A Morphometric Study of Populations of the Centaurea jacea Complex (Asteraceae) in Belgium

Abstract: The Centaurea jacea aggregate is a polymorphic polyploid complex whose taxonomic treatment is still controversial. A numerical taxonomic approach was applied to 394 individuals of known ploidy level, from 19 populations, based on the main diagnostic characters proposed in earlier revisions. Populations from xeric grasslands were not considered. Principal Component Analysis shows that variation within the complex is continuous. UPGMA Cluster Analysis based on population means supports the recognition of three groups of populations. However, the limits between these groups are blurred to a considerable extent due to extensive within-population polymorphism. It is argued that the Belgian populations of Centaurea jacea occurring in mesic grasslands should be treated as a single species, with three subspecies. The two extremes of the morphological gradient can be referred to as C. jacea subsp. jacea and C. jacea subsp. nigra, with C. jacea subsp. pratensis occupying an intermediate position. Most populations from Belgium are tetraploid, a diploid chromosome number being found only in populations of C. jacea subsp. nigra from the Ardennes massif. On average, diploids grow at higher altitude and on more acidic soils than tetraploids. Finally, a key to the three subspecies is provided.     

Hybridization between diploid Centaurea pseudophrygia and tetraploid C. jacea (Asteraceae): the role of mixed pollination,unreduced gametes,and mentor effects

We studied hybridization between the diploid Centaurea pseudophrygia and the tetraploid C. jacea by performing crossing experiments and screening natural populations using flow cytometry. The experiments confirm that the studied species exhibit strong reproductive isolation. Interspecific hybrids were formed at a low frequency, including triploids (originating from reduced gametes) and tetraploids (involving unreduced gametes of the diploids). In contrast, hybrids were almost absent among seeds and adult plants of natural mixed populations and among the offspring from experimental pollinations with a mixture of pollen of both ploidy levels. We found that mixed pollination is an important mechanism for preventing hybridization between plants of different ploidy levels and sustaining the reproduction of the tetraploids. A mentor effect (induced selfing in the presence of pollen of different ploidy levels) was observed in both diploids and tetraploids, reinforcing the reproductive isolation between cytotypes. Higher ploidy levels (pentaploid, hexaploid) involving unreduced gametes of the tetraploid species were identified. Notably, pentaploids were discovered for the first time in Centaurea sect. Jacea. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 104 , 93–106.     

Nuclear DNA content and chromosome number in some diploid and tetraploid Centaurea (Asteraceae: Cardueae) from the Dalmatia region

Given the paucity of information about genome size in the genus Centaurea, nuclear DNA content of 15 Centaurea taxa, belonging to four subgenera and six different sections, has been investigated for the first time. The sample concerns 21 populations from the Dalmatia region of Croatia. The 2C DNA content and GC percentage were assessed by flow cytometry and chromosome number was determined using standard methods. Genome size of studied Centaurea ranged from 2C=1.67 to 3.72 pg. These results were in accordance with chromosome number and especially with ploidy level that varies throughout this group; 2C DNA values ranged from 1.67 to 3.43 pg for diploid, and from 3.19 to 3.72 for polyploid taxa. No significant intraspecific variations of DNA amount were found between two subspecies of C. visiani and C. ragusina, nor between two varieties of C. gloriosa. However, some populations of C. glaberrima and C. cuspidata showed a significant difference in DNA amount. Three different basic chromosome numbers were observed in studied species (x=9, 10, and 11). The most frequent basic number was x=9. C. rupestris, C. ragusina ssp. ragusina, and C. r. ssp. lungensis possessed x=10 and C. tuberosa x=11. The species with a basic chromosome number of x=9 had a small genome size and the smallest chromosomes (on average 0.09 to 0.12 pg/chromosome) but frequently present polyploidy. Centaurea ragusina ssp. ragusina and C. r. ssp. lungensis had a mean base composition 41.3% GC.     

