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11
Multiple Approach for Plant Biodiversity
Conservation in Restoring Forests
Federico Vessella, Bartolomeo Schirone and Marco Cosimo Simeone
Department of Forest and Environment (D.A.F.) – University of Tuscia
Italy
1. Introduction
The current extinction crisis requires dramatic action to save the Earth’ s biological diversity.
In the mid-1980’s the word “biodiversity” was coined to catch the essence of research into
the variety and richness of life on Earth, that is, the variety of life expressed at many levels
(Wilson & Peter, 1986). These levels include the genetic diversity within species as well as
the array of genera, families, and still higher taxonomic levels that, taken together, comprise
communities of organisms within particular habitats and physical conditions that form
entire ecosystems. It is widely demonstrated that more species contribute to a greater
ecosystemic stability. Moreover, individuals, populations and ecosystems are tightly linked
and interact to maintain landscapes, large socio-economic systems and man’s health. As a
consequence, biodiversity maintenance is fundamental for the planet life, and should be
carried out with “passive” conservation measures implemented with “active” procedures
using the most recent progress in technique and policy. In this context, reforestation
programmes have to be considered as dynamic actions devoted to the biodiversity
conservation toward the recovery and/or the enlargement of such areas essential for
coenosis’ evolution. This concept of reforestation is relatively new and still has difficulty to
be established.
Most conservation biologists recognize that although we can not save everything, we should
at least ensure that all ecosystem and habitat types are represented within regional
conservation strategies that have been applied at a number of geographical scales, from
single watersheds to entire continents (Hummel, 1989; Eriksson et al., 1993; Caldecott et al.,
1994; Krever et al., 1994; Noss & Cooperrider, 1994; BSP et al., 1995; Dinerstein et al., 1995;
UNEP, 1995; Ricketts et al., 1999; Abell et al., 2000).
Forests are the single most important repositories of terrestrial biological diversity. They
provide a wide range of products and services to people throughout the world. Forest trees
and other woody plants help support many other organisms, and have developed complex
mechanisms to maintain high levels of genetic diversity. This genetic variation, both inter-
and intraspecific, serves a number of fundamentally important purposes. It allows trees and
shrubs to react to changes in the environment, including those brought about by pests,
diseases and climatic change. It provides the building blocks for future evolution, selection
and human use in breeding for a wide range of sites and uses. And, at different levels, it
supports the aesthetic, ethical and spiritual values of humans. Forest management for
Research in Biodiversity – Models and Applications
224
productive and protective purposes can and should be rendered compatible with
conservation through sound planning and coordination of activities at different
geographical levels. Accordingly, the conservation of these resources should be seen as an
attempt to preserve groups of genotypes or populations, and their various combinations of
genes (Gregorius, 1991). Therefore, the aim of forest resource management is to maintain
conditions in which the genetic makeup of a species can continue to evolve in response to
changes in its environment (Eriksson, 2001). At the same time, management for conservation
aims at reducing the rates of genetic erosion (FAO et al., 2004).
Different conservation strategies and practices have been developed. In situ (‘in place’)
conservation implies the continuing maintenance of a population within the environment
where it originally evolved, and to which we assume it is adapted (Frankel, 1976); this type
of conservation is most frequently applied to wild populations regenerated naturally in
protected areas or managed forests, but can include artificial regeneration whenever
planting or sowing is carried out, without directional selection, in the same area where the
seed was collected. In situ conservation in general has the advantage of conserving the
function of an ecosystem rather than just species. This means that in situ programmes for
conservation of selected target species often result in valuable conservation of a number of
associated animal and plant species (Thomson et al., 2001). Ex situ (‘out of place’)
conservation measures are mainly concerned with sampling and maintaining as much of the
genetic variation as possible that resides within and among populations of selected target
species. Ex situ conservation requires substantial levels of human intervention, in the form
either of simple seed collections, storage and field plantings or of more intensive plant
breeding and improvement approaches. Unlike breeders of agricultural crops, forest tree
breeders cannot rapidly produce new varieties, nor can they quickly breed for new
variations among populations. Therefore, the existing genetic diversity among populations
is important and fundamental to the conservation of forest genetic resources, particularly as
it may relate to maintaining genetic diversity in viable populations in the long term. This
also suggests that special attention must be given to conserving intraspecific genetic
variation in peripheral or isolated populations, as they could possess higher levels of
characteristics such as drought resistance, tolerance to various soil conditions (Stern &
Roche, 1974), or features that will help to protect them from future climate change (Muller-
Starck & Schubert, 2001). The important features of an ex situ conservation programme for
any particular species are: to be an important backup measure should other in situ
conservation means be unworkable or unavailable, to ensure that a wide range of the
diversity (phenotypic and genotypic) available in a species is conserved, and to manage the
regeneration of the species outside its original natural range (provenance) in a more
controlled way (which is likely to further develop the population(s) for use or conservation)
(Amaral & Yanchuk, 2004).
Recently, a European funded project, EUFGIS (European Information System on Forest
genetic Resources) established a web-based information system to serve as a documentation
platform for national forest genetic resources inventories and to support practical
implementation of gene conservation and sustainable forest management in Europe (further
information at http://www.eufgis.org). The main purpose was to assess pan-European
minimum requirements and data standards for the dynamic gene conservation units of
forest trees, i.e. selected areas which emphasizes the maintenance of evolutionary processes
within tree populations to safeguard their potential for continuous adaptation.
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
225
Those tools are adopted in programmes devote to afford issues such as the conversion of
forest land to other uses. Increasing pressure from human populations who aspire to higher
standards of living, without balancing the sustainability of resource utilization
underpinning such developments, raises concerns in this regard. It is inevitable that changes
of land use will occur in the future, but such changes should be planned to help ensure that
the complementary goals of conservation and development are achieved. In recent times
there has been a growing awareness in this topic, and the research community is looking for
moving away from the dominant focus on deforestation and resources’ conservation to
examine the patterns and the processes associated with reforesting landscapes (Rudel, 2005).
Developing a more comprehensive understanding of the factors that can help to promote
reforestation is therefore critical, if we are to increase useful policy interventions to arrest or
reverse deforestation, and encourage forest regrowth. Yet, it is important to recognize that
forests are embedded within larger-level ecological, socio-economic and political settings,
which have the capacity to significantly influence outcomes. Thus, discussions of context
(biophysical, geographic, ecological, socio-economic and institutional) are essential to the
development of our understanding of this area of study (Nagendra & Southworth, 2009).
This implies awareness of the availability of efficient tools to comply with traditional
management strategies, as well as action plans and guide lines at large scale. Under these
circumstances the Council of the European Union promoted a legislative tool in 1999 that
recognized social, economic, environmental, ecological and cultural functions of forests.
Both the restocking of these forests and new afforestation require a sustainable forest
management in relation to the Forestry Strategy for the European Union, that include the
use of reproductive material which is genetically and phenotypically suited to the site and
of high quality (European Council, 1999). In this context, the definition and delimitation of
Regions of Provenance have been proposed as fundamental to select reproductive material
and to approve basic material with highest possible standards.
However, an improvement of genetic knowledge about forest plants is surely required to
accomplish the requirements of the Directive, and should also contribute to better define
what inter- and intra-specific biodiversity is. One of the latest standardized molecular
approach is DNA Barcoding (Hebert et al., 2003) that identifies living organisms by joining
specific sequences of DNA and electronic information retrieval. Biodiversity
characterization and improvements in genetic knowledge would be two of the main benefits
of the widespread application of Barcoding, in terms of speed, low cost, reliability, and
improved resolution power. Besides taxonomy, a powerful research complement for
molecular ecology, diversity studies and population genetics is clearly to be expected. DNA
Barcoding may lead to many useful applications in forestry sciences, such as community
ecology (to describe plant-animal interactions and vegetation dynamics/changes),
biodiversity surveys (aimed at habitat and species protection), silviculture (to assess forest
regeneration), and nursery activities and market regulation (to establish wood, secondary
products and germplasm certification). Conversely, it must be emphasized that some
species-rich tree genera may prove very difficult to barcode, especially those in which
species circumscription is affected by complicated taxonomies, biogeographies and/or
reproductive biology.
Since plant biodiversity is strictly related to natural restoration and rehabilitation of
ecosystem functions, with respect to its health, integrity, and sustainability, all the tools
mentioned above are linked to the reforestation techniques proposed by scientists and
Research in Biodiversity – Models and Applications
226
experts from decades. New insights both in theoretical and in practical actions have been
developed as innovative methods to foresters and ecological specialists. Among these, the
Miyawaki method based on the vegetation-ecological theories, seems to be a reliable
approach that include the principles of self-organized criticality and cooperation theories in
forest ecosystems, also fulfilling the objectives anticipated by the Regions of Provenance.
