Phylogenetic relationships of Cranichidinae and Prescottiinae (Orchidaceae, Cranichideae) inferred from plastid and nuclear DNA sequences

Background and Aims

Phylogenetic relationships of subtribes Cranichidinae and Prescottiinae, two diverse groups of neotropical terrestrial orchids, are not satisfactorily understood. A previous molecular phylogenetic study supported monophyly for Cranichidinae, but Prescottiinae consisted of two clades not sister to one another. However, that analysis included only 11 species and eight genera of these subtribes. Here, plastid and nuclear DNA sequences are analysed for an enlarged sample of genera and species of Cranichidinae and Prescottiinae with the aim of clarifying their relationships, evaluating the phylogenetic position of the monospecific genera Exalaria, Ocampoa and Pseudocranichis and examining the value of various structural traits as taxonomic markers.

Methods

Approx. 6000 bp of nucleotide sequences from nuclear ribosomal (ITS) and plastid DNA (rbcL, matK-trnK and trnL-trnF) were analysed with cladistic parsimony and Bayesian inference for 45 species/14 genera of Cranichidinae and Prescottiinae (plus suitable outgroups). The utility of flower orientation, thickenings of velamen cell walls, hamular viscidium and pseudolabellum to mark clades recovered by the molecular analysis was assessed by tracing these characters on the molecular trees.

Key Results

Spiranthinae, Cranichidinae, paraphyletic Prescottia (with Pseudocranichis embedded), and a group of mainly Andean ‘prescottioid’ genera (the ‘Stenoptera clade’) were strongly supported. Relationships among these clades were unresolved by parsimony but the Bayesian tree provided moderately strong support for the resolution (Spiranthinae–(Stenoptera clade-(Prescottia/Pseudocranichis–Cranichidinae))). Three of the four structural characters mark clades on the molecular trees, but the possession of a pseudolabellum is variable in the polyphyletic Ponthieva.

Conclusions

No evidence was found for monophyly of Prescottiinae and the reinstatement of Cranichidinae s.l. (including the genera of ‘Prescottiinae’) is favoured. Cranichidinae s.l. are diagnosed by non-resupinate flowers. Lack of support from parsimony for relationships among the major clades of core spiranthids is suggestive of a rapid morphological radiation or a slow rate of molecular evolution.Key words: Cranichideae, Cranichidinae, matK-trnK, molecular phylogenetics, nrITS, Orchidaceae, Prescottiinae, resupination, trnL-trnF     

Molecular phylogenetics of subtribe Aeridinae (Orchidaceae): insights from plastid matK and nuclear ribosomal ITS sequences

We conducted phylogenetic analyses using two DNA sequence data sets derived from matK, the maturase-coding gene located in an intron of the plastid gene trnK, and the internal transcribed spacer region of 18S–26S nuclear ribosomal DNA to examine relationships in subtribe Aeridinae (Orchidaceae). Specifically, we investigated (1) phylogenetic relationships among genera in the subtribe, (2) the congruence between previous classifications of the subtribe and the phylogenetic relationships inferred from the molecular data, and (3) evolutionary trends of taxonomically important characters of the subtribe, such as pollinia, a spurred lip, and a column foot. In all, 75 species representing 62 genera in subtribe Aeridinae were examined. Our analyses provided the following insights: (1) monophyly of subtribe Aeridinae was tentatively supported in which 14 subclades reflecting phylogenetic relationships can be recognized, (2) results are inconsistent with previous classifications of the subtribe, and (3) repeated evolution of previously emphasized characters such as pollinia number and apertures, length of spur, and column foot was confirmed. It was found that the inconsistencies are mainly caused by homoplasy of these characters. At the genus level, Phalaenopsis, Cleisostoma, and Sarcochilus are shown to be non-monophyletic.     