Ecological differentiation,lack of hybrids involving diploids,and asymmetric gene flow between polyploids in narrow contact zones of Senecio carniolicus (syn. Jacobaea carniolica,Asteraceae)

Areas of immediate contact of different cytotypes offer a unique opportunity to study evolutionary dynamics within heteroploid species and to assess isolation mechanisms governing coexistence of cytotypes of different ploidy. The degree of reproductive isolation of cytotypes, that is, the frequency of heteroploid crosses and subsequent formation of viable and (partly) fertile hybrids, plays a crucial role for the long‐term integrity of lineages in contact zones. Here, we assessed fine‐scale distribution, spatial clustering, and ecological niches as well as patterns of gene flow in parental and hybrid cytotypes in zones of immediate contact of di‐, tetra‐, and hexaploid Senecio carniolicus (Asteraceae) in the Eastern Alps. Cytotypes were spatially separated also at the investigated microscale; the strongest spatial separation was observed for the fully interfertile tetra‐ and hexaploids. The three main cytotypes showed highly significant niche differences, which were, however, weaker than across their entire distribution ranges in the Eastern Alps. Individuals with intermediate ploidy levels were found neither in the diploid/tetraploid nor in the diploid/hexaploid contact zones indicating strong reproductive barriers. In contrast, pentaploid individuals were frequent in the tetraploid/hexaploid contact zone, albeit limited to a narrow strip in the immediate contact zone of their parental cytotypes. AFLP fingerprinting data revealed introgressive gene flow mediated by pentaploid hybrids from tetra‐ to hexaploid individuals, but not vice versa. The ecological niche of pentaploids differed significantly from that of tetraploids but not from hexaploids.     

Microsatellite evidence for low genetic diversity and reproductive isolation in tetraploid Centaurea seridis (Asteraceae) coexisting with diploid Centaurea aspera and triploid hybrids in contact zones

Survival of polyploids in nature depends on several factors, including competition from diploid relatives and increased genetic diversity. Unlike other reported Centaurea polyploid complexes, diploid Centaurea aspera and tetraploid Centaurea seridis coexist in hybrid zones with frequent triploid individuals. The polyploid origin of C. seridis, the genetic diversity and population structure of the three cytotypes, and the degree of genetic differentiation among them were analyzed in seven mixed‐ploidy zones, involving different subspecies and ecological conditions. Ploidy was determined by flow cytometry. Microsatellite data suggested an allopolyploid origin of C. seridis. In the contact zones, diploids and tetraploids were genetically differentiated. When compared with the related C. aspera, a low genetic diversity was observed in C. seridis, which is uncommon in tetraploids. Furthermore, although diploid individuals were grouped in a single widespread genetic cluster, tetraploids were grouped in two highly differentiated clusters and showed significant isolation by distance. This genetic pattern in C. seridis may be related to a minimal gene flow with diploid relatives and/or other genetic factors, such as rare polyploidization events, founder effects or an increased selfing rate. Neither taxonomic assignment at subspecies level, nor ecological conditions could explain the genetic differentiation between tetraploid clusters. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176 , 82–98.     

Hybridization between two polyploid Cardamine (Brassicaceae) species in North-western Spain: discordance between morphological and genetic variation patterns

BACKGROUND AND AIMS: Hybridization is an important evolutionary phenomenon, and therefore a detailed understanding of the dynamics of interspecific gene flow and resulting morphological and genetic patterns is of widespread interest. Here hybridization between the polyploids Cardamine pratensis and C. raphanifolia at four localities is explored. Using different types of data, the aim is to provide simultaneous and direct comparisons between genotype and phenotype variation patterns in the studied hybrid populations. METHODS: Evidence of hybridization has been gathered from morphology, molecular markers (amplified fragment length polymorphism and chloroplast DNA sequences), pollen viability, karyology and nuclear DNA content. KEY RESULTS: All data support extensive gene flow occurring in the hybrid populations. A wide range of morphological and genetic variation is observed, which includes both parental and intermediate types. Unbalanced pollen fertility and several ploidy levels are recorded. CONCLUSIONS: Incongruence reported between genotype and phenotype suggests that parental phenotypes are affected by introgression, and intermediate hybrid phenotypes can be genetically closer to one of the parents. Thus, it is evident that morphology, when used alone, can be misleading for interpreting hybridization, and critical evaluation of other data is needed.     

