The rare, endemic zinc violets of Central Europe originate from Violalutea Huds.

U. Hildebrandt1, K. Hoef-Emden

1, S. Backhausen

1, H. Bothe

1, M. Boz_ek2, A. Siuta2, and E. Kuta

2

1Institute of Botany, The University of Cologne, Cologne, Germany2Department of Plant Cytology and Embryology, Jagiellonian University, Cracow, Poland

Received May 27, 2005; accepted September 23, 2005Published online: February 17, 2006� Springer-Verlag 2006

Abstract. Two endemic zinc violets of the sectionMelanium Ging. occur on heavy metal soils ofCentral Europe. The form with yellow flowers isrestricted to the area between Aachen, Germany,and Li�ege, Belgium, whereas the blue zinc violetexclusively thrives on a very small location atBlankenrode, Germany. Both violets are currentlytreated as separate species. Sequences of alto-gether 674 bp of the ITS1-5.8S rDNA-ITS2 re-gions of 61 different specimens of six taxaindicated that both violets are closely related toeach other and also to Viola lutea Huds. There-fore these two zinc violets are, at best, subspeciesor even only varieties of V. lutea. Thus they aretermed V. lutea ssp. westfalica and V. lutea ssp.calaminaria in the present manuscript. Microspo-rogenesis, pollen morphology and viability of thezinc violets, particularly of the blue violet ofBlankenrode, are often defective due to disturbedmeiosis. The population of the blue violetmight not yet be stabilized genetically but cancope with the adverse effects of the heavy metalelements.

Key words: Heavy metal plants, Zinc violets,Phylogeny, ITS1-5.8S rDNA-ITS2 regions, Micro-sporogenesis, Pollen analysis, Viola lutea.

Introduction

Two violets with a very local distributionbelong to the most endangered plants inCentral Europe. Both exclusively thrive onheavy metal soils and are therefore termed zincviolets. The form with the yellow flowersoccurs between Aachen and Liege on heapsoriginating from zinc mining since the Romantimes. The plants dominate in their appearanceat the sites which led Schwickerath (1931) tolend their name to the own plant association‘‘Violetum calaminariae’’ being typical forheavy metal soils.

The form with blue color has an evennarrower distribution. It only occurs at Blan-kenrode near Paderborn in a ditch and on thesurrounding heaps which arose from miningmainly for lead in medieval times (‘‘Bleikuhleof Blankenrode’’). Adjacent to this is ameadow which formerly was regularly floodedwith heavy metal-rich water originating fromthe heaps. Both areas were separated fromeach other by a highway some years ago andspan roughly one km2.

Pl. Syst. Evol. 257: 205–222 (2006)DOI 10.1007/s00606-005-0387-4

The zinc violets have attracted the atten-tion of biologists since a long time ago (Braun1854) as they pose fascinating and only partlyresolved questions. Among these, their ende-mic distribution, their ability to cope withhighly toxic concentrations of heavy metalsand their apparent inability to undergo radi-ation to new habitats are surprising. Morerecent work has shown that roots of the zincviolets at both Blankenrode and Aachen-Liegeare strongly colonized by arbuscular mycor-rhizal fungi (Hildebrandt et al. 1999, Toninet al. 2001) which might contribute signifi-cantly to the heavy metal tolerance of theseplants (Kaldorf et al. 1999). These ornamental,perennial species can be used to colonize heavymetal heaps which has been shown for a site(heap ‘‘Welnowiec’’) in Katowice; Poland(Jedrzejczyk and Rostanski 2001).

There is no doubt that both zinc violetsbelong to the section Melanium Ging. of thegenus Viola L. but otherwise their taxonomicand phylogenetic positions have been contro-versial for years (Wisskirchen and Haeupler1998). On the one hand, a close relationship ofthe yellow zinc violet of Aachen-Liege to V.lutea Huds. seems to be evident on the basis ofthe nomenclature. The taxon was ranked as asubvariety [Viola lutea Lejeune var. eleganssubvar. multicaulis, Griesinger 1937, Hegi1965], a variety [Viola lutea var. multicaulisKoch=V. lutea ssp. calaminaria, Runge 1972],a subspecies [V. lutea Huds. ssp. calaminaria(DC.) Rothm.=Viola lutea Huds. ssp. calam-inaria (Ging.) Nauenb.], and a species [V.calaminaria (DC.) Lej. =V. calaminaria(Ging.) Lej.=V. calaminaria (Lej.) Ernst].

On the other hand, a relationship of theyellow violet to the V. tricolor complex hadalready been suspected by Braun (1854) andwas corroborated by Heimans (1936), Gadella(1963), Ernst (1968) and Kakes and Everards(1976).

As to the blue zinc violet of Blankenrode,several authors accepted the view of Ernst(1968) regarding its subspecies rank manifestedin V. calaminaria (Ging. in DC) Lej. ssp.westfalica (Ernst) J. Heimans. In a major

contribution to the field, Nauenburg (1986)grouped both violets in the different species:Viola guestphalicaNauenburg (spec. nova) (theblue form of Blankenrode) and Viola lutea ssp.calaminaria (Ging. in DC.) comb. et stat. nov.(the yellow morph). His arguments for theseparation are differences in characters asflower color, type of pollination, restrictedareas of distribution, leaf morphology partic-ularly of the stipules, and, especially, chromo-some numbers. Nauenburg confirmed previousdeterminations of 2n=52 for the blue zincviolet from Blankenrode and 2n=48 for theyellow violet from Aachen-Liege (Kakes andEverards 1976), whereas Gadella (1963) ob-tained a value of 2n=52 also for plants of thelatter region. Nauenburg suggested that theblue form from Blankerode might have arisenby autopolyploidy from V. tricolor (2n=26).Nauenburg’s separation of the violets intodifferent species is accepted in the currentbotanical literature.