The tools mentioned above face the same issues, and try to get practical solutions for
conserving and/or restoring forests. However, they approach plant biodiversity
conservation in a piecemeal fashion with projects and management plans focusing narrowly
on one or a small range of techniques that coincide with the responsibilities, philosophies,
and capabilities of the people working in a given setting.
In this chapter we would like to propose a multiple approach as a potentially powerful
system for facing the challenge of conserving, but mainly expanding forests over long time
horizons. A critical review on the methods mentioned above toward an holistic point of
view will be discussed. We believe that multidisciplinary would be the way to follow out,
and our effort regarded the setting up of a linkage between the mentioned strategies and
practices. In the next paragraphs a deeper description of Regions of Provenance, DNA
Barcoding, and reforestation using Miyawaki method will be presented, focusing on recent
achieved results, improvements and proposals. Some study cases in Italy will be also shown,
in order to shed some light on the criteria for detecting best actions in the Mediterranean
Basin. Finally, we will attempt to logically order these tools in an improved and well-
organized “toolbox”.
2. Regions of Provenance in Europe
The relationship between genetic variability and adaptability for a species is particularly
important if we refer to forest plants, because they are characterized by long life cycle and
consequently more exposed to environmental changes. Looking at biodiversity within a
single species, very important are those populations with specific adaptations that could
characterize local ecotypes. When populations are geographically separated and genetic flux
is interrupted, differentiation processes can lead to speciation.
In many countries, the uncontrolled use of germplasm of unknown origin favoured serious
phenomena of genetic erosion and pollution, in particular after the implementation of the
Regulation EEC 2080/92 which encouraged the reforestation of agricultural land. In Italy,
for instance, the Rural Development Programme 2000-2006 promoted reforestation on huge
surfaces, and many land owners joined the program. The lack of enough autochthonous
propagation material to supply the demand, leaded the operators to use plant material from
several ecologically different geographical areas; as a result , many reforestation plans failed
because of diverse pedoclimatic requirements of the adopted material, and with the rising
up of infestations by new parasites.
For these reasons, the use of high quality propagation material, phenotipically and
genetically appropriate to the plantation area is fundamental. Such principle, previously
introduced in two European Directives (EEC 404/66 and EEC 161/71) was finally integrated
in the Directive EC 105/99 about the marketing of forest reproductive material. Moreover,
the Directive establishes that the basic material for reforestation has to be harvested from
selected stands, and underlines the importance of delimiting Regions of Provenance,
defined as “the area or group of areas subject to sufficiently uniform ecological conditions in
which stands or seed sources showing similar phenotypic or genetic characters are found,
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
227
taking into account altitudinal boundaries where appropriate”. It is also specified that forest
reproductive material (seeds, cones, fruits, parts of plants, planting stocks) is classified in the
following categories: Source-identified, Selected, Qualified and Tested. About the source-
identified and the selected materials, they belong to seed sources, stands, seed orchards,
parents of family, clones or clonal mixtures located in a single Region of Provenance.
Selected materials include also the phenotypic ones, identified at population level, and
fulfilling the requirements of origin, isolation, population dimension, age and development,
homogeneity, phytosanitary status, quali-quantitative production, status and morphology.
Indeed, the Directive highlights the need for each Member State to define the Regions of
Provenance for a correct use of reproductive material, in order to ensure forest biodiversity
conservation with specific regards to the nursery activities. However, for reforestation
practices, the Directive’s contents does not suggest the use of basic material in accordance
with the Regions of Provenance.
In the last twenty years, many European countries developed management systems based
on ecoregions, adopting national measures accordingly to the Communitarian legislation.
Anyway, the delimitation of the Regions of Provenance is very elaborate, since it requires
the definition of the actual relationships between the ecological features of an area, the
ecophysiological characteristics of each species, the peculiar propagation dynamics
(pollination, dissemination, diffusion methods) of the species, and the intra-specific genetic
diversity at both the individual and the population levels.
2.1 Common criteria for the definition of the Regions of Provenance
The subdivision of the distribution range for a species in spatially and genetically
homogeneous regions complies the hypothesis of an intra-specific differentiation according
with the environmental selection effects. This argument is valid only if populations have
enough genetic variability to face, in terms of adaptability, and possibly to mild the
environmental changes that may occur within a certain physical area. Some reproductive
isolation derived from the genetic differentiation is a necessary prerequisite for allowing
adaptability processes at a local scale. Therefore, the delimitation of the Regions of
Provenance plays a key role in identifying those basic materials from which harvesting
forest reproductive materials. Despite the environmental and genetic homogeneities are
essential requirements to define different provenances, a weak point is detectable: the
genetic composition of a population, i.e. the main indicator of adaptability derived from the
evolutive processes, is commonly assessed throughout the analysis of the phenotypic
performance, while a description through the use of molecular markers would be more
appropriate. The adaptability at local environmental conditions, together with a genetic
peculiarity, are essential features to reveal the autochthony of a population, possibly
witnessed by historical documents. Recommendations by national and regional measures
underline the importance of autochthonous resources for environmental restoration, starting
from considerations about species’ adaptability. Referring to forest populations,
“autochthony” indicates the continuous occurrence of a species, in terms of genealogy, in a
defined site since the last post-glacial migration. However, adaptability, as a peculiar feature
of autochthonous populations, raises further considerations about the surface size where
populations occur, as well as the time they passed under the same environmental
conditions. For these reasons, the meaning of adaptability has been redefined several times,
but always focusing on the spatial and temporal continuity in constant environment
settings. Such quantitative characterization allows to consider the autochthony of a
Research in Biodiversity – Models and Applications
228
population as a phenomenon in terms of degree rather than presence/absence. This
suggests the need for defining the population extension, the size and structure, and the
assessment of a continuous occurrence through the time in a specific area. At the same time,
it is important to specify meta-populations’ structure preserving their reproductive
coherence by genetic flux. According to the spatial scale of investigation, the local
genealogic continuity could appear relatively low in some stands, because of punctual
extinction events or other disturbances. An appropriate spatial scale should be only
established after the understanding of the reproductive coherence within the species’
distribution range, and the analysis of the homogeneity of the environmental characteristics.
Temporal and spatial scales, and the degree of environmental heterogeneity could be
indirectly observed in the genetic structure of an autochthonous population, as the
consequence of evolutive processes of adaptability. This circumstance derived from the
presence of heterogeneity variation within a population; such variation has to be heritable,
so the availability of genetic diversity is fundamental.
As mentioned above, it is often hard to check for the main adaptability determinants, as well
as to accurately measure the features of autochthony. There is a significant mass of literature
about the most commonly applied methods for delimiting Regions of Provenance according
to the factors mentioned above (e.g. Geburek & Konrad, 2008; Kleinschmit et al., 2004;
Lindgren & Ying, 2000; Krusche & Geburek, 1991; Raymond & Lindgren, 1990). They
usually refer to the division of the territory (divisive method), if ecological parameters are
considered, instead of joining of similar populations (agglomerative method) according to
common biological features. Three clustering approaches are generally followed:
1. clusters according to homogeneous environmental conditions;
2. clusters according to genetic markers;
3. clusters according to phenotypic response.
The first procedure consists in grouping areas that share congruent ecological conditions.
The selected parameters useful to characterize these conditions are supposed to be
important for maintaining and expanding the referred species. The complexity of the
growth regulation phenomena make difficult the selection of such parameters; however,
some artificial delimitation of the Regions of Provenance based on administrative
boundaries have been adopted to facilitate the management procedures. A specific problem
raises when the reproductive coherence within a Region does not match with the potential
effects on the intra-regional genetic differentiation; this difficulty is typical of areas with
plantations, where the individuals have significant differences in terms of geographic origin,
and no genetic information resulting from the adaptation at the local conditions.
The second clustering procedure gives more emphasis on the genetic variability of every
single species, intended as peculiar feature for observing intra-specific differentiation at
level of regional areas, populations, individuals, etc., and needed for defining the Regions of
Provenance. Unfortunately, the correspondence between adaptability and neutral molecular
markers (e.g., isoenzymes, plastid and mitochondrial microsatellites) may not be sufficiently
clear to mirror differences between Regions of Provenance, and the more variable nuclear
markers (e.g., microsatellites, AFLPs) may prove too difficult and expensive for large scale
investigations (Karp et al., 1997; Duminil et al., 2007). On the other hand, adaptive traits
could be more efficiently dissected by use of QTLs analyses (Lewontin, 1984;
Borevitz and
Chory., 2004
), but the technical requirements of the method (and the complex biology of
trees) has limited the number of available information on a short number of world species.
