Marine biodiversity: a multidisciplinary journey from genes to species

The present dissertation is divided into an introduction and three different chapters. Its general organization is intended to facilitate future publications, and therefore Chapters 1, 2 and 3 are hereby reported in journal-like style and should lay the basis for independent papers to be submitted to international peer-reviewed journals as first author. On the contrary, the aim of this introduction is to focus and resume the work performed during my Ph.D. Thesis and discussed below. It starts with a description of the originally planned research project (partly covered by Chapter 1), analyzes technical problems that forced me to deviate from it, and introduces the subsequent Chapters 2 and 3. Finally, Chapter 4 resumes papers and short communications published or in press (first page only, due to copyright) during this Ph.D. programme. One of the unsolved problems in evolutionary biology is which mutations generate evolutionarily relevant phenotypic variation. Understanding what kinds of molecular changes they entail, and what are the phenotypic magnitudes and frequencies of origin, may provide important insights into evolutionary processes (adaptive evolution, balancing selection, deleterious variation and genetic drift) that influence phenotypic variation within and among population and eventually lead to speciation processes. The marine tunicate Ciona intestinalis sp. A constitutes an excellent organism for linking genomics and developmental biology. A growing interest in population biology of C. intestinalis, prompted by the paucity of laboratory lines and the ease of sampling, has recently undermined extreme levels of heterozygosity and high rates of recessive mutations (13-20% wild individuals are heterozygote carriers of recessive mutations in developmental genes). When brought to homozygosity, these naturally occurring mutations often carry phenotypes that are similar to hypothesized evolutionary transitions among ascidian families and orders, such as reduction or absence of the tadpole larval tail (e.g. genus Molgula), proximal exhalent siphon (e.g. genus Ascidia) and absence of, or supernumerary, larval sensory organs (e.g. genus Pycnoclavella). So far, results obtained in forward genetics of ascidians were mostly obtained from casual and random samplings, with no efforts in developing specific methods, such as for culturing and cryopreservation, and yet poor knowledge of several species-specific traits in the field on the other side, such as taxonomic status, demographic and reproductive dynamics. Therefore, these gaps need to be filled for optimal project design. The original aim of the present Ph.D. project was to investigate the developmental processes and genetic configurations of specific phylomimicking mutations occurring within natural populations of the ascidian Ciona intestinalis sp. A by carrying out a naturally occurring mutation study. However, due to problems of technical and biological nature, I extended it as described below. The first step was successfully achieved, and is partially explained in Chapter 1. This discusses the state-of-the-art of forward genetics in tunicates and review findings of research on naturally occurring mutants. In addition, it first reports the development and establishment of a comprehensive protocol for the study of genetic polymorphism in wild populations of Ciona intestinalis sp. A that includes newly developed procedures for sperm cryopreservation, culturing and screening, as well as the possibility of identifying subtle mutations by means of a multiple whole mount in situ hybridization with an automated system. I also show that the ecology and biology of natural populations at species-specific level holds traits that are relevant to correct project organization and conduction. Altogether, the effort hereby conduced lays the foundations for the identification of naturally occurring mutations in ascidian taxa, and may constitute an additional value when considering new marine model species for forward genetics purposes. However, after an initial survey of heterozygote carriers of developmental genes and the molecular characterization of the identified mutant larvae by ISH (in situ hybridization) and FISH (fluorescent in situ hybridization), unexpected cryobanking problems resulted in the loss of the mutants cryopreserved. This, together with field disappearance of the species, forced me to deviate from the project. Therefore, since I had originally planned to acquire knowledge at gene and species level, I have spent the remaining part of my PhD working on two conceptually linked projects. One is concerned with an evolutionary developmental study of a specific gene in chordate evolution (Chapter 2). This chapter therefore consists of an EVO-DEVO study analyzing the Magoh (Mago Nashi) gene. In particular, starting by unpublished ISH and IHC data available in the laboratory on the tunicate species Ciona intestinalis sp. A, I re-organized previous data, performed an in situ hybridization study in the phylogenetically related species Branchiostoma lanceolatum (Pallas, 1774) and analyzed sequence conservation, gene duplication and synteny in Chordates. Here I show that, despite its long history, Magoh has become part of a conserved syntenic group only in vertebrates. Genetic and genomic analyses indicate that Magoh appears to have duplicated in the mammalian lineage. Whole mount in situ hybridization and immunohistochemistry demonstrate that, similar to all other bilaterians, Magoh is regionally expressed in mesodermal and endodermal progenitor cells and tissues of the amphioxus B. lanceolatum and the tunicate C. intestinalis sp. A embryos. Further, ascidian Magoh protein undergoes early subcellular restriction and perinuclear localization in the fertilized egg, like in insects. Most importantly, Magoh is expressed in the brain of cephalochordate and ascidian larvae, where the protein accumulates in neurogenic and axonal structures. Thus, these data suggest that Magoh recruitment in neural cells is an innovation already present in the last common ancestor of Chordates. Finally, I performed a multidisciplinary study concerning the identity of the molluscan gastropod Bursa scrobilator (Linné, 1758) by using independent (molecular and morphological) species concepts (Chapter 3). This species forms established populations in the Atlantic Ocean (mainly in the Azores and Canarians), but is only known from the Mediterranean area from single isolated specimens, therefore being heralded as one of the rarest inhabitants of the Mediterranean molluscan fauna. I explored the taxonomic identity and the intraspecific variability of the species by using a combination of morphological, anatomical and molecular analyses on specimens from the entire distributional range, as to test a possible speciation pattern in the Mediterranean Sea. No difference was observed when analyzing protoconch, teleoconch and anatomy. Conversely, radulae are highly variable in shape both between and within individuals. Sequence analysis of COI and 16S shows the absence of significant differentiation among sites of occurrence, as expected for a species with teleplanic larvae. In addition, the broad dispersal potential in the investigated family, as well as the absence of a genetic structure, would suggest that B. scrobilator does not constitute a resident species of the Mediterranean fauna, but that its Mediterranean presence may be consistent with a repetitive natural dispersal of single veliger through the Gibraltar Strait, therefore calling for a numerical re-assessment of resident Mediterranean biodiversity by molecular means