Phylogenetic relationships within the moss family Bryaceae based on chloroplast DNA evidence

The Bryaceae are a large cosmopolitan family of mosses containing genera of considerable taxonomic difficulty. Phylogenetic relationships within the family were inferred using data from chloroplast DNA sequences (rps4 and trnL-trnF region). Parsimony and maximum likelihood optimality criteria, and Bayesian phylogenetic inference procedures were employed to reconstruct relationships. The genera Bryum and Brachymenium are not monophyletic groups. A clade comprising Plagiobryum, Acidodontium, Mielichhoferia macrocarpa, Bryum sects. Bryum, Apalodictyon, Limbata, Leucodontium, Caespiticia, Capillaria (in part: sect. Capillaria), and Brachymenium sect. Dicranobryum, is well supported in all analyses and represents a major lineage within the family. Section Dicranobryum of Brachymenium is more closely related to section Bryum than to the other sections of Brachymenium, as are Mielichhoferia macrocarpa and M. himalayana. Species of Acidodontium form a clade with Anomobryum julaceum. The grouping of species with a rosulate gametophytic growth form suggests the presence of a 'rosulate' clade similar in circumscription to the genus Rosulabryum. Mielichhoferia macrocarpa and M. himalayana are transferred to Bryum as B. porsildii and B. caucasicum, respectively.

Patterns of nucleotide substitution in angiosperm cpDNA trnL (UAA)-trnF (GAA) regions

Patterns of substitution in chloroplast encoded trnL_F regions were compared between species of Actaea (Ranunculales), Digitalis (Scrophulariales), Drosera (Caryophyllales), Panicoideae (Poales), the small chromosome species clade of Pelargonium (Geraniales), each representing a different order of flowering plants, and Huperzia (Lycopodiales). In total, the study included 265 taxa, each with > 900-bp sequences, totaling 0.24 Mb. Both pairwise and phylogeny-based comparisons were used to assess nucleotide substitution patterns. In all six groups, we found that transition/transversion ratios, as estimated by maximum likelihood on most-parsimonious trees, ranged between 0.8 and 1.0 for ingroups. These values occurred both at low sequence divergences, where substitutional saturation, i.e., multiple substitutions having occurred at the same (homologous) nucleotide position, was not expected, and at higher levels of divergence. This suggests that the angiosperm trnL-F regions evolve in a pattern different from that generally observed for nuclear and animal mtDNA (transitional/transversion ratio > or = 2). Transition/transversion ratios in the intron and the spacer region differed in all alignments compared, yet base compositions between the regions were highly similar in all six groups. A>-C transversions were significantly less frequent than the other four substitution types. This correlates with results from studies on fidelity mechanisms in DNA replication that predict A<->T and G<->C transversions to be least likely to occur. It therefore strengthens confidence in the link between mutation bias at the polymerase level and the actual fixation of substitutions as recorded on evolutionary trees, and concomitantly, in the neutrality of nucleotide substitutions as phylogenetic markers.

A phylogenetic reappraisal of the Peltophorum group (Caesalpinieae: Leguminosae) based on the chloroplast trnL-F, rbcL and rps16 sequence data

The monophyly of the Peltophorum group, one of nine informal groups recognized by Polhill in the Caesalpinieae, was tested using sequence data from the trnL-F, rbcL, and rps16 regions of the chloroplast genome. Exemplars were included from all 16 genera of the Peltophorum group, and from 15 genera representing seven of the other eight informal groups in the tribe. The data were analyzed separately and in combined analyses using parsimony and Bayesian methods. The analysis method had little effect on the topology of well-supported relationships. The molecular data recovered a generally well-supported phylogeny with many intergeneric relationships resolved. Results show that the Peltophorum group as currently delimited is polyphyletic, but that eight genera plus one undescribed genus form a core Peltophorum group, which is referred to here as the Peltophorum group sensu stricto. These genera are Bussea, Conzattia, Colvillea, Delonix, Heteroflorum (inedit.), Lemuropisum, Parkinsonia, Peltophorum, and Schizolobium. The remaining eight genera of the Peltophorum group s.l. are distributed across the Caesalpinieae. Morphological support for the redelimited Peltophorum group and the other recovered clades was assessed, and no unique synapomorphy was found for the Peltophorum group s.s. A proposal for the reclassification of the Peltophorum group s.l. is presented.

