Pollen evolution and its taxonomic significance in Cuscuta (dodders, Convolvulaceae)

The pollen morphology of 148 taxa (135 species and 13 varieties) of the parasitic plant genus Cuscuta (dodders, Convolvulaceae) was examined using scanning electron microscopy. Six quantitative characters were coded using the gap-weighting method and optimized onto a consensus tree constructed from three large-scale molecular phylogenies of the genus based on nuclear internal transcribed spacer (ITS) and plastid trn-LF sequences. The results indicate that 3-zonocolpate pollen is ancestral, while grains with more colpi (up to eight) have evolved only in two major lineages of Cuscuta (subg. Monogynella and clade O of subg. Grammica). Complex morphological intergradations occur between species when their tectum is described using the traditional qualitative types—imperforate, perforate, and microreticulate. This continuous variation is better expressed quantitatively as “percent perforation,” namely the proportion of perforated area (puncta or lumina) from the total tectum surface. Tectum imperforatum is likely the ancestral condition, while pollen grains with increasingly larger perforation areas have evolved multiple times. The reticulated tectum, unknown in other Convolvulaceae, has evolved in Cuscuta only in two lineages (subg. Monogynella, and clade O of subg. Grammica). Overall, the morphology of pollen supports Cuscuta as a sister to either the “bifid-style” Convolvulaceae clade (Dicranostyloideae) or to one of the members of this clade. Pollen characters alone are insufficient to reconstruct phylogenetic relationships; however, palynological information is useful for the species-level taxonomy of Cuscuta.     

Phylogenetic Relationships of American Willows (Salix L., Salicaceae)

Salix L. is the largest genus in the family Salicaceae (450 species). Several classifications have been published, but taxonomic subdivision has been under continuous revision. Our goal is to establish the phylogenetic structure of the genus using molecular data on all American willows, using three DNA markers. This complete phylogeny of American willows allows us to propose a biogeographic framework for the evolution of the genus. Material was obtained for the 122 native and introduced willow species of America. Sequences were obtained from the ITS (ribosomal nuclear DNA) and two plastid regions, matK and rbcL. Phylogenetic analyses (parsimony, maximum likelihood, Bayesian inference) were performed on the data. Geographic distribution was mapped onto the tree. The species tree provides strong support for a division of the genus into two subgenera, Salix and Vetrix. Subgenus Salix comprises temperate species from the Americas and Asia, and their disjunction may result from Tertiary events. Subgenus Vetrix is composed of boreo-arctic species of the Northern Hemisphere and their radiation may coincide with the Quaternary glaciations. Sixteen species have ambiguous positions; genetic diversity is lower in subg. Vetrix. A molecular phylogeny of all species of American willows has been inferred. It needs to be tested and further resolved using other molecular data. Nonetheless, the genus clearly has two clades that have distinct biogeographic patterns.     

Phylogenetic analysis of <Emphasis Type="Italic">IRIS</Emphasis> L. from China on chloroplast <Emphasis Type="Italic">TRN</Emphasis>L-F sequences

Phylogenetic relationships among the six Iris subgenera were reconstructed by chloroplast trnL-F sequences data using maximum likelihood. The entire matrix of aligned bases analyzed includes 1043 characters and the length of all sequences varied from 747 bp to 893 bp, and mutation sites accounted for 18.79% of the total length. The cluster analysis results accorded well with the subgeneric classification of Chinese Iris species. Results suggested rhizomes and sepals lacking ornament are ancestral characters, and subg. Xyridion and subg. Limniris are more primordial than another four subgenera. Subgenus Nepalensis and subg. Xyridion were resolved as monophyletic.     