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
229
Finally, the most recent advancements of molecular biology (identification and
characterization of ecologically important Candidate Genes, transcriptomics and
metagenomics) are highly promising but still hindered by prohibitive costs and difficulties
(Pflieger et al., 2001; Eveno et al., 2008; Derory et al., 2010).
The last procedure is based on statistical researches, starting from test areas, about
relationships between growth performance and environmental variables, such as stem
growth, bud set, flowering time, and geographic coordinates, photoperiod, altitude, etc.
These affinities are used to cluster populations according with specified range of values
calculated by statistical functions. Resulting clusters are built minimizing the distance
between the origin of propagation material and the area to under investigation.
In conclusion, all these criteria focus on reducing the risk connected with the transfer of the
material in ecologically heterogeneous areas. Such risks are evaluated in terms of adaptive
failure and undesired phenotypic traits (habitus, seed productivity, etc.), but the methods
applied are still under discussion and development. Indeed, every useful strategy devoted
to minimize the mentioned risks has to be based on a spatial delimitation consistent with the
real and/or potential adaptability of a population through the time.
The main goals to define provenances concern the species’ range, metapopulation and
subpopulation delimitation, the estimation of adaptability, and the assessment of the
adaptive effectiveness in terms of evolution. Therefore, the common methods to achieve
these goals involve a spatial-genetic clustering of at least a second generation of adult
individuals, the heterogeneity analysis of life mechanisms and functions, and the connexion
of adaptive phenotypic variability with the genetic one, developing transplant tests with
adaptive differentiation study.
In many Communitarian experiences, the definition of Regions of Provenance leaded to
delimitate large areas (e.g. oak species in Germany, Scots pine in the Baltic region), because
of the results obtained on experimental fields where the adaptive flexibility of several
populations were tested at different environmental conditions. In any case, Regions of
Provenance have to be directed to the preservation of the natural mechanisms of persistence
for a species within its range.
2.2 State of the art
In the European Union, several countries actuated the delimitation of the Regions of
Provenance for forest species after the adoption of the Directive EEC 161/71. The progress
project reports differ State by State, but practically match the requirements suggested by the
Communitarian normative. An important step concerns the mapping of species’ occurrence
in each country, and the characterization of homogeneous areas using ecological indexes. In
many cases, the Regions of Provenance have been delimited summarizing the results
obtained by the application of several clustering approaches, and taking also into account
regional and/or provincial administrative boundaries; such last conformation is particularly
useful for those countries which have commissioned the competence for environmental
topics to the local authorities.
In the following table the Member States adopting the Regions of Provenance in accordance
with the Directive 105/99 have been listed (thus, Norway has been excluded even if
adopting the Directive), providing synthetic information about used parameters and
number of target species (Table 1, modified from Alía et al., 2008). This summary table could
be subjected of updating, as the Regions of Provenance is a dynamic process still in progress
in several countries (e.g. in Italy).
Research in Biodiversity – Models and Applications
230
Countries
Parameters
AT BE CZ DE DK ES FI FR GB GR HU IT IR LT NL PL RO SE SI SK
Geographical
information
X X X X X X X X X X X X X X X X X X
Altitude X X X X X X X X X X X X X X X X X X
Climate X X X X X X X X X X X X X X X X X
Soil X X X X X X X X X X X X X X X X X
Neutral markers X X X X X X X X
Field test X X X X X X X X X
Nursery/phytotron
test
X X X X
Growth X X X X X X X
Phenology X X X X X X X X
Resistance to
disease/pests
X X X
Vegetation/
phytogeography
X X X X X X X X X
Overall adaptation X X X X X X X X X X X X
Administrative
boundaries
X X X X X X X X
N. of target species 24 37 23 50 36 56 14 53 54 10 25 78 28 9 22 10 35 17 11 7
Table 1. Overview of the criteria adopted by the countries in the European Union that have
defined the Regions of Provenance and total amount of forest target species. Short
abbreviations of country names are given according to the ISO 3166-1-alpha-2 code.
The differences highlighted between countries suggest some critical remarks. Against a
common acceptance of the Directive 105/99, there is not a pan-European strategy for the
Regions of Provenance; some difficulties could be detected such as the available information,
the possibility to exchange, and the different formats of the data. Moreover, large differences
regard the size and the methodologies for the delineation, the number of target species and the
knowledge about their biological parameters that varies from species to species.
A standardization process devoted to set up a common approach in the delineation of the
Regions of Provenance surely will take long time, as it requires both technical and political
actions. However, it is possible to suggest improvements for the methods, using additional
ecological variables and/or techniques to explore phenological and biological behaviours of
forest species not considered yet. A study case is presented in the next paragraph and
regards the delineation of Regions of Provenance in the Latium District (Italy).
2.3 New proposals and improvements for delineating Regions of Provenance: The
case of Latium
The clustering procedure according to homogeneous environmental conditions could be
considered as the easiest approach for defining Regions of Provenance, because of the large
databases about chemo-physical parameters that each country has stored since the
beginning of the last century. Generally, we have more information about the property of a
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
231
territory instead of the living species that occur there. These simple assumptions could
justify why the divisive method is often used. Nevertheless, additional variables could be
considered for improving the delimitation of the Regions of Provenance, for instance
phytoclimatic indexes as the Mitrakos Winter Cold Stress (WCS) or Summer Drought Stress
(SDS), and the Emberger coefficient (Mitrakos, 1980; Emberger, 1955). These parameters
demand for time-series climatic data, and refer to a data point network of weather stations
widespread in an area; but, it is possible to spatially extend them by using numerical and
mathematical techniques dealing with the characterization of spatial phenomena, using
geostatistic analyses that rely on statistical approaches based on random function theory to
model the uncertainty associated with spatial estimation and simulation. Using the
geostatistic methods, as implemented in many GIS softwares, it is also possible to go beyond
the interpolation problem by considering the studied phenomenon at unknown locations as
a set of correlated random variables. In the case of Latium, both Mitrakos indexes and
Emberger coefficient have been spatialized using Kriging interpolation from 85 data points
recording precipitation and temperature for 15 years at least; topography and continentality
have been also considered as supplementary variables extrapolating data from the Digital
Elevation Model (DEM) of Italy with 75x75 m grid cells. The resulting outputs have been
overlapped to other chemo-physical variables, i.e. mean annual temperature, minimum
temperature of the coldest month, maximum temperature of the warmest month, annual
precipitation, geomorphology, soil, etc. A summary layer storing all the spatialized
variables has been performed and areas with homogeneous ecological features have been
finally detected. Moreover, the boundaries of these areas have been buffered to better
represent the gradual spatial shifting from an ecological context to another (Figure 1).
According to the main phytoclimatic parameters, as well as the vegetation maps proposed
by several authors (Blasi, 1994; Tomaselli, 1973; Pavari, 1916), Latium has been divided in 3
Primary Regions of Provenance and subsequently in 17 Secondary Regions including also
the geomorphology and the soil characteristics (Figs. 2, 3). This procedure basically follows
the common strategies adopted by the other European countries, but increases the number
of variables to be considered for a deeper ecological investigation that includes plant
response to climatic conditions.
Since the evaluation of the effects of natural selection and bioclimatic responses across space
is at the base of the definition of the Regions of Provenance, a better characterization of basic
material should be achieved by combining already showed results with parameters related
to species performance (biological responses to ecological factors) and to the altitudinal
gradient (as suggested by the Directive 105/99), in order to provide homogeneous material
both for afforestation and genetic preservation. In those countries where the knowledge
about forest species’ biological and genetic features are studied from years, or for peculiar
species with great economic impact (e.g. Populus spp., Castanea sativa Mill., Quercus petraea
Liebl., Picea abies (L.) Karst., Pinus sylvestris L., Fagus sylvatica L., Quercus suber L., etc.) such
improvements have been made; in particular, as showed in Table 1, phenology, growth
performance, and neutral markers for genetic characterization are the most used
parameters. Nevertheless, it is possible to consider further investigations focusing on
bioindicator species’ behaviours, and extending the method to all forest species.
Dendroecology can contribute to these studies by improving the analysis of tree growth
response to environmental gradients, thereby refining the classifications that are based on
climate–vegetation interactions. This approach was previously taken on 17 beech forests in
central Italy (Latium and Abruzzi) to obtain horizontal and vertical gradients in tree–climate
Research in Biodiversity – Models and Applications
232
Fig. 1. Example of buffered boundary between two Regions of Provenance in Latium. The
buffer zone represents a gradual transition of the ecological characteristics from Region A to
B and vice-versa.
Fig. 2. Delineation of Primary Regions of Provenance for Latium using the improvements
cited in the text.
