Molecular and morphological evidence for hybridization between endemic Canary Island Convolvulus

Morphological data and molecular data from the chloroplast trn H- psb A region and nuclear ribosomal ITS region are used to test the hypothesis that the problematic Canary Island endemic Convolvulus floridus var. virgatus is a hybrid between the endemic species C. floridus and C. scoparius . Analysis of mean leaf length and width of 58 individuals indicates that accessions referable to C. floridus var. virgatus are intermediate between the parental taxa in leaf dimensions. Direct sequencing of the ITS region of C. scoparius and C. floridus revealed two species-specific ribotypes distinguished by 10 base differences. Examination of ITS chromatograms for putative hybrids revealed polymorphisms at those sites that are diagnostic between species in all except one putative hybrid. Morphological intermediacy and ITS additivity therefore support the hybrid status of C. floridus var. virgatus . An analysis of intraindividual ITS variation confirmed the co-occurrence of both parental ribotypes in putative hybrids and demonstrated that some hybrid individuals contained chimaeric ITS types. It is proposed that chimaeric ITS types are the result of recombination following backcrossing. Sequencing of the trn H- psb A region revealed four haplotypes. Three were, for the most part, confined to C. floridus and putative hybrids although one C. scoparius accession was also found to share this haplotype. The remaining haploype was only found in C. scoparius and putative hybrid accessions. Patterns of haplotype distribution between parental and hybrid accessions suggest multiple, bidirectional hybridization events between C. floridus and C. scoparius . The nomenclature of the hybrid is discussed and the necessary new name C.  ×  despreauxii is proposed.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 187–204.     

Allopolyploid origin of Cardamine silana (Brassicaceae) from Calabria (southern Italy): karyological, morphological and molecular evidence

Endemic Cardamine silana from Calabria (southern Italy) previously reported to be related to C. raphanifolia was found to be hexaploid. Morphological characters and AFLP data were analysed to evaluate the degree of differentiation of C. silana from closely related taxa and to find parental taxa of this polyploid. Cardamine apennina from the C. pratensis group was examined as one putative parent, as indicated in previous studies of nuclear ITS sequences, along with other related taxa based on both cpDNA and ITS sequences. Both multivariate morphometric analyses of quantitative characters and evaluation of qualitative morphological characters showed: (1) closest position of C. silana to two diploids: C. acris from the Balkan Peninsula and C. apennina from Central Italy; (2) good extent of morphological separation of C. silana from related taxa; and (3) within C. acris subspecies, C. acris ssp. vardousiae from Central Greece as closest to C. silana . Neighbour-joining tree and PCoA ordinations of AFLP data, as well as patterns of AFLP bands sharing, corroborated results of multivariate morphometrics. This evidence supports an allopolyploid origin of C. silana , with C. apennina and C. acris as parental taxa. Its origin may be dated to Pleistocene glacial events, because of the presumably wider geographical distributions of its parental taxa during more humid periods at that time.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 101–116.     

Molecular and morphological characterization of Crataegus L. species (Rosaceae) in southern Syria

The systematics of the genus Crataegus (Rosaceae) have been considered problematic owing to the fact that hybridization, introgression, polyploidy and apomixis may occur in this genus. A study of the Crataegus species from the Arab mountains, Sweida Province, Syria, has been undertaken based on both plastid DNA sequences ( trn L- trn F, psb A- trn H) and morphological data. In the investigated region, three morphologically distinguishable Crataegus species: C. azarolus var. aronia L., C.  ×  sinaica Boiss. ssp. sinaica and C. monogyna var. monogyna Jacq. were investigated. Crataegus azarolus can be clearly distinguished morphologically from C. monogyna by the colour, size and structure of fruits, the number of pyrenes, the flowering and ripening time, the density of thorns, the tree shape and also the leaf shape. According to our morphological data, in Syria, C.  ×  sinaica is variable and could represent a hybrid of C. azarolus  ×  C. monogyna ; the cpDNA sequence analysis showed sequences corresponding to C. monogyna as the plausible mother of the hybrid.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 255–263.     