However, Nauenburg’s classification doesnot seem to be unequivocal. He himself posesthe blue zinc violet from Blankenrode outsideof the V. calaminaria group and in juxtaposi-tion to the V. lutea group (see Fig. 1 on page 3of his thesis, Nauenburg 1986). In the Blan-kenrode violets, the width of the flowers, thediameters of the stems and the form of thestipules are very variable and different at bothstands (ditch and meadow, Kakes andEverards 1976) and thus hardly provide crite-ria for taxonomic differentiation. As to thecolor, Kakes and Everards (1976) suggestedthat a simple mutation in one allele results indifferent colors of the blue and the yellowviolets. Further genealogical investigations onviolets from Blankenrode indicated that bothzinc violets are evolutionary young and closelyrelated taxa (Kakes 1979).

Based on classical cytogenetic results(Clausen 1931, Fothergill 1938, Erben 1996)andmodernmolecular data (Ballard et al. 1999,Yockteng et al. 2003), violets of the sectionMelanium (the Pansies) are regarded as amonophyletic and derived group of the genusViola. Interspecific hybridization is frequent

206 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

Fig. 1. Rooted maximum likelihood tree of ITS1 and ITS2 sequences (322 bp) of the genus Viola. Hybanthusconcolor served as outgroup taxon. Most of the sequences of the section Melanium Ging. did not separate.However, a V. tricolor clade was supported by moderate to low bootstrap values. The V. lutea ssp. westfalicaand V. lutea ssp. calaminaria sequences did not cluster with V. tricolor. Support values in order from left toright: maximum likelihood bootstrap/ maximum parsimony bootstrap/ neighbor-joining bootstrap/ posteriorprobabilities (Bayesian analysis); bold face, newly sequenced specimen; scale bar, substitutions per site; )lnL =2209.53

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 207

even between species of different chromosomenumbers often resulting in fertile hybrids (Clau-sen 1931, Fothergill 1938, Erben 1996). This isthe case also with the blue (Blankenrode) andyellow (Aachen-Liege) violets, resulting inhybrids of mixed colors with sometimes evenwhite and enlarged petals (Hildebrandt et al.1999, and unpublished observations). Alto-gether, no rigid criteria have been forwardedto date to justify a definitive separation of thetwo zinc violets into different species.

Modern molecular techniques provide anew avenue to resolve the controversial issue ofthe phylogenetic and taxonomic relationship ofthe zinc violets. In the current study, the DNAof both ITS sequences with the intervening 5.8SrDNA region (altogether 674 bp) has beencharacterized. In addition, pollen developmentand morphology of the zinc violets have beenstudied. In the section Melanium of the violets,pollen grains are multiaperturate and theaperture number is not correlated with theploidy level (Nadot et al. 2000). The currentcharacterization of the pollen morphology andof their viability assessed by a staining tech-

nique, will provide new insights into the evo-lutionary and taxonomic affinities of the zincviolets.

Material and methods

Plant material. Several related taxa of the sectionMelanium were investigated: Seeds, leaves or budsof ten taxa including two hybrids were collected atthe sites indicated in Table 1. Few seeds of V. luteassp. westfalica were originally collected at Blanken-rode, Germany, but then multiplied in a privategarden in Erftstadt-Bliesheim, Germany, where noother violets occur at least within a radius of1.5 km. V. lutea ssp. calaminaria was collected froma meadow close to the sports field in Breinigerberg,Germany, outside of the nature protected area.Seeds from both zinc violets were also purchased(www.rareplants.de). The two hybrids (V. lutea ssp.westfalica � V. lutea ssp. calaminaria) originatedspontaneously from the growth of both parents inclose proximity in the garden of the BotanicalInstitute of the University of Cologne. V. lutea ssp.calaminaria · V. arvensis grew spontaneously in theabove mentioned meadow close to Breinigerberg.Their hybrid nature was suspected from the colorof their flowers which was subsequently confirmed

Table 1. Origin of plant material

Plants collected Locality GeographicCoordinates(WGS84)

Collectiondate

Viola lutea ssp. westfalica Blankenrode, Germany(seeds multiplied in own garden)

N 51o 110 4500

E 08o 030150006/08/04

V. lutea ssp. calaminaria Breinigerberg, Germany,meadow at the sport fields

N 50o 4303200

E 06o 130220004/08/04

V. lutea Huds. blue andyellow morph

Hohneck, Vosges, Francemountains

N 48o 020 2800

E 06o 580 800027/06/04

V. arvensis Murr. Erftstadt-Bliesheim,Germany rape field

N 50o 4602300

E 06o 500360004/08/04

V. tricolor L. var. tricolor Boleslaw near Olkusz, Poland,calamine heaps

N 50o 170 3000

E 19o 280 200009/11/04

V. tricolor L. var. tricolor Siebertal, Harz mountains,Germany, heavy metal heaps

N 51o 110 4500

E 10o 270230028/08/04

V. tricolor L. var. dunense Terschelling, The Netherlands,west border of the island, sand dunes

N 53o 220 8000

E 05o 140500023/06/04

V. lutea ssp. calaminaria ·V. lutea ssp. westfalica

Cologne, Germany, house gardenof the Botanical Institute

N 50o 540 2400

E 06o 540360006/08/04

V. lutea ssp. calaminaria ·V. arvensis

Breinigerberg, Germany,meadow at the sport fields

N 50o 4303200

E 06o 130220004/08/04

V. biflora Spitzingsee, Ldkrs. Miersbach, Germany N 48o 4806800

E 16o 060320005/06/04

V. reichenbachiana Spitzingsee, Ldkrs. Miersbach, Germany N 48o 4806800

E 16o 060320005/06/04

V. reichenbachiana Garden of the Botanical Institutein Cologne, Germany

N 50o 5402400

E 06o 540360001/07/04

208 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

by sequencing. Seeds of V. tricolor from calamineheaps in Bolesław near Olkusz were kindly pro-vided by Dr. Grazyna Szarek-Łukaszewska fromthe Polish Academy of Sciences in Cracow. Theexact locations of the sites are given in Table 1. Inthe figures, letters indicate the plant collected andthe number the clone analyzed, e.g., a15 meansplant a was examined and, from this, clone number15 was sequenced.

