Cladistic and phenetic analysis of relationships in Vicia subgenus Vicia (Fabaceae) by morphology and isozymes

 Variation of 80 multistate morphological characters and isozymes encoded by 13 loci among 23 vetch species of the type subgenus of the genus Vicia in comparison with V. dumetorum, V. pisiformis and V. sylvatica of the subgenus Cracca is described and analyzed with cladistic parsimony and phenetic neighbour-joining methods by using two different ways of coding. Morphological analyses showed the subgenus Vicia monophyletic and revealed subgroups in a general agreement with traditionally recognized sections, except showing V. faba nested within section Narbonensis and ambiguity in the position of V. lathyroides and V. bithynica. Parsimony analysis of orthozymes as presence/absence characters revealed in the subgenus two basic monophyletic clades: 1) V. faba and three species of the section Peregrinae, V. michauxii, V. aintabensis and V. peregrina, in one subclade linked with species of the Narbonensis and Hyperchusa sections together with V. pisiformis of subgenus Cracca in a second subclade; 2) species belonging to sections Vicia, Sepium, Pseudolathyrus and Lathyroides together with V. sylvatica of the subgenus Cracca. Neighbour-joining analysis of orthozymes revealed the same two basic groups, differing only in the relative position of some species in subclusters. Both isozyme analyses showed paraphyly of the subgenera Vicia and Cracca. Parsimony analysis of orthozymes as character states of isozymes yielded a largely unresolved strict consensus cladogram of 209 most parsimonious trees, and reweighting of characters failed to produce a stable tree. Phylogenetic congruence and discordance among morphological and isozyme analyses, coding ways, homoplasy and weighting of characters are discussed. Received November 20, 2001 Accepted January 31, 2002     

Nuclear DNA contents, rDNAs, and karyotype evolution in subgenus Vicia: III. The heterogeneous section Hypechusa

Summary. Nuclear DNA contents, automated karyotype analyses, and sequences of internal transcribed spacers from ribosomal genes have been determined in the species belonging to section Hypechusa of the subgenus Vicia. Karyomorphological results and phylogenetic data generated from the comparison of rDNA (genes coding for rRNA) sequences showed that sect. Hypechusa is not monophyletic; however, some monophyletic units are apparent (one including Vicia galeata, V. hyrcanica, V. noeana, and V. tigridis, another including V. assyriaca, V. hybrida, V. melanops, V. mollis, and V. sericocarpa), which partly correspond to morphology-based infrasectional groups. The relationships among these species and the species in sections Faba, Narbonensis, Bithynicae, and Peregrinae have been also investigated. Correspondence and reprints: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, via San Camillo de Lellis, 01100 Viterbo, Italy.     

Cytological and molecular characterization of Vicia esdraelonensis Warb. & Eig: a rare taxon

Summary. Vicia esdraelonensis, a rare taxon belonging to section Hypechusa of subgenus Vicia, was recovered and analyzed by cytological, karyological, and molecular methods, with the aim of both characterizing this species and furthering our knowledge of the phylogeny of subgenus Vicia. Automated karyotype analysis, nuclear DNA content, and chromatin organization were determined by the Feulgen reaction, as well as chromosome banding after double staining with 4′,6-diamidino-2-phenylindole (DAPI) and chromomycin A3. The chromosome number and the nuclear DNA content were in agreement with the values of the species of section Hypechusa. The GC- and AT-rich preferential sites were determined by chromomycin A3 and DAPI staining. Karyomorphological parameters indicated that V. esdraelonensis is in an intermediate position in the spatial representation of the species of section Hypechusa on the basis of symmetry indices, as well as in the dendrogram of linkage distance constructed on 37 chromosome parameters. Molecular data based on internal transcribed spacer sequences show that V. esdraelonensis can doubtlessly be included in section Hypechusa and document its closeness to V. noeana. A cladistic analysis combining the molecular data set with karyological characters is also reported. Karyological, cytological, and molecular data allow characterization of the V. esdraelonensis genome and provide information about the phylogenetic position of this species within the Hyrcanicae series of section Hypechusa. Correspondence and reprints: Dipartimento di Biologia, Università di Pisa, Via L. Ghini 5, 56126 Pisa, Italy.     

