Multivariate morphometric study of theCardamine pratensis group (Cruciferae) in the Carpathian and Pannonian area

A multivariate morphometric study of theCardamine pratensis group is presented, based on 84 population samples collected from the Carpathian and Pannonian area in the Czech Republic, Slovakia, Poland, Ukraine, Hungary, and Romania. Among the multivariate methods, principal component analysis, cluster analysis, and classificatory and canonical discriminant analysis were used. The analysis of chromosome numbers from all populations studied showed wide variation. The morphometric study showed that not all groups of populations characterised by their chromosome numbers and geographical criteria are morphologically, and thus taxonomically, distinguishable. Besides the morphologically well characterised speciesCardamine dentata andC. rivularis, the following species were recognised in the area studied:C. matthioli, C. majovskii andC. pratensis. Within the last species, besides the typical populations, two diploid types are provisionally recognised: type ucranica and type rivularis auct..     

Infraspecific differentiation in the Balkan diploidCardamine acris (Brassicaceae): Molecular and morphological evidence

Molecular and morphological variation in the Balkan diploidCardamine acris was studied by amplified fragment length polymorphism (AFLP) and multivariate morphometric analyses. Principal coordinate analysis and neighbour-joining analysis of the AFLP data showed three genetically differentiated groups of populations corresponding to their geographic origin. Following the results of molecular analysis three subspecies were recognized by a combination of morphological characters as shown by separate analyses of field-collected and cultivated plants. These subspecies are allopatric;C. acris subsp.acris occurs in most parts of the Balkan Peninsula, and newly described subsp.pindicola and subsp.vardousiae are endemic to the Northern Pindhos Mts. in Northwestern Greece, and Central Greece, respectively. The differentiation into geographically isolated subspecies is most probably caused by long-term isolation of populations in different mountain ranges of the Balkan Peninsula, possibly initiated during the cold periods of the Pleistocene.     

Natural hybridization in Cardamine (Brassicaceae) in the Pyrenees: evidence from morphological and molecular data

While researching Cardamine (Brassicaceae) in the Pyrenees, putative hybrid plants were found at two natural sites. Pollen grain viability, AFLP, and multivariate morphometric analyses were performed in order to assess the plants' presumed hybrid origin, establishing that natural hybridization between the diploids C. crassifolia and C. amara ssp. pyrenaea had occurred. A new diploid nothospecies, C. × enriquei (2 n = 2x = 16), is described. Examination of 18 morphological characters showed the intermediacy of the hybrid between the parental taxa in most characters. AFLP analyses of C. amara ssp. amara , ssp. austriaca , ssp. olotensis and C. raphanifolia , demonstrated the close position of the hybrid to C. crassifolia , and revealed that the highest number of markers were shared with the parents. Polymorphism found in the AFLP pattern of the hybrid suggested recurrent origin, segregation and/or backcrosses, although assessment of pollen viability indicated high male sterility. The hybridization event reported here represents the second documented case between the C. pratensis group and C. amara . An account of the nomenclature of C. crassifolia is also presented, including lectotypification of relevant names.  © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 275–294.     

Morphometric and RAPD study of theMelampyrum sylvaticum group in the Sudeten, the Alps and Carpathians

TheMelampyrum sylvaticum group is a heterogeneous group at the inter- and infra-specific level. A wide range of morphological characters was examined on specimens collected for this study. The corolla size, the corolla colour and the anther length are considered the most important morphological characters at the interspecific taxonomic level. The morphological and molecular variation of theMelampyrum sylvaticum group in the Sudeten, Alps and Carpathians was analyzed. The morphometric study of European populations ofMelampyrum sylvaticum group partly clarified the pattern of the variation of selected characters in this region. The RAPD analysis manifests a very similar trend of the population grouping. The combined analysis of morphological and molecular data gives the most explicit results. Central European yellow flowering populations ofM. sylvaticum group form two distinctive groups. The first one is an exclusive type of the Alps and of the Hercynian Mountains (except the Sudeten). The second group has its centre of distribution in the eastern part of the Carpathians. This type was described asMelampyrum herbichii from the present Ukrainian Carpathians and was regarded to be endemic to the Eastern and Southern Carpathians. Populations from the Western Carpathians and Sudeten Mts. are polymorphic. They seem to be closer toM. herbichii based on the RAPD markers, whereas the morphology of most populations from these regions is closer toM. sylvaticum s.str. The bract shape seems to be important for discrimination between both groups of populations along with the flower characters and a modest ecological differentiation. The Eastern CarpathianMelampyrum saxosum with white corolla is very close toM. herbichii based on the studied morphological characters.     