DNA analysis. Leaves of the collected plantswere ground to a fine powder in liquid nitrogen,and DNA was isolated using CTAB (cetyl-trimethylammoniumbromide) according to Doyleand Doyle (1990). The method of White et al.(1990) was applied to amplify the DNA regionbetween the end of the 18S rRNA and the beginningof the 28S rRNA (including ITS1, 5.8S rRNA, andITS2 regions) using the primers ITS4 and ITS5. ThePCR products with a length of approximately680 bp were separated electrophoretically on a 1%(v/v) agarose gel (GibcoBRL ultrapure) followed byDNA staining with ethidium bromide and photo-graphing. PCR-products were cut off the gel andeluted with the MinElute PCR purification kit(Qiagen, Hilden, Germany) according to the man-ufacturer’s protocol. Cloning and sequencing wasexactly as described in Landwehr et al. (2002).

Phylogenetic analyses. The sequence align-ments done by ClustalX (Thompson et al. 1997)were refined by eye using the multiple sequencealignment editor SeaView (Galtier et al. 1996). Fourdata sets were subjected to phylogenetic analyses:an alignment using both ITS1 and ITS2 sequencescomprising the genus Viola andHybanthus concoloras an outgroup utilizing 43 specimens and 322positions, including one V. lutea ssp. westfalica andone V. lutea ssp. calaminaria sequence (for Fig. 1),an alignment of ITS1 (85 specimens and 237positions), an alignment of the ITS2 (80 specimens,194 positions) and an alignment of concatenatedITS1, 5.8S rDNA and ITS2 sequences using 75specimens and 674 positions (for the data ofTable 2). The first alignment consisted predomi-nantly of ITS1 and ITS2 sequences from GenBankexcept for seven new sequences in the claderepresenting the section Melanium Ging. The otherthree data sets were focused on the section Mela-nium Ging. and included 14 outgroup samplescomprising sequences of Viola reichenbachiana(eight specimens) and V. biflora (six specimens).These outgroup taxa were omitted from the data

sets for the unrooted analyses. The ingroup con-sisted of V. arvensis, V. lutea ssp. calaminaria, V.lutea ssp. westfalica, V. lutea (from Hohneck,France) and V. tricolor sequences. All data setspassed the test for homogeneity of base frequenciesacross taxa. The sequences newly obtained in thepresent study were deposited to EMBL/GenBank/DDBJ with the accession numbers for Viola arven-sis: DQ055340-47, V. biflora: DQ055348-53, V.lutea ssp. calaminaria from the house garden ofthe Botanical Institute in Cologne: DQ055354-55,V. lutea ssp. calaminaria from Breinigerberg:DQ055356-62, V. lutea from Hohneck. France:DQ055363-77, V. lutea ssp. westfalica from thehouse garden of the Botanical Institute in Cologne:DQ055378-79, V. lutea ssp. westfalica from Blan-kenrode, thereafter multiplied in the private gardenin Erftstadt-Bliesheim: DQ055380-81, V. reichenba-chiana from the garden of the Botanical Institute inCologne: DQ53055382-83, V. reichenbachiana fromSpitzingsee: DQ055384-89, V. tricolor var. tricolorfrom Siebertal: DQ 055390-98, V. tricolor var.dunense from Terschelling: DQ055399-405, V. tri-color var. tricolor from Bolesław: DQ055406-14.

To determine the evolutionary model fittingbest, the data according to the Akaike informationcriterion (AIC), Modeltest 3.6, was used (Posadaand Crandall 1998). All data sets for rooted orunrooted trees were subjected to maximum likeli-hood, unweighted maximum parsimony and dis-tance/neighbor-joining analyses using PAUP*4.0b10 (Swofford 2002). Bootstrap analyses wererun with 200 subsamples (maximum likelihood), 500subsamples (maximum parsimony; except for thedata set of Fig. 1: 200 replicates) or 1000 subsam-ples (distance/neighbor-joining). In both maximumlikelihood and distance/neighbor-joining analyses,the evolutionary model proposed by Modeltest 3.6was used. In addition, the rooted and unrootedconcatenated sequences (=ITS1, 5.8S rDNA andITS2 sequences together), as well as the unrootedalignment of the ITS1, were subjected to Bayesiananalyses using MrBayes 3 (Ronquist and Huelsen-beck 2003; 5 million generations, sample frequency:every 100th generation, four chains; burn-in deter-mined according to the ‘‘sump’’ plot). The align-ments used in this study are available from one ofthe authors (K. H.-E.) on request.

