Workpackage 5: The influence of hydrology on sexual and vegetative
regeneration strategies in floodplain species
Participating partners:
University of Cambridge (UK) (1)
University of Lethbridge (Canada) (4)
Centre National de la Recherche Scientifique –Toulouse (F) (5)
3.1 Objectives

Completion of genetic analyses of 450+ P. nigra leaf samples from the Garonne
study sites. Statistical analysis of this data and field survey data.

Write up of the Garonne study with the aim of sending off a manuscript by month 33.
The write up prompted the cross-checking of some of the genetics data and the need
for additional dendrochronological measurements. Statistical analyses of data and
technical drawings also needed some refining.

Produce a conceptual model of the influence of flow regimes on regeneration
strategies.

Completion of genetics analyses of 400 leaf samples from the Drôme study site.
Completion of GIS study to describe disturbance in channelised and non-channelised
river reaches from a series of aerial photographs and production of a map of leaf
collection points. Analysis of combined datasets. Write up of Drôme study with the
aim of sending off a manuscript for publication by month 33.
3.2 Methodology and scientific achievements
3.2.1 Drôme River
Molecular analysis of plant material
This work is completed, but results are difficult to interpret. This has slowed progress on
joint analysis and publication of work.
Mapping work + GIS study describing disturbance in contrasting river reaches
Maps of the relative positions of leaf sample collection points have been produced. GIS
work has been completed using available aerial photographs from the past decade. The
results reveal a trend for most erosion of established pioneer stands to occur on the upstream
tips. When not swept away, changes to established pioneer stands were more significant in
the channelised than non-channelised reaches and there was a greater turnover rate of
vegetated islands in channelised than non-channelised reaches.
3.2.2. Garonne River
Molecular analyses of plant material
Microsatellite analyses of over 450 P. nigra samples have been successfully completed using
5 primers pairs (dinucleotide repeats). Sizes of amplified alleles were determined and
visualised by automated florescent detection with an ABI 3100 autosequencer. A high
number of alleles per locus (between 12 and 32 alleles) and wide allelic ranges among these
suggested that the 5 microsatellite loci selected for this study were sufficient to cover the
polymorphism present in the Garonne population. Replicate samples and re-runs of the same
leaf samples revealed the high repeatability of this molecular genetic technique. Identical
genotypes were scored where allele sizes at all five microsatellite loci were identical or the
same with one base pair size difference at no more than a single loci.
Statistical analysis of field survey and genetic data sets + relating genetic results to
indicators of hydrological disturbance
One way ANOVA’s were applied to test for significant differences in a number of stand
descriptors (e.g. stand density, stem diameters, shoot height…) between ‘young’, ‘middleaged’ and ‘old’ stands. The frequency of identical genotypes in the different stand age
categories was also tested using One-way ANOVA’s. Relationships between indicators of
stand disturbance (i.e. angle of slant of trees, frequency and magnitude of clumped growth)
and the incidence and frequency of identical genotypes within stands in each age group were
investigated using Pearson’s correlation coefficient.
Frequency of identical genotypes within young, middle-aged and old stands
A high proportion of black poplar stands contained replicate genotypes; i.e. 50 % of
young stands, 100% of middle-aged stands and 50% of old stands. However, when
considering the entire data set, the total number of unique, non-replicated genotypes remains
high; i.e. out of 450 trees sampled, 351 represented distinct, non-replicated genotypes, 98
represented replicated genotypes and 1 sample yielded no results. The high numbers of
unique genotypes despite high apparent frequency of clonal replication, is explained by low
levels of replication of any given ‘clonal’ genotype within stands; i.e. clonal genotypes were
replicated 1.3 times on average. High numbers of copies of replicated genotypes within
stands were otherwise relatively rare with only seven instances of three identical genotypes
detected in the same stand and one instance of four identical genotypes detected within the
same stand.
The number of different genotypes differed between young, middle-aged and old
stands. In young stands, the number of genotypes found among the 15 trees sampled in each
stand was significantly greater (p<0.05) than in the middle–aged or old stands, suggesting
that there is less clonal replication in young stands. There was no significant difference
between middle-aged and old stands in this respect, where the numbers of genotypes per
stand averaged 12.5 and 13.1, respectively. In terms of the number of replicated genotypes
found among the 15 trees sampled per stand, significantly more (p<0.01) were found in the
middle-aged than the young, or old stands. The greater number of replicated genotypes in
middle-aged stands also tended to be represented by a significantly (p<0.05) greater number
of stems than replicated genotypes in the young or old stands.
The location of replicate genotypes within stands (nearest neighbours in 85% of
cases) suggested that flood-training was primarily responsible for the promotion of
vegetative copies of distinct genotypes. However, translocated fragments of parental material
also contributed to the number of replicated genotypes within and among stands, travelling
several kms downstream in some instances.
Frequency of identical genotypes and indicators of stand disturbance
In relating the angle of slant of trees and the number of identical genotypes detected,
