Transcript C. waldsteiniana

Campanula Waldsteiniana and Pyramidalis aggs.:
Not more than Neighbours - or Truly Relatives?
Zlatko Liber1, Sanja Kovačić1, Mirta Tkalec1, Toni Nikolić1, Gerald Schneeweiss2
1Department
of Botany and Botanical Garden, Faculty of Science, University of Zagreb, Marulićev trg 22, HR-10000 Zagreb, Croatia
2Institute of Botany and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria
Material and Methods
Morphology
HETEROPHYLLA
+ C. tommasiniana
“isophylloids”
HETEROPHYLLA
ISOPHYLLA +
C. waldsteiniana
“isophylloids”
(C. pyramidalis)
0
2
4
6
8
10
Linkage Distance
Campanula velebitica
Campanula justiniana
Campanula rotundifolia (1)
Campanula rotundifolia (2)
Campanula hercegovina
Campanula scheuchzeri
Campanula witasekiana
Campanula romanica
Campanula xylocarpa
Campanula gentilis
Campanula tatrae
Campanula moravica
Campanula cochleariifolia
Fig. 2. 50% majority rule
consensus tree derived from
restriction site data of trnT-trnF
cpDNA regions
Fig. 1. Dendrogram based on the flower
morphological traits
Chloroplast trnT-trnF region
For the chloroplast DNA analysis total DNA was isolated from 100 mg of the fresh
leaves tissue using Dneasy® Plant Mini Kit (Qiagen). The chloroplast DNA regions
between trnT and trnF genes (about 1800 bp) were amplified. The amplified products
were digested with 10 different restriction enzymes and separated on 1,8 % agarose
gel. The ambiguous, mostly low molecular weight restriction fragments were
additionally resolved using HPLC system (Agilent 1100 Series). The obtained restriction
patterns were transformed to binary data and Maximum parsimony analyses were
conducted using PAUP 4.0b10.
Nuclear ITS region
Total genomic DNA was extracted from silica-gel dried (rarely air dried) leaf material,
following the CTAB extraction protocol. The ITS1-5.8S-ITS2-regions of the nuclear
ribosomal DNAs were amplified and cycle sequenced using BigDye terminator kit and
ABI PRISM 377 DNA autosequencer (Applied Biosystems). Maximum parsimony
analyses were conducted using PAUP 4.0b10.
Cluster f or 68 Cases/65 v ars., Manhattan+UPGMA
C. pyramidalis
C. velebitica
C. waldsteiniana
10
“isophylloids”
C. tommasiniana
15
C. justiniana
20
C. fenestrellata
25
HETEROPHYLLA
30
C. scheuchzeri
35
Linkage Distance
European
amphi-Adriatic
and
Western Balkan region is floristically
exceptionally rich, among other,
comprising at least 85 species and
subspecies of Campanula genus,
without many poorly known lower
(incipient) taxa and hybrids. Among the
most interesting in the region are
endemic indigenous lineages, such as
the aggregates Waldsteiniana (C.
waldsteiniana and C. tommasiniana)
and Pyramidalis (C. pyramidalis, C.
versicolor and C. secundiflora). Being
neither isophyllous nor heterophyllous,
but a little bit of both, relationships of
Waldsteiniana and Pyramidalis to other
campanuloids in the region puzzle
botanists for a long time.
Comparative
morphological
and
ecological
data,
isoenzymes,
restriction-site variation of PCRamplified cpDNA fragments and ITS15.8S-ITS2- sequences of the nuclear
ribosomal DNA were used to provide
better insights into these fine
relationships. Data were analysed
using multivariate statistic and cladistic
methods. The results shown here are
preliminary: parts of several more
complex studies that include more than
80 Campanula taxa of this region. This
poster deals exclusively with the
results
and
taxa
related
to
Waldsteiniana
and
Pyramidalis
lineages.