Anthropogenic disturbance as a driver of microspatial and microhabitat segregation of cytotypes of Centaurea stoebe and cytotype interactions in secondary contact zones

Background and Aims

In a mixed-ploidy population, strong frequency-dependent mating will lead to the elimination of the less common cytotype, unless prezygotic barriers enhance assortative mating. However, such barriers favouring cytotype coexistence have only rarely been explored. Here, an assessment is made of the mechanisms involved in formation of mixed-ploidy populations and coexistence of diploid plants and their closely related allotetraploid derivates from the Centaurea stoebe complex (Asteraceae).

Methods

An investigation was made of microspatial and microhabitat distribution, life-history and fitness traits, flowering phenology, genetic relatedness of cytotypes and intercytotype gene flow (cpDNA and microsatellites) in six mixed-ploidy populations in Central Europe.

Key Results

Diploids and tetraploids were genetically differentiated, thus corroborating the secondary origin of contact zones. The cytotypes were spatially segregated at all sites studied, with tetraploids colonizing preferentially drier and open microhabitats created by human-induced disturbances. Conversely, they were rare in more natural microsites and microsites with denser vegetation despite their superior persistence ability (polycarpic life cycle). The seed set of tetraploid plants was strongly influenced by their frequency in mixed-ploidy populations. Triploid hybrids originated from bidirectional hybridizations were extremely rare and almost completely sterile, indicating a strong postzygotic barrier between cytotypes.

Conclusions

The findings suggest that tetraploids are later immigrants into already established diploid populations and that anthropogenic activities creating open niches favouring propagule introductions were the major factor shaping the non-random distribution and habitat segregation of cytotypes at fine spatial scale. Establishment and spread of tetraploids was further facilitated by their superior persistence through the perennial life cycle. The results highlight the importance of non-adaptive spatio-temporal processes in explaining microhabitat and microspatial segregation of cytotypes.     

When hybrids are not hybrids: a case study of a putative hybrid zone between Cattleya coccinea and C. brevipedunculata (Orchidaceae)

Cattleya coccinea and C. brevipedunculata (Orchidaceae) are closely related species distinguished primarily by geographical distribution, vegetative morphology and flowering period. Both species inhabit high‐elevation regions in south‐eastern Brazil, but are traditionally associated with different habitats, located in cloudy forests and campos rupestres (rocky fields), respectively. We used morphometrics and genetic variation of microsatellite markers to test the occurrence of a hybrid zone between these species located in Parque Estadual do Ibitipoca (PEI), Brazil. Morphological data reveal a continuum of variation between the putative taxa, influenced mainly by characters of leaf, pseudobulb and peduncle. However, genetic data do not support the occurrence of hybridization and introgression in PEI, showing that it is a pure population of C. brevipedunculata. Differences in vegetative characters among individuals from cloudy forests and campos rupestres suggest that morphological variation may be related to phenotypic plasticity in response to environmental light fluctuations, an unknown situation for this species. These results highlight the inconsistency of morphology for the identification of hybrids and the role of vegetative characters as a possible complicating factor for the taxonomy of these species, as they are subject to environmental influence.     

Towards a better understanding of polyploid Sorbus (Rosaceae) from Bosnia and Herzegovina (Balkan Peninsula), including description of a novel,tetraploid apomictic species

Sorbus subgenus Soraria encompasses taxa originating from spontaneous hybridization between members of subgenera Aria and Sorbus disjunctly distributed across Europe and Asia. Using molecular data (amplified fragment length polymorphisms, plastid DNA sequences and nuclear microsatellites), flow cytometry (allowing for the determination of ploidy and mode of reproduction) and morphology, we disentangled the relationships among polyploid cytotypes and explored their relationships with their diploid ancestors. Among others, we focused on a large, geographically isolated hybrid population in Bosnia and Herzegovina. Molecular and morphological analyses confirmed the distinct position of this population in relation to its parental (S. aria and S. aucuparia) and other hybridogenous taxa originating independently from the same parents in different parts of Europe. After establishing its genetic and morphological divergence, we describe the isolated Bosnian population as a new tetraploid apomictic species, S orbus bosniaca , discuss its taxonomic status and propose conservation measures to protect the locus classicus of this new Balkan endemic. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 670–685.     