Morphometric analysis of varieties of Acacia caven: (Leguminosae, Mimosoideae): Taxonomic inferences in the context of other Argentinean species

The aims of this study were to evaluate the degree of morphological differentiation between six varieties of Acacia caven and to examine their taxonomic validity in the context of other Argentinean species of the genus. To accomplish these purposes, morphological traits have been analyzed using multivariate methods (non parametric ANOVA, phenetic analysis and principal component analysis) on the varieties of A. caven and other six species of the genus, represented in Argentina. The phenogram obtained showed two principal clusters, one grouping all the species of subg. Acacia and the other grouping the species of subg. Aculeiferum. This result agreed with Vassal's infrageneric classification. However, the results of the principal PCA gathered the seven species here included in three groups, which were consistent with Bentham's infrageneric treatment. The ANOVA method indicated that most of the morphometric characters used were statistically sound for differentiation between varieties of A. caven. Further studies, including more species and characters, must be performed in order to clarify the position of Acacia boliviana and the relationships between A. caven and A. curvifructa.     

Phylogeny and infrageneric classification of Correa Andrews (Rutaceae) on the basis of nuclear and chloroplast DNA

This paper presents phylogenies of the small but ecologically and horticulturally important Australian genus Correa (Rutaceae). Consensus phylogenies generated using parsimony were congruent with their counterparts generated by Bayesian analysis, although usually less well resolved. The phylogeny generated from the second internal transcribed spacer region of the nuclear ribosomal DNA supported the monophyly of Correa and identified two well supported clades (one comprising C. lawrenceana and C. baeuerlenii and the other containing all other species of the genus). Phylogenetic reconstructions based on the combined trnL-trnF spacer and the trnK intron (including the matK gene) regions of chloroplast DNA also supported the monophyly of Correa and of the C. lawrenceana/C. baeuerlenii clade, but the topology among the other species differed markedly from that in the ITS-based phylogeny. The major clades identified in the chloroplast phylogenies seemed to follow geographic patterns rather than species boundaries, with different samples of C. glabra bearing chloroplast genotypes from different clades. These patterns are likely to be because of independent evolution of the chloroplast and nuclear genomes, and are typical of cases of introgressive hybridisation among species or incomplete lineage sorting of chloroplast genomes leading to incongruence between chloroplast and nuclear phylogenies. Thus, the phylogenies based on nuclear DNA should reflect species relations better than the chloroplast phylogeny in Correa, and we propose a new subgeneric classification of the genus on the basis of the ITS-based phylogeny and morphology. Correa subgenus Persistens Othman, Duretto and G.J. Jord., containing C. lawrenceana and C. baeuerlenii, is formally described.     

Phylogenetic relationships in Iranian <Emphasis Type="Italic">Scutellaria</Emphasis> (Lamiaceae) based on nuclear ribosomal ITS and chloroplast <Emphasis Type="Italic">trn</Emphasis>L-F DNA data

The genus Scutellaria (Lamiaceae, Scutellarioidae) includes taxa with a wide range of pharmacological traits, which make this genus economically important. Previous taxonomical studies on Scutellaria were mainly based on morphological and chromosomal characters. The infrageneric classification of the genus has been the subject of a long controversy among authors, and delimitation of taxa remains problematic. In this study, the phylogeny of the genus was evaluated using nrDNA ITS and trnL-F sequences, with a focus on the 42 Iranian taxa belonging to subgenera Apeltanthus and Scutellaria. In both ITS and trnL-F trees, there were two main clades within the genus corresponding to the two subgenera Scutellaria and Apeltanthus. In the subg. Scutellaria, the sect. Anaspis was monophyletic, but the monophyly of sect. Scutellaria was not supported. In this subgenus, S. galericulata was located away from other species in both trees, supported the isolation of this species from the sect. Scutellaria and inclusion in the sect. Galericularia as was previously considered by some authors. In subg. Apeltanthus, sect. Apeltanthus and sect. Lupulinaria were not monophyletic; furthermore, the monophyly of subsect. Lupulinaria was not supported. Based on the results of this study, it can be suggested that the sectional classification of both subgenera Apeltanthus and Scutellaria should be revised.     