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
233
Fig. 3. Map of Secondary Regions of Provenance for Latium derived from phytoclimatic,
vegetation and chemo-physical variables.
relationships, thus providing the basis to assess bioclimatic units in terms of the leading
dendroclimatic signals (Piovesan et al., 2005). Evidence from tree-ring analysis
demonstrated that tree growth is strictly related to elevation, generating distinct beech forest
types. In agreement with previous studies (e.g. Biondi, 1992; Biondi & Visani, 1996; Dittmar
et al., 2003), distinctive radial growth–climate relationships uncovered in the tree-ring
network are organized along altitudinal and latitudinal gradients. Since beech could be
considered a good bioindicator, i.e. its dendroecological features are significantly related to
elevation, comparisons were extended to all the Latium forest surfaces, including the
altitudinal belt where beech is not present in the landscape, i.e. a non-beech belt with
bioclimatic traits that generally do not allow the growth of beech. The following results
could be considered a starting point for the selection of basic material used in genetic and
provenance studies, to accomplish the definition of the Regions of Provenance for Latium
previously showed. It is a new approach that checks the agreement between the
dendroclimatic classification and the phenological traits analyzed by remote sensing
measurements, expressed by the normalized difference vegetation index (NDVI).
The NDVI allows decadal (10 day) monitoring of terrestrial vegetation, at regional to global
scales, using the spectral reflectance measurements acquired in the red and near-infrared
regions. These spectral reflectances are themselves ratios of the reflected over the incoming
radiation in each spectral band. NDVI reflects the chlorophyll and carotenoid content in the
Research in Biodiversity – Models and Applications
234
leaves (Tucker & Sellers, 1986), but it is also related to leaf area index (LAI) (Fassnacht et al.,
1997) and the fraction of photosynthetically active radiation absorbed by leaves (fPAR)
(Veroustraete & Myneni, 1996). The NDVI expresses the greenness of a pixel, and it is a
good remote sensing methodology to detect interannual and seasonal changes in forest
ecosystems. Using the GIS software an NDVI time series spanning 11 years (1998–2008) was
developed. The data have a spatial resolution of 1x1km
2
. To detect the area covered by
broadleaved forests in Latium, the Corine Land Cover (CLC) database (3.1.1 classes—
Broadleaf woods) was used. Only pixels (1x1km
2
) with a forest area above 60% were used in
the analysis. Raster data were reprojected to the same coordinate system of the subset vector
grid map obtained, to overlap the CLC forest surface with satellite images. NDVI mean
values were calculated for each selected cell and partitioned using k-mean clustering. Four
fixed a priori clusters (referred to in the text as NDVI classes) were chosen to test the
correspondence with the four bioclimatic zones obtained by the dendroclimatic
classification.
The NDVI class assigned to each cell was graphically overlapped with bioclimatic
altitudinal belts, showing a good spatial correspondence between results obtained by the
dendroecological and the NDVI classification (Figure 4). The relative frequency distribution
of NDVI clustered cells per altitudinal belt ranged between 61% and 92% (Figure 5).
Fig. 4. Map of the spatial overlapping between normalized difference vegetation index
(NDVI) classes and altitudinal belts detected using the dendroclimatic approach.
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
235
Physical parameters, such as aspect or edaphic conditions, could play a fundamental role
where other non-correspondent NDVI class cells were present. This confirmed the general
role of elevation as a key factor in controlling both the growth and phenological
behaviour of forest stands in central Italy. The NDVI varied greatly among months and
NDVI classes, stressing the difference in photosynthetic activities throughout the growing
season of distinct forest bioclimatic belts; in particular the growing season length
shortened according to increasing elevation (Figure 6). These results assess that it is
possible to link tree-ring climate signals to phenology for each altitudinal belt by
combining the two methods, adding important clues to the further comprehension and
modelling of the bioclimatic organization of these forests. The two methods were
mutually validated, and therefore would be useful in defining Regions of Provenance as
agglomerative approach. The main benefit is in providing an automated approach at local
spatial scale useful to map these regions. The coupled dendroecological application and
NDVI can offer a prompt, economic and operative tool to check and manage
homogeneous ecological areas, objectively identifying Regions of Provenance according to
plant responses. Moreover, this approach could be combined with other biological and
genetic parameters, e.g. growth performance, resistance to diseases, DNA markers, for a
wider scenario of species’ behaviour. At the same time, matching the full dataset of
ecological, biological and genetic variables a more completed delineation of Regions of
Provenance could be achieved (Alessandrini et al., 2010).
Fig. 5. Pie charts of percentage correspondence between assigned normalized difference
vegetation index (NDVI) classes and tree-ring altitudinal belts. The panel below the charts
shows the number of cells per class and belt.
Research in Biodiversity – Models and Applications
236
Fig. 6. Annual normalized difference vegetation index (NDVI) profile of the four classes
obtained by k-means clustering.
3. DNA barcoding approach: A new challenge for species identification and
conservation
DNA barcoding is a standardized molecular approach to label living organisms by joining
specific sequences of DNA and electronic information retrieval (Hebert et al., 2003), and it
has recently become an increasingly attractive tool for species identification in terms of
accuracy, speed, cost and functionality. Ideally, a universal barcode system would be a
valuable resource to provide objective and worldwide comparable results, which can be
efficiently used in turn to compile biodiversity surveys in local floras (Lahaye et al., 2008;
Gonzalez et al., 2009; Kress et al., 2009, 2010). Additionally, the method allows the analysis
of poor, fragmented samples at any life stage (Chase et al., 2005) and it can be easily
repeated even by non-taxonomist specialists. The primary goals of barcoding are thus
species identification of known specimens and discovery of unnoticed species to enhance
taxonomy for the benefit of science and society (Kress & Erickson, 2008). The term “DNA
barcode” refers to a short DNA sequence-based identification system which may be
constructed of one locus or several loci used together as a complementary unit (Kress &
Erickson, 2007). Necessary prerequisites of DNA barcodes are ease of application across a
broad range of taxa, sufficient sequence variation to distinguish between species, and
absence of intra- and inter-specific diversity overlaps which would prevent rank definition.
Many studies have proved the efficacy of the mitochondrial cytochrome c oxidase 1 (COI or
cox1) gene sequence in barcoding animal groups such as birds (Hebert et al., 2004), fishes
(Ward et al., 2005), spiders (Greenstone et al., 2005), lepidopterans (Hajibabaei et al., 2006),
and amphibians (Smith et al., 2008), as well as in red algae (Robba et al., 2006) and fungi
(Seifert et al., 2007). In plants, the difficulty of finding a single-locus barcode has suggested a
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
237
multilocus approach, focusing on the plastid genome as currently the most effective strategy
(see Hollingsworth et al., 2009, and citations therein), although there is still much debate
concerning the most suitable regions to be used. From the broad pool of loci recently
considered (Kress et al., 2005; Chase et al., 2007; Newmaster et al., 2008; Ford et al., 2009), the
greatest interest was aroused by seven candidate plastid loci: rpoB, rpoC1 and rbcL (three
easy-to-align coding regions), a section of matK (a rapidly evolving coding region), and
trnH-psbA, atpF-atpH and psbK-psbI (three rapidly evolving intergenic spacers).
Various biological contexts (e.g., sampling strategies) have been used to compare the
performance of plant barcoding loci, and/or the efficacy of the method. A sound assessment
of the universality of regions is usually given by the “species pairs” and “floristic”
approaches. The former involves analysing pairs of related species from multiple
phylogenetically divergent genera and may be defined as a “methodological” protocol; the
latter involves sampling multiple species within a given geographical area and represents an
example of how barcoding might be applied in practice. However, only limited insights into
species-level resolution is usually provided by both approaches, as individual genera are
not sampled in depth to provide estimates of intra- and interspecific genetic distances to
achieve species identification. Conversely, a third method, the “taxon-based” approach,
involves sampling multiple species within a given taxonomic group, in a global
geographical context. This provides limited insights into universality and local applicability,
but offers more definitive information on discrimination power at species level. To date, the
species pairs (e.g. Kress et al., 2005; Kress & Erickson, 2007), and the taxon-based (e.g.
Newmaster et al., 2008; Newmaster & Ragupathy, 2009) sampling designs have provided
useful insights into the potential performance of varying combinations of barcoding loci,
whereas the floristic approach (e.g. Fazekas et al., 2008; Lahaye et al., 2008; Gonzalez et al.,
2009), has showed strong potential applicability in as many diverse research fields as
biodiversity inventories, community assembly, food and medicine identification, ethno- and
forensic botany. Based on the relative ease of amplification, sequencing, multi-alignment,
and on the amount of variation displayed (sufficient to discriminate among sister species
without affecting their correct assignation through intra-specific variation), the most
frequently recommended marker combinations for broad future applications appear to be:
rbcL + trnH-psbA (Kress & Erickson, 2007), matK + trnH-psbA (Newmaster et al., 2008;
Lahaye et al., 2008), rbcL + trnH-psbA + matK, and rbcL + matK (Consortium for the
barcode of Life, Plant Working Group [CBOL PWG], 2009).
