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.     

Five molecular markers reveal extensive morphological homoplasy and reticulate evolution in the Malva alliance (Malvaceae)

The Malva alliance is a well-defined group with extensive morphological homoplasy. As a result, the relationships among the taxa as well as the evolution of morphological traits have remained elusive and the traditional classifications are highly artificial. Using five molecular markers (nuclear ITS, plastid matK plus trnK, ndhF, trnL-trnF, psbA-trnH), we arrived at a phylogenetic hypothesis of this group, the genera Alcea, Althaea and Malvalthaea being studied here for the first time with molecular data. Althaea and, in particular, Lavatera and Malva are highly polyphyletic as currently circumscribed, because their diagnostic characters, the number and degree of fusion of the epicalyx bracts, evolve in a highly homoplasious manner. In contrast, fruit morphology largely agrees with the molecularly delimited groups. Hybrid origins confirmed for the genus Malvalthaea and for Lavatera mauritanica and hybridization in the group of ruderal small-flowered mallows underline the importance of reticulate evolution in shaping the history of this group and complicating the interpretation of morphological evolution.     

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.     

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.     

A molecular phylogenetic study of Hemsleya (Cucurbitaceae) based on ITS, rpl16, trnH-psbA, and trnL DNA sequences

This paper presents the first molecular phylogeny of the genus Hemsleya using nuclear ITS and plastid trnH-psbA, rpl16, and trnL DNA sequences to examine the relationships among Hemsleya species. Phylogenetic relationships were elucidated using a combined analysis of all four datasets, however, the number of parsimony-informative characters was still insufficient to resolve all relationships. Parsimony and Bayesian trees were highly congruent. Twenty-three species of Hemsleya split into two major clades corresponding to two subgenera, i.e., subg. Graciliflorae and subg. Hemsleya. These results are partly in agreement with Li’s sectional classification. However, the molecular data are inconsistent with Li’s classification at the subsectional level. The molecular phylogeny revealed a striking overall correlation between the phylogenetic relationships of the species and their geographical distribution. The Kangdian ancient landmass could be the center of origin of the genus.     

An ITS-based phylogenetic framework for the genus Vorticella: finding the molecular and morphological gaps in a taxonomically difficult group

Vorticella includes more than 100 currently recognized species and represents one of the most taxonomically challenging genera of ciliates. Molecular phylogenetic analysis of Vorticella has been performed so far with only sequences coding for small subunit ribosomal RNA (SSU rRNA); only a few of its species have been investigated using other genetic markers owing to a lack of similar sequences for comparison. Consequently, phylogenetic relationships within the genus remain unclear, and molecular discrimination between morphospecies is often difficult because most regions of the SSU rRNA gene are too highly conserved to be helpful. In this paper, we move molecular systematics for this group of ciliates to the infrageneric level by sequencing additional molecular markers—fast-evolving internal transcribed spacer (ITS) regions—in a broad sample of 66 individual samples of 28 morphospecies of Vorticella collected from Asia, North America and Europe. Our phylogenies all featured two strongly supported, highly divergent, paraphyletic clades (I, II) comprising the morphologically defined genus Vorticella. Three major lineages made up clade I, with a relatively well-resolved branching order in each one. The marked divergence of clade II from clade I confirms that the former should be recognized as a separate taxonomic unit as indicated by SSU rRNA phylogenies. We made the first attempt to elucidate relationships between species in clade II using both morphological and multi-gene approaches, and our data supported a close relationship between some morphospecies of Vorticella and Opisthonecta, indicating that relationships between species in the clade are far more complex than would be expected from their morphology. Different patterns of helix III of ITS2 secondary structure were clearly specific to clades and subclades of Vorticella and, therefore, may prove useful for resolving phylogenetic relationships in other groups of ciliates.     