Slide preparation for chromosome analy-

sis. Seeds were germinated in Petri dishes on wetfilter paper. Seedlings were treated with saturated

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 209

Table

2.Support

values

inthedifferentevolutionary

modelsfortheViola

clades

Regionanalyzed

ITS1

ITS2

ITS1+IT

S2+

5.8SrD

NA

UnrootedIT

S1

UnrootedIT

S2

UnrootedIT

S1+

ITS2+

5.8SrD

NA

Number

ofspecim

ens

85

80

75

71

66

61

No.ofpositionsanalyzed

237

194

674

237

194

674

AIC

test

results

GTR

HKY+I

GTR+I

TVM

TVM

TVM

Bayesiananalysis

--

GTR+I

GTR

-GTR

Support

values

in%

ML/M

P/N

JML/M

P/N

JML/M

P/N

J/PP

ML/M

P/N

J/PP

ML/M

P/N

J/PP

ML/M

P/N

J/PP

V.luteassp.calaminaria

V.luteassp.westfalica

69/83/52

-/-/-

73/52/54

83/77/83/0.67

-/-/-

80/74/91/0.79

V.arvensis

80/-/87

69/69/73

98/98/97/1.00

71/73/87/0.90

57/-/57

100/92/98/1.00

V.tricolor

-58/66/52

85/86/81/0.93

-61/64/69

84/82/84/1.00

V.tricolorwithV.luteassp.

calaminariad3

84/100/71

-83/87/81/0.87

97/92/91/1.00

-90/86/91/1.00

Outgroup

V.biflora

100/100/100

100/100/100

100/100/100/1.00

--

-

V.reichenbachiana

86/-/94

89/93/94

89/99/99/0.97

--

-

Root

100/100/100

100/99/100

100/100/100/1.00

--

-

Note:AIC

,evolutionary

model

fittingbestthedata

accordingto

theAIC

test

inModeltest;Bayes,evolutionary

model

settingsforBayesian

analyses;

GTR,generaltimereversible

model

(Rodrıguez

etal.1990);HKY,Hasegawa-K

ishino-Y

anomodel

(Hasegawaet

al.1985);I,

pro-

portionofinvariable

sites;

ITS1,internaltranscribed

spacer1;IT

S2,internaltranscribed

spacer2;ML,bootstrapsupport

from

maxim

um

likelihoodanalysis;MP,bootstrapsupport

from

maxim

um

parsim

onyanalysis;NJ,

bootstrapsupport

from

distance/neighbor-joininganalysis;

PP,posteriorprobabilitiesinferred

from

Bayesiananalysis;root,branch

oftheoutgroup(V

.biflora

+V.reichenbachiana);TVM,transversion

model

(Rodrıguez

etal.1990)

210 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

solution of a-bromonaphthalene for 24 h at 4�Cand then fixed with acetic alcohol (96% ethanol :glacial acetic acid, 3:1) for 24 h. Chromosomeswere stained with 2% acetic orcein or with Schiff’sreagent following Feulgen’s procedure (Singh2003). Dissected root tips were squashed in a dropof 45% acetic acid by the cover glasses which werethen removed with dry ice. Slides were air-driedand mounted in Canada balsam (Aldrich).

Slide preparation for the study of microsporo-

genesis. Young flower buds (3–7 mm length) of V.lutea ssp. westfalica and V. lutea ssp. calaminariawere fixed in ethanol/acetic alcohol (3:1) andstained either with 2% acetic orcein, with Schiff’sreagent or with DAPI (40,6-Diamidino-2-phenylin-dole, Aldrich) (Singh 2003). Isolated anthers weresquashed and slides for storages were prepared asabove.

Determination of pollen viability and hetero-

morphism. To establish pollen viability and pollenheteromorphism, semi-opened flowers of all inves-tigated taxa were fixed in acetic alcohol (3:1). Whenpollen grains were stained with 1% acetocarmine,viable pollen grains (cytoplasm and nuclei) stainedred whereas degenerated, shrunken, empty (with-out cytoplasm and nuclei), non-viable pollen grainsremained colorless. Pollen grains also were differ-entially stained using Alexander’s mixture of threedyes (malachite green, acid fuchsin and orange G,Singh 2003). Viable and sterile pollen could easilybe differentiated by their contrasting colors – viablecolored red with the pollen wall green, whereassterile pollen grains were totally green.

Results

Phylogenetic analyses. The Genbank dataprovided ITS1 and ITS2 sequences of only 42Viola species which could be used withoutambiguity. ITS1 and ITS2 sequences (referringto positions 4–240 of Viola eugeniae ITS1sequence AY148233 and positions 1-150 of V.eugeniae ITS2 sequence AY148253) were sub-jected to analysis using the highly divergentHybanthus concolor as an outgroup. However,only 322 bp of this data set could be includedin the phylogenetic analyses since indels had tobe removed. The results confirmed theconclusion by Yockteng et al. (2003) that theviolets of the section Melanium show only fewsubstitutions in the analyzed DNA region and

are very closely related (Fig. 1). That V.tricolor was a separate entity was supportedby moderate values (maximum likelihood:66%; maximum parsimony: 54%; neighbor-joining: 84%; Bayesian analysis: 0.97), butdiverged from an otherwise completely unre-solved comb-like, separate clade consisting ofV. lutea, V. lutea ssp. westfalica, V. lutea ssp.calaminaria and other Viola species of thesection Melanium (Fig. 1). Clearly, V. luteassp. westfalica and V. lutea ssp. calaminaria didnot cluster in the V. tricolor clade (Fig. 1).