Molecular phylogenetics of Paphiopedilum (Cypripedioideae; Orchidaceae) based on nuclear ribosomal ITS and plastid sequences

Phylogenetic relationships in the genus Paphiopedilum were studied using nuclear ribosomal internal transcribed spacer (ITS) and plastid sequence data. The results confirm that the genus Paphiopedilum is monophyletic, and the division of the genus into three subgenera Parvisepalum, Brachypetalum and Paphiopedilum is well supported. Four sections of subgenus Paphiopedilum (Pardalopetalum, Cochlopetalum, Paphiopedilum and Barbata) are recovered as in a recent infrageneric treatment, with strong support. Section Coryopedilum is also recovered, with low bootstrap but high posterior probability values for support of monophyly. Relationships in section Barbata remain unresolved, and short branch lengths and the narrow geographical distribution of many species in the section suggest that it possibly underwent rapid radiation. Mapping chromosome and genome size data (including some new genome size measurements) onto the phylogenetic framework shows that there is no clear trend in increase in chromosome number in the genus. However, the diploid chromosome number of 2n = 26 in subgenera Parvisepalum and Brachypetalum suggests that this is the ancestral condition, and higher chromosome numbers in sections Cochlopetalum and Barbata suggest that centric fission has possibly occurred in parallel in these sections. The trend for genome size evolution is also unclear, although species in section Barbata have larger genome sizes than those in other sections. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 176–196.     

Phylogenetics of Cranichideae with emphasis on Spiranthinae (Orchidaceae, Orchidoideae): evidence from plastid and nuclear DNA sequences

DNA sequences from plastid rbcL and matK genes and the trnL-F region, as well as the nuclear ribosomal ITS region, were used to evaluate monophyly and subtribal delimitation of Cranichideae and generic relationships in Spiranthinae. Cranichideae are moderately supported as monophyletic, with Chloraeinae and Pterostylis-Megastylis indicated as their collective sisters. Within Cranichideae, Pachyplectroninae and Goodyerinae form a well-supported monophyletic group sister to a "core spiranthid" clade that includes, according to their branching order, Galeottiellinae, Manniellinae, and a Prescottiinae-Cranichidinae-Spiranthinae subclade. Inclusion of Galeottiella in Spiranthinae, as in previous classifications, renders the latter paraphyletic to all other spiranthid subtribes. Cranichidinae and Spiranthinae (minus Galeottiella) are monophyletic and strongly supported, but Prescottiinae form a grade that includes a strongly supported prescottioid Andean clade and a weakly supported Prescottia-Cranichidinae clade sister to Spiranthinae. Well-supported major clades in Spiranthinae identified in this study do not correspond to previous alliances or the narrowly defined subtribes in which they have been divided recently. Morphological characters, especially those that have been used for taxonomic delimitation in Cranichideae, are discussed against the framework of the molecular trees, emphasizing putative synapomorphies and problems derived from lack of information or inadequate interpretation of the characters.     

Molecular phylogenetics of Diseae (Orchidaceae): a contribution from nuclear ribosomal ITS sequences

We present here the first molecular phylogeny of tribe Diseae (Orchidoideae: Orchidaceae). Nuclear ribosomal ITS1, 5.8S rDNA, and ITS2 sequences were compared for 30 Diseae, 20 Orchideae, and four Cranichideae and Diurideae outgroups. ITS - rDNA sequences exhibited a transition:transversion ratio of 1.3 and extensive ITS length polymorphism. Phylogenetic analyses using maximum parsimony identified seven major core orchidoid groups. The branching order of the five Diseae and two Orchideae clades was weakly supported but indicated paraphyly of Diseae, with Disperis sister to the rest, followed by successive divergence of Brownleea, Disinae, Coryciinae sensu stricto (s.s.), Satyriinae, and terminated by Orchidinae plus Habenariinae. Within the monophyletic Disinae, Herschelia and Monadenia were nested within a paraphyletic Disa and clustered with D. sect. Micranthae. Within monophyletic Satyriinae, Satyridium rostratum plus Satyrium bicallosum was sister to the rest of Satyrium, and then Satyrium nepalense plus S. odorum was distinct from a cluster of six species. Coryciinae are paraphyletic because Disperis is sister to all other core orchidoids. Coryciinae s.s. are sister to Satyriinae plus Orchideae, with Pterygodium nested within Corycium. Maximum likelihood analysis supported possible affinities among Disinae, Brownleeinae, and Coryciinae but did not support monophyly of Diseae or an affinity between Disinae and Satyriinae. Morphological characters are fully congruent with the well-supported groups identified in the ITS phylogeny.     