3.1 DNA Barcoding of forest tree species
In forestry science, DNA barcodes is highly promising for the detection, monitoring and
management of biodiversity (von Crautlein et al., 2011). In addition to resolving many
taxonomic uncertainties, enhancing clear and more accurate biodiversity assessments, DNA
barcoding may provide a boost to efficient management and conservation practices, mainly
focusing on community ecology (to describe plant-animal interactions and vegetation
dynamics/changes, to discriminate native vs. alien germplasm), biodiversity inventories
(aimed at habitat and species protection), silviculture (to assess forest regeneration), and
nursery activities and market regulation (to establish wood, secondary products and
germplasm certification). The applications might be particularly relevant to manage
correctly the over-exploited and the newly identified species, to adequately protect those
having limited ranges and relatively small population sizes, as well as for mending
damaged landscapes by planning and monitor congruent reforestation programmes.
Research in Biodiversity – Models and Applications
238
Indeed, one of the future challenges for DNA barcoding in plants is to increase the number
of practical studies, and validation of the method for forestry purposes is still to be
demonstrated. Priority should be given to the use of markers with universal primers and
uniform PCR conditions. Under these criteria, the most updated recommendation from the
CBOL PWG is that rbcL+matK is adopted as the core DNA barcode for land plants (CBOL
PWG, 2009), with trnH-psbA (the next best performing plastid locus) as a supplementary
barcode option for difficult plant groups. However, success in angiosperms is often
perceived by the majority as the most important issue. For gymnosperms (and cryptogams)
the universality criterion has received little consideration up to date, and clade
specific/multiple primer sets were often used to evaluate matK and other putative barcode
markers (including rbcL and rpoC1). For instance, in the few currently available
gymnosperm-based barcoding studies, only 24% PCR success was obtained in Cycads (Sass
et al., 2007) with matK universal primers, whereas Hollingsworth et al. (2009) and Ran et al.
(2010) obtained 100% PCR and sequencing success in Araucaria and Picea by use of a
combined set of specific primers and under non-standard PCR conditions. More recently, a
taxon based study on Taxus was attempted with new matK specific primers (Liu et al., 2011).
Clearly, matK universality across both gymnosperms and angiosperms is still a matter of
concern, while rbcL and trnH-psbA have repeatedly shown strong rates of sequence
recovery in both clades but their use still requires some technical adjustments (see for
instance Hollingsworth et al., 2009).
The efficacy of the method is still under question, too. Pooled sequence data from 445
angiosperm, 38 gymnosperm, and 67 cryptogam species indicated that overall species
discrimination was successful in 72% of cases (CBOL PWG, 2009), in agreement with the
upper limit of ca. 70% resolution pointed out in previous studies (Fazekas et al., 2009;
Hollingsworth et al., 2009). Large-scale plant diversity inventories conducted at a local or
regional context matched this limit or revealed even higher percentages, although
absence/scarcity of gymnosperms in their datasets is still noticeable. Irrespective to the
statistical methods used to cluster sequences into taxonomic units, and to the marker
combinations used, <70% of species resolution was achieved on 254 angiosperm species
from an environmental sampling in Amazonia (Gonzalez et al., 2009), ca. 71% on 92
primarily angiosperm species (including 7 conifers) from selected locations of Southern
Ontario (Fazekas et al., 2008), ca. 90% on 32 angiosperm species and over 1000 orchid
species from two national parks (Lahaye et al., 2008), and 93-98% on 143 and 296
angiosperm species in community studies in tropical forest dynamics plots in Puerto Rico
and Panama (Kress et al., 2009, 2010). However, it has been shown that woody plant
lineages have consistently lower rates of molecular evolution as compared with herbaceous
plant lineages (Smith & Donoghue, 2008), suggesting that the application of DNA barcoding
concepts should be more difficult for tree than for non-woody floras (Fazekas et al., 2009).
Moreover, the discrimination rate of plastid barcoding loci varies greatly among different
plant lineages. In tree species, no resolution was achieved in 12 Quercus (Piredda et al.,
2011), 18 Betula and 26 Salix species (von Crautlein et al., 2011), whereas 30%, 63% and 100%
were achieved in Berberis (16 species), Alnus (26 species), and Compsoneura (8 species),
respectively (Roy et al., 2011; Ren et al., 2010; Newmaster et al., 2008). In Gymnosperms, all
extant five Taxus species (Liu et al., 2011) were fully discriminated with a non-standard
barcode (trnL-F); in 32 Picea species (Ran et al., 2010), the highest rate of successful
discrimination was 28.57% for a three-locus barcode (trnH-psbA, matK, atpF-atpH). A
slightly higher percentage was obtaine
d by Hollingsworth et al. (2009) in Araucaria (32%).
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
239
Available data show that some limitations are predictable, matching the view of Fazekas et
al. (2009). Limitations are mostly due to polyploidy, hybridization/introgression
phenomena, shares of ancestral polymorphism, which would prevent the correct match
between DNA variation at the plastid level and species identity. Such phenomena probably
affect many tree species; in addition, trees are known to have markedly slower mutation,
nucleotide substitution and speciation rates than other plants, seemingly owing to longer
generation times and slower metabolic rates (see Petit & Hampe, 2006 for a review). At the
same time, biogeographic patterns of species, lineages and area relationships can strongly
affect the resolution of taxa. Together with this assumption, the barcoding efficiency of tree
taxa is still to be demonstrated, and it appears to be most hardly challenged by the peculiar
evolutionary history and intrinsic biology of each taxon, and in those areas where recent
explosive radiations have taken place, or where a high number of only slightly diversified
congenrics co-exist.
3.2 Barcode application in the Italian flora
A summary of explorative data on the foreseeable barcoding efficacy in the Mediterranean
area, with specific regard to Italian forest flora is reported in Table 2. With the aim to
provide a test for future in situ applications of DNA barcodes by evaluating the efficacy of
species discrimination under the criteria of uniformity of methods and natural co-
occurrence of the species in the main forest ecosystems, we examined whether four marker
regions (trnh-psba, rbcL, rpoc1, matK) proposed by the Consortium for the Barcode Of Life
matched species taxonomy in a biodiversity survey of Italian forested land.
Seventy-eight species were included in a floristic study, including 53 Angiosperm and 25
gymnosperm species (trees, shrubs and vines from the Alpine timberline to the
Mediterranean sea dunes; 68 native and 10 introduced/naturalized taxa); in addition, taxon-
based studies were performed on Quercus (15 species, 30 individuals), Acer (8 species, 15
individuals) and Pinus (10 species, 30 individuals). individuals) and Pinus (10 species, 30
individuals). We observed total universality of the rbcL+trnH-psbA marker combination
across all taxa, and an overall 78.4% of species discrimination, with 100% in gymnosperms
and 66.7% in Angiosperms,whereas matK and rpoC1 showed incomplete, or limited,
applicability due to some primer specificity, Differences in the biology/evolutionary history
of tree genera are represented by the contrasting results obtained in the three taxon-based
studies: Quercus exhibited an exceptional 0% of species resolution, whereas Acer and Pinus
reached 100% discrimination success. As a main result, the barcoding approach provided
molecular tools for the identification of all taxa co-occurring in most of the Italian forest
ecosystems, from the Alpine timberline, to montane, submontane, humid/riparian,
Mediterranean evergreen forest/maquis and sea dunes, including some ubiquitous vines
and shrubs, with the exception of oaks and willows. The approach was also useful for the
molecular identification of all the rare endemics investigated (Fontanesia phylliraeoides, Acer
lobelii, Abies nebrodensis, Pinus heldreichii ss
p. leucodermis), and all native vs. allochtonous
germplasm (Aesculus hippocastanum, Quercus rubra, Acer negundo, Abies pinsapo, A.
cephalonica, Pinus radiata, P. brutia, Cupressus arizonica, Pseudotsuga menziesii, Gingko biloba).
Concerning the intraspecific taxa, ssp. nigra was clearly differentiated from all other Pinus
nigra subspecies, as well as ssp. turbinata within Juniperus phoenicea. Lastly, two vines and
four shrubs were efficiently discriminated from co-occurring arboreal taxa. Investigated taxa
could be efficiently barcoded in most ecosystems, with the exception of those forests where
a high number of willows and oak species co-occurred.