The resolution of the sequences withinsection Melanium needed to be improved byanalyzing a more comprehensive set of data(Figs. 2, 3). By combining ITS1, 5.8S rDNAand ITS2, the number of positions in thephylogenetic analyses could be increased to674 bp altogether. Several V. reichenbachianaand V. biflora sequences served as an out-group. In phylogenetic trees then obtained,the blue zinc violet from Blankenrode and theyellow one from Aachen-Liege unequivo-cally clustered with V. lutea and not withV. tricolor or V. arvensis (Figs. 2, 3). In thephylogenetic analyses of the ITS2 data set,however, the divergent ITS2 sequence ofV. lutea ssp. westfalica clone a2 was foundclose to the root, but clustered in the V. luteaclade in the ITS1 and in the concatenated(=ITS1, 5.8S rRNA and ITS2 together) dataset (Table 2). In the rooted data sets (Fig. 2),the position of the root was not resolved andthe V. lutea ssp. westfalica/V. lutea ssp.calaminaria/V. lutea clade was weakly sup-ported only in the ITS1 phylogeny (Fig. 2; forsupport values see Table 2). However, anexclusion of the outgroup taxa from theanalyses obviously increased the support forthe blue zinc violet/yellow zinc violet/V. luteaclade in the ITS1 phylogeny and in thephylogeny inferred from the concatenatedsequences (Table 2; Fig. 3). In the rootedITS1 phylogeny, bootstrap support was 69%(maximum likelihood), 83% (maximumparsimony) and 52% (distance/neighbor-join-ing) for this clade. In contrast, in theunrooted ITS1 phylogeny, bootstrap values

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 211

Fig. 2. Rooted maximum likelihood tree of ITS1, 5.8S rDNA and ITS2 sequences (674 bp) of 75 specimens ofeight taxa (14 outgroup and 61 ingroup specimens). The Viola lutea/ Viola lutea ssp. calaminaria/ ssp.westfalicaclade showed up but only the maximum likelihood bootstrap supported this clade moderately (73%). TheV. arvensis and V. tricolor clades were well to significantly supported in all analyses. Numbers of support valuesin the order from left to right: maximum likelihood bootstrap/ maximum parsimony bootstrap/ distance/neighbor-joining bootstrap/ posterior probabilities; scale bar = substitutions per site; )lnL = 2015.33

212 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

increased to 83% (maximum likelihood), 77%(maximum parsimony) and 83% (distance/neighbor-joining). In the unrooted concate-

nated data set, bootstrap values increased to80% (maximum likelihood) and 74% (maxi-mum parsimony) and 91% (distance/neigh-

Fig. 3. Unrooted maximum likelihood tree of concatenated ITS1, 5.8S rDNA and ITS2 sequences (674 bp) of61 specimens of six taxa. Support values for the Viola lutea/Viola lutea ssp. calaminaria/ ssp. westfalica cladeobviously increased compared to the rooted phylogeny in Fig. 2. Numbers for support values as in Fig. 2. Scalebar = substitutions per site; -lnL = 1510.13

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 213

bor-joining). Bayesian analysis resulted in aposterior probability of 1.00 for the V. luteassp. westfalica/V. lutea ssp. calaminaria /V. lu-tea clade in the unrooted concatenated dataset.

The ITS2 sequence of the yellow zinc violetfrom Breinigerberg V. lutea ssp. calaminariaclone d3 clustered together with the V. luteassp. westfalica/V. lutea ssp. calaminaria/V.lutea clade, whereas the ITS1 sequencegrouped with V. tricolor (trees not shown butavailable on request). This sequence may havebeen a hybrid from the yellow zinc violet andV. tricolor with recombination between theITS1 and ITS2 region. As a consequence, thesequence from the yellow zinc violet fromBreinigerberg clone d3 diverged basally fromthe V. tricolor clade in the phylogenetic trees,as inferred from the concatenated sequences(Figs. 2, 3).

The number of chromosomes of zinc

violets. In the present study chromosomenumbers of V. lutea ssp. westfalica, V. luteassp. calaminaria were counted in mitoticmetaphases of root tip meristematic cellsand on pollen meiosis. 4–6 metaphase plateswere analyzed for each taxon. The investi-gated taxa differed in chromosome numberwith 2n=52 in V. lutea ssp. westfalica and2n=48 in V. lutea ssp. calaminaria (notdocumented). A more detailed karyologicalanalysis was difficult to achieve because ofthe small chromosome size, ranging from1.4–3.4 lm in both taxa. In addition, thechromosomes were uniform in morphology,representing the metacentric type in majority(with the centromere located in the median orsub-median region).

Microsporogenesis of zinc violets. Micro-sporogenesis was examined in 270 cells ofV. lutea ssp. westfalica and in 259 cells ofV. lutea ssp. calaminaria. Meiosis was regularin V. lutea ssp. calaminaria resulting in tetradformation. No irregularities were found in theanalyzed stages of the first and second meioticdivisions (Fig. 4G–J). In contrast, irregulari-ties occurred in both first and second meioticdivisions in V. lutea ssp. westfalica. In each of

30 analyzed samples of metaphase I, despite ofmultivalent formation (trivalents, quadriva-lents, Fig. 4C), univalents laying out of theequatorial plate were visible (Fig. 4A–B). Inanaphase I, chromosome congression wasdisturbed resulting in chromosomes laggingbehind (Fig. 4D). Triads and polyads (Fig. 4F)accompanied tetrads due to the disturbedmeiosis. In tetrads and polyads, individualchromosomes were visible in microspores(Fig. 4E–F).

Viability and heteromorphism of pollen

grains. The determination of the pollen viabil-ity assayed by the acetocarmine test (Table 3;Fig. 5) indicated that all investigated specieshave rather high frequency of stainable pollengrains, being the highest (� 97%) in V. tricolorfrom calamine heaps in Boleslaw and in theyellow morph of V. lutea from the Vosges. Theyellow zinc violets have very high percentage ofstainable pollen grains (95%), whereas onlyabout 77% of them were stained in the blueplants originating from Blankenrode. In addi-tion, mature pollen grains in the blue zinc violetvary conspicuously in size (Fig. 5A). Althoughsmall and large (even giant) pollen grains werestained, they were cytologically unbalancedand will not be able to germinate as is deducedfrom our own knowledge with other plantspecies. Pollen viability was evidently lower(48% and 25%, respectively) in the two hybridsV. lutea ssp. calaminaria � V. lutea ssp.westfalica and V. lutea ssp. calaminaria �V. arvensis (Table 3). Empty, degeneratingpollen grains accompanied stainable ones,which varied in size (Fig. 5H–I), indicatingthat meiosis in the hybrid forms was disturbed.