Molecular phylogenetics of Meliaceae (Sapindales) based on nuclear and plastid DNA sequences

Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32 genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial), and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Furthermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.     

Molecular phylogenetics of the subtribeGentianinae (Gentianaceae) inferred from the sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA

The internal transcribed spacer (ITS) regions of 18S–25S nuclear ribosomal DNA from representatives of 23 species of the subtribeGentianinae and one outgroup species (Centaurium capitatum) were analyzed by polymerase chain reaction amplification and direct DNA sequencing. Within the taxa analyzed, the length of the ITS1 region varied from 221 to 233 bp, ITS2 from 226 to 234 bp. Of the aligned sequences of 497 positions, 151 sites involved gaps or nucleotide ambiguity, 133 were invariable and 213 showed divergence. In pairwise comparisons among the taxa of the subtribeGentianinae and the outgroup, sequence divergence ranged from 1.3% to 34.1% in ITS1, from 0 to 28.1% in ITS2 and from 0.6% to 27.5% in combined ITS1 and ITS2. Phylogenetic trees generated from ITS sequences were highly resolutive and principally concordant with morphological classifications for the major phylogenetic divisions in the subtribe. An ancient divergence leading to two evolutionary lines was suggested in the subtribe by both DNA sequence and morphological data. One line encompasses the generaGentiana, Crawfurdia andTripterospermum, morphologically characterized by their glands on the base of ovary and their plicate corolla, while the other line involves all other members of the subcribe surveyed, characterized by their epipetalous glands and simple corolla without plicae.Megacodon, with glands on the base of ovary but without plicae on its corolla, was revealed to be more related to the latter group than to the former.Comastoma, Gentianella andGentianopsis were shown to be well-defined monophyletic genera.Pterygocalyx showed much closer affinity toGentianopsis than to any other genus. Some conflictions were detected in the genusSwertia.     

Molecular phylogeny of Chinese Rubia (Rubiaceae: Rubieae) inferred from nuclear and plastid DNA sequences

Rubia L. is the type genus of the coffee family Rubiaceae and the third largest genus in the tribe Rubieae, comprising ca. 80 species restricted to the Old World. China is an important diversity center for Rubia, where approximately half of its species occur. However, its internal phylogenetic relationships are still poorly understood. The objective of the present study is to contribute to the phylogenetic relationships within Rubia, using the nuclear internal transcribed spacer and six plastid markers and focusing on species from China. Twenty-seven species of Rubia were sampled to infer their phylogeny using Maximum parsimony, Maximum likelihood, and Bayesian analyses. The monophyly of Rubia is supported, provided that R. rezniczenkoana Litv. is excluded from Rubia and transferred to Galium as a new combination: G. rezniczenkoanum (Litv.) L. E Yang & Z. L. Nie. Within Rubia, two clades are clearly supported. They correspond to the traditional sect. Rubias.l. (A) and sect. Oligoneura Pojark. (B), and are morphologically mainly separable by their pinnate (A) versus palmate (B) leaf venation. Plesiomorphic features are the pinnate leaf veining in sect. Rubia s.l. and the occurrence of some species with opposite leaves and true stipules in sect. Oligoneura. In sect. Oligoneura one can assume an evolution from species with opposite leaves and true stipules (as in the R. siamensis Craib group) to those with whorls of two leaves and two leaf-like stipules (as in ser. Chinenses and the R. mandersii Collett & Hemsl. group) and finally with whorls of 6 or even more elements (as in ser. Cordifoliae). The correlation between clades recognized by DNA analyses and available differential morphological features is partly only loose, particularly in the group of R. cordifolia with 2×, 4×, and 6× cytotypes. This may be due to rapid evolutionary divergence and/or hybridization and allopolyploidy.     

