Download 1 Natural hybridization – crossings in nature between individuals

Natural hybridization – crossings in nature between individuals belonging to different species
(Harrison 1993) – is one of the central themes for the scientific community. The importance of
natural hybridization is given by its evolutionary consequences: formation of new species
(Rieseberg 1997, Annual Review of Ecology and Systematics), increase of interspecific genetic
variation (Anderson 1948), transfer of favourable genes (alleles) between species (Arnold 2004,
Plant Cell), and invasion of one species into the range of other species (Petit et al. 2004, New
Phytologist). The renewed interest in studying the interspecific gene flow, in particular in a series of
plant and animal organisms, appears to be in strong connection with big advances in molecular
biology and statistical tools (Bayesian analysis) as well (e.g. Burgarella et al. 2009, Heredity;
Pritchard et al. 2000, Genetics; Evanno et al. 2005, Molecular Ecology).
Introgressive hybridization – incorporation of genes of one species into the gene pool of other
species via repeated backcrossings – has been invoked to explain the high levels of phenotypic
variation in many plant genera. Among the tree species, maybe the most known example is the
Quercus species complex. Due to the difficulties of delimiting oak species, Otto Schwarz – the
monographer of European oaks – named the genus Quercus „crux botanicorum” (Schwarz 1937,
Monographie der Eichen Europas). The observation of intermediate morphological forms between
oak species, in particular after leaf descriptors, has been considered as an evidence of hybridization.
However, recent studies indicated that individuals showing intermediate morphologies are not
necessarily first-generation hybrids (F1) and introgressed forms. By contrast, individual trees
showing a typical morphology for one or another oak species maybe in reality hybrids (e.g. Curtu et
al. 2007, BMC Evolutionary Biology, Lepais et al. 2009, Molecular Ecology; Penaloza-Ramirez et
al. 2010, Annals of Botany). Consequently, the genetic analyses are essential for a better estimation
of hybridization occurrence.
Many types of genetic markers – visible, detectable characters controlled by the alleles of single
genes – have been used for studying hybridization. Useful information about past hybridization
events has been obtained from the chloroplast DNA genome (cpDNA), which is maternally
inherited, as in most of the angiosperm species (Petit et al. 2004). However, the nuclear markers,
which are biparentally inherited (e.g. isozymes, AFLPs, simple sequence repeats - SSRs) are much
more indicated in studies on hybridization. In particular, the highly polymorphic DNA markers
(over 20-30 alleles per locus in oak populations) allowed a better detection of hybridization events
in the genome (Curtu et al. 2007; Plant Biology). For instance, identification of hybrids up to the
second generation was made based on a set of only 10 simple sequence repeats (Lepais et al. 2009,
Molecular Ecology; Viscosi et al. 2009, Pant Biosystems). In the last period, the availability of
expressed sequence tags (EST) for oaks has increased (e.g. within the European Network of
Excelence EVOLTREE: Evolution of Trees as Drivers of Terrestrial Biodiversity) which will lead to
the development of new molecular markers. This last category of genetic markers which are located
in the transcribed regions of the genome (EST-SSRs), may reveal o higher genetic differentiation
among oak species (Pashley et al. 2006, Journal of Heredity; Ellis and Burke 2007, Heredity). By
using these markers the identification of later generation hybrids will be more facile.
Few studies have estimated the actual level of hybridization by means of paternity analyses
(Curtu et al. 2009, BMC Evolutionary Biology; Savini et al. 2008, Plant Biology; Streiff et al. 1999
Molecular Ecology). These analyses were made on acorns collected from mother trees distributed
throughout mixed oak forests. However, the study of actual patterns of hybridization has been
confined to a single year and in the absence of any data about the flowering phenology. The
existence of prezygotic mechanisms (e.g. phenology) may influence the level and direction of
hybridization.
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The majority of the studies on natural hybridization has been focused on one pair of species, in the
case of genus Quercus, on pedunculate (Quercus robur) and sessile oak (Q. petraea) (e.g. Bacilieri
et al. 1996, Evolution; Gugerli et al. 2007, Annals of Botany). A few studies have investigated the
interspecific gene flow in multispecies oak hybrid zones (Dod şi Afzal-Rafii, 2004, Evolution; Curtu
et al. 2007, BMC Evolutionary Biology; Lepais et al. 2009, Molecular Ecology). The single genetic
study on hybridization among the Romanian oaks has been carried out in Bejan-Deva Oak Reserve
(Curtu 2006). Bejan forest represents a zone of secondary contact between four oak species from
genus Lepidobalanus (white oaks). In the present project we propose an extension of the study site
at Bejan and the additional sampling of 8 pure populations, two of which for every white oak
species occurring in Romania. The material originating from four oak species (Q. robur, Q. petraea,
Q. frainetto and Q. pubescens) will be analysed by means of SSRs and EST-SSRs markers. The
input of reference pure populations for each oak species in the assignment procedure will enable us
a better classification, based on the multilocus genotypes, in pure species, first generation hybrids
and introgressed forms. A set of nuclear genetic markers will be tested for species delimitation and
hybrid identification among the Romanian white oaks. To better characterize the extend and
dynamics of natural hybridization, a sample of populations with various ecological settings is
needed (Viscosi et al. 2009, Pant Biosystems, Buerkle 2009, Molecular Ecology). Consequently, the
present study will include oak populations from different ecological regions distributed throughout
Romania.
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