Genetic diversity and differentiation of allopolyploid Dactylorhiza (Orchidaceae) with particular focus on the Dactylorhiza majalis ssp. traunsteineri/lapponica complex

Genetic differentiation of Dactylorhiza majalis ssp. traunsteineri from the Alps, Scandinavia, and Britain was studied and compared with other allotetraploid members of the systematically challenging genus Dactylorhiza . One-hundred and eleven populations from altogether 18 taxa were analysed for eight polymorphic plastid markers and two size-variable fragments from the nuclear internal transcribed spacer (ITS) region. In total, 60 plastid haplotypes and six ITS alleles were found among the 737 individuals analysed. No clear differentiation between populations of ssp. traunsteineri from the three regions was revealed. However, ssp. traunsteineri was genetically differentiated from Dactylorhiza baumanniana , Dactylorhiza elata , and D. majalis ssp. sphagnicola , although the majority of allotetraploid taxa remained inseparable. Judging from the degree of concerted evolution in ITS, D. majalis ssp. alpestris may be regarded as a relatively old allotetraploid, whereas ssp. baltica and ssp. purpurella may be considerably younger. Based on plastid data, the Alp region had the highest genetic diversity followed by Scandinavia and Britain. The geographic distribution of haplotypes provided support for possible refugial areas around the Alps and for several independent immigration routes into Scandinavia after the last ice age.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 97 , 52–67.     

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.     

Molecular characterization of two natural hybrids from the Iberian Peninsula: <Emphasis Type="Italic">Ranunculus × luizetii</Emphasis> and <Emphasis Type="Italic">R. × peredae</Emphasis> (Ranunculaceae)

Two nothospecies, Ranunculus × luizetii and R. × peredae (Ranunculaceae), were analyzed and discussed. For this purpose, Amplified Fragment Length Polymorphism (AFLP) markers, nuclear rDNA sequences (ITS1, 5.8S and ITS2) and pollen viability were conducted. The profiles of these hybrid samples were compared to their putative progenitors. Several additive polymorphic sites detected in the ITS sequences of the hybrid samples (R. × luizetii and R. × peredae) also confirmed their derived origins from ribotypes of their parental taxa (R. parnassiifolius subsp. parnassiifolius × R. pyrenaeus; R. amplexicaulis × R. cabrerensis subsp. cabrerensis, respectively). Despite the lack of exclusive AFLP markers reported in both hybrids, presumably due to effects of introgression, the concerted evolution of many rDNA polymorphisms towards either of the parental ribotypes indicated their ancient origin. Pollen fertility estimation in R. × luizetii presented a mean value of 60.58%, which showed that hybrid samples are well established and fertile. However, a larger difference was observed in R. × peredae, where the mean value of pollen fertility was very low (18.91%).     

Molecular evidence for repeated hybridization events involved in the origin of the genus × Crepidiastrixeris (Asteraceae) using RAPDs and ITS data

We investigated the hybrid origin of × Crepidiastrixeris denticulato-platyphylla using RAPDs and ITS sequence data. The putative parents Paraixeris denticulata and Crepidiastrum platyphyllum represent separate species, irrespective of geographical origin. The occurrence of species specific RAPD markers from P. denticulata and C. platyphyllum in × C. denticulato-platyphylla established unambiguously a hybrid origin between the two taxa. This was in line with the occurrence of a combination of morphological characters such as plant habit and floret numbers. The parent taxa differed from each other by 7 nucleotide substitutions and 2 indel events in the ITS region. The hybrids showed sequence additivity and most likely represent F₁ plants, with the exception of two plants which were of possible F₂ origin, possessing either the ITS sequences of one parent only, or one predominant ITS type. The hybrids occurred in two out of three localities where the parents occurred sympatrically. This fact, together with the short life-span of the plants, suggests that × C. denticulato-platyphylla exists as a result of repeated, frequent hybridization between the parent species.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 151 , 333–343.     