Introgression and phylogeography of Betula nana (diploid), B. pubescens (tetraploid) and their triploid hybrids in Iceland inferred from cpDNA haplotype variation

Aim The objective was to find direct genetic evidence supporting introgressive hybridization between tetraploid tree birch (Betula pubescens) and diploid dwarf birch (B. nana), via triploid hybrids, and to investigate an association between the introgression and phylogeographical distribution of Icelandic birch. Location Samples were collected from 463 trees in 12 woodlands in Iceland and eight locations in Norway, Sweden, Scotland and Greenland. Methods Ploidy status of individual trees was determined by chromosome counting. Variation in the chloroplast genome was assessed using polymerase chain reaction‐restriction fragment length polymorphism. The geographical distribution of the haplotypes was mapped. The haplotype variation and introgression ratios (IG) were analysed statistically. Results Thirteen haplotypes were identified among Icelandic samples. The most common haplotype (T, 49% occurrence) was present in all ploidy groups and in all woodlands. All common haplotypes were shared between the triploid group and the parental species, indicating introgressive hybridization. This was supported by the statistical analysis of IG indices and the variation components. Considerable differences existed among samples, shaped by isolation by distance and local introgression. An east–west phylogeographical distribution in Iceland was observed. Main conclusions Despite extensive introgression across species and ploidy levels, a biogeographical pattern has been observed, and this may indicate different population histories or multiple origins of Icelandic birch. The chloroplast haplotype diversity found in Iceland resembles that found in birch populations from northern Scandinavia.     

Morphological variation among Betula nana (diploid), B. pubescens (tetraploid) and their triploid hybrids in Iceland

BACKGROUND AND AIMS: Introgressive hybridization between two co-existing Betula species in Iceland, diploid dwarf birch B. nana and tetraploid downy birch B. pubescens, has been well documented. The two species are highly variable morphologically, making taxonomic delineation difficult despite stable ploidy levels. Here an analysis is made of morphological variation within each ploidy group with an aim to establishing a reliable means to distinguish the species. METHODS: Plant materials were collected from 14 woodlands in Iceland. The plants were identified based on 2n chromosome numbers. Morphological variation in species-specific characters within each ploidy group was analysed qualitatively and quantitatively. The morphological index was based on eight discrete characters, whereas the multivariate analysis was based on nine leaf variables. KEY RESULTS: Of the 461 plants examined, 9.5 % were found to be triploid hybrids. The three ploidy groups were morphologically distinguishable but their variation overlapped. The diploid, triploid and tetraploid groups had average scores of 1.3, 4.1 and 8.3, respectively, in the morphology index scale from 0 (B. nana) to 13 (B. pubescens). A linear discriminant analysis also revealed significant separation among the three ploidy groups and the model assigned 96 % and 97 % of the B. nana and B. pubescens individuals correctly. The triploid hybrids were difficult to predict since only half of them could be assigned correctly. Leaf length was the most useful variable identifying triploid hybrids. Geographical patterns within the ploidy groups could partly be explained by differences in mean July temperature. CONCLUSIONS: Hybridization between B. nana and B. pubescens is widespread in Iceland. The species can be distinguished from each other morphologically, and from the triploid hybrids. The overlapping morphological variation indicates bidirectional introgression between the two species via triploid hybrids. Iceland could be considered a birch hybrid zone, harbouring genetic variation which may be advantageous in subarctic regions.     

Comparative allozyme and microsatellite population structure in a narrow endemic plant species, Centaurea corymbosa Pourret (Asteraceae)

Centaurea corymbosa Pourret (Asteraceae) is a narrow endemic species known only from six populations located in a 3-km2 area in the south of France. Earlier field experiments have suggested that pollen and seed dispersal were highly restricted within and among populations. Consistent with the field results, populations were highly differentiated for five allozyme loci and among-population variation fitted an isolation-by-distance model. In the present study, we investigated the genetic structure of C. corymbosa using six microsatellite loci. As with allozymes, microsatellites revealed no within-population structure and a large differentiation among populations. However, allozyme loci were less powerful than microsatellites in detecting the extent of gene flow assessed by assignment tests. The patterns of structuration greatly varied among loci for both types of marker; we suggest that differences in single-locus pattern could mainly be an effect of stochastic variation for allozymes and an effect of variation in mutation rate for microsatellites. In contrast to the multilocus results, the two most polymorphic microsatellite loci did not show any isolation-by-distance pattern. Our results suggest that highly variable loci might not always be the best suited markers to quantify levels of gene flow among populations.     