Evolutionary history of the polyploid complex of Cardamine amara (Brassicaceae): isozyme evidence

 In samples from 56 populations of Cardamine amara, representing four diploid subspecies (subspp. amara, opicii, balcanica, and pyrenaea) and two tetraploid subspecies (subspp. austriaca and olotensis) from different parts of the European distribution area, four enzyme systems with 23 alleles were studied. These data, together with previous morphological and karyological data, suggest that the distribution and variation pattern within the species was strongly influenced by the last glacial period and postglacial migrations. Cardamine amara subsp. pyrenaea is monomorphic for a unique allele, and subsp. balcanica has a unique allele, too which, however, is not fixed in all populations of the taxon. Both taxa seem to be relic ones, although otherwise subsp. balcanica in respect of allelic spectrum much resembles subsp. amara. The other two diploid subspecies, subsp. amara and subsp. opicii, are not characterised by presence of unique alleles but differ in allele frequencies. The two tetraploid subspecies have different evolutionary histories. C. amara subsp. austriaca seems to be an autopolyploid derivative of subsp. amara which colonised open space offered by retreating glaciers in the Eastern Alps. C. amara subsp. olotensis from the Iberian Peninsula represents most probably a polyploid of preglacial time. Received June 22, 2001; accepted May 17, 2002 Published online: September 13, 2002     

Origin of the disjunct tetraploid Cardamine amporitana (Brassicaceae) assessed with nuclear and chloroplast DNA sequence data

Seventy-four nucleotide sequences from the ITS regions of nuclear ribosomal DNA and 76 from the trnL-trnF spacer of chloroplast DNA were used to address the origin of tetraploid Cardamine amporitana, the conspecifity of central Italian and northeastern Spanish populations, and the possible cause for such geographic disjunction. Because of the complex lineage relationships in Cardamine, the sampling included 22 taxa. In the results, both data sets are highly congruent in supporting a close relationship of C. amporitana to the widespread Eurasian C. amara. Low genetic variability in northeastern Spanish populations of C. amporitana suggests long-distance dispersal from central Italy. The interior position of the single northeastern Spanish haplotype in a statistical parsimony network of trnL-trnF haplotypes however does not support this scenario and invokes other plausible phylogeographic explanations. The disappearance of geographically intermediate populations and genetic impoverishment by migration and isolation, both probably associated with Quaternary climatic oscillations, appears as an alternative hypothesis to explain the phylogeographic pattern. A recent hybridization event is reported between C. amporitana and a diploid from the C. pratensis group in central Italy on the basis of additive polymorphisms in ITS for all the 22 distinguishing nucleotides.     

Genetic control of Silver fir isozymes (Abies alba Mill.) of the Ukrainian Carpathian Mountains

Genetic control of GOT, GDH, DIA, MDH, ME, SOD, FDH, ADH, ACP, LAP enzymes has been studied in the seed megagametophytes of Silver fir (Abies alba Mill.) from four natural populations of the Ukrainian Carpathian mountains. The distinct electrophoretic division has been obtained for the 21 loci products. The analysis of allele segregation in the heterozygous trees confirms monogenic inheritance of the revealed variants.     

A biometric study of some representatives of the subsection <Emphasis Type="Italic">Nivales</Emphasis> (Prantl) Luferov of the genus <Emphasis Type="Italic">Ranunculus</Emphasis> L. (Ranunculaceae)

The results of a study of morphological character variability in the populations of R. altaicus s. l., R. sulphureus subsp. exaltatus and R. schmakovii are presented. Univariate and multivariate analysis methods have been applied: a principal components method, discriminant and variance analysis. As a result, two Altai buttercup subspecies have been distinguished in the territory of the Altai mountains (R. altaicus subsp. altaicus, R. altaicus subsp. frigidus), as well as the new taxa R. sulphureus subsp. exaltatus and R. schmakovii     

Molecular and morphometric variation in chromosomally differentiated populations of the grasshopper Sinipta dalmani (Orthopthera: Acrididae)

Sinipta dalmani is an Argentine grasshopper whose chromosome polymorphisms have been widely studied through cytogenetic, morphometric, and fitness component analyses. The present work analysed molecular and morphometric variation in seven chromosomally differentiated populations from Entre Rios and Buenos Aires provinces to analyse population structure. Molecular studies were performed studying RAPD loci and morphometric analyses were carried out measuring five morphometric traits. Genetic variability was high in all studied populations and was characterized by a decrease in H as a function of latitude and temperature. Both conventional F(ST) analysis and Bayesian approach for dominant marker showed that there were significant genetic differences among all populations, between provinces, and among populations within provinces. Entre Rios populations showed higher mean numbers of migrants per generation as well as low genetic differentiation and high gene flow with almost all populations whereas Buenos Aires populations may be considered as a result of a more recently colonization. There is considerable morphometric variation between populations and this variation correlates with latitude and temperature. Our results suggest that selection contributes to phenotypic differentiation among populations by moulding the differences in trait means whereas genetic drift is responsible for differences in the matrix of variance-covariance. The gene flow detected is insufficient to prevent phenotypic and chromosome divergences.     

