Phylogeny of Crocus (Iridaceae) based on one chloroplast and two nuclear loci: Ancient hybridization and chromosome number evolution

Crocus consists of about 100 species distributed from western Europe and northern Africa to western China, with the center of diversity on the Balkan Peninsula and in Asia Minor. Our study focuses on clarifying phylogenetic relationships and chromosome number evolution within the genus using sequences of the chloroplast trnL-F region, the nuclear ribosomal DNA internal transcribed spacer (ITS) region, and a part of the nuclear single-copy gene pCOSAt103.In a combined dataset of ITS and trnL-F sequences, 115 individuals representing 110 taxa from both subgenera and all sections and series of Crocus were analyzed with Bayesian phylogenetic inference. For pCOSAt103 79 individuals representing 74 Crocus taxa were included, and for the majority of them PCR amplicons were cloned and up to eight clones per individual were sequenced to detect allopolyploidization events. Romulea species were included as outgroup in both analyses. Characteristics of seed surface structures were evaluated by scanning electron microscopy.Phylogenetic analysis of ITS/trnL-F data resulted in a monophyletic genus Crocus, probably monophyletic sections Crocus and Nudiscapus, and inferred monophyly for eight of the 15 series of the genus. The C. biflorus aggregate, thought to be consisting of closely related subspecies, was found to be polyphyletic, the taxa occurring within three major clades in the phylogenetic tree. Cloning of pCOSAt103 resulted in the detection of homoeologous copies in about one third of the taxa of section Nudiscapus, indicating an allotetraploid origin of this section. Reconstruction of chromosome number evolution along the phylogenetic tree using a probabilistic and a parsimony approach arrived at partly contradictory results. Both analyses agreed however on the occurrence of multiple polyploidization and dysploidy events. B chromosomes evolved at least five times independently within the genus, preferentially in clades characterized by karyotype changes.     

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.     

First molecular phylogeny of the pantropical genus Dalbergia: implications for infrageneric circumscription and biogeography

The genus Dalbergia with c. 250 species has a pantropical distribution. In spite of the high economic and ecological value of the genus, it has not yet been the focus of a species level phylogenetic study. We utilized ITS nuclear sequence data and included 64 Dalbergia species representative of its entire geographic range to provide a first phylogenetic framework of the genus to evaluate previous infrageneric classifications based on morphological data. The phylogenetic analyses performed suggest that Dalbergia is monophyletic and that it probably originated in the New World. Several clades corresponding to sections of these previous classifications are revealed. Taking into account that there is not a complete correlation between geography and phylogeny, and the estimation that the Dalbergia stem and crown clades are 40.4–43.3 mya and 3.8–12.7 mya, respectively, it is plausible that several long distance dispersal events underlie the pantropical distribution of the genus.     

Molecular phylogeny of Anaphalis (Asteraceae, Gnaphalieae) with biogeographic implications in the Northern Hemisphere

Anaphalis is the largest Asian genus in the tribe Gnaphalieae (Asteraceae) and has its greatest species diversity in the eastern Himalayas. The nuclear internal and external transcribed spacers were sequenced for Anaphalis species, with an emphasis on the eastern Himalayan taxa to examine the monophyly and construct the phylogenetic relationships of and within the genus. The results suggest that all species of Anaphalis are nested with Helichrysum, showing a close relationship with a Mediterranean–Asian group of Helichrysum. Although the monophyly of Anaphalis is only weakly supported, two clades within the genus are well recognized, each consisting of two subgroups. The inferred phylogenetic relationships within Anaphalis correspond to the shape of leaf base, rather than the morphology of the capitula and phyllaries that are usually used for species delimitation and classification in the genus. All four subgroups of Anaphalis are common and diversified in the eastern Himalayas with multiple dispersals out of this region. The sole North American species of Anaphalis is best hypothesized to be the result of long-distance dispersal or overland migration via Bering land bridge from Asia. Our analyses suggest that the extant distribution of Anaphalis has most likely resulted one radiation into the eastern Himalayas followed by repeated independent dispersals and/or radiations mostly into eastern Asia but also into the western Himalayas, North America, and southeast Asia.     