Cytoplasmic diversity,phylogenetic relationships and molecular evolution of Tunisian Citrus species as inferred from mutational events and pseudogene of chloroplast trnL-trnF spacer

The genus Citrus L. is among the most important fruit trees in the world. In this report, cytoplasmic polymorphism of twenty seven Tunisian Citrus cultivars was explored using the chloroplast trnL-trnF intergenic spacer. Chloroplast sequences showed variation in length and nucleotide content. Haplotype and nucleotide diversity showed low variations. Molecular phylogenetic tree identifies Citrus maternal origins and demonstrates two major groups distinguishing between mandarin and pummelo groups. The trnL-trnF intergenic spacer showed one copy of pseudogene of the original trnF gene in 27 Citrus species at position 275 bp with a size varying from 49 to 63 bp. The anticodon domain was identified as the most conserved element, but one transversion (T−>C) was found in the D-domain. Meanwhile, one transversion (T−>A) and one transition (T−>G) were found in the T-domain. Neutrality tests (Tajima, Fu & Li and Fu) which revealed positive and non-significant values and Pi and θ_W assume a neutral model of evolution and advocated a constant population size. The study demonstrates the resolving power of trnL-trnF sequence data to prove both pummelo and mandarin gene pool’s contribution in the development of Tunisian secondary species and inferring their genetic and phylogenetic relationships.     

Species, genomes, and section relationships in the genus Arachis (Fabaceae): a molecular phylogeny

The economically important genus Arachis (Fabaceae) comprises 80 species restricted to South America. One monograph on the genus divided it into nine sections and included an intuitive assessment of evolutionary relationships. There is no comprehensive phylogenetic study of the genus. To test the current systematic treatment of the genus, we reconstructed a phylogeny for Arachis using nuclear ITS and plastid trnT–trnF sequences from 46 species representing all nine sections. ITS cloning of the allotetraploid species of section Arachis indicated the presence of A and B genome alleles and chimeric sequences. Our study revealed that species from section Extranervosae were the first emerging lineage in the genus, followed by sections Triseminatae and Caulorrhizae, and two terminal major lineages, which we refer to as erectoides and arachis. The lineage erectoides comprises members of sections Erectoides, Heteranthae, Procumbentes, Rhizomatosae, and Trierectoides. Species in the arachis lineage form two major clades, arachis I (B and D genomes species and the aneuploids) and arachis II (A genome species). Our results substantiated the sectional treatment of Caulorrhizae, Extranervosae, and Triseminatae, but demonstrated that sections Erectoides, Procumbentes, and Trierectoides are not monophyletic. A detailed study of the genus Arachis with denser taxon sampling, additional genomic regions, plus information from morphology and cytogenetics is needed for comprehensive assessment of its systematics.     

Chemical and phylogenetic relationships among Aristolochia L. (Aristolochiaceae) from southeastern Brazil

In this report, a molecular phylogeny based on the plastid gene matK and on the region between the genes trnL and trnF, and a chemical relationship based on the pattern of sesquiterpenes, is proposed for Aristolochia species from southeastern Brazil. We found that Aristolochia is a monophyletic genus and species considered to be derived contain labdanoic acids (LAs) in their leaves. The phenetic relationship recovered with sesquiterpenes did not agree with the phylogenetic relationships for Aristolochia, and three main clusters were recognized, namely, germacrene-D, germacrene-C and Z-caryophyllene groups. The presence of different sesquiterpene structures in species that are phylogenetically closely related may reflect adaptations to avoid predation of herbivores specialized in feeding on Aristolochiaceae plants.     

Phylogenetic analysis of Lappula Moench (Boraginaceae) based on molecular and morphological data

Lappula Moench includes ca. 70 species and exhibits a wide range of nutlet variation. Currently, the evolutionary relationships among species of Lappula have not been examined; therefore, to elucidate phylogenetic relationships and morphological evolution within Lappula and related genera, we conducted phylogenetic analyses with matrices that include 48 species as well as four DNA regions (ITS, trnL-trnF, rpS16 and psbA-trnH) and 18 morphological characters. These matrices were analyzed using maximum parsimony, maximum likelihood, and Bayesian inference methods. Analyses of the combined molecular and morphological data result in a phylogeny that is better resolved than that based solely on molecular sequence data. Phylogenetic results suggest that the current infrageneric classification of Lappula, at least at the subsectional and series level, is artificial. The evolutionary patterns of 18 morphological characters are investigated in a phylogenetic context. In Lappula, nutlet homomorphism and small corollas are resolved as ancestral, while nutlet heteromorphism and larger corollas are derived.     