Root character evolution and systematics in Cranichidinae, Prescottiinae and Spiranthinae (Orchidaceae, Cranichideae)

Background and Aims: Previous studies have suggested that velamen characteristicsare useful as taxonomic markers in Orchidaceae. Members of tribeCranichideae have been assigned to two velamen types constructedbased on combinations of characters such as the presence ofsecondary cell-wall thickenings and pores. However, such charactershave not been analysed on an individual basis in explicit cladisticanalyses. Methods: The micromorphology of roots of 26 species of Cranichideae wasexamined through scanning electron microscopy and light microscopy,scoring the variation and distribution of four characters: numberof velamen cell layers, velamen cell-wall thickenings, presenceand type of tilosomes, and supraendodermal spaces. The lastthree characters were analysed cladistically in combinationwith DNA sequence data of plastid trnK/matK and nuclear ribosomalinternal transcribed spacer (ITS) regions and optimized on theresulting phylogenetic tree. Key Results: Thickenings of velamen cell walls group Prescottiinae with Spiranthinae,whereas tilosomes, documented here for the first time in Cranichideae,provide an unambiguous synapomorphy for subtribe Spiranthinae.Supraendodermal spaces occur mostly in species dwelling in seasonallydry habitats and appear to have evolved three times. Conclusions: Three of the four structural characters assessed are phylogeneticallyinformative, marking monophyletic groups recovered in the combinedmolecular–morphological analysis. This study highlightsthe need for conducting character-based structural studies toovercome analytical shortcomings of the typological approach.     

Molecular phylogeny of Alnus (Betulaceae), inferred from nuclear ribosomal DNA ITS sequences

The nuclear ITS region of 19 species of Alnus was amplified and sequenced. The inferred molecular phylogeny shows that all species of the genus Alnus form a monophyletic group close to Betula and that the fundamental dichotomy within the genus lies between the subgenera Alnaster and Gymnothyrsus, sensu Murai (1964). The subgenus Alnaster appears to be basal in the genus, based on archaism of morphological features, and branching close to the root of the trees due to low ITS divergence from genus Betula. The monophyly of the section Clethropsis is not supported by the present data: Alnus nepalensis is positioned in the subgenus Gymnothyrsus, away from A. nitida and A. maritima. Surprisingly, A. formosana sect. Japonicae is closely tied to A. maritima sect. Clethropsis, with which it shares few morphological traits, and is separate from A. japonica sect. Japonicae with which it shares many traits. An increase in substitution rate is noted in the group comprising A. formosana, A. maritima and A. nitida relative to the rest of the genus, which appears to have had, on the average, a very slow mutation rate. Alnus glutinosa, the designated type for the genus, appears to be representative of the genus both for morphological characters and evolutionary rate. North-East Asia is comforted in its position of origin of the genus since not only does it have the highest number of species and representatives in all deep branching lineages, there are also fewer transcontinental migrations when a North-East Asian ancestor is postulated than when a North American ancestor is postulated.     

Molecular phylogeny of Solms-laubachia (Brassicaceae) s.l., based on multiple nuclear and plastid DNA sequences, and its biogeographic implications

The Hengduan Mountains region of south-west China is a noted biodiversity,hotspot, but the geographic origins and historical assembly of its rich endemic flora, including the sky-island species of Solms-laubachia Muschl. (Brassicaceae), have been little studied. Previous molecular studies on the phylogeny of Solms-laubachia showed it to be paraphyletic, leading to considerable expansion not only of its taxonomic limits, but also its geographic range, with the inclusion of taxa from outside the Hengduan region. However, these studies provided little resolution of interspecific relationships, preventing inferences about historical biogeography within the clade. In the present study, new sequence data from two nuclear genes (LEAFY and G3pdh) and two chloroplast intergenic spacers (petN-psbM and psbM-trnD) were combined with existing markers to increase phylogenetic signals. Phaeonychium villosum (Maxim.) Al-Shehbaz was found to be nested within Solms-laubachia s.l. In general, phylogenetic relationships appear to be a good predictor of geography, with the Hengduan Mountain endemics embedded in a paraphyletic grade of species from the western Himalayas and central Asia, but they also imply morphological homoplasy. Incongruence was detected between the nuclear and chloroplast gene trees, perhaps resulting from incomplete lineage sorting of ancestral polymorphisms. The crown age of Solms-laubachia s.l. was estimated to be approximately 1.42-3.68 mya, using Bayesian relaxed molecular clock analysis. Historical biogeographic analysis using a parametric dispersal-extinction-cladogenesis model inferred central Asia and the western Himalayas as most probable ancestral range of Solms-laubachia s.l., and estimated higher rates of eastward expansion than westward during the diversification of descendant lineages. In summary, our results suggest that Solms-laubachia s.l. originated during the Pliocene in central Asia, and subsequently migrated eastward into the Hengduan Mountains, colonizing sky-island, alpine scree-slope habitats that may have provided novel ecological opportunity and accelerated speciation, ultimately establishing this region as the present center of diversity of the genus.     