Phylogeny and biogeography of Alyssum (Brassicaceae) based on nuclear ribosomal ITS DNA sequences

The genus Alyssum consists of about 195 species native to Europe, Asia and northern Africa. All species were assigned to six sections. Previous molecular phylogeny studies indicate that Alyssum is polyphyletic. However, the divergence time and dispersal of the genus are not well studied. In this study, the phylogenetic relationships within the genus Alyssum were studied with nrDNA ITS sequences obtained from five sections. The divergence time was estimated by fossil calibration and the biogeography was examined by spread analysis. The phylogeny indicated two main lineages: lineage 1 includes the section of Alyssum, Gamosepalum and Psilonema; lineage 2 includes the section of Odontarrhena, Meniocus and Clypeola. The phylogenetic relationship was not congruent with the previous sectional classifications. The age of Alyssum was dated to the upper Miocene. Molecular data suggested the diversification of Alyssum in Mediterranean areas and wide-ranging distribution such as North Africa, eastward into Central Asia and immigration into North America. Climatic aridification and arid/semiarid areas established in the Pliocene/Pleistocene could have provided favourable conditions for the migration and diversification of Alyssum.     

Molecular phylogenetics,character evolution and systematics of the genus Micranthes (Saxifragaceae)

With c. 85 species, the genus Micranthes is among the larger genera of the Saxifragaceae. It is only distantly related to the morphologically similar genus Saxifraga, in which it has frequently been included as Saxifraga section Micranthes. To study the molecular evolution of Micranthes, we analysed nuclear ribosomal (internal transcribed spacer, ITS) and plastid (trnL–trnF) DNA sequences in a comprehensive set of taxa comprising c. 75% of the species. The molecular phylogenetic tree from the combined dataset revealed eight well‐supported clades of Micranthes. These clades agree in part with previously acknowledged subsections or series of Saxifraga section Micranthes. As these eight groups can also be delineated morphologically, we suggest that they should be recognized as sections of Micranthes. New relationships were also detected for some species and species groups, e.g. section Davuricae sister to sections Intermediae and Merkianae, and M. micranthidifolia as a member of section Micranthes. Species proposed to be excluded from the genus Micranthes for morphological reasons were resolved in the molecular tree in Saxifraga. Many morphological characters surveyed were homoplasious to varying extents. Micromorphological characters support comparatively well the clades in the phylogenetic tree. An updated nomenclature and a taxonomic conspectus of sections and species of Micranthes are provided. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 178 , 47–66.     

Biology of the ectomycorrhizal genus Rhizopogon. VI. Re-examination of infrageneric relationships inferred from phylogenetic analyses of ITS sequences

Rhizopogon (Basidiomycota, Boletales) is a genus of hypogeous fungi that form ectomycorrhizal associations mostly with members of the Pinaceae. This genus comprises an estimated 100(+) species, with the greatest diversity found in coniferous forests of the Pacific northwestern United States. Maximum parsimony analyses of 54 nuclear ribosomal DNA internal transcribed spacer (ITS) sequences including 27 Rhizopogon and 10 Suillus species were conducted to test sectional relationships in Rhizopogon and examine phylogenetic relationships with the closely related epigeous genus, Suillus. Sequences from 10 Rhizopogon type collections were included in these analyses. Rhizopogon and Suillus were both monophyletic. Rhizopogon section Rhizopogon is not monophyletic and comprised two clades, one of which consisted of two well supported lineages characterized by several long insertions. Rhizopogon sections Amylopogon and Villosuli formed well supported groups, but certain species concepts within these sections were unresolved. Four species from section Fulviglebae formed a strongly supported clade within section Villosuli. Subgeneric taxonomic revisions are presented.     

Evolutionary history of Gymnocarpos (Caryophyllaceae) in the arid regions from North Africa to Central Asia

Gymnocarpos has only about ten species distributed in the arid regions of Asia and Africa, but it exhibits a geographical disjunction between eastern Central Asia and western North Africa and Minor Asia. We sampled eight species of the genus and sequenced two chloroplast regions (rps16 and psbB–psbH), and the nuclear rDNA (ITS) to study the phylogeny and biogeography. The results of the phylogenetic analyses corroborated that Gymnocarpos is monophyletic, in the phylogenetic tree two well supported clades are recognized: clade 1 includes Gymnocarpos sclerocephalus and G. decandrus, mainly the North African group, whereas clade 2 comprises the remaining species, mainly in the Southern Arabian Peninsula. Molecular dating analysis revealed that the divergence age of Gymnocarpos was c. 31.33 Mya near the Eocene and Oligocene transition boundary, the initial diversification within Gymnocarpos dated to c. 6.69 Mya in the late Miocene, and the intraspecific diversification mostly occurred during the Quaternary climate oscillations. Ancestral area reconstruction suggested that the Southern Arabian Peninsula was the ancestral area for Gymnocarpos. Our conclusions revealed that the aridification since mid‐late Miocene significantly affected the diversification of the genus in these areas.     