Taxonomic relationships between V. faba and its relatives based on nuclear and mitochondrial RFLPs and PCR analysis

Summary The taxonomic relationships between 52 accessions of 12 Vicia species and three accessions of Lathyrus were examined using nuclear RFLP- and PCR-generated data. Two hundred and sixty informative restriction fragments or amplification products were analysed by single linkage analysis, average cluster analysis, and the Wagner parsimony method. Dendrograms constructed from each type of analysis showed similar overall topologies and could be divided into three parts corresponding respectively to the Lathyrus outgroup, the species grouped in the section Faba/narbonemis complex, and the species belonging to the sections Hypechusa and Peregrinae. With few exceptions, the majority of accessions belonging to one species grouped together before branching to other species. An analysis of mitochondrial DNA phenotypes was both consistent with and complemented the results from the nuclear data. Overall, the species relationships show a good correlation with the classification of Maxted et al. but suggest that V. faba is more closely aligned to species from the sections Hypechusa and Peregrinae than to those in the narbonensis complex. In addition, the position of two new species, V. kalakhensis and V. eristaloides, as members of the narbonensis complex was supported by the molecular data, which also allowed a preliminary classification for recently collected Vicia accessions.     

Nuclear DNA contents, rDNAs, and karyotype evolution in Vicia subgenus Vicia: II. Section Peregrinae

Summary. Nuclear DNA contents, automated karyotype analyses, and sequences of rDNA spacers have been determined for the species of Vicia belonging to sect. Peregrinae, as well as for V. mollis. The phylogenetic data generated from the comparison of rDNA sequences and karyomorphological results would both indicate that Vicia mollis is a sister group to sect. Peregrinae. The relationships among the species belonging to the Peregrinae section and species enclosed in sections Faba, Narbonensis, and Bithynicae have been also investigated: a clade including V. mollis and sect. Peregrinae is a sister group to a clade including V. bithynica and sect. Narbonensis. With our choice of outgroup, Vicia faba (including subsp. paucijuga) is external to the above mentioned inclusive group. Correspondence and reprints: Dipartimento di Agrobiologia ed Agrochimica, Università della Tuscia, via San Camillo de Lellis, 01100 Viterbo, Italy.     

Pollen morphology of some taxa of Vicia L. subgenus Vicia (Fabaceae) from Turkey

In this study, the pollen morphology of 11 taxa belonging to Atossa (Alef.) Asch. & Graebner, Hypechusa (Alef.) Asch. & Graebner, Peregrinae Kupicha, Wiggersia (Alef.) Maxted, Vicia L. and Narbonensis (Radzhi) Maxted sections of the genus Vicia L. subgenus Vicia (Fabeae, Fabaceae) naturally growing in Turkey has been studied using Light Microscopy (LM) and Scanning Electron Microscopy (SEM) to evaluate the taxonomic relevance of pollen characters. Twelve morphometric characters are analysed with one-way analysis of variance and Tukey’s honestly significant difference test for multiple comparisons. Of the taxa studied V. narbonensis var. narbonensis (sect. Narbonensis) has the largest pollen grains (P = 51.98 μm × E = 30.52 μm) and V. lathyroides (sect. Wiggersia) has the smallest pollen grains (P = 27.71 μm × E = 20.14 μm). The pollen grains are subprolate to perprolate (P/E = 1.16–2.11), but the prolate shape occurs in the majority of the taxa. The regular pollen grains of all taxa are trizonocolporate, isopolar, and released in monads. Ornamentation of the mesocolpium is psilate-perforate in V. lathyroides (sect. Wiggersia), reticulate-rugulate in V. narbonensis var. narbonensis (sect. Narbonensis), (micro)reticulate in V. sericocarpa var. sericocarpa (sect. Hypechusa), V. sativa subsp. sativa (sect. Vicia) and V. grandiflora var. grandiflora (sect. Vicia), and reticulate-perforate in the remaining taxa. The apocolpium and colpus area are psilate or perforate in all taxa except V. sericocarpa var. sericocarpa (sect. Hypechusa) and V. sativa subsp. sativa (sect. Vicia), which exhibit the obscurely reticulate-perforate pattern. Several palynological features have taxonomic importance in sectional level: polar axis, equatorial diameter, pollen shape (P/E ratio), colpus length, colpus width, size of pori, porus length/width ratio, lumina diameter, muri thickness and ornamentation. The results also indicate that pollen characters can be useful in distinguishing the examined taxa.     