Counting the number of apertures revealeda striking pollen heteromorphism. The aper-ture number varied from 3–6 depending onspecies. 4-apertured pollen grains were mostfrequent in all analyzed taxa with the exceptionof V. arvensis where 5-apertured pollen grains(63%) prevailed (Figs. 5C, 6). Three-aperturedpollen grains were not observed, neither in V.arvensis, V. tricolor var. dunense nor in V. luteassp. calaminaria � V. arvensis. They weresporadically formed in the yellow zinc violet

214 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

(0.4%) and V. tricolor from calamine heaps inBolesław (0.4%). The highest frequency of 3-apertured pollen grains was observed in theblue zinc violet Viola lutea ssp. westfalica(10%) and in V. lutea ssp. westfalica � V. luteassp. calaminaria (7%).

Discussion

The ITS1 and ITS2 sequence alignment of 510positions by Yockteng et al. (2003) indicatedthat the Pansies (Viola section Melanium) areof monophyletic origin, but the reduced diver-gence within the Pansies did not allow theauthors to draw any further definitive conclu-sion regarding the evolutionary relationshipwithin these violets. In that study, apparently

all sequences published were utilized, and theseauthors obtained their information from directsequencing of the PCR products withoutcloning. For a first phylogenetic analysis inthe present study, only previously publishedsequences without ambiguous positions wereused, but the taxon sampling had to be furtherreduced due to partial sequences in the data-bases. In addition, the inclusion of distantlyrelated taxa (esp. of Hybanthus concolor)reduced the number of positions for phyloge-netic analyses to only 322 nucleotides (Fig. 1).The analysis of this short data set confirmedthe conclusion of Yockteng et al. (2003) thatthe genetic divergence in the section Melaniumis low. The bootstrap values in Fig. 1 alreadyindicate a differentation, though weakly

Fig. 4. Microsporogenesis and tetrad formation in Viola lutea ssp. westfalica A–F and Viola lutea ssp.calaminaria G–J. A–B Metaphase I with univalents (arrows), C Metaphase I with trivalents (doublearrowheads) and quadrivalent (arrowhead), D Anaphase I with lagging chromosomes (arrow), E Tetrad withsingle chromosomes in microspores (arrows), F Hexad, one microspore smaller than others (arrow), singlechromosomes visible in microspores, G Diad, H Metaphase II, I Late anaphase II, J Late telophase II. A-F -DAPI staining, G–J - acetic orcein staining. Bars in A,B,D, G–J=10 lm; in C=5 lm; in E,F=30 lm

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 215

supported, of the V. lutea comb (including V.lutea ssp. calaminaria and V. lutea ssp. west-falica) from V. tricolor sequences. The dataalso call for a more profound reexamination ofthe taxonomy of Viola lutea and its relativessuch as V. eugeniae and the others listed inFig. 1. To obtain a better resolution, 61sequences of different Melanium taxa includingV. lutea, V. arvensis, V. tricolor, V. lutea ssp.calaminaria, V. lutea ssp. westfalica weresubjected to ITS1-5.8S rDNA-ITS2 analysisof 674 positions altogether in the presentstudy. Both the rooted (Fig. 2) and particu-larly the unrooted (Fig. 3) tree unequivocallyshowed a separation of the blue and yellowviolets from V. arvensis and, notably, from V.tricolor. The maximum likelihood, unweightedmaximum parsimony as well as the neighbor-joining analyses revealed that both zinc violetstogether with V. lutea form a cluster which isseparated from V. tricolor by bootstrap valuesclose to 100%. Thus, we conclude that the blueand yellow zinc violets are, at best, subspeciesor even only forms (varieties) of V. lutea. Theproposal of Nauenburg (1986) to classify theviolet of Blankenrode as a separate, ownspecies (V. guestphalica) does not appear to

be justified. Despite some arguments regardingthe nomenclature raised by Nauenburg (1986)we propose to adopt names from Ernst (1968)and to designate the zinc violets as V. luteaHuds. ssp. calaminaria (Ging.) Nauenb. for theyellow violet of Aachen-Liege and V. lutea ssp.westfalica (comb. et stat. nov. for the bluemorph of Blankenrode).

The counting of the chromosome numbersconfirmed the correctness of more recent deter-minations, particularly of those of Nauenburg(1986). The chromosome number of V. luteassp. westfalica (2n=52) which was just thedouble of that ofV. tricolorneeds comment.Thelow genetic differentiation combined with com-plex cytological evolution indicates a rapidradiation of the section Melanium in Europeand Northern Africa, being an effect of reticu-late evolution (Yockteng et al. 2003). Hybrid-ization between different violet species isparticularly abundant within the section Mela-nium (Clausen 1931, Erben 1996). Fertilehybrids have been described to occur even withodd numbers (Valentine 1975). Possible basicchromosomenumbers for the sectionwere givenfrom karyological data by different authors.Clausen (1931) proposed x @ 6, Ballard (1996,

Table 3. Pollen viability assessed by staining using the acetocarmine dye

Taxon No. offlowers (n)