Research in Biodiversity – Models and Applications
240
Among the species-rich genera, those which would benefit most from molecular
identification (Quercus, Salix) because of their complex morphology, showed little or no
variation at the plastid genome. Remarkably, none of the markers used could resolve 12
Italian Quercus species below the sectional level (i.e., Sclerophyllodrys, Cerris and Quercus),
due to large haplotype sharing between closely related species. On the other hand, intra-
specific variation in Italian conifers appears to correspond to some regional patterns
reflecting important prints of species survival during glaciations and post-glacial
recolonization (Follieri, 2010). Specific haplotypes were found in Southern Italy (Apulia),
Central Italy (Tuscany, Latium), Northern Italy (Eastern and Western Alps), and main
islands (Sicily), all falling within the 52 biodiversity refugia recently indicated on a regional
scale in the Mediterranean basin (Medail & Diadema, 2009). Variation in the barcoding loci
also evidenced the occurrence of two distinct haplotypes of Taxus baccata in Italy, one shared
with other European provenances and a second exclusive of South East Italy. Finally, our
results confirmed the genetic diversity existing between Southern and Central Italy
provenances of Cupressus sempervirens (Bagnoli et al., 2009), and divergence between Eastern
and Western Alps provenances of Picea abies (Collignon & Favre, 2000), as well as between
Eastern and Western Mediterranean provenances of P. halepensis (Korol et al., 2002), all
previously detected with other molecular markers.
Major Clade Familia Genus Species
in Italy
Species
investigated
Species
identification
Notes
Angiosperms Aceraceae Acer
7 8* Yes
Possible haplotype
sharing between A.
obtusatum and A.
monspessulanum
Oleaceae Ligustru
m
1 1 Yes
Olea 1 1 Yes
Fraxinus 3 3 Yes
Phyllirea
3 3 Yes
Possible haplotype
sharing between P.
angustifolia and P.
latifolia
Fontanesi
a
1 1 Yes
Fagaceae Fagus 1 1 Yes
Castanea 1 1 Yes
Quercus
10-14 15* No
No species resolution
at National scale
Salicaceae Populus
3 2 Yes
Possible haplotype
sharing between P.
nigra and P. alba
Salix
>30 2 No
No species resolution
at National scale (**)
Ulmaceae Ulmus 3 1 Yes
Rosaceae Prunus
9 1 Yes
Possible haplotype
sharing
Craetegus
2-3 1 Yes
Possible haplotype
sharing
Rosa >20 2 Yes Possible haplotype
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
241
Major Clade Familia Genus Species
in Italy
Species
investigated
Species
identification
Notes
sharing
Rubus
>20 2 Yes
Possible haplotype
sharing
Betulaceae Corylus 1 1 Yes
Alnus 4 1 Yes
Araliaceae Hedera 1 1 Yes
Sapindaceae Aesculus 0 1* Yes
Cannabaceae Humulus 1 1 Yes
Moraceae Ficus 1 1 Yes
Morus 0 1* Yes
Tamaricaceae Tamarix 10 1 Yes
Gimnosperms Pinaceae Pinus
8 10* Yes
Possible haplotype
sharing between P.
mugo and P.
sylvestris
Larix 1 1 Yes
Pseudotsu
ga
0 1* Yes
Abies 2 4* Yes
Picea
1 1 Yes
No species resolution
at National scale (**)
Cupressaceae Juniperus 4 4 Yes
Cupressus 1 2* Yes
Taxaceae Taxus 1 1 Yes
Gingkoaceae Gingko 0 1* Yes
Table 2. Barcoding efficacy on some of the most important tree species in Italy. Asterisk
indicate non native species included (*), and results implemented with literature data (**).
We therefore conclude that, despite some failures, the DNA barcoding approach will
continue to be useful in some applications, especially when applied at local contexts, with
some plant groups and for some peculiar investigations. Ideally, an important technological
advancement to improve the method would include the achievement of primer universality
for the main plastid markers, and eventually the opportunity to cope information from both
organellar DNA and the more informative nuclear genome.
Organisms identification is essential to many disciplines, and the scientific community has
recently come to realize the importance of integrated approaches to organism identification
(Steele & Pires, 2011). Indeed, conservation planners and government agencies would need
well defined species boundaries to protect ecosystems and writing effective laws (Primack,
2008), and restoration ecologists must accurately identify native plant species suitable for
rebuilding damaged ecosystems (Guerrant et al., 2004). As well, conservation biologists
must be able to correctly identify plant species for fighting invasive, reseeding restoration
areas with appropriate species, monitor the regeneration processes of a community after
their intervention, protecting native and/or threatened ecosystems by preserving all life
forms. Finally, seed harvesters and germplasm traders must ensure the end-users that the
right species are produced before distribution to the public. Nevertheless, the role that
DNA barcoding might play in these views still relies heavily on experimentation and tests.
Research in Biodiversity – Models and Applications
242
Our data suggest that forest biodiversity can be efficiently barcoded at a local level, or in
well characterized regions of the world which have comparatively low numbers of species;
conversely, the barcoding efficiency of tree taxa might rather be under question in large
areas where peculiar genera (e.g., Betula, Quercus, Salix, etc.) occur with multiple species.
Future large breadth taxon-based studies will help clarify the efficacy of DNA barcoding to
inspect the biological diversity of forest tree species. However, factors suggested to
contribute toward limiting the efficacy of barcoding tree species such as longevity, complex
reproductive strategies, and slow mutation and speciation rates (Petit & Hampe, 2006) may
not affect the barcoding efficacy at a local context.
4. From conservation to restoration: The Miyawaki method
It is widely known that global climatic changes, together with recent rapid urbanization and
industrialization, have been the main anthropogenic effects worldwide in destroying natural
environments, changing land use, reducing biodiversity, and modifying ecosystems. They
suggest the need for performing more environmental conservation strategies, as well as
using innovative environmental recovery activities. We have seen in the Introduction as in
situ gene conservation measures ecosystem functions and species interactions, rather than
individual tree species; however their conservation may require specific management
measures, which could be ensured through the establishment of genetic conservation areas.
From a theoretical point of view, a network of genetic resource conservation areas should be
an efficient way to conserve the genetic resources of target species, if they follow the
patterns of distribution of genetic variation (Eriksson et al., 1995). Practical experience
suggests that sound management of genetic resources must include conservation efforts
based on two overlapping strategies: management of natural forests with due respect to
their genetic resources, and the establishment of networks of smaller gene conservation
areas (Thomson et al., 2001). Nevertheless, it should be remembered that in situ conservation
is only a technical option in a broader approach to conservation of the diversity between
species and within species. In several cases, conserving forest trees in situ may be the only
method that is socially and economically possible. In other cases, a combination of protected
areas, managed reserves, clone banks, research plantations and breeding programmes may
be better suited to different conditions and objectives.
In the last years, the greatest challenge is to move from the conservation of existing
resources, toward a rationale restoration ecology, increasing efforts to rehabilitate degraded
lands. Often the preliminary objective is to re-establish tree cover for environmental
purposes, especially for control of soil erosion and for watershed protection. Facing these
items, scientists have developed new insights both in theoretical and in practical actions for
restoration and reconstruction of natural ecosystems (Clewell & Aronson, 2008; Falk et al.,
2006; Jordan et al., 1987; Perrow & Davy 2002a, b; Soulé & Wilcox, 1980; Miyawaki, 1975,
1981). Natural restoration is strictly related to increased sustainability and includes
rehabilitation of ecosystem functions, enlargement of specific ecosystems, and enhancement
of biodiversity restoration (Stanturf & Madsen, 2004). At the ecological level, restoration is
also defined as “an intentional activity that initiates or accelerates recovery of an ecosystem
with respect to its health, integrity and sustainability” (Society for Ecological Restoration
International Science & Policy Working Group [SER], 2002). Degraded plant communities
are generally quite difficult or sometimes impossible to restore (Van Diggelen & Marrs,
2003). More than 200 years of reforestation practice has demonstrated that forest recovery
takes a very long time, frequently with unsatisfying results. Nowadays, it is possible to plant
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
243
plantations of several species, but the transition from the simple plantation to a forest
community able to evolve and sustain itself, according to the natural successional pattern, is
still a rare event. Moreover, a number of “regreening” projects in the past have paid scant
attention to the source of planting materials used and their biological requirements, and
have failed because of poor species choice. Use of non-local seed sources of indigenous
species can result in the contamination of genepools of nearby populations (Thomson, 2001).