Phylogenetic relationships betweenVicia faba (Fabaceae) and related species inferred from chloroplasttrnL sequences

The chloroplasttrnL intron from 46 differentVicia accessions, representing five of the nine sections of the genusVicia subg.Vicia sensuMaxted (1991a) were amplified by the Polymerase Chain Reaction (PCR) using oligonucleotide primers homologous to conserved regions intrnL. The products fell into two distinct groups; those of approximately 250 nt and those of around 450 nt in length. Of these, products from 17 differentVicia species were cloned and their nucleotide sequences determined. Multiple alignments were assembled and phylogenetic trees constructed by the weighted least-squares distance method. ALathyrus latifolius trnL intron sequence was used as an outgroup. The resulting trees clearly group and separate the sectt.Narbonensis, Bithynica andFaba species but were less able to distinguish species from sectt.Hypechusa andPeregrinae. Based on these sequence data,V. faba appears to be more distant from sect.Narbonensis than sectt.Hypechusa andPeregrinae. The results are in general agreement with a recent treatment ofVicia subg.Vicia (Maxted 1993) and lend further support to placingV. faba in the monospecific sect.Faba.     

Taxonomy, evolution and domestication of Vicia in relation to aphid resistance

Thirty Vicia species and 28 V. faba cultivars were evaluated for Aphis fabae resistance by measuring rates of aphid population increase (r_m) on the host plants. A subset of these species (22) and cultivars (five) were evaluated for resistance to Acyrthosiphon pisum and Megoura viciae. For the same subset of 22 Vicia species and all three aphid species, positive correlations were observed between the concentration and numbers of leaf and stem non-protein amino acids and the level of aphid resistance. Correlations were also observed between trichome density, on some organs of the host, and both nymphal survival and development time. Aphid resistance, together with these possible physical and chemical resistance mechanisms, showed a pattern which was described in relation to the taxonomy of the genus Vicia and the degree of domestication of the host. The least advanced Vicia species were most resistant whilst the more specialised species and those most closely related to V. faba and V. faba itself were most susceptible. Within each taxonomic group (subgenus or section), the most domesticated species were least resistant. The merits of utilising the resistance found in some V. faba cultivars and in other wild Vicia species were considered.     

Isozyme evidence on the specific distinctness and phylogenetic position of <Emphasis Type="Italic">Vicia incisa</Emphasis> (Fabaceae)

Vicia incisa is a taxonomically controversial species that has been also treated as a subspecies of V. sativa because of a great morphological similarity. The phylogenetic position of V. incisa is uncertain because various DNA markers have provided contradictory results. Isozymes of V. incisa encoded by 15 loci and resolved with the use of polyacrylamide gel electrophoresis (PAGE) are described and compared with those of seven related species belonging to sections Vicia, Sepium, Lathyroides and Pseudolathyrus in order to get new evidence about its taxonomic rank and phylogenetic position. Phylogenetic relationships are analyzed with maximum parsimony and neighbour joining methods. Vicia incisa is shown to differ from all three subspecies of V. sativa including, sativa, cordata and nigra, by alternate variants of ten isozymes out of 15 analysed. Instead, V. incisa has much more similarity to V. grandiflora and V. sepium by sharing eight isozyme variants which differ from the subspecies of V. sativa. The most parsimony and neighbour joining analyses of the isozyme variation placed V. incisa as basally linked to the V. grandiflora and V. sepium species couple in the clade of section Sepium (= sect. Atossa), while the subspecies of V. sativa together with V. lathyroides formed a separate clade of section Vicia. The isozyme data provide further support to the species status of V. incisa.     

Infrageneric variation of the low molecular weight carbohydrate composition of the seeds of the genusVicia (Leguminosae)

The low molecular weight carbohydrate compositions of the seeds of 29 species ofVicia, namelyV. amoena, V. amurensis, V. bifolia, V. dumetorum, V. fauriei, V. japonica, V. nipponica, V. pisiformis, V. pseudo-orobus, V. sylvatica, V. unijuga, V. venosa, V. cassubica, V. orobus, V. cracca agg.,V. hirsuta, V. villosa agg.,V. tetrasperma,V. oroboides, V. sepium, V. cuspidata, V. grandiflora, V. lathyloides, V. sativa agg.,V. bithynica, V. faba, V. narbonensis, V. hybrida andV. lutea were determined by gas liquid chromatography. The carbohydrate compositions were found to be species-specific. Principal component analysis of the carbohydrate composition data showed that these species can be divided into three groups. Although, as far as the examined species were concerned, these groups were not correlated with the known subgenera, significant correlation between the groups and the known sections was detected in the subgenusVicia. The carbohydrate composition character would be important to clarify the relationships among closely related taxa of the genusVicia.     

