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Genotyping and phylogeography of the
Salix alba
S. fragilis

complex: evidence from nuclear

intron polymorphisms

Le Quang Trung

Algemene Plantkunde en Natuurbeheer (APNA)

(Plant Science and Nature Management)

Vrije Universiteit Brussel



PhD thesis

Willows including
Salix alba
S. fragilix

complex are widespread over Europe and play
important roles in biodiversity and in availability of energy resources for human beings.
The discrimination of hybrid and pure source
s, their distribution and relatedness are
difficult. Phylogeographical structure of willows remains unclear. Popular molecular
markers such as AFLP, RAPD, and ISSR have failed to clearly distinguish first generation
hybrids from introgressed hybrids in thi
s complex. cp
DNA marker has revealed no clear
phylogeographic structure of

species so far. Therefore, nuclear intron length
polymorphisms (ILPs), which have been recently developed and applied, could be the
markers of choice for this tetraploid compl

In this study, to obtain PCR
based ILP markers, consensus primers were designed based
on sequences of 5 homologous genes and 3 gene families of

sp. and technically
followed the approach of exon
primed intron
crossing PCR (EPIC
PCR). Among

developed consensus primer pairs, only C4H2, designed from the second and third exon
sequences of the

gene family of

sp. coding for cinnamate 4
(C4H) of the lignin biosynthesis pathway, gave rise to ILPs in willow species of
lix alba


. ILPs treated as diagnostic alleles at species level were observed among
nuclear intron length polymorphisms of three

genes and allowed unambiguous
detection of the first generation and introgressed hybrids in populations.

analysis of a female
S. alba

with a male introgressant with
S. fragilis


codominant inheritance of each intron. Sequences of the diagnostic alleles of both
species were >98% similar to those found in the first generation hybrids.


intron length polymorphic markers also allowed to identify multilocus
genotypes of
Salix alba
S. fragilis
, their first generation hybrids (F1) and introgressed
hybrids at European level. Sixteen multilocus genotypes of
S. alba
, 10 of
S. fragilis
, 9 of
first generation hybrids, 12 of introgressed hybrids with
S. alba
, and 5 of introgressed
hybrids with
S. fragilis

were obtained with one subset of 13 diagnostic specific alleles
markers for
S. fragilis
, and another subset of 9 for
S. alba
. Clusterin
g analysis revealed
five distinct genotype groupings for
S. alba
S. fragilis

and their first generation hybrids,
introgressants with
S. alba

and introgressants with
S. fragilis
. On a total of 961 examined

samples across Europe, the relative abundanc
e showed that
S. alba

accounted for
S. fragilis

for 14% and the various hybrids for 17%. Genotypic diversity estimates
at ecoregion level indicated that whereas only


were detected in southern
European peninsulas, both pure

species and

their hybrids were found in the
Western and Central highland regions, only
S. fragilis

in the Carpathians, and only F1
hybrids in the Central plains of Sweden. The abundance of
S. alba

genotypes was highest
in the Hellenic and Eastern balkan region (15 of

a total 16 genotypes) followed by 12 in
the Central highlands and 11 in Italy and the Alps, whereas only 6 were detected in the
Western plains a
nd Highlands and 5 in the Iberian

region. Among 16
S. alba

genotypes, a1 was the most common (56%) a
cross European ecoregions while
S. fragilis

genotypes were different between ecoregions. A neighbour joining tree revealed that the
S. alba

genotypes in the Iberian

peninsula was closer to those from the Western plains
and highlands whereas those from Ital
y and
Alps were more related to those in the
nearby Central highlands than from the Hellenic and Eastern Balkans. In addition to
potential of genotyping
S. alba
S. fragilis

complex, ILPs showed efficient in genotyping
some other

species and hybri
ds as well.

The intraspecific variation of nuclear

intron haplotypes, which was one of the
three allele markers in a common
S. alba

multilocus genotype a1 allowed to interpret the
divergent pattern of
S. alba

in Europe. The haplotype diversity o
f the nuclear

intron was high (Hd =0.871), most probably as a result of frequent recombination.
Though genetic differentiation between
S. alba

populations in Europe was low (Gst =
0.09), a significance of Gst within ecoregions revealed that
S. alb

populations in the
Hellenic and Eastern Balkan were most divergent between three southern peninsulas,
and that populations in the Central highlands were more divergent than those in the
Western plains and highlands. This divergent pattern of sequencing v
ariation agreed with
that based on multilocus genotypic diversity in
S. alba


above. The
significance of Gst when estimated between
S. alba

populations at European level
(P<0.001) implies the recolonization and isolation by distance between

in Europe during the postglacial period. The intraspecific variation of nuclear

intron haplotypes also permitted to predict the phylogeographical pattern of
S. alba

Europe during this time. Seventeen

intron haplotypes ne
sted from 123
sequences into three haplotypes groups, conveniently named
, and
. A significant
restricted gene flow with isolation by distance (P <0.001) and bootstrap values (ranging
from 56
65%) between these three groups indicated that the postglac
ial recolonization of
S. alba

in the Western and the Central Europe possibly occurred from all three southern
refugia, but most likely located in the Balkans, and rarely in Italy. A non
(P>0.05) of this geographical distance test between popul
ations and high gene flow
estimates (Nm = 2.86) implied that a putative refugium in the Iberic region might not
contribute to recolonization and isolation by distance, but in fact there might have been a
gene flow occurrence from several populations in thi
s refugium, mainly to the Western
plains and highlands and several localities in the Central region. Showing the highest
genetic differentiation between populations (Gst = 0.12; P = 0.032), and containing its
most common haplotype group

all over Europe (
namely present in 36 out of a total 49
investigated localities), the Hellenic and Eastern Balkans may be regarded as the main
refugium of the white willows
S. alba

in Europe.

The high amount of polymorphism in length of the nuclear cyp73 intron markers th
allowed to elucidate genotypes of the
S. alba
S. fragilis

complex at large geographical
distance, and those of other

species and hybrids as well. The high level of
intraspecific variation in their sequences also permitted to interpret the northwar
expansion pattern of
S. alba

in Europe during the postglacial period. The nuclear

intron markers were therefore considered to be more advantageous than
markers known so far in these approaches.