Evolution of the polyploid north-west Iberian Leucanthemum pluriflorum clan (Compositae,Anthemideae) based on plastid DNA sequence variation and AFLP fingerprinting

Background and Aims

The genus Leucanthemum is a species-rich polyploid complex from southern and central Europe, comprising 41 species with ploidy ranging from 2x to 22x. The present contribution aims at reconstructing the evolutionary history of a geographically isolated species group (the L. pluriflorum clan) from the north-west Iberian Peninsula comprising the diploid L. pluriflorum, the tetraploids L. ircutianum subsp. pseudosylvaticum and L. × corunnense (a putative hybrid taxon based on crossing between L. pluriflorum and L. merinoi), and the hexaploids L. sylvaticum and L. merinoi.

Methods

Chromosome number variation (determined flow cytometrically) and sequence variation were analysed for two intergenic spacer regions on the plastid genome (psbA-trnH and trnC-petN) for individuals from 54 populations in combination with amplified fragment length polymorphism (AFLP) fingerprinting of 246 representative individuals from these populations.

Key Results

Plastid sequence data revealed that all surveyed members of the L. pluriflorum clan possess plastid haplotypes that are closely related to each other and distinctly separated from other Leucanthemum species. AFLP fingerprinting resulted in allopolyploid fragment patterns for most of the polyploid populations, except for the tetraploid L. × corunnense and a further tetraploid population in northern Galicia, which cluster with the diploids rather than with the other polyploids. In silico modelling of (auto)tetraploid AFLP genotypes further corroborates the allopolyploid nature of L. ircutianum subsp. pseudosylvaticum, L. sylvaticum and L. merinoi.

Conclusions

The present study provides evidence for recognizing one diploid (L. pluriflorum), one autotetraploid (L. corunnense), one allotetraploid (L. pseudosylvaticum) and one allohexaploid (L. sylvaticum with the two geographically and ecologically differentiated subspecies subsp. sylvaticum and subsp. merinoi) in the L. pluriflorum clan. It also has implications for the understanding of biogeographical patterns in the Iberian Peninsula.     

Reproductive isolation between oviparous and viviparous lineages of the Eurasian common lizard Zootoca vivipara in a contact zone

Contact zones between two evolutionary lineages are often useful for understanding the process of speciation because the observed genetic pattern reflects the history of differentiation. The Eurasian lacertid lizard Zootoca vivipara is a potentially interesting model for studying the role of reproductive mode in the speciation of squamate reptiles because it has both oviparous (Zootoca vivipara carniolica) and viviparous (Zootoca vivipara vivipara) populations that have recently been shown to be genetically distinct. We studied a newly‐discovered syntopic area of these two Zootoca subspecies in the central Italian Alps using genetic markers to investigate the level of introgression between them. Patterns of genetic differentiation in a fragment of the mitochondrial DNA cytb gene and a set of nuclear microsatellites show that the speciation process is complete in this area, with no evidence of recent introgression. Phylogenetic and genotypic divergence suggests that the two subspecies have experienced long independent evolutionary histories, during which genetic and phenotypic differences evolved. The possible roles of biogeography, reproductive mode, and cytogenetic differentiation in this speciation process are discussed. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, 114 , 566–573.     

Natural hybridization between a clonally propagated crop, cassava (Manihot esculenta Crantz) and a wild relative in French Guiana

Because domestication rarely leads to speciation, domesticated populations often hybridize with wild relatives when they occur in close proximity. Little work has focused on this question in clonally propagated crops. If selection on the capacity for sexual reproduction has been relaxed, these crops would not be expected to hybridize with their wild relatives as frequently as seed-propagated crops. Cassava is one of the most important clonally propagated plants in tropical agriculture. Gene flow between cassava and wild relatives has often been postulated, but never demonstrated in nature. We studied a population of a wild Manihot sp. in French Guiana, which was recently in contact with domesticated cassava, and characterized phenotypes (10 morphological traits) and genotypes (six microsatellite loci) of individuals in a transect parallel to the direction of hypothesized gene flow. Wild and domesticated populations were strongly differentiated at microsatellite loci. We identified many hybrids forming a continuum between these two populations, and phenotypic variation was strongly correlated with the degree of hybridization as determined by molecular markers. Analysis of linkage disequilibrium and of the diversity of hybrid pedigrees showed that hybridization has gone on for at least three generations and that no strong barrier prevents admixture of the populations. Hybrids were more heterozygous than either wild or domesticated individuals, and phenotypic comparisons suggested heterosis in vegetative traits. Our results also suggest that this situation is not uncommon, at least in French Guiana, and demonstrate the need for integrated management of wild and domesticated populations even in clonally propagated crops.     