Molecular phylogenetics of the sexually deceptive orchid genus Ophrys (Orchidaceae) based on nuclear and chloroplast DNA sequences.

We present a phylogenetic analysis of the major lineages of the sexually deceptive orchid genus Ophrys based on nuclear ribosomal (nr) DNA (internal transcribed spacer region) and noncoding chloroplast (cp) DNA (trnL-trnF region) sequences. Sequence divergence within and among major Ophrys lineages was low for both nrDNA and cpDNA sequences. Separate analyses resulted in similar but poorly resolved trees. An incongruence length difference test revealed that nrDNA and cpDNA data sets were not incongruent. A combined analysis resulted in a better-resolved phylogenetic hypothesis of relationships among the major Ophrys lineages. Our data strongly support a division of Ophrys into two groups. These groups do not correspond to the earlier proposed sections Euophrys and Pseudophrys and are thus in conflict with traditional classifications. Our results support a well-resolved monophyletic group that contains the geographically widespread O. bombyliflora, O. speculum, O. tenthredinifera, and the O. fusca-lutea lineage. Relationships in the other group are poorly resolved. Based on our observations that taxa with identical sequences at presumably rapidly evolving loci clearly differ in floral morphology, we hypothesize that the diversity in the genus Ophrys is the result of a recent radiation in this orchid lineage.     

Phylogenetic analysis of Chloraeinae (Orchidaceae) based on plastid and nuclear DNA sequences

The phylogenetic relationships of subtribe Chloraeinae, a group of terrestrial orchids endemic to southern South America, have not been satisfactorily investigated. A previous molecular phylogenetic analysis based on plastid DNA supported the monophyly of Chloraeinae and Gavilea, but showed that Chloraea is non‐monophyletic and that the sole species of Bipinnula analysed is sister to Geoblasta. However, that analysis included only 18 of the 73 species belonging to this subtribe. Here, the phylogenetic relationships of Chloraeinae were assessed by analysing aproximately 7500 bp of nucleotide sequences from nuclear ribosomal internal transcribed spacer (ITS) and plastid DNA (rbcL, matK, trnL‐trnF, rpoB‐trnC) for 42 species representing all four currently accepted genera of Chloraeinae and appropriate outgroups. Nuclear and plastid data were analysed separately and in combination using two different methods, namely parsimony and Bayesian inference. Our analyses support the monophyly of Chloraeinae and their inclusion in an expanded concept of Cranichideae, but none of the genera of Chloraeinae that includes more than one species is monophyletic. Gavilea and Bipinnula are paraphyletic, with Chloraea chica nested in Gavilea and Geoblasta penicillata in Bipinnula. As currently delimited, Chloraea is polyphyletic. The taxonomic changes proposed recently are for the most part not justifiable on phylogenetic grounds, except for recognition of the monotypic genus Correorchis. The lack of resolution for the relationships among species of ‘core’Chloraea suggests a relatively recent diversification of this group. The current generic classification is in need or revision, but additional study is advisable before carrying out further taxonomic changes. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 168 , 258–277.     

Molecular phylogenetics of Phyllanthaceae: evidence from plastid MATK and nuclear PHYC sequences

Plastid matK and a fragment of the low-copy nuclear gene PHYC were sequenced for 30 genera of Phyllanthaceae to evaluate tribal and generic delimitation. Resolution and bootstrap percentages obtained with matK are higher than that of PHYC, but both regions show nearly identical phylogenetic patterns. Phylogenetic relationships inferred from the independent and combined data are congruent and differ from previous, morphology-based classifications but are highly concordant with those of the plastid gene rbcL previously published. Phyllanthaceae is monophyletic and gives rise to two well-resolved clades (T and F) that could be recognized as subfamilies. DNA sequence data for Keayodendron and Zimmermanniopsis are presented for the first time. Keayodendron is misplaced in tribe Phyllantheae and belongs to the Bridelia alliance. Zimmermanniopsis is sister to Zimmermannia. Phyllanthus and Cleistanthus are paraphyletic. Savia and Phyllanthus subgenus Kirganelia are not monophyletic.     

