic ecosystems of Lake Skadar basin. In case of detecting new morphospecies or (pseudo-)cryptic species, it will be attempted to define them not only at the molecular level but also with morphological (including geometrical morphometry) and ultrastructural (SEM) characters. It will result in description of species new to science. The key result of this task will be construction of the publicly accessible COI barcode library in BOLD and selection of individuals for amplification of other molecular markers for the phylogenetic analyses (Key Activity 2).
Key Activity 2: Revealing phylogenetic/biogeographical affinities of selected taxa based on the whole set of molecular markers (amplification, sequencing, data analysis). Modern approach to phylogenetic studies requires analyses of several independent markers. Based on the available literature, authors of the project selected preliminarily the following set of nuclear markers (28S rDNA, 18s rDNA, ITS1, eEF1a1 or H3) as well as additional mitochondrial marker (16S rDNA) supplementing COI. This enables comparative analysis of relations between species and phylogenetic lineages for the 10 MOTUs representing all the major taxonomic groups. It allows for revealing molecular diversity of the analyzed taxa and their interpretation in the geographical context and possible common patterns in phylogeny. This way it enables deeper interpretation of the results obtained within the Task 1. This key activity will provide the baseline data for the Key Activity 3.
Key Activity 3: Defining the spatio-temporal framework for the colonization of Lake Skadar basin. Final analysis and interpretation of results in the context of geological history and paleography of the Lake Skadar basin. Completing this task requires construction of credibly calibrated chronograms, estimation of divergence and evolutionary rate shifts as well as insight into historical phylogeography of the model taxa. Final analysis of the whole dataset and the comparative interpretation of results in the paleogeographic (and geological) history of the region will allow for the verification of the main hypotheses of the project.