On the other hand, the mere superficial appearance of vegetation restoration should be
avoided. It is essential to restore the natural vegetation using a combination of native species
that conform to the potential trend of the habitat and to try to restore the whole specific
ecosystem of a region (Miyawaki, 1992). Currently, most forest reforestation programs
adopt a scheme of planting one or more early successional species; after successful
establishment, they are gradually replaced by intermediate species (either naturally or by
planting), until late successional species arise. This pattern tries to simulate natural
processes of ecological succession, from pioneer species to climax vegetation. However, it
requires several silvicutural practices and normally takes a long time; because we live in a
world where industry and urbanization are developing very rapidly, improvement of an
alternative reforestation technique that reduces these times could be a useful tool
(Miyawaki, 1999).
One reliable forest restoration method is the “native forests by native trees”, based on the
vegetation–ecological theories (Miyawaki, 1993a, b, 1996, 1998b; Miyawaki & Golley, 1993;
Miyawaki et al., 1993; Padilla & Pugnaire, 2006) proposed by Prof. Akira Miyawaki and
applied first in Japan. Restoring native green environments, multilayer forests, and natural
biocoenosis is possible, and well-developed ecosystems can be quickly established because
of the simultaneous use of intermediate and late successional species in plantations. The
Miyawaki method involves surveying the potential natural vegetation (sensu Tüxen, 1956)
of the area to be reforested and recovering topsoil to a depth of 20–30 cm by mixing the soil
and a compost from organic materials. In this way, the time of the natural process of soil
evolution, established by the vegetational succession itself, is reduced. Tree species must be
chosen from the forest communities of the region in order to restore multilayer natural or
quasi-natural forests. For a correct choice, based on reconstructing the potential natural
vegetation, several analyses (e.g., phytosociological investigation) are required. Detection of
the soil profile, topography, and land utilization can improve our grasp of the potential
natural vegetation. After these field surveys, all intermediate and late successional species
are mixed and densely planted, with as many companion species as possible (Kelty, 2006;
Miyawaki, 1998a), and soil between them is mulched. In fact, biocoenotic relationships
involve autoregulations between species, favouring a dynamic equilibrium and avoiding
any further silvicultural practice and need no insecticides or herbicides (with some
exceptions). Indeed, in the Miyawaki method, the principles of self-organized criticality and
cooperation theories have been essentially applied (Bak et al., 1988; Callaway, 1997;
Camazine et al., 2003; Padilla & Pugnaire, 2006; Sachs et al., 2004). If compared to traditional
methods, some known restrictions regard the requirement of specialists for botanical and
ecological investigation of the sites, a higher need of manpower for planting, and higher
costs of plant material due to the plant density. On the other hand, no human care is
required after 1-2 years from planting, the undergrowth with late-successional species is
immediately on site, and forest stands become quickly part of the natural ecosystems.
Moreover, the theoretical principle at the base of the definition of Regions of Provenance
might be considered almost included in the Miyawaki method, as it suggests to use seed
Research in Biodiversity – Models and Applications
244
from the nearest natural populations. Figure 7 shows a schematic overview of the
comparison between classical succession theory and the one proposed by Miyawaki.
Fig. 7. Comparison between classical succession theory and the new one proposed by
Miyawaki (redrawn from Miyawaki, 1999).
4.1 The adaptability of Miyawaki method to the Mediterranean environment: a case
study
It has been demonstrated that multilayer quasi-natural forests can be built in 15–20 years in
Japan and 40–50 years in Southeast Asia by ecological reforestation based on the system of
natural forests. Results obtained by application of the Miyawaki method in about 550
locations in Japan, as well as in Malaysia, Southeast Asia, Brazil, Chile, and in some areas of
China, were found to be successful, allowing quick environmental restorations of strongly
degraded areas (Miyawaki, 1989, 1999). Until now, the Miyawaki method has been applied
in countries characterized by cold-temperate and tropical climatic regimes, which do not
experience seasonality, i.e. winter cold and summer aridity stress (cf. Mitrakos, 1980) with
potential risk of desertification (increased by global change). Thus, the Mediterranean
context could be considered an interesting test to assure the effectiveness of such a method
in other important biomes, even with high biodiversity hotspots. Nevertheless, it could be
interesting for the Mediterranean Basin, because complete environment restoration takes
longer time than in tropical or cold-temperate climates. To estimate the effectiveness of
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
245
Miyawaki method in such different circumstances without altering its theoretical principles,
several changes were introduced and tested in two experimental plots in Sardinia (Italy) in
1997, on target sites where traditional reforestation approaches are widely used but have
mostly failed (Schirone, 1998). First, the soil condition of the planting sites was not adjusted,
so no recovery of the 20-30 centimetre-deep topsoil with compost from organic materials has
been done, but only a labouring of the pre-existent soil. Tillage was used to improve soil
water storage over the winter and reduce water stress during the summer. Between the
selected species, some autochthonous early-successional ones were planted (e.g. Pinus
pinaster L., and shrubs) to improve plant community resilience, and no weeding after
planting was done. Mulching was provided experimenting straw as in the original method,
but also other types of materials (saw mill residuals, dry and green materials), and tested
planting densities were assessed to 8600 and 21000 plants/hectare respectively. A particular
care was dedicated to the choice of the best planting season, and watering was provided
once soon after planting. Figure 8 summarizes the Miyawaki method as implemented in the
mentioned experiments.
Fig. 8. Schematic overview of the Miyawaki method modified for Mediterranean
environment. Dark grey text boxes describe main processes; bold texts refer to the changes
to the original method.
To estimate the efficiency of this adapted Miyawaki method to Mediterranean, three surveys
were performed in 1998, 1999, and 2009 in both experimental plots. Moreover, comparisons
Research in Biodiversity – Models and Applications
246
were done with two nearby coeval sites where traditional reforestation techniques were
applied to better understand the differences in plants growth, forest composition, and
vegetation cover in percentage (Schirone et al., 2011). The results after 12 years from the
planting showed a more rapid development of trees on the Miyawaki plots, in particular
early-successional species, as well as a stable assessment of species’ occurrence with high
level of biodiversity (Table 3). The benefits over previous methods are remarkable and
comparable with those obtained by Miyawaki in Asia and South America. At the same time,
the changes made to better fit the method to the Mediterranean environment seem to be
particularly useful. For instance, adding some autochthonous early successional species to
the intermediate- and late-successional ones the system resilience was improved; this
solution was already tested by Miyawaki in Brazil, even if no benefits were recorded
(Miyawaki & Abe, 2004). Looking for an optimal high plant density, it was assessed that
cooperative processes (e.g. mutual shading) prevail over competitive ones (Callaway, 1997).
In fact, low plant density has been traditionally retained as appropriate in arid and semiarid
environments in order to avoid competition for water resources between plants (Caramalli,
1973; Bernetti, 1995), but a higher one reduces, for instance, the impact of acorn predators,
thus encouraging oak regeneration, i.e., the main late-successional forest species in
Mediterranean environments (Gómez et al., 2003); high plant density can also favour root
anastomosis processes, that seem to influence coenosis’ stability and reforestation success
(Kramer & Kozlowski, 1979). In addition, excellent plant stock remains fundamental for
planting success in harsh environments (Palacios et al., 2009). Finally, these results could
offer a chance to introduce a new method into the Mediterranean context that is able to
reduce the time for a complete environmental restoration.
Species survival in
Miyawaki sites
Height ± (Stand. Dev.)