Conflicting patterns of genetic and morphological variation in European Gentianella section Gentianella

Genetics in European Gentianella Moench sect. Gentianella were investigated using internal transcribed spacer (ITS) nrDNA, trn L-F cpDNA and Amplified Fragment Length Polymorphisms (AFLP) fingerprint data to analyse relationships within the section. Both a cladistic and a phenetic approach, as well as nonhierarchical analysis (PCA) and partitioning of molecular variance (AMOVA), were used. Sequence data did not resolve groups within the section. They reflected, however, geographical isolation between central European and eastern Carpathian ( G. lutescens and G. bulgarica ) as well as Caucasian samples ( G. caucasea ). AFLP data revealed a strong differentiation within G. germanica between populations of the Alps and north-western middle Europe, although there is hardly any morphological differentiation of taxonomic significance. Morphological similarities between G. aspera and G. pilosa were not reflected in genetics, and no support was found for G. aspera . Genetic similarities were not in line with morphology in G. lutescens and G. bulgarica . AFLP data only partly resolve relationships among taxa. High genetic similarities, as well as partitioning of molecular variance among taxa/populations of eastern middle Europe ( G. austriaca , G. bohemica , G. germanica , G. stiriaca ) and intermediate morphological characters in G. bohemica and G. stiriaca , might indicate reticulate evolution. The overall low sequence divergence within the section points to a very young origin of all taxa.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 175–187.     

Reticulate evolution and phylogeography in Asarum sect. Asiasarum (Aristolochiaceae) documented in internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA

Phylogenetic analyses were performed for the taxonomically complicated group, Asarum sect. Asiasarum, using internal transcribed spacers (ITS) of nuclear ribosomal DNA. Direct sequences for 99 samples of a total of 14 taxa and geographic races and cloning analyses for 32 of these samples provided new insights that extensive reticulate evolution has occurred in this group. Eight taxa had slight or no polymorphism of the ITS sequences. On the other hand, the other five taxa had polymorphic ITS sequences composed of two ribotypes that were completely the same or almost the same as the sequences recognized in the taxa with only slight or no polymorphism, and were probably of diploid hybrid origin and to have retained their parental ribotypes. In terms of biogeographic implications, at least four interactions including migration, hybridization, and introgression, were presumed between the Japanese Archipelago and the continents, two times via a southern route, from the Korean Peninsula, and two times via a northern route, from Sakhalin or directly from the Eurasian continent.     

Taxonomic status and relationships of bracken ferns (Pteridium: Dennstaedtiaceae) of Laurasian affinity in Central and North America

Detailed quantitative comparisons confirm and extend the discrimination of four major morphotypes amongst brackens of Laurasian affinity in Central and North America. These are recognized here at subspecies level as: Pteridium aquilinum sspp. feei , pubescens , latiusculum , and pseudocaudatum . Measurements of spore size indicate that sporophytes of P. aquilinum ssp. feei are diploid (2 n  = 104), as are sspp. pubescens , latiusculum , and pseudocaudatum . Phenetic cluster analysis based on DNA fingerprinting by arbitrarily primed polymerase chain reaction groups these four subspecies as genomically more similar to each other than to any taxa from other geographical regions. The chloroplast haplotype of ssp. feei is the same as that of sspp. latiusculum , pseudocaudatum , and pubescens with respect to the absence of both of the short direct repeats in the rps 4– trn S region (haplotype A), whereas the European ssp. aquilinum (haplotype B) has one of these repeats, and the Southern Hemisphere brackens P. arachnoideum and P. esculentum (haplotype C) have the other.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 157 , 1–17.     

Relationships in Patagonian species of Berberis (Berberidaceae) based on the characterization of rDNA internal transcribed spacer sequences

Sequence analysis of the internal transcribed spacer (ITS) of the 18S(ITS1)-5.8S-26S(ITS2) rDNA region was performed in order to analyse the phylogenetic relationships between 13 Patagonian species of the genus Berberis (Berberidaceae). The divergence values between the pairwise sequence in the studied Patagonian species were in the range 2.9–22.9%. The lengths of the ITS1 and ITS2 sequences were in the range 227–231 bp and 220–224 bp, respectively, and the 5.8S sequence was 159 bp throughout all species . B. microphylla sensu Landrum does not appear to be monophyletic based on current sampling. Indeed, we suggest that B. microphylla should be distinguished from B. buxifolia , B. parodii , and B. heterophylla . ITS sequences, together with data obtained from morphological, biochemical, amplified fragment length polymorphism, and cytological characterizations, support the existence of diploid and polyploid hybrid speciation in the genus.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 321–328.     