Genetic differentiation of the marbled white butterfly, Melanargia galathea, accounts for glacial distribution patterns and postglacial range expansion in southeastern Europe

Isolation of Mediterranean species in the southern European peninsulas during the cold glacial phases often resulted in differentiation of several genetic lineages confined to the respective peninsulas. However, whilst there is good genetic evidence for multiple refugia in Iberia, there are only limited data available for the Balkans. Therefore, we wish to examine the hypothesis of a strong genetic structuring within southeastern Europe for the existence of multiple Balkan differentiation centres and/or several leading edges. As a model we use the marbled white butterfly, Melanargia galathea. We studied 18 allozyme loci of 564 individuals from 16 populations distributed over a large part of southeastern Europe. The single populations showed moderately high genetic diversity and no northward decline of genetic diversity was detected. The overall genetic differentiation between populations was considerable (F(ST) 7.0%). Cluster analysis discriminated three genetic groups: (i) a western flank in the former Yugoslavia, parts of eastern Austria and Hungary; (ii) an eastern flank with populations from Bulgaria and Romania (south of the southern Carpathians and eastern Carpathians); and (iii) the eastern Carpathian Basin. Hierarchical variance analysis distributed 53% of the variance among populations between these three groups. One sample from the Greek-Bulgarian border clustered within the eastern flank, but showed some tendency towards the eastern Carpathian Basin populations. Two populations from Carinthia clustered together with the eastern Carpathian Basin ones and a population from Styria showed an intermediate genetic composition between the three groups. Most probably, the eastern and the western flank groups are due to postglacial range expansion from the northeastern and the northwestern edges of the glacial differentiation centre (so-called leading edges). The eastern Carpathian Basin group may have resulted from postglacial expansion from northern Greece through valley systems of the central Balkan peninsula, maybe even expanding westwards north of the Balkan mountains reaching some parts of eastern Austria (e.g. Carinthia). Therefore, the Balkanic refugium of M. galathea may or may not have been continuous along the coastal areas of the Mediterranean, but must have been strongly genetically structured.     

The Iberian endemic species <Emphasis Type="Italic">Ranunculus cabrerensis</Emphasis> Rothm.: an intricate history in the <Emphasis Type="Italic">Ranunculus parnassiifolius</Emphasis> L. polyploid complex

The orophilous plant, Ranunculus parnassiifolius L., is a polyploid complex that is widespread throughout the southern European mountains, where at least five taxa have traditionally been recognized. The aim of this study was to test whether R. parnassiifolius subsp. cabrerensis should be treated as an independent species of the R. parnassiifolius polyploid complex and constitutes, therefore, an evolutionary line in itself. To disentangle its evolutionary history and taxonomy, we used genome size estimation based on flow cytometric measurements (FCM), multivariate morphometric analyses, polymerase chain reaction-restriction fragment length polymorphisms (PCR–RFLPs) and subsequent sequencing of cpDNA regions (trnS, trnR-atpA, atpH-atpI). This study provides molecular and morphological evidence for the recognition of R. cabrerensis at the species level, rather than as an intraspecific taxon of R. parnassiifolius. Furthermore, it is concluded that those plants previously known as R. parnassiifolius subsp. muniellensis should be systematized at the subspecies level as R. cabrerensis subsp. muniellensis. This contribution highlights the benefit of combining diverse approaches to obtain knowledge about relict populations and for the implementation of suitable conservation measures.     