Detection of diploid males in a natural colony of the cleptobiotic bee Lestrimelitta sp (Hymenoptera, Apidae)

When working at quantifying the genome size of stingless bees, it was observed that males of Lestrimelitta sp possessed the same amount of nuclear DNA as the females. Thus, we used flow cytometry (FCM) and cytogenetic analysis to confirm the ploidy of these individuals. The males analyzed proved to be diploid, since, through cytometric analysis, it was demonstrated that the mean genome size of both males and females was the same (C = 0.463 pg), and, furthermore, cytogenetic analysis demonstrated that both had 2n = 28 chromosomes.     

Gene flow and differential mortality in a contact zone between two Albinaria species (Gastropoda; Clausiliidae)

This paper reports on the disruption of normally effective ecological and behavioural barriers, at a contact zone on Monemvasia peninsula (Peloponnese, Greece), between parapatric populations of the land snail species Albinaria discolor and Albinaria campylauchen. Detected outcomes were the increase of gene flow between these two species at the contact zone, the occurrence of rare alleles, as well as the occurrence of morphologically and ethologically intermediate A. campylauchen specimens. Furthermore, there was high genetic variability along with deficiency of heterozygotes, probably due to inbreeding. Additionally, the study of mortality of these populations, during the adverse summer aestivation period, indicated that A. campylauchen individuals with an inappropriate combination of morphological and behavioural characteristics suffered heavily. The maladaptation and disadvantage of these specimens revealed the possible action of a postzygotic isolation mechanism between these two morphologically, molecularly and electrophoretically distinct species, that can, however, mate under laboratory conditions. Finally, the above situation is discussed in terms of exogenous versus endogenous selection.     

Wild and agronomically important Agave species (Asparagaceae) show proportional increases in chromosome number,genome size,and genetic markers with increasing ploidy

Agave (Asparagaceae) includes cultivated and wild varieties of henequen used for hard fibre production. As part of a breeding programme to improve Agave production, species with different ploidy levels were genetically characterized: two diploids [A. tequiliana Weber and the hybrid H11648 ((A. amaniensis Trel. & Nowell × A. angustifolia Haw.) × A. amaniensis)], a triploid (A fourcroydes Lem. var. kitam ki), a tetraploid (A. angustifolia var. letona), three pentaploids (A. fourcroydes var. sac ki, A. fourcroydes var. yaax ki, and A. sisalana Perrine), and two hexaploids (A. angustifolia var. chelem ki from two locations). Chromosome spreading was used to determine the chromosome number, flow cytometry was employed to measure the genome size, and fluorescent in situ hybridization was performed using 45S and 5S ribosomal DNA (rDNA) and the telomeric sequences (TTAGGG)_n and (TTTAGGG)_n as genetic markers. There were proportional increases with ploidy level of the following: (1) chromosome number (from diploid 2n = 2x = 60 to hexaploid 2n = 6x = 180), including the number of large and small chromosomes in the bimodal karyotype of Agave; (2) genome size, with a mean monoploid genome size (1Cx) of 7.5 pg (range, 7.36–7.61 pg); and (3) the number and distribution of 45S and 5S rDNA loci, with one locus of each per basic, monoploid genome. Thus there was complete additivity in genome structure with increasing ploidy, as reported in some angiosperm polyploids. However, as other analyses of polyploids have revealed a decrease in 1Cx values with increased ploidy, possible explanations for the observed genomic stability were considered. With the (TTAGGG)_n probe, the signal was localized at the telomeres, consistent with published data showing that many species in the order Asparagales have this type of telomere sequence. It is speculated that sporadic telomeric signals using the (TTTAGGG)_n probe are probably derived from either errors in telomerase activity or relic ancestral‐type telomeric sequences. © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society, 2008, 158 , 215–222.