Morphological variation in pollen grains of <Emphasis Type="Italic">Mucuna</Emphasis> (Leguminosae): new biogeographic and evolutionary patterns

Mucuna comprises 105 species with an overall pantropical distribution and is divided into three subgenera: M. subg. Mucuna, M. subg. Stizolobium and M. subg. Macrocarpa. Although phylogenetic studies have supported the occurrence of three main clades, evolutionary relationships among them are not fully resolved. The objective of this study was to examine pollen grain morphology from representatives of all three subgenera and map these onto the phylogenetic trees generated by analysis of other characters. Pollen grain surface, form, size, and aperture number were compared. A Bayesian inference tree using matK sequences was constructed. The results indicate that the representatives of M. subg. Macrocarpa have the smallest pollen grains in the genus (a synapomorphic character here identified for this subgenus) and that species of subgenus Mucuna (those with umbelliform inflorescences) have the largest pollen grains. Additional morphological diversity of the pollen grain surface was noted: reticulate and/or micro-reticulate (in all three subgenera), perforate, gemmate or verrucose (only in M. subg. Mucuna). For all studied taxa, the pollen grains are triaperturate, except for two species of M. subg. Mucuna, which have tetraperturate pollen. The phylogenetic tree obtained using the matK marker resolved M. subg. Stizolobium as the earliest diverging lineage in Mucuna. Based on this phylogeny, a reticulate ornamentation pattern of the pollen surface may represent the ancestral state for the genus, while the larger pollen size and the foraminate, gemmate, and verrucose ornamentations are derived characteristics within the genus. These putative derived ornamentations have been observed only in neotropical species.     

Diversity and evolution of the gynoecium in Cuscuta (dodders, Convolvulaceae) in relation to their reproductive biology: two styles are better than one

The gynoecium of 122 species and 14 varieties of Cuscuta (dodders) was investigated by light and scanning electron microscopy to assess its diversity and evolution and to provide a morphological foundation for understanding the different reproductive strategies encountered in the genus. Data were optimized into a consensus tree constructed from three large-scale molecular phylogenies of the genus based on nuclear ITS and plastid trnL-F sequences. The number of styles combined with the stigma shape are the only floral/fruit characters that enable the separation of Cuscuta subgenera. In addition, gynoecium morphology is useful for delimiting species in some clades. The one-style gynoecium of subg. Monogynella is mostly likely ancestral whereas gynoecia with two styles are derived in subgenera Cuscuta and Grammica. Gynoecia with two styles encountered in the latter subgenera provide a greater morphological complexity and flexibility for various reproductive strategies. In subg. Cuscuta, both the equal styles and stigmas continue to elongate and modify their position after the flowers open, until pollination occurs. In subg. Grammica, the two unequal styles may cause a spatial separation of the sexes in the flower, herkogamy, while the two stigmas mature sequentially and have a differential timing of their receptivity for pollen. A nectary consisting of a ring of modified stomata at the base of the ovary, the equivalent of the hypogynous nectary disc present in many Convolvulaceae, was observed for the first time in all Cuscuta species. The vasculature of the styles is reduced, represented mostly by phloem; xylem is present only in subg. Monogynella. Some gynoecial characters, for example papillae diameter, stigma surface area, stigma width, and style width were moderately correlated with pollen volume, pollen polar?Cequatorial ratio and tectum perforation. Gynoecium features suggest that Cuscuta is allied with the ??bifid clade?? (Dicranostyloideae) in Convolvulaceae.     