Molecular phylogeny and taxonomic implications of Asarum (Aristolochiaceae) based on ITS and matK sequences

The genus Asarum (Aristolochiaceae) encompasses approximately 120 species from five sections. Taxonomic controversies concerning the genus Asarum and/or its intrageneric classification remain unresolved. In particular, sect. Heterotropa accounts for a large percentage of the genus (80 of 120 species) and is well diverged in the Sino–Japanese Forest subkingdom. Reconstruction of Heterotropa phylogeny and estimation of its divergence times would provide significant insight into the process of species diversity in the Sino–Japanese floristic region. This study encompassed 106 operational taxonomic units (OTUs), and phylogenetic analyses were conducted based on internal transcribed spacer (ITS) and matK sequences. Although the matK sequences provided informative results solely for section Geotaenium, phylogenetic trees based on ITS regions yielded a clear result for several sections. Three sections, Asarum, Geotaenium and Asiasarum, were supported as robust monophyletic groups, whereas Heterotropa had low support. Sect. Hexastylis was revealed to be polyphyletic, suggesting taxonomic reconstruction would be needed. Sect. Heterotropa comprises two clades, which correspond to species distribution ranges: mainland China and the island arc from Taiwan to mainland Japan via the Ryukyu Islands. It is notable that the common ancestry of the latter clade in the eastern Asian islands was highly supported, suggesting that the present species diversity of Heterotropa was initially caused by allopatric range fragmentation in East Asia.     

Phylogeny of Impatiens (Balsaminaceae): integrating molecular and morphological evidence into a new classification

Impatiens L. is one of the largest angiosperm genera, containing over 1000 species, and is notorious for its taxonomic difficulty. Here, we present, to our knowledge, the most comprehensive phylogenetic analysis of the genus to date based on a total evidence approach. Forty‐six morphological characters, mainly obtained from our own investigations, are combined with sequence data from three genetic regions, including nuclear ribosomal ITS and plastid atpB‐rbcL and trnL‐F. We include 150 Impatiens species representing all clades recovered by previous phylogenetic analyses as well as three outgroups. Maximum‐parsimony and Bayesian inference methods were used to infer phylogenetic relationships. Our analyses concur with previous studies, but in most cases provide stronger support. Impatiens splits into two major clades. For the first time, we report that species with three‐colpate pollen and four carpels form a monophyletic group (clade I). Within clade II, seven well‐supported subclades are recognized. Within this phylogenetic framework, character evolution is reconstructed, and diagnostic morphological characters for different clades and subclades are identified and discussed. Based on both morphological and molecular evidence, a new classification outline is presented, in which Impatiens is divided into two subgenera, subgen. Clavicarpa and subgen. Impatiens; the latter is further subdivided into seven sections.     

Molecular Phylogeny of Asian Meconopsis Based on Nuclear Ribosomal and Chloroplast DNA Sequence Data

The taxonomy and phylogeny of Asian Meconopsis (Himalayan blue poppy) remain largely unresolved. We used the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and the chloroplast DNA (cpDNA) trnL-F region for phylogenetic reconstruction of Meconopsis and its close relatives Papaver, Roemeria, and Stylomecon. We identified five main clades, which were well-supported in the gene trees reconstructed with the nrDNA ITS and cpDNA trnL-F sequences. We found that 41 species of Asian Meconopsis did not constitute a monophyletic clade, but formed two solid clades (I and V) separated in the phylogenetic tree by three clades (II, III and IV) of Papaver and its allies. Clade V includes only four Asian Meconopsis species, with the remaining 90 percent of Asian species included in clade I. In this core Asian Meconopsis clade, five subclades (Ia–Ie) were recognized in the nrDNA ITS tree. Three species (Meconopsis discigera, M. pinnatifolia, and M. torquata) of subgenus Discogyne were imbedded in subclade Ia, indicating that the present definition of subgenera in Meconopsis should be rejected. These subclades are inconsistent with any series or sections of the present classifications, suggesting that classifications of the genus should be completely revised. Finally, proposals for further revision of the genus Meconopsis were put forward based on molecular, morphological, and biogeographical evidences.     