Mitochondrial and chloroplast DNA-based phylogeny of Pelargonium (Geraniaceae)

Overall phylogenetic relationships within the genus Pelargonium (Geraniaceae) were inferred based on DNA sequences from mitochondrial(mt)-encoded nad1 b/c exons and from chloroplast(cp)-encoded trnL (UAA) 5' exon-trnF (GAA) exon regions using two species of Geranium and Sarcocaulon vanderetiae as outgroups. The group II intron between nad1 exons b and c was found to be absent from the Pelargonium, Geranium, and Sarcocaulon sequences presented here as well as from Erodium, which is the first recorded loss of this intron in angiosperms. Separate phylogenetic analyses of the mtDNA and cpDNA data sets produced largely congruent topologies, indicating linkage between mitochondrial and chloroplast genome inheritance. Simultaneous analysis of the combined data sets yielded a well-resolved topology with high clade support exhibiting a basic split into small and large chromosome species, the first group containing two lineages and the latter three. One large chromosome lineage (x = 11) comprises species from sections Myrrhidium and Chorisma and is sister to a lineage comprising P. mutans (x = 11) and species from section Jenkinsonia (x = 9). Sister to these two lineages is a lineage comprising species from sections Ciconium (x = 9) and Subsucculentia (x = 10). Cladistic evaluation of this pattern suggests that x = 11 is the ancestral basic chromosome number for the genus.

Phylogenetic relationships withinPelargonium sect. Peristera (Geraniaceae) inferred from nrDNA and cpDNA sequence comparisons

Phylogenetic analysis of nrDNA ITS and trnL (UAA) 5′ exon-trnF (GAA) chloroplast DNA sequences from 17 species ofPelargonium sect.Peristera, together with nine putative outgroups, suggests paraphyly for the section and a close relationship between the highly disjunct South African and Australian species of sect.Peristera. Representatives fromPelargonium sectt.Reniformia, Ligularia s. l. andIsopetalum (the St. Helena endemicP. cotyledonis) appear to be nested within thePeristera clade. The close relationship between the South African and AustralianPeristera is interpreted as being caused by long-range dispersal to Australia, probably as recent as the late Pliocene.

Land plants and DNA barcodes: short-term and long-term goals

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.

Cyclamen: time, sea and speciation biogeography using a temporally calibrated phylogeny

Aim The Mediterranean region is a species-rich area with a complex geographical history. Geographical barriers have been removed and restored due to sea level changes and local climatic change. Such barriers have been proposed as a plausible mechanism driving the high levels of speciation and endemism in the Mediterranean basin. This raises the fundamental question: is allopatric isolation the mechanism by which speciation occurs? This study explores the potential driving influence of palaeo-geographical events on the speciation of Cyclamen (Myrsinaceae), a group with most species endemic to the Mediterranean region. Cyclamen species have been shown experimentally to have few genetic barriers to hybridization. Location The Mediterranean region, including northern Africa, extending eastwards to the Black Sea coast. Methods A generic level molecular phylogeny of Myrsinaceae and Primulaceae is constructed, using Bayesian approximation, to produce a secondary age estimate for the stem lineage of Cyclamen. This estimate is used to calibrate temporally an infrageneric phylogeny of Cyclamen, built with nrDNA ITS, cpDNA trnL-F and cpDNA rps16 sequences. A biogeographical analysis of Cyclamen is performed using dispersal-vicariance analysis. Results The emergence of the Cyclamen stem lineage is estimated at 30.1-29.2 Ma, and the crown divergence at 12.9-12.2 Ma. The average age of Cyclamen species is 3.7 Myr. Every pair of sister species have mutually exclusive, allopatric distributions relative to each other. This pattern appears typical of divergence events throughout the evolutionary history of the genus. Main conclusions Geographical barriers, such as the varying levels of the Mediterranean Sea, are the most plausible explanation for speciation events throughout the phylogenetic history of Cyclamen. The genus demonstrates distributional patterns congruent with the temporally reticulate palaeogeography of the Mediterranean region.

