STUDIO DELLA VARIABILITA’ E DELLA CONNETTIVITA’ GENETICA IN POPOLAZIONI RELITTE DI Platanus orientalis UNIVERSITA’ DEGLI STUDI DI NAPOLI FEDERICO II DOTTORATO DI RICERCA IN BIOLOGIA AVANZATA XXVIII CICLO STUDIO DELLA VARIABILITA’ E DELLA CONNETTIVITA’ GENETICA IN POPOLAZIONI RELITTE DI Platanus orientalis Coordinatore del corso Ch.mo Prof. Luciano Gaudio Tutor Candidata Ch.mo Prof. Salvatore Cozzolino Dott.ssa Rosita Rinaldi 1 STUDIO DELLA VARIABILITA’ E DELLA CONNETTIVITA’ GENETICA IN POPOLAZIONI RELITTE DI Platanus orientalis ABSTRACT Genetic diversity and connectivity in relict populations of Platanus orientalis. Mediterranean ecosystem, due to its complex paleoclimatic history, hosts important residual of past vegetation that survives with a scattered natural range by occupying restricted ecological niches. Massive changes in vegetation have occurred recurrently during the succession of glacial and interglacial periods in Europe. Several members of the Tertiary tree flora disappeared from the European continent during Quaternary glaciations, with the exception of few relict species that survived in refuge areas in southern and south-eastern Mediterranean. For these relict species information about the distribution of genetic diversity and populations genetic structure is fundamental for determining in situ and ex situ conservation strategies. In particular, these relict species, often distributed in small and scattered populations are expected to display very low levels of diversity due to the effect of genetic drift and to the restricted inter-populations gene flow. For these species the genetic characterization of individuals, if coupled with information from their spatial distribution, age and pattern of relationship among individuals, can allow a fine-scale genetic spatial structure of the relict populations. This is of special interest because the spatial arrangement of related individuals within populations provides also insights into pollen and seed dispersal patterns. Finally, because pollination is often between near neighbours, an understanding of fine-scale genetic structure may allow predictions concerning the likelihood of selfing or biparental inbreeding. Among relict tree species, Platanus orientalis L. is an Euro-Asiatic floristic element of warm riparian forests and southern Italy represents the westernmost limit of its distribution. Here we compared levels of genetic diversity and connectivity within and among populations of P. orientalis selected from the peripheral and central part of its distribution. For this aim we used microsatellites markers in order to: (1) measure genetic diversity at different levels (individual, population, species); (2) analyse gene flow within and among populations; (3) perform paternity and parentage analysis to evaluate the contribution of seed and pollen dispersion to population connectivity. The analysis was conducted considering seven microsatellites loci for a total of 429 individuals from 17 populations distributed in South Italy, Greece, Albania, Turkey and Bulgaria selected as representative of peripheral and central distribution. The total number of detected alleles was 38. Populations from central distribution always show polymorphism in all examined loci while most of loci were often found fixed in the peripheral Italian populations. These latter were also the ones with the lowest values of observed and expected heterozygosity (the lowest value, Ho=0.15541 and He=0.29551, found in the Campanian population). The genetic differentiation between populations, estimated as proportion of total diversity, was calculated through the coefficient of Fst. Fst values were higher among the Italian populations (average Fst=0.3547) than among populations from the central distribution (average Fst =0.1213) indicating a greater isolation and then an higher differentiation among the Italian populations. These results were also supported by AMOVA analysis that showed most of the observed genetic variation occurring within population (63%) and not among 2 STUDIO DELLA VARIABILITA’ E DELLA CONNETTIVITA’ GENETICA IN POPOLAZIONI RELITTE DI Platanus orientalis populations (37%). According to this analysis, the small population size and the low genetic connectivity detected among Italian populations support a scenario where genetic drift plays a major role in shaping the diversity of P. orientalis populations at the peripheral of its range. Paternity analyses on seeds experimentally tested the effective seed and pollen dispersion within and among isolated populations. Plastid DNA sequencing supported a scenario of two main phylogeographic lineages as also outlined by population genetic analyses with Sicilian populations connected to east Mediterranean populations and Italian peninsular population connected to a continental Greek lineages. The genetic bottleneck and the low connectivity among the peripheral populations could potentially determine extinction of these residual populations. 3 STUDIO DELLA VARIABILITA’ E DELLA CONNETTIVITA’ GENETICA IN POPOLAZIONI RELITTE DI Platanus orientalis INDICE INTRODUZIONE ............................................................................................................................................... 5 BIODIVERSITA’ E HOTSPOT NELL’AREA MEDITERRANEA ............................................................................. 5 STORIA GEOLOGICA DELL’AREA DEL MEDITERRANEO ............................................................................... 12 STORIA PALEOCLIMATICA DEL MEDITERRANEO ........................................................................................ 15 STORIA DELLA VEGETAZIONE NEL MEDITERRANEO ................................................................................... 21 TAXA ATTUALMETE A DISTRIBUZIONE RELITTA ......................................................................................... 30 BIOGEOGRAFIA STORICA DELLA FAMIGLIA DELLE PLATANACEAE ............................................................. 37 IL GENERE PLATANUS ................................................................................................................................. 41 MITI E LEGGENDE LEGATE AL PLATANO ..................................................................................................... 47 PRINCIPALI FATTORI CHE INFLUENZANO LA DIVERSITA’ GENETICA DELLE POPOLAZIONI RELITTE DI Platanus orientalis E SCOPO DELLA TESI .................................................................................................... 60 MATERIALI E METODI ...................................................................................................................................71 ESTRAZIONE DEL DNA GENOMICO DA MATERIALE FRESCO E CAMPIONI D’ERBARIO .................................71 ELETTROFORESI SU GEL D’AGAROSIO ...........................................................................................................73 AMPLIFICAZIONE DELLE REGIONI MICROSATELLITARI ..................................................................................74 ELETTROFORESI CAPILLARE SU MICROSATELLITI ..........................................................................................77 ANALISI DEGLI ELETTROFEROGRAMMI: ASSEGNAZIONE ALLELICA E DETRMINAZIONE DEL GENOTIPO.....79 ANALISI STATISTICHE SU DATI MICROSATELLITARI .......................................................................................81 LIBRARY MICROSATELLITARE ........................................................................................................................84 ESPERIMENTI DI GERMINAZIONE ..................................................................................................................88 ANALISI DI PATERNITA’ ..................................................................................................................................91 AMPLIFICAZIONE DEI MARCATORI PLASTIDIALI VIA PCR .............................................................................92 REAZIONI ED ANALISI DI SEQUENZA .............................................................................................................95 ESTRAZIONE DEL DNA PLASTIDIALE ..............................................................................................................95 NEXT GENERATION SEQUENCING (NGS) .......................................................................................................97 RISULTATI E DISCUSSIONE ............................................................................................................................98 AREE DI CAMPIONAMENTO ..........................................................................................................................98 ANALISI DELLA VARIAZIONE ALLELICA MICROSATELLITARE ..........................................................................99 ANALISI DEL DNA PLASTIDIALE ....................................................................................................................125 CONCLUSIONI ..............................................................................................................................................134 BIBLIOGRAFIA .............................................................................................................................................137 4 STUDIO DELLA VARIABILITA’ E DELLA CONNETTIVITA’ GENETICA IN POPOLAZIONI RELITTE DI Platanus orientalis INTRODUZIONE BIODIVERSITA’ E HOTSPOT NELL’AREA MEDITERRANEA Il termine biodiversità è un neologismo entrato nell’uso quotidiano circa