An expanded phylogeny of Cuphea (Lythraceae) and a North American monophyly

A phylogenetic analysis of the New World genus Cuphea was conducted employing sequences from the nuclear rDNA internal transcribed spacer (ITS) and chloroplast trnL-trnF spacer and rpl16 intron. The analysis expands the number of Cuphea species from 53 in an earlier ITS study to 70 and adds two chloroplast data sets in order to generate a more complete and robust phylogeny and to test a previous result that suggested the presence of a large North American clade. Results reaffirm the monophyly of Cuphea with Pleurophora as the sister genus and recover a basal divergence event that mirrors the two subgenera of the current classification. Phylogenies of the two chloroplast regions are largely unresolved beyond the initial dichotomy and some resolution at the terminal and subterminal nodes. Based on the ITS phylogeny, five major clades are recognized. Subgenus Cuphea (Clade 1), defined morphologically by the synapomorphic loss of bracteoles, is sister to the much larger subg. Bracteolatae (Clades 2–5). Clades 2–4, comprising the South American and Caribbean species, grade successively to Clade 5, an exclusively North American lineage of 29 species. Among the 12 sections included in the study, only section Trispermum, a subclade of Clade 4, is monophyletic. Section Pseudocircaea is nested within Clade 3, which is largely equivalent to section Euandra. The North American endemic clade includes four sections, of which none are recovered as monophyletic in this study.     

Molecular phylogenetic analysis of the genus Abies (Pinaceae) based on the nucleotide sequence of chloroplast DNA

A phylogenetic study of firs (Abies Mill.) was conducted using nucleotide sequences of several chloroplast DNA regions with a total length of 5580 bp. The analysis included 37 taxa, which represented the main evolutionary lineages of the genus, and Keteleeria davidiana. According to phylogenetic reconstruction, the Abies species were subdivided into six main groups, generally corresponding to their geographic distribution. The phylogenetic tree had three basal clades. All of these clades contained American species, and only one of them contained Eurasian species. The divergence time calibrations, based on paleobotanical data and the chloroplast DNA mutation rate estimates in Pinaceae, produced similar results. The age of diversification among the basal clades of the present-day Abies was estimated as the end of the Oligocene-beginning of Miocene. The age of the separation of Mediterranean firs from the Asian-North American branch corresponds to the Miocene. The age of diversification within the young groups of Mediterranean, Asian, and “boreal” American firs (A. lasiocarpa, A. balsamea, A. fraseri) was estimated as the Pliocene-Pleistocene. Based on the phylogenetic reconstruction obtained, the most plausible biogeographic scenarios were suggested. It is noted that the existing systematic classification of the genus Abies strongly contradicts with phylogenetic reconstruction and requires revision.     

Historical biogeography of the genus Rhadinaea (Squamata: Dipsadinae)

Multiple geological and climatic events have created geographical or ecological barriers associated with speciation events, playing a role in biological diversification in North and Central America. Here, we evaluate the influence of the Neogene and Quaternary geological events, as well as the climatic changes in the diversification of the colubrid snake genus Rhadinaea using molecular dating and ancestral area reconstruction. A multilocus sequence dataset was generated for 37 individuals of Rhadinaea from most of the biogeographical provinces where the genus is distributed, representing 19 of the 21 currently recognized species, and two undescribed species. Our analyses show that the majority of the Rhadinaea species nest in two main clades, herein identified as “Eastern” and “Southern”. These clades probably diverged from each other in the early Miocene, and their divergence was followed by 11 divergences during the middle to late Miocene, three divergences during the Pliocene, and six divergences in the Pleistocene. The ancestral distribution of Rhadinaea was reconstructed across the Sierra Madre del Sur. Our phylogenetic analyses do not support the monophyly of Rhadinaea. The Miocene and Pliocene geomorphology, perhaps in conjunction with climate change, appears to have triggered the diversification of the genus, while the climatic changes during the Miocene probably induced the diversification of Rhadinaea in the Sierra Madre del Sur. Our analysis suggests that the uplifting of the Trans‐Mexican Volcanic Belt and Chiapan–Guatemalan highlands in this same period resulted in northward and southward colonization events. This was followed by more recent, independent colonization events in the Pliocene and Pleistocene involving the Balsas Basin, Chihuahuan Desert, Pacific Coast, Sierra Madre Occidental, Sierra Madre Oriental, Sierra Madre del Sur, Trans‐Mexican Volcanic Belt, and Veracruz provinces, probably driven by the climatic fluctuations of the time.     