the positive correlation coefficients derived suggest that the greater the angle of slant of
trees, the greater the frequency of identical genotypes in stands, although this relationship
was significant (p<0.01) only when combining the young and middle-aged stands or when
considering the old stands independently (p<0.1). A weak positive correlation was also
observed between trees in stands displaying clumped growth and the incidence of identical
genotypes; the relationship, however, was only found to be significant (p<0.05) among
middle-aged stands and where stands of all age categories were combined (p<0.1).
3.2.3 Conceptual model of the influence of flow regimes on regeneration strategies
Figure 1.
SEXUAL RECRUITS
(unique genotypes)
ASEXUAL RECRUITS
(replicated genotypes)
Young
Middle-aged
Old
B.
Percentage of Sexual vs. Asexual Recruits
Percentage of Sexual vs. Asexual Recruits
A.
MEDIUM DISTURBANCE
HIGH DISTURBANCE
SEXUAL RECRUITS
(unique genotypes)
ASEXUAL RECRUITS
(replicated genotypes)
Young
Age of individuals
Middle-aged
Old
Age of individuals
A. Low frequency, medium impact, early spring floods promote seedling recruitment and some
vegetative recruitment. Seedling mortality is high during the first years of recruitment, while
vegetative expansion gradually increases during this period with each successive flood.
Competition occurs between seed-derived trees and vegetative sprouts in the middle-aged
phase. Vegetative sprouts also compete among themselves resulting in some mortality of
vegetative recruits and consequently an increase in the proportion of unique genotypes in the
old stands and hence in the established populations.
B. High frequency, high magnitude and/or irregular flooding events reduce the starting number
of seed-recruited individuals, but favour asexual recruitment. Asexual recruits further reduce
the number of seed-recruited individuals in young and middle-aged stands through
competition. The proportion of asexual recruits continues to increase in middle-aged stands
as successive floods promote vegetative expansion. A decline in the number of replicated
genotypes occurs eventually, however, in the middle-aged and old stands when vegetative
recruits compete amongst themselves. The result is a slight increase in the proportion of
unique genotypes in the old stands.
3.3 Socio-economic relevance and policy implication
The policy implications of these results relate to the planning stages of initiatives
aiming at improving river flow management to sustain floodplain biodiversity and
particularly where there is a will to sustain the genetic diversity of naturally regenerating
populations of black poplar and other related early successional floodplain woodland species
(e.g. Salix spp.). The conceptual model (figure 1) clearly illustrates the potential loss to
genetic diversity resulting from frequent, irregular and/or high magnitude flooding events
and this loss may be more significant for willow species which have excellent re-sprouting
abilities, but comparatively poor seedling survivorship. Flood-training will be promoted in
favour of recruitment from seed where (i) flooding occurs during periods of seedling
establishment in the early summer, (ii) there is a high frequency of medium to high
magnitude flooding events. Since the risks of frequent, sustained disturbance are greatest
along channelised river reaches, ideally the active alluvial zone should be as wide as possible
to increase the ‘heterogeneity of disturbance’; the effect would be that some areas of the
floodplain would remain undisturbed for sufficiently long time periods to allow significant
numbers of seedlings to become established.
Where monitoring of the genetic diversity of populations is considered these results
indicate the importance of taking tree age and location into account. Sampling should thus,
be undertaken for a single age category, samples should be collected widely across the
population and sample points should be widely spaced (5m+) to avoid sampling in clonal
clumps. Small scale sampling can give an idea of the relative proportions of sexual to
asexual recruits, or this can be extrapolated from the findings in this study. In making final
estimates of the genetic diversity of a population and/or a Minimum Viable Population
estimate for a given site, the age distribution of the population and expected clonal frequency
should be included in the estimate.
3.4 Discussion and Conclusions
While it has been demonstrated that P. nigra reproduces by both sexual and asexual
regeneration strategies in a number of riparian contexts (channelised and non-channelised
river reaches of a braided river system and along a semi-constrained reach of a meandering
river), asexual regeneration through re-sprouting was not found to be a dominant
regeneration strategy in any of these situations. Asexual regeneration tended instead to be
highly localised although the number of replicated genotypes (clones) and the number of
stems representing each of these replicated genotypes varied with respect to stand age and
the particular hydrological disturbance history at a given site. Overall, for similar age groups,
there were fewer identical genotypes present in the braided Drôme River compared to the
meandering Garonne River. This might be explained by the more erratic shifts in flow
direction along the Drôme River (vegetation is subjected to high flow for limited periods and
if in a vulnerable position, displaced) compared with the Garonne River where changes in
river flow direction are less dramatic and thus, vegetation must sustain the fluctuating forces
of river flow over longer periods at a given point which inevitably leads to flood-training in
areas subject to the highest flow rates.
3.5 Plan and objectives for the next period

Sending off manuscript of Garonne work for publication and starting work on a
second related publiucation.

Reopening discussions (May 2003) on solutions to problem results and potential
publications to be produced from the Drôme work.