The ITS region of groundwater amphipods: length,secondary structure and phylogenetic information content in Crangonyctoids and Niphargids

The phylogenetic analysis of groundwater amphipods is challenging due to the lack of suitable morphological characters. However, molecular phylogenies based on the 18S and 28S nuclear genes of two Crangonyctoidea species endemic to Iceland, Crymostygius thingvallensis and Crangonyx islandicus, support the taxonomy of these species on the basis of morphological characters. Molecular analyses suggest that the genus Crangonyx is paraphyletic, with the species that is found in Eurasia being highly divergent genetically from the species present in North America and Iceland. Studies of the phylogenetic relationships within the genus Niphargus also warrant further work. The nuclear ITS2 region has recently been proposed as a barcoding marker for plants and animals. In addition, ITS2 has been used to build phylogenies at high taxonomic levels by including its secondary structure. In this study, we want to evaluate the applicability of the ITS region for this group of species and describe its characteristics. The taxonomy of C. thingvallensis, as well as the paraphyly of the genus Crangonyx, is supported herein by phylogenies based on the ITS2 variation. The secondary structure and the length of the ITS2 sequences of the Crangonyctoidea and the Niphargidae species studied are highly variable and are characterized by duplications. The ITS2 sequence of Niphargus plateaui is the longest metazoan sequence deposited in the ITS2 database so far. Although saturation was observed in the nucleotide variation of this marker, the addition of the secondary structure information for the reconstruction of the phylogeny did not add support to the phylogenetic trees. The ITS1 region, which is known to be more variable than ITS2 and bears a large duplication within C. islandicus, was found to be less useful for phylogenetic reconstruction.     

Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi

Fungal species belonging to the genus Melampsora (Basidiomycota, Pucciniales) comprise rust pathogens that alternate between Salicaceae and other plant hosts. Species delineation and identification are difficult within this group due to the paucity of observable morphological features. Several Melampsora rusts are highly host-specific and this feature has been used for identification at the species level. However, this criterion is not always reliable since different Melampsora rust species can overlap on one host but specialize on a different one. To date, two different species recognition methods are used to recognize and define species within the Melampsora genus: (i) morphological species recognition, which is based solely on morphological criteria; and (ii) ecological species recognition, which combines morphological criteria with host range to recognize and define species. In order to clarify species recognition within the Melampsora genus, we applied phylogenetic species recognition to Melampsora poplar rusts by conducting molecular phylogenetic analyses on 15 Melampsora taxa using six nuclear and mitochondrial loci. By assessing the genealogical concordance between phylogenies, we identified 12 lineages that evolved independently, corresponding to distinct phylogenetic species. All 12 lineages were concordant with host specialization, but only three belonged to strictly defined morphological species. The estimation of the species tree obtained with Bayesian concordance analysis highlighted a potential co-evolutionary history between Melampsora species and their reciprocal aecial host plants. Within the Melampsora speciation process, aecial host may have had a strong effect on ancestral evolution, whereas telial host specificity seems to have evolved more recently. The morphological characters initially used to define species boundaries in the Melampsora genus are not reflective of the evolutionary and genetic relationships among poplar rusts. In order to construct a more meaningful taxonomy, host specificity must be considered an important criterion for delineating and describing species within the genus Melampsora as previously suggested by ecological species recognition.     

Parahellenia,a new genus segregated from Hellenia (Costaceae) based on phylogenetic and morphological evidence

Previous studies recognized three major lineages of the family Costaceae: a South American clade, an Asian clade and a Costus clade. However, the genus Hellenia within the Asian clade has been shown to be non-monophyletic and its morphology has not been studied carefully. Therefore, the complete plastid genomes of Hellenia species were obtained and the monophyly of Hellenia was tested through four different datasets in this study. Plastid phylogenomic analyses of Costaceae revealed that Hellenia is strongly supported as paraphyletic. Two major clades are recovered, namely the Hellenia s.s. subclade and the Parahellenia subclade. Phylogenetic analyses based on an enlarged taxon sampling of the Asian clade using a two chloroplast markers dataset (trnK intron and trnL-F spacer) confirmed the paraphyly of Hellenia. Meanwhile, morphological analyses suggested that members of the Parahellenia subclade differ from the remaining Hellenia species in many characters including inflorescences, bracts, stigma, axillary buds, floral tubes and labellum. According to the present molecular and morphological evidence, the latter subclade is recognized as a new genus, Parahellenia. Two new species are described, four new combinations are made, and identification keys are also provided.     