Molecular phylogenetics and morphological evolution of St. John’s wort (Hypericum; Hypericaceae)

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John’s wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America.     

Phylogenetic relationships among New World Scrophularia L. (Scrophulariaceae): new insights inferred from DNA sequence data

The genus Scrophularia L. (Scrophulariaceae) comprises 200?C300 species, of which approximately 19 are distributed in North America and the Greater Antilles. To investigate phylogenetic and biogeographic relationships of the New World species, two intergenic spacers (trnQ-rps16 and psbA-trnH) of chloroplast DNA and nuclear ribosomal ITS were sequenced. Phylogenetic analyses revealed three distinct New World clades that correspond to their geographical distribution and are corroborated by morphological characters. Phylogenetic inference indicates the eastern American S. marilandica L. as sister to all Antillean species; for colonization of the Caribbean archipelago, a late Miocene dispersal event from the North American mainland is assumed. There is evidence for a hybrid origin of the most widespread North American species, S. lanceolata Pursh. The results further suggest that S. nodosa L. is sister to all New World and three Japanese species of Scrophularia. The latter form an Eastern Asian?CEastern North American (EA-ENA) disjunction with six New World species. We propose an eastern Asian origin for the New World taxa of Scrophularia. Divergence times estimated using a relaxed molecular clock model suggest one or more Miocene migration events from eastern Asia to the New World via the Bering Land Bridge followed by diversification in North America.     

Molecular kinship analyses of the agaricoid Russulaceae: Correspondence with mycorrhizal anatomy and sporocarp features in the genus Russula

A data set of LSU DNA sequences of mainly European Russula and Lactarius species was subjected to molecular phylogenetic analysis. Species could be allocated to six clades, with an unresolved phylogeny. One of these clades represents the genus Lactarius. The only analysed species of the section Archaeinae (Russula) was placed basal to both genera. Thus Lactarius appears to be derived from Russula. Russula was divided into four clusters, corresponding to the sections Plorantinae and Nigricantinae, subgenus Heterophyllidia including the section Foetentinae, and a cluster representing the remaining subgenera of the “Genuinae”. Even though the resulting groups can be considered as valid classificatory groups, species associations resulting from molecular analyses neither support the division of Russula into the subgenus Compacta (including the sections Nigricantinae, Plorantinae, and Archaeinae) and the “Genuinae” (including all remaining taxa), nor do they support previously proposed evolutionary lineages within the “Genuinae”. Ribosomal ITS DNA sequences of Russula species were analysed to achieve better infrageneric resolution. The results are discussed in relation to current classification systems and to what is known about the mycorrhizae formed by Russula species. While the systematic value attached to many macroscopic and microscopic sporocarp features was not supported by sequence data, mycorrhizal anatomy is in good correspondence with many of the results from the phylogenetic analysis.     

Phylogenetic relationships within Chamaecrista sect. Xerocalyx (Leguminosae, Caesalpinioideae) inferred from the cpDNA trnE-trnT intergenic spacer and nrDNA ITS sequences

Chamaecrista belongs to subtribe Cassiinae (Caesalpinioideae), and it comprises over 330 species, divided into six sections. The section Xerocalyx has been subjected to a profound taxonomic shuffling over the years. Therefore, we conducted a phylogenetic analysis using a cpDNA trnE-trnT intergenic spacer and nrDNA ITS/5.8S sequences from Cassiinae taxa, in an attempt to elucidate the relationships within this section from Chamaecrista. The tree topology was congruent between the two data sets studied in which the monophyly of the genus Chamaecrista was strongly supported. Our analyses reinforce that new sectional boundaries must be defined in the Chamaecrista genus, especially the inclusion of sections Caliciopsis and Xerocalyx in sect. Chamaecrista, considered here paraphyletic. The section Xerocalyx was strongly supported as monophyletic; however, the current data did not show C. ramosa (microphyllous) and C. desvauxii (macrophyllous) and their respective varieties in distinct clades, suggesting that speciation events are still ongoing in these specimens.