Morphometric and allozyme variation in central and southern Greek populations ofMicrotus (Terricola) thomasi

The endemic Balkan vole taxonMicrotus (Terricola) thomasi (Barrett-Hamilton, 1903) exhibits great karyological variability in Greece. In this study, populations belonging to two different karyotypic forms (‘atticus’ and ‘thomasi’) are examined both morphometrically and electrophoretically. A total of 140 individuals ofM. (T.) thomasi were collected from 6 localities of south and central Greece. For the morphometric analysis, 27 variables (external body and cranial characters) were examined and evaluated according to multivariate analyses (PCA, MANOVA, CVA and CLUS). For the electrophoretic analysis, 18 putative genetic loci were examined and the allozymic data were treated by the biostatistical package BIOSYS-1. According to the results obtained, all the populations studied show little overall morphometric variability, whereas they are characterized by high electrophoretic variability. The populations studied are not grouped according to the karyotypic form. In almost all the cases, in the two UPGMA-dendrograms plotted according to morphometric and electrophoretic distances (Mahalanobis’ and Nei’s distances, respectively), the populations branching together belong to different karyotypic forms. Conclusively, the morphometric and electrophoretic results of this study revealed that the two karyotypic forms should not be considered separate species or subspecies, as it has been proposed by some authors in the past, and the populations studied can be considered only as different local populations of the rather variable vole speciesM. (T.) thomasi.     

Comparative ITS and AFLP analysis of diploid Cardamine (Brassicaceae) taxa from closely related polyploid complexes

BACKGROUND AND AIMS: Diploid representatives from the related polyploid complexes of Cardamine amara, C. pratensis and C. raphanifolia (Brassicaceae), were studied to elucidate phylogenetic relationships among the complexes and among the individual taxa included. METHODS: Two independent molecular data sets were used: nucleotide sequences from the internal transcribed spacers (ITS) of nrDNA, and amplified fragment length polymorphism (AFLP) markers. Seventeen diploid taxa from the studied groups were sampled. KEY RESULTS: Both ITS and AFLP analyses provided congruent results in inferred relationships, and revealed two main lineages. While the C. amara group, consisting of C. wiedemanniana and four subspecies of C. amara, was resolved as a well-supported monophyletic group, taxa from the C. pratensis and C. tenera groups (the latter representing diploid taxa of the complex of C. raphanifolia) all appeared together in a single clade/cluster with no support for the recognition of either of the groups. Intra-individual polymorphisms and patterns of nucleotide variation in the ITS region in C. uliginosa and C. tenera, together with the distribution of AFLP bands, indicate ancient hybridization and introgression among these Caucasian diploids. CONCLUSIONS: The lack of supported hierarchical structure suggests that extensive reticulate evolution between these groups, even at the diploid level, has occurred (although an alternative explanation, namely ancestral polymorphism in ITS data, cannot be completely excluded). Several implications for the investigation of the polyploid complexes of concern are drawn. When tracing origins of polyploid taxa, a much more complex scenario should be expected, taking into account all relatives as potential parents, irrespective of the group in which they are classified.     

Cyanus triumfettii subsp. triumfettii (Compositae) does not occur in the western Carpathians and adjacent parts of Pannonia: karyological and morphological evidence

The pronounced morphological variation of the Cyanus triumfettii group (syn. Centaurea triumfettii s. latissimo) in the western Carpathians and Pannonia has resulted in confusing and contradictory taxonomic treatments. We focus on the taxonomic identity of populations which have been treated as C. triumfettii subsp. triumfettii in this area. Karyological and morphological data were used to compare 47 populations (746 plants) from the western Carpatho–Pannonian area with 8 populations (105 plants) from the type locality of C. triumfettii subsp. triumfettii and its vicinity in the southern part of the western Alps. Chromosome counting and flow cytometry confirmed the diploid level for all analysed populations. Multivariate morphometric analyses revealed a clear separation between Alpine populations and populations in the western Carpathians and Pannonia. The results proved that C. triumfettii subsp. triumfettii does not occur in the western Carpathians and Pannonia. Instead, populations in this area are provisionally treated as C. triumfetti subsp. axillaris , C. triumfettii subsp. dominii and C. triumfettii subsp. strictus . The complex morphological variation of these subspecies is discussed and the need for further taxonomic studies is emphasized.     

A new species of Armeria (Plumbaginaceae) from southern Spain with molecular and morphometric evidence on its origin

The taxonomic status of the populations of Armeria from the Horconera massif (Cordoba province), previously misidentified or referred to without an explicit identification, is re-examined on the basis of morphological and molecular data. A multivariate analysis indicates that they can be discriminated from morphologically and geographically close taxa using morphometric characters. According to these results, and taking into account the previous evidence that supports extensive reticulation in the genus, a new species, A. trianoi , is formally described. Molecular evidence from our previous works, in particular the nuclear ribosomal ITS sequence data, provides the frame for a discussion on the origin of the new entity. It is suggested that A. trianoi may be the result of introgression of sympatric populations of A. villosa subsp. longiaristata into a pre-existing taxon, possibly A. filicaulis var. minor presently occurring in Sierra Tejeda some 50 km apart.     