No. ofpollen grains analyzed

Stainable pollen grains %

Min. Max. Average ±standarddeviation

V. lutea ssp. westfalica 5 707 63 86 77 ± 9

V. lutea ssp. calaminaria 4 711 88 99 95 ± 5

V. lutea Huds. blue morph 5 640 52 96 75 ± 16

V. lutea Huds. yellow morph 4 536 93 99 97 ± 5

V. arvensis Murr. 4 507 73 100 93 ± 13

V. tricolor L. var. tricolor,heap, Poland

4 478 93 99 97 ± 3

V. tricolor L. var. dunense 4 560 64 97 87 ± 16

Hybrid V. lutea ssp. calaminaria �V. lutea ssp. westfalica

5* 451 0 66 48 ± 35

Hybrid V. lutea ssp. calaminaria �V. arvensis

5* 476 0 44 25 ± 23

*including 2 sterile flowers; 100% of empty, non-viable, shrunken pollen grains.Standard deviations were calculated from the percentage of stainable pollen grains in anthers of analyzedflowers.

216 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

after Yockteng et al. 2003) x = 5, 6 or x = 10,11, Erben (1996) x = 11, Ballard et al. (1999)x=5–17,Yockteng et al. (2003) x=5or x=7.Currently it is believed that the primitive basicchromosome number in angiosperms and evenin all land plants is x= 7 (Bennett 2004). Loweror higher basic numbers have to arise bychromosome mutations or hybridization(Grant 1981, Bennett 2004). Therefore V. tri-color with 2n=26 can be recognized as poly-ploid (tetraploid) but not as a diploid cytotype.A haploid chromosomal set of n=13 could bean effect of hybridization of putative parentalspecies with different basic chromosome num-bers e.g. 7 and 6. Hybridization following bypolyploidization resulted in a fully fertile allo-

polyploid generating in V. tricolor. If so, Violalutea ssp. westfalica could be thought to havearisen by autopolyploidy from V. tricolor (sim-ple multiplication of V. tricolor genome byproduction of unreduced gametes) and thuswasan alloautopolyploid. However, this wouldresult in a highly unstable species with irregularmeiosis and reduced pollen fertility and seedsettings. Therefore, we strongly favor the sug-gestion that V. lutea ssp. westfalica as well asV. lutea ssp. calaminaria have evolved not byautopolyploidy from V. tricolor but originatedfrom V. lutea via hybridization or mutations(structural chromosome mutations or genemutations). It is noteworthy here that thechromosome number of V. lutea ssp. sudetica

Fig. 5. Pollen morphology and pollen heteromorphism. A Viola lutea ssp. westfalica, pollen grains differ in size,degenerated empty pollen grains visible, B V. lutea ssp. calaminaria, 4- and 5-apertured pollen grains,C V. arvensis, 4- and 5-apertured pollen grains, D V. lutea blue morph, 4- and 5-apertured pollen grains,E V. lutea yellow morph, 3-, 4- and 5-apertured pollen grains, F Viola tricolor var. dunense, 4-apertured pollengrains, G V. tricolor var. tricolor from calamine heap in Bolesław, Poland, 4- and 5-apertured pollen grains,H V. lutea ssp. calaminaria � Viola lutea ssp. westfalica, 3- and 4- apertured pollen grains, empty degeneratedpollen grains visible, I V. lutea ssp. calaminaria � V. arvensis, 4- and 5-apertured pollen grains, empty,degenerated pollen grains visible. Bar in all figures = 5 lm

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 217

has been confirmed to be 2n=50 (Krahulcovaet al. 1996). Violets particularly of the sectionMelanium but also in others either have nocrossing barriers or genetic isolation is veryweak (Clausen 1931; Erben 1996; Kakes 1977;1979;Kakes andEverards 1976;Marcussen andBorgen 2000). If the two zinc violets aredescendants of V. lutea, we propose two possi-ble explanations for their evolution:

a) Both zinc violets could have been directlyderived from V. lutea without hybridizationwith other species developing as ecotypes atboth stands. Such microevolutionary pro-cesses were described for other species onheavy metal soils (Ernst 1999, Wierzbickaand Rostanski 2002). V. lutea ssp. calami-nariawith the same chromosome number asV. lutea (2n = 48) reproduces normally(Pilarska et al., unpublished results). How-ever, in the case of V. lutea ssp. westfalica,the harsh conditions in the areas of Blan-kenrode with the soil contaminated by leadand zinc can negatively influence the repro-ductive processes, mainly meiosis, bothmicrosporogenesis (this communication)and also female gametophyte and embryo

formation (Siuta et al. 2005). The observedabnormalities leading to reduced pollen andembryo viability might be the consequenceof growth in the deleterious heavymetal soil(which is retained with the growth in non-polluted soil in the private garden). Similarabnormalities have been described also forother plant species outside the genus Viola(see Czapik 2002 and references cited,Biskup and Izmaiłow 2004). Indeed, in thecase of V. lutea ssp. westfalica, many seedsdo not germinate (Kakes 1977, Siuta et al.2005). Nonetheless, the around 30% offertile seeds might suffice for V. lutea ssp.westfalica to survive. Apparently formednon-vegetatively Offsprings have occasion-ally been observed in our own gardenpopulation.

b) Both zinc violets may have arisen fromintrogression of V. lutea with other Violaspecies, possibly with V. tricolor. Suchhybrids between these two species havebeen described (Clausen 1931, Fothergill1938, Krahulcova et al. 1996), however,not from heavy metal stands. Progeny ofhybrid swarms between V. lutea and

Fig. 6. Pollen heteromorphism of investigated taxa of Viola section Melanium. Hybrid 1=V. lutea ssp.calaminaria � Viola lutea ssp. westfalica; hybrid 2 = V. lutea ssp. calaminaria � V. arvensis. The numbersbehind the bars (3, 4, 5, 6 upper abcissa) indicate the number of apertures

218 U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe

V. tricolor can have the chromosome num-bers of either parent but also intermediate,or even higher as well as lower chromo-some numbers are also possible. Repeatedback-crosses may have produced the cur-rent zinc violets which are genetically closeto V. lutea with few characters only of theother parent(s). Hybrid origin of the bluezinc violet with two or even more Violaspecies involved was postulated by Kakes(1979). The hypothesis was based on irreg-ular male meiosis observed in specimensfrom Blankenrode.