DNA barcoding of a large genus, Aspalathus L. (Fabaceae)

The DNA barcode potential of three regions (the nuclear ribosomal ITS and the plastid psbA-trnH and trnT-trnL intergenic spacers) was investigated for the plant genus Aspalathus L. (Fabaceac: Crotalarieae). Aspalathus is a large genus (278 species) that revealed low levels of DNA variation in phylogenetic studies. In a 51-species dataset for the psbA-trnH and ITS regions, 45%, and 16% of sequences respectively were identical to the sequence of at least one other species, with two species undiscriminated even when the two regions were combined. In contrast, trnT-trnL, discriminated between all species in this dataset. In a larger ITS and trnT-trnL dataset. including a further 82 species. 7 species in five pairwise comparisons remained Undiscriminated when the two regions were combined. Four of the five pairs of species not discriminated by sequence data were readily distinguished using a combination of qualitative and quantitative morphological data. The difficulty of barcoding in this group is increased by the presence of intraspecific variation in all three regions studied. In the case of psbA-trnH, three intraspecific samples had a sequence identical to at least one other species. Overall, psbA-trnH. currently a candidate for plant barcoding, was the least discriminatory region in our study.

Phylogenetic utility of more axillary growth (MAX4 ) - like genes: a case study in Digitalis / Isoplexis (Plantaginaceae)

We present the first assessment of phylogenetic utility of a potential novel low-copy nuclear gene region in flowering plants. A fragment of the MORE AXILLARY GROWTH 4 gene (MAX4, also known as RAMOSUS1 and DECREASED APICAL DOMINANCE1), predicted to span two introns, was isolated from members of Digitalis/Isoplexis. Phylogenetic analyses, under both maximum parsimony and Bayesian inference, were performed and revealed evidence of putative MAX4-like paralogues. The MAX4-like trees were compared with those obtained for Digitalis/Isoplexis using ITS and trnL-F, revealing a high degree of incongruence between these different DNA regions. Network analyses indicate complex patterns of evolution between the MAX4 sequences, which cannot be adequately represented on bifurcating trees. The incidence of paralogy restricts the use of MAX4 in phylogenetic inference within the study group, although MAX4 could potentially be used in combination with other DNA regions for resolving species relationships in cases where paralogues can be clearly identified.

Phylogenetic utility of MORE AXILLARY GROWTH4 (MAX4)-like genes: a case study in Digitalis/Isoplexis (Plantaginaceae)

We present the first assessment of phylogenetic utility of a potential novel low-copy nuclear gene region in flowering plants. A fragment of the MORE AXILLARY GROWTH 4 gene (MAX4, also known as RAMOSUS1 and DECREASED APICAL DOMINANCE1), predicted to span two introns, was isolated from members of Digitalis/Isoplexis. Phylogenetic analyses, under both maximum parsimony and Bayesian inference, were performed and revealed evidence of putative MAX4-like paralogues. The MAX4-like trees were compared with those obtained for Digitalis/Isoplexis using ITS and trnL-F, revealing a high degree of incongruence between these different DNA regions. Network analyses indicate complex patterns of evolution between the MAX4 sequences, which cannot be adequately represented on bifurcating trees. The incidence of paralogy restricts the use of MAX4 in phylogenetic inference within the study group, although MAX4 could potentially be used in combination with other DNA regions for resolving species relationships in cases where paralogues can be clearly identified.

Diversification of a large genus in a continental biodiversity hotspot: temporal and spatial origin of Muraltia (Polygalaceae) in the Cape of South Africa

Phylogenetic relationships in the largely South African genus Muraltia (Polygalaceae) are assessed based on DNA sequence data (nuclear ribosomal ITS, plastid atpB-rbcL spacer, trnL intron, and trnL-F spacer) for 73 of the 117 currently recognized species in the genus. The previously recognised subgenus Muraltia is monophyletic, but the South African endemic genus Nylandtia is embedded in Muraltia subgenus Psiloclada. Subgenus Muraltia is found to be sister to subgenus Psiloclada. Estimates show the beginning of diversification of the two subgenera in the early Miocene (Psiloclada, 19.3+/-3.4 Ma; Muraltia, 21.0+/-3.5 Ma) pre-dating the establishment of the Benguela current (intermittent in the middle to late Oligocene and markedly intensifying in the late Miocene), and summer-dry climate in the Cape region. However, the later increase in species numbers is contemporaneous with these climatic phenomena. Results of dispersal-vicariance analyses indicate that major clades in Muraltia diversified from the southwestern and northwestern Cape, where most of the species are found today.