Molecular phylogeny of the endemic fern genera Cyrtomidictyum and Cyrtogonellum (Dryopteridaceae) from East Asia

Cyrtomidictyum Ching and Cyrtogonellum Ching are two eastern Asian endemic genera whose taxonomic affinities and phylogenetic relationships have long been controversial. The main uncertainty surrounds the separation of the two genera from the species-rich genus Polystichum. Here we present a phylogenetic study focusing on the phylogenetic relationships of these polystichoid ferns. We reconstructed the relationships based on DNA sequence variation in four chloroplast genome regions, rbcL, atpB, and the intergenic spacers (IGS) rps4-trnS and trnL-trnF. Maximum likelihood and Bayesian inference analyses confirm earlier results that were based on less comprehensive taxon sampling and either only a single gene (rbcL) or two IGS (rps4-trnS and trnL-trnF). Cyrtomidictyum is the sister of the clade of polystichoid ferns that includes Cyrtogonellum, Cyrtomium subser. Balansana and three sections of Polystichum. Cyrtogonellum groups with several species of Polystichum, and constitutes the sister taxon to Polystichum sect. Sphaenopolystichum. We support the recognition of Cyrtomidictyum as circumscribed initially, rather than expansion of the genus to include either several Polystichum species or Cyrtogonellum, some Polystichum and Cyrtomium species. The monophyly of Cyrtomidictyum is supported by morphological characters such as once-pinnate leaves, free venation, prolongated leaf apices, and exindusiate sori. Two synapomorphic indels in the chloroplast genome, one 15-bp deletion in rps4-trnS, and one 3-bp insertion in trnL-trnF further differentiate Cyrtomidictyum from other polystichoid ferns. The close affinity of Cyrtogonellum to section Sphaenopolystichum of Polystichum s.s. is highly supported by molecular data. However, no shared morphological characters or molecular indels have been detected, although the distinctness of Cyrtogonellum is shown by a 13-bp insertion in the rps4-trnS alignment.     

Molecular phylogeny of the genus <Emphasis Type="Italic">Bolusiella</Emphasis> (Orchidaceae,Angraecinae)

Recent molecular studies have suggested the monophyly of Bolusiella, a small orchid genus comprising five species and one subspecies from Continental Africa, but sampling has been limited. Using the species delimitation presented in the recent taxonomic revision of the genus, this study aimed to confirm the monophyly of Bolusiella and assess the interspecific relationships using a comprehensive sampling and various analytical methods. DNA sequences of one nuclear spacer region (ITS-1) and five plastid regions (matK, rps16, trnL–trnF, trnC–petN, and ycf1) from 20 specimens representing all five species of the genus were analyzed using static homology, dynamic homology, and Bayesian methods. The monophyly of both the genus Bolusiella and each of its five species was confirmed, corroborating the previously published taxonomic revision. The use of dynamic homology methods was not conclusive for this particular group. The results of the total evidence analysis (combining all six sequence regions) using the dynamic homology approach yielded a slightly different hypothesis regarding interspecific relationships (namely the exchange of B. talbotii and Bolusiella iridifolia as the earliest diverging lineage), probably because the nodes in question are supported by a small subset of conflicting characters, compared to the hypotheses resulting from the static homology and Bayesian methods, which are congruent with the results of previous studies.     

Phylogenetic split of Larix: evidence from paternally inherited cpDNA trnT-trnF region

A molecular phylogeny of Larix comprising 12 species was constructed from the sequence analysis of the paternally inherited cpDNA trnT-trnF region of 46 individuals. The most parsimonious tree split Larix into three sister clades: one clade was composed of two North American species, the other two were short-bracted and long-bracted species of Eurasia respectively except that L. sibirica was clustered in the long-bracted clade. The difference between the present cpDNA phylogeny and previous nrDNA ITS phylogeny in the position of L. sibirica seems to suggest that ancient cytoplasmic gene flow might exist between sections Larix and Multiserialis. The short-bracted L. laricina and long-bracted L. occidentalis have an identical sequence of the trnT-trnF region, which implies that the bract length divergence among North American larches might have occurred recently or chloroplast capture happened during the early differentiation of the two species. The cpDNA results also shed some light on the biogeography of Larix.     