Allopolyploid origin of Cardamine asarifolia (Brassicaceae): incongruence between plastid and nuclear ribosomal DNA sequences solved by a single-copy nuclear gene

Interspecific hybridization and polyploidization have played central roles in plant diversification. However, technical difficulties in the analyses of low-copy genes have limited the study of the origins of hybrid and polyploid plants. Here, we present a phylogenetic analysis of the hexaploid Cardamine asarifolia, distributed in the southern European Alps and northern Apennines. Our study included all relevant taxa of the genus found in Europe. A marked discrepancy was revealed between the trnL-trnF region of cpDNA and internal transcribed spacer (nrDNA ITS) sequences. To solve the incongruence, we sequenced a single-copy nuclear CHS gene (chalcone synthase) using a novel method to design homoeologue-specific PCR primers to bypass artefacts caused by artificial recombination of homoeologues during PCR and/or cloning. Three homoeologues were isolated from C. asarifolia, providing evidence for its allopolyploid origin. One homoeologue, showing the same phylogenetic position as the ITS sequences, most likely originated from an extinct parent. Furthermore, we documented recurrent polytopic hybridizations between C. asarifolia and diploid C. amara. The allohexaploidization and the following hybridization with a diploid species exemplify the ongoing dynamic processes of speciation in the genus Cardamine.     

Comparative karyotypic analysis in the Alstroemeria hookeri Lodd. (Alstroemeriaceae) complex sensu Bayer (1987)

Alstroemeria L. (Alstroemeriaceae) is an American genus of monocots with two principal distribution centers in Chile and Brazil. In Chile, it is represented by about 32 species, most of them in central Chile, an area known for its high level of endemism. The "complex" Alstroemeriahookeri is endemic to Chile, where it is distributed from the Coquimbo to the Bío-Bío Region. We analyzed the karyotypes of 36 populations of this complex along its natural distribution. Ten metaphases per population were used for chromosome measurements. All analyzed subspecies presented a well defined asymmetric karyotype. The populations of A. hookeri subsp. hookeri collected in the coastal range of the Bío-Bío Region and the populations from the Central Valley of this Region (Pangal del Laja) presented striking morphological differences in the karyotype, mainly on chromosome 3. The population of A. hookeri subsp. recumbens from Pichicuy showed a polymorphism on chromosome 7, which differed from the other analyzed populations of this subspecies. Phenetic analysis suggested that A. hookeri subsp. cummingiana, which showed a more symmetrical karyotype and did not grow in sandy soil, should be alocated to A. cummingiana rather than considered as part of the hookeri complex.     

Taxonomic status of Ononis tridentata (Fabaceae) from Morocco,resolved by multivariate morphometric analyses

In this study, we present the results of morphological studies on the SW Mediterranean Ononis tridentata aggregate, in order to establish the identity and taxonomic status of the only known Moroccan population. By using multivariate morphometric techniques (principal component analysis and discriminant analysis), we examined the diagnostic potential of 14 morphological characters. Moroccan individuals of O. tridentata are well characterised by their long inflorescence axis and calyx lobes frequently toothed among other distinguishing characters. As the Moroccan population is distinct in multivariate morphometric space and occupies an isolated geographical distribution, we recommend the taxonomic status of subspecies: O. tridentata subsp. mauretanica (Maire) Agulló, Juan, M.A. Alonso, Terrones & M.B. Crespo stat. nov.     

Taxonomy and cytogeography of Cardamine raphanifolia and C. gallaecica (Brassicaceae) in the Iberian Peninsula

For a long time it was considered that Cardamine raphanifolia is widely distributed with several infraspecific taxa in the Mediterranean region. Recent studies showed that C. raphanifolia is restricted to the northern Iberian Peninsula. Cardamine gallaecica from NW Spain was regarded to be closely related, but was originally regarded as C. amara, and later described as C. raphanifolia subsp. gallaecica. In this study, we validate the treatment of C. raphanifolia and C. gallaecica as distinct species, and show their clear differentiation based on morphometric and molecular AFLP analyses. Moreover, both species are clearly distinct from C. amara subsp. pyrenaea, which has an overlapping distributional range with C. raphanifolia. Hexaploid (2n = 48) and octoploid cytotypes (2n = 64) of C. raphanifolia, and tetraploid (2n = 32) and hexaploid (2n = 48) cytotypes of C. gallaecica were found. Due to their weak morphological and genetic differentiation, these polyploid races are not classified as separate taxa.