Exploring the history of Serapias politisii (Orchidaceae), a narrow endemic on the opposite coasts of the Otranto channel: insights from molecular investigations

Knowledge of genetic spatial structure may provide insights into the causes of population disjunctions in plants. Serapias politisii is a narrow endemic with only a few populations scattered along the opposite coasts of the Otranto strait (southern Adriatic Sea). It was originally considered to be of hybrid origin between S. vomeracea ssp. laxiflora and S. parviflora, a possibility suggested also by a DNA phylogenetic study that grouped Italian and Greek populations in two distinct clades. In this study we have carried out additional plastid DNA sequencing and an AFLP analysis of the three taxa. Whereas the geographical distribution of four plastid DNA haplotypes supports the likelihood of a double hybrid origin or of a plastid capture, AFLP data do not support such a hypothesis, because S. politisii shows several private alleles, some of which are shared by Italian and Greek populations. In light of the floristic specificity of the coasts bordering the Otranto strait, we consider that the present‐day disjunction of S. politisii could have originated either by a long‐distance seed dispersal or by a fragmentation of an old polymorphic population. The pairwise mismatch distribution excludes a recent expansion of the populations examined. In light of recent evidence concerning the Mediterranean Lago‐Mare period and the westward haplotype evolution detected in some Euro‐Mediterranean trees, we argue that fragmentation of a previously continuous population could be an intriguing possibility. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162 , 572–580.     

The taxonomic status and relationships of bracken ferns (Pteridium: Dennstaedtiaceae) from sub-Saharan Africa

Two morphotypes of bracken fern in the genus Pteridium Gled. ex Scop. occur in sub-Saharan Africa. The more common and widespread of these is assigned in contemporary systematic treatments to the European subspecies, P. aquilinum (L.) Kuhn ssp. aquilinum . We show that this African form is separable morphometrically and genomically from the European subspecies, and reinstate its earlier name P. aquilinum ssp. capense (Thunb.) C.Chr. The second African bracken, with a more localized tropical distribution mainly in the drainage basins of the Congo and Zambezi River systems, is confirmed as P. aquilinum ssp. centrali-africanum Hieron. ex R.E. Fr. We reject suggestions that this taxon be treated as a full species. Phenetic cluster analysis based on use of Arbitrarily Primed Polymerase Chain Reaction (A-P PCR) and Inter Simple Sequence Repeat (ISSR) fingerprinting of the nuclear genome provides evidence that the two African brackens are more closely related to each other than to other taxa, and sister to a grouping of the European sspp. aquilinum and pinetorum . The two African subspecies share solely with ssp. aquilinum a distinctive chloroplast haplotype carrying a 5-base direct repeat in the rps 4 –trn S region, confirming the close phyletic relationship between sspp. aquilinum , capense and centrali-africanum .  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 311–321.     

Natural hybridization does not dissolve species boundaries in commercially important sea cucumbers

The study of species boundaries in areas of sympatry provides important insight into speciation processes. We investigated whether (i) two sympatric holothurians, Holothuria scabra and H. s. var. versicolor constituted species, and (ii) specimens of intermediate phenotype hybrids. Results from allozyme and 16S mtDNA sequence analyses indicated these two sea cucumbers to be distinct but young biological and phylogenetic species. Several private allozyme alleles existed and a Bayesian analysis grouped varieties into separate clusters. MtDNA sequences hardly varied within each taxon, and nine single bp changes were diagnostic between these two taxa. Allozyme allele frequencies in individuals of intermediate phenotype were intermediate to those of H. scabra and H. s.  var.  versicolor , most private alleles were present and heterozygote frequencies were higher than in either species. Ancestry coefficients modelled for these individuals were close to 0.5, indicating that the two taxa contributed equally to their genome. MtDNA sequences were identical to those of either species. We conclude that individuals of intermediate phenotype represent F1 hybrids. The presence of hybrids demonstrates that the opportunity for introgression exists, but is not realized, as backcrossing and introgression were not supported by the data. Thus, the genetic integrity of either holothurian species remains intact through an unknown postzygotic mechanism, possibly hybrid sterility.  © 2005 The Linnean Society of London, Biological Journal of the Linnean Society , 2005, 85 , 261–270.     