MOLECULAR AND MORPHOMETRIC DATA PINPOINT SPECIES BOUNDARIES IN HALIMEDA SECTION RHIPSALIS (BRYOPSIDALES,CHLOROPHYTA)1

Molecular systematic studies have changed the face of algal taxonomy. Particularly at the species level, molecular phylogenetic research has revealed the inaccuracy of morphology‐based taxonomy: Cryptic and pseudo‐cryptic species were shown to exist within many morphologically conceived species. This study focused on section Rhipsalis of the green algal genus Halimeda. This section was known to contain cryptic diversity and to comprise species with overlapping morphological boundaries. In the present study, species diversity within the section and identity of individual specimens were assessed using ITS1–5.8S–ITS2 (nrDNA) and rps3 (cpDNA) sequence data. The sequences grouped in a number of clear‐cut genotypic clusters that were considered species. The same specimens were subjected to morphometric analysis of external morphological and anatomical structures. Morphological differences between the genotypic cluster species were assessed using discriminant analysis. It was shown that significant morphological differences exist between genetically delineated species and that allocation of specimens to species on the basis of morphometric variables is nearly perfect. Anatomical characters yielded better results than external morphological characters. Two approaches were offered to allow future morphological identifications: a probabilistic approach based on classification functions of discriminant analyses and the classical approach of an identification key.     

Cytology of Vicia species. X. Karyotype evolution and phylogenetic implication in Vicia species of the sections Atossa, Microcarinae, Wiggersia and Vicia

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF% indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses.     

Electrophoretic seed albumin patterns inVicia species of sectt.Hypechusa andPeregrinae (Fabaceae)

This work is a continuation of electrophoretic investigations aimed at revealing a wild relative ofVicia faba. Electrophoretic analysis (PAGE) of seed albumins covered 52 accessions representing eightVicia species of sect.Hypechusa and two species of sect.Peregrinae. Most of the examined species showed an intraspecific variation due to differences between accessions and/or individual variation within accessions. In spite of the intraspecific variation, marked interspecific differences were recorded. However, none of the investigated species displayed electrophoretic seed albumin patterns similar to those reported earlier forV. faba. Contribution of the obtained results to characterization of the examined taxa is discussed.     

Phylogenetic significance of stylar features in genus Vicia (Leguminosae): an analysis with molecular phylogeny

Tribe Fabeae consists of five genera, Lathyrus (160 spp.), Lens (4–6 spp.), Pisum (2–3 spp.), Vavilovia (monotypic), and Vicia (160 spp.), and shows a diversity in stylar features. At least six different stylar types are known in the tribe. In order to reclassify the tribe at the rank of genus, we tried to discover apomorphies in stylar features using a molecular phylogenetic study. We surveyed internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA of representative species, selected from each group having different types of styles in the tribe. We paid particular attention in sampling to members of Vicia section Vicilla, as stylar features are heterogeneous within this section. Consequently, our sample set included 15 species of section Vicilla, 23 species of other Fabeae, and two species of Trifolieae, which were used as a sister group of Fabeae. Based on our analysis, we found that a laterally compressed style and an abaxially tufted hairy style would be advanced against a dorsiventrally compressed style and an evenly hairy style, respectively, in genus Vicia. The species group, which shares the latter apomorphy, is composed of 56 species and was dispersed into 11 sections of two subgenera in the recent system of genus Vicia. We consider future revision of Fabeae should treat this species group as a single higher taxon.     

Reappraisal of Primula ranunculoides (Primulaceae), an endangered species endemic to China,based on morphological,molecular genetic and reproductive characters

The taxonomic status of Primula ranunculoides and other members of section Ranunuculoides is reappraised here based on data from morphological, reproductive and molecular characters. Multivariate analysis of morphological characters indicates that P. ranunculoides is a coherent species that can be distinguished from its sectional congeners P. cicutariifolia and P. merrilliana by the characters of simple kidney‐shaped outer leaves and the unique clonal reproductive ability by which apices of the scape differentiate into bulblets at the late phases of flowering. Recognition of P. ranunculoides at the specific level is also supported by palynological characters, breeding system, cross pollination results and molecular phylogenetic analysis of nrDNA internal transcribed spacer sequences. A taxonomic revision of section Ranunuculoides is presented and a possible mechanism of speciation discussed. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 338–349.     

