Abstract of the Ph. D. thesis of Paulo Guilherme Leandro de Oliveira
Isoenzyme Analysis for the Identification of Hybrids of Cork Oak and Holm Oak
University of Évora, Portugal
2006
In view of the persisting uncertainties on the hybrid origin of cork oak trees that produce the so-called "preguenta" cork, a question impossible to ignore in the recurring discussion over the mixed stands with cork oak and holm oak in Portugal, and in order to obtain credible estimates of the hybridization rates in these stands, the present study aimed at establishing genetic criteria for the discrimination between the two species and their hybrids.
This study was part of the PAMAF Project 8153 (Instituto Nacional de Investigação Agrária) approved in 1997, coordinated by Prof. Carola Meierrose, of the Biology Department, University of Évora, in which the University of Évora, the National Forestry Station (Estação Florestal Nacional) and the Regional Office for Agriculture in Alentejo (Direcção Regional de Agricultura do Alentejo) took part. The option taken was to analyse enzymatic markers, under the hypothesis that interspecific variations in some of them would be diagnostic between the two species and their hybrids, in part because isoenzymes are very suitable for large-scale screenings, and also because at the time practically no DNA analysis was made on cork oak, not even internationally.
To monitor the occurrence of new hybrids, three mixed
stands were selected and in 1998 acorns were collected from trees of both
species, which were sown and raised in a nursery. The same was done in reference
stands ("pure", either of cork or holm oak) located at distances from the other
species that would a priori preclude any contact. The seedlings that were
obtained made for most of the analysed material, the remainder including the
adults from the mixed stands, to detect possible hybridization within that
generation and, through the verification of the genotypes in the progenies, to
confirm the kinship, important because the acorns were collected on the ground.
Thus each adult within the mixed stands was marked and numbered. Finally,
another reference were the hybrids themselves, and the project set out to make
an inventory of them, while sampling the progenies from some of them.
Integration with studies on
flowering The same project coupled isoenzyme analysis with records
of flowering and fruiting on the adults. It was assumed that the opportunity for
hybridization to occur increases with a higher overlap between the flowering
time spans of each species each year. Holm oaks precede cork oaks by a few
weeks, but this separation is only between average dates, is variable from year
to year, for example in response to different temperature profiles, and is of
little practical significance because of the large intraspecific scatter among
individuals, in their flowering timings, in both species. Hence, marking the
adults in each mixed stand also allowed monitoring the phenology of flowering in
each individual for the year of 1998, for the event that, based on the timing
proximity of one tree with those of the other species, its progeny could be
considered more or less likely to contain hybrids. Specific component of this study This thesis refers mainly to the isoenzyme analysis
component of the project. With a methodological implementation projecting a
future capacity for processing large samples, genetic markers were defined for
the discrimination between cork oak and holm oak, and their hybrids, and their
analysis among the progenies, beyond the purpose of detecting hybrids, also
allowed the preliminary characterization of the biology of reproduction in the
two species and the hybrids. Most of the enzyme activities tested in leaf extracts
from cork oak or holm oak produced some signal in at least one separation
system, but only a minority (11 in 35) were considered consistent. Of the
remainder, 9 gave good results in one single attempt and might be confirmed on
future trials. In seed extracts the results were essentially the same as those
in leaves, but at least 1 was consistent only in the former. Some activities
were detected only in leaves from other species of dicotyledons, but not in
those from species of Quercus, thus excluding that the extraction method
in itself caused a failure in detecting them. There were 8 enzyme activities that, after routine
analysis in leaf extracts and seeds from the reference and mixed stands, and
hybrids, could contribute for the dircrimination between cork oak, holm oak and
their hybrids: diaphorase (DIA), malate dehydrogenase (MDH), glutathione
reductase (GsR), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase
(PGD), 6-phosphoglucose isomerase (PGI) and esterases
(EST). Other activities appear to show the same potential, but
are yet to be confirmed under routine analyses: high isoelectric point esterases
(EST) and fluorogenic acid phosphatases (ACP-F), and in seeds only, L-glutamate
dehydrogenase (GDH). Genetic interpretations for the polymorphisms in DIA,
GsR, PGM, PGI and (partly) MDH) were achieved. Applications of the enzyme markers electrophoretic
analysis From the routine analyses primarily designed for the
validation of discriminant markers and the detection of hybrids, it was possible
to: Confirm the detected interspecific, and part of the
intraspecific, contaminants; partially characterise 9 hybrids, one among them
(SM2) possibly the product of a backcross between a hybrid and a cork oak; in
the absence of hybrids in the mixed stands progenies of 1998, compute a high
boundary for the relevant probability of hybridization, for which the most