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.     

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.     

Comparative phylogeography of capitulate <Emphasis Type="Italic">Campanula</Emphasis> species from the Balkans,with description of a new species, <Emphasis Type="Italic">C. daucoides</Emphasis>

Capitulate inflorescence is a specific, strongly adaptive and rare feature in the genus Campanula. We studied morphologically eight capitulate Campanula taxa from the Balkans (1537 individuals/52 populations) and one more species from Caucasus at the molecular level (using chloroplast markers trnG^UCC-trnS^GCU and psbA-trnH, 130 individuals/58 populations) to assess their relations and evolutionary histories. Although all studied taxa were well circumscribed at both the morphological and molecular levels (except morphologically distinct but genetically invariable C. moesiaca which acquired its single haplotype via past cytoplasmic introgression from C. cervicaria), their relations inferred from the two datasets were incongruent possibly due to the homoplasy of morphological characters frequently reported in Campanula. Interspecific hybridization and introgression affected majority of studied species and may be more common in Campanula than previously thought. These processes, along with incomplete lineage sorting and retention of ancestral polymorphisms, hampered our phylogenetic reconstructions and prevented us to fully resolve species relations, and to support monophyletic origin of capitulate Campanula species. Nonetheless, several cryptic taxa were delineated, and C. daucoides was described as a new capitulate Campanula species. Different evolutionary histories and multiple glacial refugia were inferred for all species represented by multiple samples (except C. moesiaca). According to our dating, their speciation was in most cases triggered by various geo-historic events such as the uplift of the Alpide belt, Messinian Salinity Crisis, or desiccation of the Pannonian Sea/Pliocene Lakes from the central Balkans, while their further diversification was mainly driven by the onset of the Quaternary and cycles of glacials/interglacials.     

Bertilia — A new monotypic genus in the Senecioneae (Asteraceae) from South Africa

The unusual disjunct distribution of the yellow-flowered, radiate, Northern Cape endemic Emilia hantamensis J.C.Manning & Goldblatt, relative to the other Emilia species in southern Africa, prompted investigation into its phylogenetic position and relationships. Phylogenies based on the ITS and trnL–trnF regions reveal that it is not a member of the genus Emilia but belongs in a well-supported clade with Bolandia, a genus comprising five species mainly from the Western and Northern Cape, and the monotypic genera Stilpnogyne and Mesogramma. It is placed sister to Bolandia and its cypselas match those of Bolandia in shape, colour and indumentum, except for a distinct ridge of hairs on the inner rib of the ray cypselas. It is further distinguished from Bolandia by its annual habit and conical disc floret style apices and obtuse to rounded ray floret style apices. It is recognised and described here as a new monotypic genus, Bertilia Cron with the single species B. hantamensis (J.C.Manning & Goldblatt) Cron.     

Gagea bohemica (Liliaceae), a highly variable monotypic species within Gagea sect. Didymobulbos

Abstract

Gagea bohemica s.l. (Liliaceae) consists of two morphologically extreme forms (saxatilis and bohemica) which depending on the varying opinions of the authors are considered as separate species, subspecies or as a single variable species. In this study, morphological (form and length of perianth segments, pilosity of pedicles and peduncles) and molecular data (cpDNA trnL–trnF IGS) of 55 samples corresponding to 43 populations from Western Europe to the Mediterranean area and eastern Caucasus were investigated. G. bohemica (Zauschn.) Schult. & Schult. f. represents a highly variable monotypic species from both morphological and karyological (2n = 24, 36, 48, 60, 72) aspects. Based on the molecular and morphological data presented here, a differentiation between specific and infraspecific taxa is not possible. We hypothesize that the morphological variability of this species is largely influenced by different site conditions and ploidy level.     