Molecular variation and speciation in Antitrichia curtipendula s.l. (Leucodontaceae, Bryophyta)

Molecular variation in Antitrichia curtipendula (Hedw.) Brid. s.l. was studied based on the nuclear internal transcribed spacer (ITS) and the chloroplast markers trn L- trn F and rpl 16, and analysed by neighbour joining (for ITS; recombination present), maximum parsimony (chloroplast markers) and TCS (haplotype network). Old World plus E North American populations belong to a different lineage than those of W North America. These are molecularly well differentiated and are treated as A. curtipendula and A. gigantea (Sull. & Lesq.) Kindb. Two distinct groups of Old World haplotypes are separated by one 'missing' haplotype and are interpreted as cryptic species. Tropical African populations share one ITS deletion and form a lineage within one of the cryptic species. Molecular variation within A. gigantea , within each of the two cryptic Old World plus E North American (except tropical African) haplotype groups, and among tropical African populations are of similar magnitude, suggesting that analogous mechanisms and similar time spans explain the found variation. Events related to Pleistocene climatic oscillations are suggested as having caused this differentiation within each group, whereas the African lineage was probably split off before this. Identical tRNA-Gly sequences were found in 33 specimens; new primers were designed for rpl 16 and ITS 1+2.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 341–354.     

A revision and phylogenetic analysis of Stapeliopsis (Apocynaceae)

A survey of morphological characters is carried out for Stapeliopsis . The information obtained from this is combined with molecular data from the plastid trn L-F DNA region and ITS1 of the nuclear encoded 18S−26S rRNA cistron, to obtain a hypothesis of the evolutionary relationships among the species. It is shown that Stapeliopsis is monophyletic in a combined molecular and morphological analysis. Stapeliopsis is sister to a clade containing Huernia , Orbea and Tromotriche . The species of Stapeliopsis group into two clades. One contains S. khamiesbergensis , S. neronis and S. urniflora , and this is highly supported. The remaining species fall into an unsupported clade in which S. exasperata is sister to the others. The genera Hermanschwartzia Plowes and Neopectinaria Plowes are rejected. It is shown that a synapomorphy for Stapeliopsis is the laterally flattened inner corona-lobes, which touch the anthers only at their bases. Eight species of Stapeliopsis are recognized, with no subgeneric divisions.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 148 , 125–155.     

Morphological differentiation among closely related species with disjunct distributions: a case study of Mediterranean Cyclamen L. subgen. Psilanthum Schwarz (Primulaceae)

In Cyclamen subgenus Psilanthum , previous authors have recognized three species ( C. repandum, C. balearicum and C. creticum ), with C. repandum subdivided into three subspecies (ssp. repandum , ssp. peloponnesiacum and ssp. rhodense ), and two varieties (var. vividum and var. peloponnesiacum ) within C. repandum ssp. peloponnesiacum . To examine the validity of this classification and the degree of morphological differentiation among closely related endemic taxa with disjunct distributions, we undertook analyses of three quantitative and eight qualitative morphological traits of flowers and leaves, most of which have previously been used without statistical comparison in the classification of the different taxa. Cyclamen balearicum showed high levels of morphological differentiation from all other taxa in subgenus Psilanthum consistent with its specific status. In contrast, morphological differentiation between C. creticum and subspecies of C. repandum was similar to that among the three subspecies of C. repandum . When compared with a recent molecular phylogeny of this subgenus, our results suggest that C. creticum may best be described as a geographical subspecies of C. repandum . The two varieties of C. repandum ssp. peloponnesiacum showed few significant differences for individual traits and their overall morphology is very similar to each other. The morphology of plants at three sites on Corsica strongly suggest, consistent with molecular data, the occurrence of hybrid populations between C. balearicum (outside of its previously recognized distribution) and local C. repandum .  © 2002 The Linnean Society of London, Botanical Journal of the Linnean Society , 2002, 139 , 133–144.