Phylogeny of Pelargonium (Geraniaceae) based on DNA sequences from three genomes

Phylogenetic hypotheses for the largely South African genus Pelargonium L'Hér. (Geraniaceae) were derived based on DNA sequence data from nuclear, chloroplast and mitochondrial encoded regions. The datasets were unequally represented and comprised cpDNA trnL-F sequences for 152 taxa, nrDNA ITS sequences for 55 taxa, and mtDNA nad1 b/c exons for 51 taxa. Phylogenetic hypotheses derived from the separate three datasets were overall congruent. A single hypothesis synthesising the information in the three datasets was constructed following a total evidence approach and implementing dataset specific stepmatrices in order to correct for substitution biases. Pelargonium was found to consist of five main clades, some with contrasting evolutionary patterns with respect to biogeographic distributions, dispersal capacity, pollination biology and karyological diversification. The five main clades are structured in two (subgeneric) clades that correlate with chromosome size. One of these clades includes a "winter rainfall clade" containing more than 70% of all currently described Pelargonium species, and all restricted to the South African Cape winter rainfall region. Apart from (woody) shrubs and small herbaceous rosette subshrubs, this clade comprises a large "xerophytic" clade including geophytes, stem and leaf succulents, harbouring in total almost half of the genus. This clade is considered to be the result of in situ proliferation, possibly in response to late-Miocene and Pliocene aridification events. Nested within it is a radiation comprising c. 80 species from the geophytic Pelargonium section Hoarea, all characterised by the possession of (a series of) tunicate tubers.

Nomenclature in the dock. Overclassification leads to instability: a case study in the horticulturally important genus Cyclamen (Myrsinaceae)

Nine different classifications have been produced in the last 70 years for the horticulturally valuable genus Cyclamen, a small genus with fewer than 30 species. These classifications, generated by intuitive methods and cladistic analyses, incorporated a total of four infrageneric ranks above that of species and were based on data from morphology, cytology and DNA sequencing. Our results, based on cladistic analyses of three independent data sources − nrDNA ITS, cpDNA trnL intron and morphological data − reveal good resolution only in nrDNA sequence data. However, when these three data sources are combined they provide stronger resolution and support for three major clades, only one of which, subgenus Psilanthum, has been consistently supported in previous classifications. The differing infrageneric classifications produced in Cyclamen result from varying taxon sampling, differing interpretation of morphological data, changes in the sources and analysis of data, and inconsistent application of names. Extensive subdivision of small genera in the absence of adequate data that could provide evidence for consistent patterns of relationship is premature and leads to a proliferation of names.© 2004 The Linnean Society of London, Botanical Journal of the Linnean Society, 2004, 146, 339-349.

Is there such a thing as Kalmia x Rhododendron?

Two putative hybrids between Kalmia and Rhododendron, their suspected progenitor species and related taxa were submitted to DNA sequencing of cpDNA trnL-F and nrDNA ITS regions in order to test whether there was DNA sequence evidence both for hybridization per se and for the direction of the cross should one be evident. Comparison of eight DNA sequences from these putative hybrids with Rhododendron and Kalmia species showed clear evidence of origin within Rhododendron. No evidence of Kalmia DNA was detected. These putative intergeneric hybrids appear to be mutants of Rhododendron and not of hybrid origin.

Mapania multiflora, a distinctive new species of Cyperaceae (Mapanioideae) from Borneo

Mapania multiflora is described and illustrated. It is vegetatively similar to taxa with broad leaves and pseudopetioles, such as M. cuspidata. However, it is reproductively similar to sect. Thoractostachyum with a paniculate inflorescence and furrowed fruit. The DNA is similar to M. bancana in sect. Thoractostachyum, in the three sampled cpDNA regions: atpH-F, trnL-F and psbA-trnH. However, it is identical to none of these due to its unique combination of vegetative, reproductive and molecular characteristics.