Interspecific relationships and origins of Taxaceae and Cephalotaxaceae revealed by partitioned Bayesian analyses of chloroplast and nuclear DNA sequences

The precise delimitation of Taxaceae and Cephalotaxaceae is not totally resolved. Some contradicting taxonomic proposals have been published, which demonstrates the difficulties in establishing a natural classification of the families and especially in proposing a relevant treatment within the genera Taxus and Cephalotaxus. The aims of this study are to contribute to the phylogeny and specific delineation of the two conifer families on the basis of molecular data. A cladistic analysis of the sequences of five chloroplast (matK, rbcL, trnL, trnL-trnF spacer, and psbA-trnH spacer) and one nuclear (ITS) molecular markers was carried out, both individually and in combination, by distance, parsimony, likelihood, and Bayesian methods. The results confirm that the two families are monophyletic. In the genus Taxus, T. floridana is the first-branching taxon; T. brevifolia and T. globosa cluster together and are sister to T. baccata; the endemic T. yunnanensis clusters with T. wallichiana in subclade B and is only distantly related with the other four Taxus species in China (subclade A); T. fuana is closer to T. baccata than to other Taxus species. Torreya jackii and A. formosana are the first-branching species within Torreya and Amentotaxus, respectively. C. koreana and C. wilsoniana could be treated as two varieties of C. harringtonia. The ancestral distribution area of Taxaceae and Cephalotaxaceae is restricted either to Southwest China or Southeast China by DIVA analysis. The relaxed molecular clock indicates that the deepest divergences in Taxus go back to the late-Cretaceous. psbA-trnH, rbcL third codon position, and matK first codon position contributed most to the separation of taxa in Discriminant function analysis. Our results confirm, on a basis of multiple molecular markers and a complete sampling of basic species, the suggested monophyly of Taxaceae and Cephalotaxaceae and propose interspecific relationships within each group, with profound nomenclatural and taxonomic implications. Combination of partitioned Bayesian analysis and likelihood-based methods produced a more robust phylogenetic hypothesis for the two studied families.     

Multilocus phylogenetic reconstruction informing polyploid relationships of <Emphasis Type="Italic">Aconitum</Emphasis> subgenus <Emphasis Type="Italic">Lycoctonum</Emphasis> (Ranunculaceae) in China

Polyploidization has long been recognized as one of the most important driving forces of plant evolution. Aconitum subgenus Lycoctonum (Ranunculaceae) has a wide distribution range and well-known background of polyploidy, thereby providing a potentially valuable model to explore polyploid origin and evolutionary history. However, the phylogeny of subg. Lycoctonum has not yet been completely resolved. In the current study, 29 species including diploid, tetraploid and hexaploid species were sampled in subg. Lycoctonum. Using four cpDNA regions (ndhF-trnL, psbA-trnH, psbD-trnT and trnT-L) and two nrDNA regions (internal transcribed spacer, ITS, and external transcribed spacer, ETS), phylogenetic relationship was first reconstructed for the polyploid species within subg. Lycoctonum. In combination with nuclear diversification rate estimation, cpDNA haplotype network, ancestral area reconstruction as well as morphological and karyotypic evidence, potential origin and divergence time were further assessed among the polyploid species. Hybridization was inferred for A. angustius and A. finetianum was suggested to be the potential maternal progenitor, due to their close phylogenetic relationship, highly similar morphologies and overlapping distribution range. Local origin was inferred for tetraploids in the Hengduan Mountains (HDM) with eight groups of chromosomes of four homeologous, which diverged approximately 3.00 Ma in the same period of the orogeny of the HDM. The hexaploid A. apetalum was inferred to suffer from geographical isolation due to the formation of the Qinghai–Tibetan Plateau (QTP) and the HDM. Hybridization and heterogeneous habitats in the HDM were suggested to play an important role in the polyploidization in subg. Lycoctonum.