Bothriospermum longistylum sp. nov. (Boraginaceae) from north China

Based on field investigations as well as morphological and molecular systematic studies we found a new species of Bothriospermum which can be easily distinguished from all other known congeneric species by its unlobed faucal appendages and very long styles. We conducted molecular phylogenetic analyses based on a combined dataset from nrITS, plastid rps16, trnL‐trnF and trnG‐trnS regions. The results indicated that the new species indeed belongs in Bothriospermum and that it is well separated from other congeneric clusters, but that the genus Bothriospermum is paraphyletic with Nihon and Thyrocarpus clusetered within it. As a result, Bothriospermum longistylum sp. nov. is described and illustrated here. A revised key to species of Bothriospermum and a distributional map are also presented.     

Phylogenetic status of Vavilovia formosa (Fabaceae-Fabeae) based on nrDNA ITS and cpDNA sequences

The phylogenetic status of the monotypic genus Vavilovia was studied using nrDNA ITS and cpDNA trnL-F and trnS-G regions. The results from the analysis of each dataset and the combined dataset, revealed that Vavilovia is closely related to Pisum, forming a group that is sister to Lathyrus. The molecular data and some morphological and biological characteristics strongly indicate that Vavilovia should be subsumed under Pisum, as Pisum formosum.     

The phylogenetic placement of hypocrealean insect pathogens in the genus Polycephalomyces: An application of One Fungus One Name

Understanding the systematics and evolution of clavicipitoid fungi has been greatly aided by the application of molecular phylogenetics. They are now classified in three families, largely driven by reevaluation of the morphologically and ecologically diverse genus Cordyceps. Although reevaluation of morphological features of both sexual and asexual states were often found to reflect the structure of phylogenies based on molecular data, many species remain of uncertain placement due to a lack of reliable data or conflicting morphological characters. A rigid, darkly pigmented stipe and the production of a Hirsutella-like anamorph in culture were taken as evidence for the transfer of the species Cordyceps cuboidea, Cordyceps prolifica, and Cordyceps ryogamiensis to the genus Ophiocordyceps. Data from ribosomal DNA supported these species as a single group, but were unable to infer deeper relationships in Hypocreales. Here, molecular data for ribosomal and protein coding DNA from specimens of Ophiocordyceps cuboidea, Ophiocordyceps ryogamiensis, Ophiocordyceps paracuboidea, Ophiocordyceps prolifica, Cordyceps ramosopulvinata, Cordyceps nipponica, and isolates of Polycephalomyces were combined with a broadly sampled dataset of Hypocreales. Phylogenetic analyses of these data revealed that these species represent a clade distinct from the other clavicipitoid genera. Applying the recently adopted single system of nomenclature, new taxonomic combinations are proposed for these species in the genus Polycephalomyces, which has been historically reserved for asexual or anamorphic taxa.     

Phylogeny of Androcymbium (Colchicaceae) based on morphology and DNA sequences

The inclusion of species of Colchicum within Androcymbium in a previous cpDNA phylogeny of the Colchicaceae, questioned the monophyly of Androcymbium, and it was proposed to unite the two genera in Colchicum. Here we expand the previous phylogenetic analyses of Androcymbium by increasing the taxon sampling and adding more data. The analysis include 29 of the 57 species of Androcymbium, more cpDNA (trnL intron, trnL-trnF IGS, trnY-trnD IGS, and trnH-psbA IGS), and nDNA (RNApol2 intron 23) regions, and morphological and life-history traits data. Both parsimony and Bayesian inference were used. According to our data there is no reason to expand Colchicum to include Androcymbium, but we support the inclusion of Bulbocodium and Merendera within Colchicum. Morphology and life history traits are the main arguments in favor of recognizing Androcymbium as a well-circumscribed genus. In the phylogeny two Androcymbium groups are clearly differentiated: (1) one including species from Western and Eastern South Africa, Namibia, and North Africa, and (2) one including species from the north west of South Africa and south of Namibia.