ISOPHYLLA
C. velebitica (1)
C. justiniana (14)
C. scheuchzeri (8)
C. albanica (32)
C. marchesetti (7)
C. velebitica (33)
C. justiniana (11)
C. rotundifolia (6)
C. velebitica (2)
C. velebitica (3)
C. caespitosa (15)
C. herzegovina (23)
C. tommasiniana (16)
C. tommasiniana (17)
C. velebitica (4)
C. justiniana (12)
C. justiniana (13)
C. herzegovina (10)
C. velebitica (5)
C. rotundifolia (9)
C. jordanovii (31)
C. velebitica (22)
C. waldsteiniana (18)
C. waldsteiniana (19)
C. waldsteiniana (20)
C. reatina (24)
C. debarensis (21)
C. pyramidalis (25)
C. pyramidalis (27)
C. pyramidalis (26)
C. pyramidalis (28)
C. pyramidalis (30)
C. pyramidalis (29)
ISOPHYLLA
Introduction
Edraianthus
Campanula fenestrellata (1)
Campanula fenestrellata (2)
Campanula poscharskyana
Campanula garganica
Campanula portenschlagiana
Campanula reatina
Campanula pyramidalis
Campanula versicolor
Campanula tommasiniana
Campanula waldsteiniana
Campanula carpatica
Campanula isophylla
Tree Diagram f or 33 Cases, Unweighted pair-group av erage, Euclidean distances
C. poscharskyana
Campanula waldsteiniana
Quantitative floral characteristics were compared to establish the pattern of mutual
relations on individual and population levels. Up to 20 fresh Campanula flowers per
species were collected from the living plants at the natural localities. To each flower 9
morphological variables were measured on digital photographs using CARNOY 2.0
image analyser. Only the results of cluster analysis made in STATISTICA 6.0 package
are shown here.
5
Fig. 3. Part of 50% majority
consensus tree derived from
sequence data
rule
ITS
C. tommasiniana
Results and
Discussion
Results
of
comparative
floral
morphology (Fig. 1) indicated that the
quantitative
flower-characteristics
could be valuable in distinguishing the
main Campanula groups, placing
Waldsteiniana and Pyramidalis inbetween
the
isophyllous
and
heterophyllous lineages. Molecular
methods further confirmed that the
endemic aggregates Waldsteiniana
and Pyramidalis are not members of
isophyllous or heterophyllous lineages, but are clearly isolated and
placed intermediary (Figs. 2 and 3).
Similar result could be observed
according to the analyses of the
isoenzymatic
activity
(Fig.
4):
“isophylloid” lineages are separated
from both isophyllous and heterophyllous taxa and grouped together.
Though quite distant in overall
morphology, small amphi-Adriatic
lineages Waldsteiniana and Pyramidalis share certain characteristics with
both isophyllous and hetero-phyllous
campanulas, but are separated from
both lineages. Moreover, the results of
molecular (RFLP cpDNA, ITS nrDNA)
and isoenzymatic analyses indicate
that Waldsteiniana and Pyramidalis
could also be truly – though distantly –
mutually related. Perhaps the nearest
relatives of this peculiar “isophyllous”
aggregates should be searched for
among the old, relic taxa such as
Campanula carpatica, C. raineri, C.
morettiana etc.
0
Fig. 4. Dendrogram based on the
isoenzymes data
Isoenzymes
Campanula versicolor
Fresh mature leaves of eight Campanula species were homogenized in Tris-HCl buffer
pH 8.0 with addition of PVP and centrifuged (30000g at 4C) for 60 min. Proteins were
resolved by 10 % vertical anodic PAGE electrophoresis in native conditions. Gels were
stained for superoxide dismutase, pyrogallol peroxidase, ascorbate peroxidase and
esterase. The obtained restriction patterns were transformed to binary data. Different
bands encoding the same isoenzymes were numbered by the relative mobilities of the
enzymes they specify. Statistical data evaluations contain cluster analysis were
conducted using STATISTICA 6.0 package.
Campanula pyramidalis
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