It is somewhat surprising that introgressionfavored the formation of V. lutea formsrestricted to the polluted areas but not ofV. tricolor. Ecotypes of V. tricolor thriving onheavy metal soils which are known to theauthors from stands in Poland, in the GermanHarz mountains or in Bad Bleiberg in Austria.V. tricolor is either not (Kakes and Everards1976) or only rarely (Haeupler and Schonfelder1988) found in the wider surroundings of eitherBlankenrode or Aachen-Liege. V. arvensis,however, occurs abundantly in this vicinity.An hybrid between Viola lutea ssp. westfalicaand V. arvensis, termed V. preywischiana wasdescribed to occur at the edge of the heavymetalditch of Blankenrode (Nauenburg 1987). How-ever, V. arvensis is apparently heavy-metal-sensitive, and selection, therefore, favors thetolerant V. lutea ssp. westfalica population(Kakes and Everards 1976). If heavy metalecotypes ofV. tricolordid occur in the vicinity ofBlankenrode or Aachen-Liege in the past, itwould be surprising that hybridization favoredV. lutea but not the heavy metal tolerantpopulations of V. tricolor.

Pollen heteromorphism is common in morethan 85% of all Pansies and is not correlatedwith the chromosome number or the ploidylevel of the species (Dajoz 1999, Nadot et al.2000). The 3-aperturated pollen morph is theancestral condition and the occurrence of 4-, 5-or 6- aperturated pollen grains are considered asderived (Nadot et al. 2000). V. lutea from theVosges as well as V. lutea ssp. calaminaria have

multiple pollen morphs, and their high chro-mosome number might also indicate their morerecent evolution. Aperture number was re-ported to be positively correlated with thegermination speed and negatively with the lifeexpectance (Dajoz 1999). The high aperturenumber thus could represent an ecologicaladvantage in the mountain climate. Similarly,V. lutea ssp. calaminaria has high aperturenumbers which could be taken as (faint) indi-cation that it is derived from V. lutea. TheBlankenrode violet has, however, 85% of 4-apertured pollen grains and only 15% with 3apertures. This could mean that pollen tubesmust have a longer life span for fertilizationunder the harsh environmental conditions ofBlankenrode. The low pollen and seed fertilitymay not only be a consequence of the heavymetal toxicity but could also indicate that thepopulation in Blankenrode has not yet fullydifferentiated genetically. All this may explainwhyV. lutea ssp. westfalica does not swarm andcan only thrive in the harsh condition which istoo adverse for other violets.

Comments on the current local distributionof the zinc violets may be of interest. Nowa-days V. lutea occurs, for example, at Hohneckin the Vosges mountains, with both blue andyellow morphs. Distribution at Hohneck ispatchy, with blue plant colonies being sepa-rated from yellow morphs by distances of some50–100 m forming local populations. Interme-diate color forms can also be found indicatingthat cross pollination occurs between popula-tions. V. lutea may have had a much widerdistribution in Central Europe during orimmediately after the last glacial period thanat present. The general rise in temperature andthe gradually improving growth conditionsmay have forced V. lutea, due to poorcompetitiveness to survive only on heavy metallocations in the plains of Central Europe.There they have developed to form a blueswarm in Blankenrode and a yellow one inAachen-Liege. In the wider neighborhood ofBlankenrode, other glacial relicts of disjunctdistribution occur, e.g. Arabis alpina at the

U. Hildebrandt et al.: The rare, endemic zinc violets of Central Europe 219

Bruchhauser Steine near Brilon (Runge 1972).Alternatively, the zinc violets may have devel-oped recently: the ditch in Blankenrode andthe surrounding heaps were formed by Pb-mining in the medieval times. Miners togetherwith their tools may have introduced seeds orother propagules from distant locations. Thelarge percentage of defective pollen grainsfurther supports the view that the populationin Blankenrode has developed in historicaltimes and has not been stabilized geneticallyon the heavy metal soil.

The discussion about the phylogenetic andtaxonomic affinities of the violets is not justacademic. Violets form cytotoxic cyclotids,varying in concentration between the species.These may have pharmacological applicationsacting as antitumor agents (Lindholm et al.2002, Svangard et al. 2004). Thus the taxo-nomic resolution and particularly the preser-vation of their endangered species are ofconsiderable interest for future potential appli-cations.

The authors are indebted to Dr. M. GeoffreyYates, Lewes, England, for improving the English.This work was kindly supported by the partnershipbetween the Universities of Cracow and Cologne.Continuous support by grants from the DeutscheForschungsgemeinschaft (to H. B.) and the Jagiel-lonian University in Cracow (to E.K) is alsogratefully acknowledged.

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Addresses of the authors: Ulrich Hildebrandt,Kerstin Hoef-Emden, Stefanie Backhausen,Hermann Bothe (e-mail: hermann.bothe@uni-koeln.de), Institute of Botany, The University of Cologne,Gyrhofstrasse 15, 50923 Cologne, Germany. MonikaBoz_ek, Aneta Siuta, Elz_bieta Kuta, Department ofPlant Cytology and Embryology, Jagiellonian Uni-versity, 52 Grodzka str., 31-044 Cracow, Poland.

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