Campanula Waldsteiniana and Pyramidalis aggs.:

1
Campanula Waldsteiniana and Pyramidalis aggs.
Not more than Neighbours - or Truly Relatives?
Zlatko Liber1, Sanja Kovacic1, Mirta Tkalec1,
Toni Nikolic1, Gerald Schneeweiss2
1Department of Botany and Botanical Garden,
Faculty of Science, University of Zagreb,
Marulicev 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 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.
Introduction
Edraianthus Campanula fenestrellata (1) Campanula
fenestrellata (2) Campanula poscharskyana Campanul
a garganica Campanula portenschlagiana Campanula
reatina Campanula pyramidalis Campanula
versicolor Campanula tommasiniana Campanula
waldsteiniana Campanula carpatica Campanula
isophylla 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
Results and Discussion
ISOPHYLLA
HETEROPHYLLA C. tommasiniana
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 PCR-amplified cpDNA
fragments and ITS1-5.8S-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.
isophylloids
HETEROPHYLLA
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 in-between the
isophyllous and heterophyllous lineages.
Molecular methods further confirmed that the
endemic aggregates Waldsteiniana and Pyramidalis
are not members of isophyllous or heterophyllous
line-ages, 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
hetero-phyllous taxa and grouped together.
Though quite distant in overall morphology, small
amphi-Adriatic lineages Waldsteiniana and
Pyrami-dalis 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.
ISOPHYLLA C. waldsteiniana
isophylloids (C. pyramidalis)
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.
isophylloids
C. justiniana
C. scheuchzeri
HETEROPHYLLA
C. tommasiniana
C. pyramidalis
C. waldsteiniana
ISOPHYLLA
C. velebitica
C. poscharskyana
C. fenestrellata
Fig. 3. Part of 50 majority rule consensus tree
derived from ITS sequence data
Fig. 4. Dendrogram based on the isoenzymes data
Isoenzymes 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.
E-mail zlatko.liber_at_botanic.hr