Numerical analyses of seed morphology and its taxonomic significance in the tribe Nigelleae (Ranunculaceae)

We examined the seed morphology of Nigella, Garidella and Komaroffia to aid systematics and taxonomy. The seeds of 21 taxa in 20 species from Mediterranean and West Asian countries were studied using both zoom stereo and scanning electron microscopy (SEM). Principal coordinate analysis (PCoA) and unweighted average linkage analysis (UPGMA) were performed to assess the contribution of seed characters to the taxonomy of the tribe. Representative pictures from zoom stereo microscopy and SEM studies and measurement of seeds are presented. Most studied species showed unique seed surface sculpturing features, and seed shape, size and surface sculpturing separated the studied taxa into five main groups. However, the variation in seed shape and color were found to be in conflict with the generic taxonomy while macromorphological characters were found more congruent with the sectional taxonomy than with the generic taxonomy. Five main types of seed ornamentations – rugulose + striate, granulate, smooth, rugulose and mucronulate – were identified. Patterns in seed micromorphology strongly supported the sectional level taxonomy of Nigella. Two of the three sections of the genus were found to be well differentiated in terms of seed characters. Furthermore, seed morphology alone provide good support to Garidella as a distinct genus, but shows less taxonomic value for Komaroffia.     

Genetic diversity and relationships in some <Emphasis Type="Italic">Vicia</Emphasis> species as determined by SDS-PAGE of seed proteins

To evaluate the genetic diversity of some Vicia species, seed proteins of 160 accessions (30 of Vicia faba, 15 of V. narbonensis, 82 of V. sativa and 25 of V. ervilia and 8 accessions of other Vicia species) were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dendrogram showed that the two outcrossing species V. faba and V. villosa were the most distant among all species (average percent disagreement value PDV 0.47 and 0.45, respectively). The tree was divided into small clusters of two species each. V. narbonensis fell in one cluster with V. michausai (at PDV = 0.35) and V. lutea (var. hirta) fell in one cluster with V. serococorpes (at PDV = 0.32) whereas, V. ervilia fell in one cluster with V. sativa (at PDV = 0.27). The V. sativa subspecies, however, were closely related (PDV < 0.1). In general, this study did not prove any relationship between the studied storage proteins and the geographical distribution or ecological needs of the studied accessions.     

Karyological and molecular characterisation of subgenus Vicia (Fabaceae)

In the present report, we have analysed the subgenus Vicia by karyological and molecular approaches with the aim to clarify the relationships among Vicia species included in this subgenus by previously evidenced morphological investigations. Multivariate analysis using several karyomorphological parameters in addition to symmetry indices has allowed the construction of a dendrogram of linkage distances very useful to compare and to include in a phylogenetic tree obtained by internal transcribed spacer (ITS) rDNA sequences. Moreover, a separate analysis was performed combining our molecular data on ITS sequences with those reported in the literature for the section Vicilla. Our analyses partly confirm the monophyletic status of the various sections in which the subgenus Vicia has been divided, however questioning, in some cases, the real need to maintain all the nine sections so far accepted and the placement of some individual species in the two subgenera Vicia and Vicilla.     

SEED COAT MORPHOLOGY IN CORDYLANTHUS (SCROPHULARIACEAE) AND ITS TAXONOMIC SIGNIFICANCE

A study of seed coat sculpturing in Cordylanthus (Scrophulariaceae—Rhinantheae) using the scanning electron microscope (SEM) shows that seed surface patterns are characteristic and constant for a given species or a group of species. Seeds of 23 species were examined and classified into four types (irregularly crested, deeply reticulate, shallowly reticulate, and irregularly striate) based on differences in the reticulated seed coat. Anatomical studies of sections with the light microscope show that the reticular patterns result from enlargement of the epidermal cells of the integument, followed by formation of characteristic wall patterns through lignification. Seed characters furnish useful data for formulating the taxonomy of Cordylanthus both on the sectional and subsectional level and for the delimitation of certain species. Seed coat morphology offers evidence for including the genus Dicranostegia in Cordylanthus and for maintaining C. hispidus and C. palmatus as separate species. The distinctness of two seed coat types within section Cordylanthus suggests two major phylogenetic lines within this section.