Campanula Waldsteiniana and Pyramidalis aggs.:

Not more than Neighbours - or Truly Relatives?

Cluster for 68 Cases/65 vars., Manhattan+UPGMA

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IntroductionIntroduction

Material and MethodsMaterial and Methods

Results and Results and DiscussionDiscussion

C. tommasinianaC. tommasiniana

Campanula pyramidalis

Campanula waldsteinianaCampanula waldsteiniana

Fig. 1. Dendrogram based on the flower morphological traits

Fig. 4. Dendrogram based on the isoenzymes data

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.

MorphologyQuantitative 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.

IsoenzymesFresh 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.

Nuclear ITS regionTotal 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.

Chloroplast trnT-trnF regionFor 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.

Fig. 2. 50% majority rule consensus tree derived from restriction site data of trnT-trnF cpDNA regions

Fig. 3. Part of 50% majority rule consensus tree derived from ITS sequence data

Tree Diagram for 33 Cases, Unweighted pair-group average, Euclidean distances

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Linkage Distance

C. pyramidalis (29)C. pyramidalis (30)C. pyramidalis (28)C. pyramidalis (26)C. pyramidalis (27)C. pyramidalis (25)C. debarensis (21)

C. reatina (24)C. waldsteiniana (20)C. waldsteiniana (19)C. waldsteiniana (18)

C. velebitica (22)C. jordanovii (31)C. rotundifolia (9)

C. velebitica (5)C. herzegovina (10)

C. justiniana (13)C. justiniana (12)

C. velebitica (4)C. tommasiniana (17)C. tommasiniana (16)

C. herzegovina (23)C. caespitosa (15)

C. velebitica (3)C. velebitica (2)

C. rotundifolia (6)C. justiniana (11)C. velebitica (33)

C. marchesetti (7)C. albanica (32)

C. scheuchzeri (8)C. justiniana (14)

C. velebitica (1)

“isophylloids” (C. pyramidalis)

ISOPHYLLA +C. waldsteiniana

HETEROPHYLLA + C. tommasiniana

EdraianthusCampanula fenestrellata (1)Campanula fenestrellata (2)Campanula poscharskyanaCampanula garganicaCampanula portenschlagianaCampanula reatina

Campanula pyramidalisCampanula versicolorCampanula tommasinianaCampanula waldsteinianaCampanula carpaticaCampanula isophylla

Campanula velebiticaCampanula justinianaCampanula rotundifolia (1)Campanula rotundifolia (2)Campanula hercegovinaCampanula scheuchzeriCampanula witasekianaCampanula romanicaCampanula xylocarpaCampanula gentilisCampanula tatraeCampanula moravicaCampanula cochleariifolia

ISOPHYLLA

“isophylloids”

HETEROPHYLLA

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“isophylloids”

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, Croatia2Institute of Botany and Botanical Garden, University of Vienna, Rennweg 14, A-1030 Vienna, Austria

E-mail zlatko.liber@botanic.hr

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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.

Campanula versicolor