Cytogenetic and molecular characterization of lacertid lizard species from the Iberian Peninsula

[Abstract] Reptiles, with their great diversity of sex-determining systems, have long been regarded as a model group for studying the evolution of sex determination and sex chromosomes. They also hold a key phylogenetic position to elucidate the organization and evolution of amniote genomes. This PhD thesis aims to contribute to this understanding by investigating sex chromosomes and karyotype evolution in lacertid lizards, with a focus on rock lizard species (genus Iberolacerta) endemic of the Iberian Peninsula. Firstly, we applied classical and molecular cytogenetic methods to identify and characterize previously unknown ZW sex chromosomes in the species I. monticola. Secondly, we developed whole-chromosome paints from I. monticola to detect chromosomal rearragements and test the homology of sex chromosomes among closely related lacertid species. These results revealed a high degree of karyotype conservation, but a rapid and independent differentiation of sex chromosomes, and even a putative cryptic event of sex chromosome turnover. Finally, we explored the mode of evolution of two satellite DNA families shared by all eight Iberolacerta species. Both satellite DNAs showed complex and disparate evolutionary patterns, and a highly dynamic behaviour which may be correlated with chromosomal rearragements and karyotype diversification in this genus.[Resumen] Los reptiles, con su extraordinaria diversidad de sistemas de determinación del sexo, constituyen uno de los grupos más atractivos para el estudio de la evolución del genoma y los cromosomas sexuales en amniotas. Esta tesis pretende ampliar el conocimiento en esta área investigando la evolución del cariotipo y de los cromosomas sexuales en lacértidos, y principalmente en lagartijas del género Iberolacerta endémicas de la Península Ibérica. En primer lugar, se emplearon técnicas de citogenética clásica y molecular para caracterizar el par sexual ZW, previamente no identificado, en la especie I. monticola. En segundo lugar, se desarrollaron sondas cromosómicas en I. monticola para detectar reordenaciones cromosómicas y evaluar la homología de los cromosomas sexuales en otras especies de lacértidos. Estos resultados revelaron un alto grado de conservación de los cariotipos, pero también procesos independientes de diferenciación de los cromosomas sexuales, e incluso un posible evento de sustitución del par sexual. Por último, se analizó el modo de evolución de xii dos familias de ADN satélite presentes en las ocho especies del género Iberolacerta. Ambas familias mostraron patrones evolutivos distintos y complejos, y un elevado dinamismo que podría estar asociado a la diversificación del cariotipo en este género.[Resumo] Os réptiles, coa súa extraordinaria diversidade de sistemas de determinación do sexo, constitúen un dos grupos máis atractivos para o estudo da evolución do xenoma e os cromosomas sexuais en amniotas. Esta tese pretende ampliar o coñecemento nesta área investigando a evolución do cariotipo e dos cromosomas sexuais en lacértidos, e principalmente en especies de lagartas do xénero Iberolacerta endémicas da Península Ibérica. En primeiro lugar, empregáronse técnicas de citoxenética clásica e molecular para caracterizar o par sexual ZW, previamente non identificado, na especie I. monticola. En segundo lugar, elaboráronse sondas cromosómicas en I. monticola para detectar reordenacións cromosómicas e avaliar a homoloxía dos cromosomas sexuais noutras especies de lacértidos. Estes resultados revelaron un alto grao de conservación dos cariotipos, pero tamén procesos independentes de diferenciación dos cromosomas sexuais, e mesmo un posible evento de substitución do par sexual. Finalmente, analizouse o modo de evolución de dúas familias de ADN satélite presentes nas oito especies do xénero Iberolacerta. Ámbalas dúas familias amosaron patróns evolutivos distintos e complexos, e un elevado dinamismo que podería estar asociado á diversificación do cariotipo neste xénero

Cytogenetic characterisation of the razor shells "Ensis directus" (Conrad, 1843) and "E. minor" (Chenu, 1843) (Mollusca: Bivalvia)

[Abstract] The European razor shell Ensis minor (Chenu 1843) and the American E. directus (Conrad 1843) have a diploid chromosome number of 38 and remarkable differences in their karyotypes: E. minor has four metacentric, one metacentric–submetacentric, five submetacentric, one subtelocentric and eight telocentric chromosome pairs, whereas E. directus has three metacentric, two metacentric–submetacentric, six submetacentric, six subtelocentric and two telocentric pairs. Fluorescent in situ hybridisation (FISH) using a major ribosomal DNA probe located the major ribosomal genes on one submetacentric chromosome pair in both species; FISH with a 5S ribosomal DNA (5S rDNA) probe rendered one chromosomal (weak) signal for E. minor and no signal for E. directus, supporting a more dispersed organisation of 5S rDNA compared to the major ribosomal genes. The vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome, and no interstitial signals were detected. In this work, a comparative karyological analysis was also performed between the four Ensis species analysed revealing that the three European species studied so far, namely E. minor, E. siliqua (Linné 1758) and E. magnus Schumacher 1817 show more similarities among them than compared to the American species E. directus. In addition, clear karyotype differences were found between the morphologically similar species E. minor and E. siliqua.Ministerio de Educación y Ciencia; CTM2007-28919-E/MA

Karyological Characterization of the Endemic Iberian Rock Lizard, Iberolacerta Monticola (Squamata, Lacertidae): Insights into Sex Chromosome Evolution

[Abstract] Rock lizards of the genus Iberolacerta constitute a promising model to examine the process of sex chromosome evolution, as these closely related taxa exhibit remarkable diversity in the degree of sex chromosome differentiation with no clear phylogenetic segregation, ranging from cryptic to highly heteromorphic ZW chromosomes and even multiple chromosome systems (Z1Z1Z2Z2/Z1Z2W). To gain a deeper insight into the patterns of karyotype and sex chromosome evolution, we performed a cytogenetic analysis based on conventional staining, banding techniques and fluorescence in situ hybridization in the species I. monticola, for which previous cytogenetic investigations did not detect differentiated sex chromosomes. The karyotype is composed of 2n = 36 acrocentric chromosomes. NORs and the major ribosomal genes were located in the subtelomeric region of chromosome pair 6. Hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes and interstitially in 5 chromosome pairs. C-banding showed constitutive heterochromatin at the centromeres of all chromosomes, as well as clear pericentromeric and light telomeric C-bands in several chromosome pairs. These results highlight some chromosomal markers which can be useful to identify species specific diagnostic characters, although they may not accurately reflect the phylogenetic relationships among the taxa. In addition, C-banding revealed the presence of a heteromorphic ZW sex chromosome pair, where W is smaller than Z and almost completely heterochromatic. This finding sheds light on sex chromosome evolution in the genus Iberolacerta and suggests that further comparative cytogenetic analyses are needed to understand the processes underlying the origin, differentiation and plasticity of sex chromosome systems in lacertid lizards.Xunta de Galicia; PGIDIT03RFO10301PRXunta de Galicia; PGIDIT06RFO10301PRItalia. Ministero dell'istruzione, dell'università e della ricerca; PRIN 200