conservative estimate is P < 0,043%; characterise adults and seedlings with
atypical phenotypes, the number of which appears to be very low; characterise
samples from 45 "preguenta" cork trees, with two markers (DIA and PER) giving
signs of possible introgression of holm oak genes in some of them; quantify the
specificity and sensitivity of each marker, used in the routine analyses, for
distinguishing hybrids and holm oaks from cork oak; determine the pollen origin
for each seedling from the hybrids SM1, SM2 and SES; analyse the intraspecific
frequencies at the loci Pgm (holm oak) and Pgi-B (cork oak), thus
showing the ability to treat the three mixed stands as one population, in each
species, and the computation of various population genetic estimators; reveal
disparities between the distribution frequencies in pollen, female gametophytes
and seedlings, especially at Pgm, allowing the estimation of neutral
values for Ne in the order of 5 to 7 in both species; estimate the
percentage of unrelated pollen at 81% in holm oak (Pgm) and 84% in cork
oak (Pgi-B). The analysis of cork oak seedlings from provenances of
the 7 countries spanning the species range has not revealed any isoenzyme
pattern contradicting the specificity of the hybridization markers defined on
the basis of the reference stands (Santiago do Cacém, Azeiteiros and Testa) and
mixed stands (Mitra, Feijoas do Ramos and Alfaiates). The application of the same extraction protocol to other
species in the order Fagales showed a more or less striking variation of the
isoezyme patterns between taxonomic levels (variably according to the
activities) and confirmed the potential of application of these markers to
discrimination work between other taxa. The present work contains the following progress in the
initially proposed research: 1. Discovery and validation of isoenzyme markers for the
distinction between cork oak and hybrids and their
descendants; 2. Confirmation, with genetic markers, of the hybrid
status of the trees known as "cerqueiro" oaks; 3. Estimate of a very low hybridization rate (less than
0.1%) in the mixed stands studied; 4. Detection, in cork oak as well as in holm oak, of
markers of introgression from the other species; 5. Preliminary analysis of cork oaks that produce
"preguenta" cork; 6. Preliminary analysis of hybrid progenies, including
the demonstration that pollen grains from either of the two parental species are
involved, as well as a relatively high selfing rate; 7. Preliminary analysis of the allelic frequencies in two
marker loci, one of each species, and derivation of a relatively low neutral
value of Ne; 8. Demonstration of the same methodology in other species
of the genus Quercus and related taxa. Genetic demonstration of the "cerqueiro" oaks hybrid
character The hybrid status of the "cerqueiro" oaks was verified
based on genetic markers (isoenzymes), thus contributing to establish these
markers as efficient means to detect hybrids between cork oak and holm oak. The
fact that this approach is feasible on leaf material removes limitations that
might be posed by age or season. Fertility of the "cerqueiro" hybrids and production of
recombinants Knowing the heredity for the discriminating isoenzymes,
although with some limtations, allowed the identification in the progenies of 3
hybrids of the type of pollen from which each descendant derived, and from that
the realization that the hybrids are fertile with pollen either of cork oak,
holm oak or hybrids, the latter to a proportion apparently very high for what is
expected in Quercus. Although inefficient, because of the small number of
markers, to verify the conjecture of introgressed holm oak genes in the
"preguenta"-producing cork oak trees, the results from a limited sampling of
such trees, along with demonstration of fertility of these hybrids with cork
oak, favour this conjecture. The absence of hybrids among the progenies from cork and
holm oaks in the mixed stands allowed only the calculation of a high boundary
for their actual incidence, estimated at very low values. The biology of
reproduction of both species, based on the evidence from intraspecific
polymorphic markers, leads to postulate that, along with the a priori
relevant factor for hybridization that topographic proximity between individuals
of the two species must be, an overlap of flowering times must also exist but is
not sufficient; the individual compatibility between mating trees must also play
some role. Therefore, the incidence of hybridization is predicted to be very
low, but it might occur at higher rates in foci where for several years all
these factors coincide. The frequency at which it is said that another type of
cork oak called "negral" occurs in Alentejo is not compatible with the estimates
made, hence it is supposed that this designation is not for real
hybrids. The present study allows to envisage several lines of
investigation, where the enzyme markers used in large scale in the present
study, and others that in the meantime are proven useful, will play a relevant
role. The verification of the incidence of hybridization in mixed stands, based
on a sampling spanning several years and encompassing different provenances, the
study of the hybrids in their biology of reproduction, and of the ritidome
development in their recombinant progenies, are very interdependent. The
isoenzyme markers can be also of great use in the research on the presumed holm
oak introgression in cork oak trees that produce "preguenta" cork and in the
"negral" type. University of Évora, on November
2006