Species n
i
n
f
n
f
/n
i
(%) MS-1 MS-2 TRS-1 TRS-2
Acer monspessulanum L. 51 2 3.92% 40 ± (14.1) 0 - -
Arbutus unedo L. 61 41 67.21% 32.7 ± (4.1) 0
500 ±
(35.8)
110 ±
(20.6)
Castanea sativa Mill. 42 1 2.38% 10 0 - -
Cedrus atlantica Endl. - - - - - -
162 ±
(54.6)
Celtis australis L. 59 3 5.08%
26.7 ±
(28.9)
- - -
Erica arborea L. - - - - -
115 ±
(12.7)
130 ±
(18.6)
Fraxinus ornus L. 17 1 5.88% 250 - - -
Ilex aquifolium L. 237 23 9.70%
45.2 ±
(30.6)
0 - -
Juniperus oxicedrus L. 45 30 66.67% -
36.2 ±
(18.5)
- -
Laurus nobilis L. 41 3 7.32% 30 ± (17.3) 0 - -
Ligustrum vulgare L. 139 33 23.74%
32.8 ±
(52.6)
30 ±
(8.16)
- -
Malus domestica Borkh. 40 7 17.50% 100 ± (45.5) 0 - -
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
247
Myrtus communis L. 114 5 4.39% 10 10 ± (1.4) - -
Phyllirea angustifolia L. 1 1 100.00% 70 0 - -
Phyllirea latifolia L. 203 0 0.00% - 0 - -
Pinus pinaster L. 428 288 67.29%
433.2 ±
(143.6)
325.5 ±
(38.6)
376.4 ±
(73)
425.7 ±
(25.1)
Pyrus communis L. 41 20 48.78% 71 ± (65.1)
60 ±
(61.2)
- -
Quercus ilex L. 694 255 36.74%
34.2 ±
(32.1)
40.8 ±
(36.2)
69.4 ±
(23.2)
146.2 ±
(38.1)
Quercus pubescens Willd. 361 124 34.35%
23.6 ±
(27.5)
10 ± (5.3) - -
Quercus suber L. 632 103 16.30%
174.3 ±
(49.6)
77.5 ±
(51.9)
- -
Rosmarinus officinalis L. 46 15 32.61%
89.3 ±
(33.9)
0-
80 ±
(14.9)
Salvia officinalis L. 9 0 0.00% 0 0 - -
Sorbus torminalis (L.) Crantz 42 12 28.57% 35 ± (50)
40 ±
(12.9)
- -
Spartium junceum L. 74 29 39.19%
110.7 ±
(62.2)
0- -
Taxus baccata L. 377 9 2.39% 33.3 ± (38) 0 - -
Thymus vulgaris L. 24 0 0.00% - 0 - -
Viburnum tinus L. 84 3 3.57% 10 0 - -
Table 3. Total number of individuals in the Miyawaki sites, at the beginning of the
experiment (n
i
, 1997), after 12 years (n
f
, 2009), percentage of species’ survival (n
f
/n
i
), and
comparison of plant height (cm) between Miyawaki sites (MS-1, MS-2) and the traditional
reforested ones (TRS-1, TRS-2) in 2009. Dashes indicate species not planted, and zero values
refer to planted species that did not survive in 2009. Successional position of each species is
indicated by the row color: white (early successional), light grey (middle-successional), dark
grey (late-successional).
5. Conclusion
The conservation of biodiversity has become a major concern for resource managers and
conservationists worldwide, and it is one of the foundation principles of ecologically
sustainable forestry (Carey & Curtis, 1996; Hunter, 1999). Many efforts were dedicated to set
aside networks of reserves and protected areas advocated by scientists, governments, etc. to
preserve the extraordinary biodiversity that characterizes forest ecosystems, perpetuating
their integrity, their evolutionary patterns and yet providing social and environmental
benefit . At the same time, a strategic value has been assigned also to biodiversity in terms of
genetic resources, through the conservation of plant populations in their natural habitats (in
situ) to better evolve and adapt to physical environmental trends and to changes in the web
of interactions with other life forms. Generally, the simplest way forward in economic and
political terms is for countries to locate genetic resources in existing protected areas, as this
likely to provide benefits to local people communities. However, despite the critical role of
conservation sites, a large debate arose about the combination of protection, management,
Research in Biodiversity – Models and Applications
248
and restoration of forests and woodland landscapes as pivotal starting points of sustainable
development in many of the world’s ecoregions (e.g. Pierce et al., 2003; Norton, 2003;
Aldrich et al., 2004; Loucks et al., 2004). At pan-European level, several legislative tools
emphasized the need of facing habitat fragmentation, biodiversity loss, genetic pollution,
and invasive species use, throughout the definition of certified basic material and
ecologically homogeneous areas.
Some strategies have been included in the Directive 105/99, with the definition of Regions
of Provenance and the requirements for an appropriate marketing of forest reproductive
material. Unfortunatently, there was an heterogeneous achievement of the Directive by the
European countries in time, as well as in adopting common methods. Mainly according to
the available data, the chosen parameters for detecting the Regions of Provenance differed
case by case. However, it is also evident that both agglomerative and divisive approaches
could be improved by adding further variables and/or methods. Nowadays, the need for
models implemented with biological parameters is suggested by a changing climate, in
which bioclimatic shifts could characterize vegetation arranged along altitudinal gradients
or at ecotonal boundaries (e.g. Peñuelas & Boada, 2003; Steltzer & Post, 2009). Data analysis
at different temporal scales could allow to understand the effects of climate trends on
species success and survival, and thus to choose the most appropriate genetic material for
reforestation actions. In this view, genetic approaches must certainly be refined and made
uniform through countries in order to speed up detection of diversity and comparability of
results (Aguinagalde et al., 2005). At the same time, given the rapid pace of environmental
degradation in many biologically species-rich parts of the world, a clear organism
identification is essential for restoration experts to define species’ distribution range, native
plants for restoring damaged ecosystems or afforesting new ones, invasive species to fight.
Moreover, it is important to check the phases of the regeneration processes of a community
after an intervention, and protect native and/or threatened ecosystems. These items could
be achieved by using a standardized molecular approach as DNA Barcoding, once its actual
efficacy is demonstrated with preliminary study cases.
Recently, the need to understand the development and the spatial dynamics of pattern in
ecological phenomena leaded to the concepts of landscape ecology, i.e. broad scale
investigations strictly linked to the vegetation occurring at local scale. The Committee of
Ministers of the Council of Europe adopted the European Landscape Convention on 2000,
aiming to promote European landscape protection, management and planning and to
organise European cooperation (European Council, 2000). The Convention is the first
international treaty exclusively devoted to all aspects of European landscape, but the
importance of reforestation and genetic fundamentals of landscape is not well considered
yet (Granke et al., 2008).
Since the main goal is to guarantee not only simple conservation measures, but also the
expansion of forest surfaces throughout reforestation actions, we need methods able to
provide forest quality and reduce the time for a complete environmental restoration. This is
particularly true in those areas where the environment has been modified and exploited by
humans over the course of thousands of years, as in the Mediterranean Basin. In particular,
forests have experienced many processes that have led to degradation and consequent soil
loss as reported since the fourth century B.C. by Plato in Critias. The Miyawaki method
could take up the challenge, but its effectiveness will be increased if it is joined with other
tools, like well defined Regions of Provenance, in situ and/or ex situ networks of reserves for
Multiple Approach for Plant Biodiversity Conservation in Restoring Forests
249
providing the most suitable genetic resources, and DNA Barcoding to assess and monitor
the trend of the intervention.
It is undoubted that we have to move toward a holistic approach, in order to improve the
present methods with as many criteria as possible, and define a unique project design. For
these reasons, a toolbox based on this multidisciplinary concept is presented as ideal
guideline attending the gained experiences in the Mediterranean Basin (Figure 9).
Fig. 9. Theoretical example of reforestation process implemented with the tools (bold texts)
discussed in the chapter.
Computer-based methods existed since 1980 to assist tree species and provide information
about uses, distribution, environment, and silviculture; nowadays The Forestry
Compendium developed by CAB International (CABI) is probably the most impressive tool
that has been developed (CABI, 2010). However, further developments of this tool should
include information on selection systems about requirements of particular genotypes,
including provenances, hybrids, clones, and genetically modified material choice. The
toolbox we propose is composed of the mentioned actions and methods, including latest
informatics supports, and it has been developed to be applied in reforestation activities,
starting from the delimitation of the Regions of Provenance with the detection of adequate
seed sources, the correct identification of plant species, the environmental and vegetation
surveys, the selection of certified basic materials, up to the reforestation technique and the
checks after planting. Basically, each mentioned step poses a specific question, and the
toolbox would provide the answer or the best tool to achieve it. However, this proposal is a
preliminary tentative to create a logic framework of actions that will require a validation
measure also throughout a socio-economic analysis to estimate the costs of each step. For
instance, it would be useful to understand the costs for data capture and development of
further biological indexes retrieved from satellite images, the expenses for extracting and
analysing DNA, including molecular markers and Barcoding of plants, the costs of
manpower and plantlets for the reforestation practices.
Research in Biodiversity – Models and Applications
250
6. Acknowledgements
We are indebted to Regional Forest Directorate of Sardinia for conceding the logistic support
in providing the Miyawaki test areas. Special thanks to Dr. Carmine Sau, Dr. Antonello
Salis, and Dr. Francesco Mazzocchi for their valuable help and commitment on the field
work performed in Pattada Municipality. The study about NDVI remote sensing analysis
was partially supported by the project PRIN 2007AZFFAK and developed in collaboration
with Prof. Gianluca Piovesan and Dr. Alfredo Alessadrini. Special thanks to Dr. Despina
Paitaridou of the Greek Ministry of the Environment for providing information about the
Regions of Provenance in Greece. For DNA Barcoding bioinformatic study in Italy, we are
grateful to Dr. Roberta Piredda, Dr. Laura Armenise, and Dr. Alessio Papini. Our warmest
thanks to Prof. Rosanna Bellarosa, Prof. Luca Santi, Dr. Avra Schirone, Mr. Armando
Parlante, Mr. Luigi Sandoletti, and Ms. Giulia Sandoletti.
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