Molecular phylogeny and radiation time of Erysiphales inferred from the nuclear ribosomal DNA sequences

Phylogenetic relationships of Erysiphales within Ascomycota were inferred from the newly determined sequences of the 18S rDNA and partial sequences of the 28S rDNA including the D1 and D2 regions of 10 Erysiphales taxa. Phylogenetic analyses revealed that the Erysiphales form a distinct clade among ascomycetous fungi suggesting that the Erysiphales diverged from a single ancestral taxon. The Myxotrichaceae of the Onygenales was distantly related to the other onygenalean families and was the sister group to the Erysiphales calde, with which it combined to form a clade. The Erysiphales/Myxotrichaceae clade was also closely related to some discomycetous fungi (Leotiales, Cyttariales and Thelebolaceae) including taxa that form cleistothecial ascomata. The present molecular analyses as well as previously reported morphological observations suggest the possible existence of a novel evolutionary pathway from cleistothecial discomycetous fungi to Erysiphales and Myxotrichaceae. However, since most of these fungi, except for the Erysiphales, are saprophytic on dung and/or plant materials, the questions of how and why an obligate biotroph like the Erysiphales radiated from the saprophytic fungi remain to be addressed. We also estimated the radiation time of the Erysiphales using the 18S rDNA sequences and the two molecular clockes that have been previously reported. The calculation showed that the Erysiphales split from the Myxotrichaceae 190–127 myr ago. Since the radiation time of the Erysiphales does not exceed 230 myr ago, even when allowance is made for the uncertainty of the molecular clocks, it is possible to consider that the Erysiphales evolved after the radiation of angiosperms. The results of our calculation also showed that the first radiation within the Erysiphales (138–92 myr ago) coincided with the date of a major diversification of angiosperms (130–90 myr ago). These results may support our early assumption that the radiation of the Erysiphales coincided with the evolution of angiosperm plants. Contribution No. 152 from the Laboratory of Plant Pathology, Mie University     

Molecular phylogenetics of Ruscaceae sensu lato and related families (Asparagales) based on plastid and nuclear DNA sequences

Background

Previous phylogenetics studies of Asparagales, although extensive and generally well supported, have left several sets of taxa unclearly placed and have not addressed all relationships within certain clades thoroughly (some clades were relatively sparsely sampled). One of the most important of these is sampling within and placement of Nolinoideae (Ruscaceae s.l.) of Asparagaceae sensu Angiosperm Phylogeny Group (APG) III, which subfamily includes taxa previously referred to Convallariaceae, Dracaenaaceae, Eriospermaceae, Nolinaceae and Ruscaceae.

Methods

A phylogenetic analysis of a combined data set for 126 taxa of Ruscaceae s.l. and related groups in Asparagales based on three nuclear and plastid DNA coding genes, 18S rDNA (1796 bp), rbcL (1338 bp) and matK (1668 bp), representing a total of approx. 4·8 kb is presented. Parsimony and Bayesian inference analyses were conducted to elucidate relationships of Ruscaceae s.l. and related groups, and parsimony bootstrap analysis was performed to assess support of clades.

Key Results

The combination of the three genes results in the most highly resolved and strongly supported topology yet obtained for Asparagales including Ruscaceae s.l. Asparagales relationships are nearly congruent with previous combined gene analyses, which were reflected in the APG III classification. Parsimony and Bayesian analyses yield identical relationships except for some slight variation among the core asparagoid families, which nevertheless form a strongly supported group in both types of analyses. In core asparagoids, five major clades are identified: (1) Alliaceae s.l. (sensu APG III, Amarylidaceae–Agapanthaceae–Alliaceae); (2) Asparagaceae–Laxmanniaceae–Ruscaceae s.l.; (3) Themidaceae; (4) Hyacinthaceae; (5) Anemarrhenaceae–Behniaceae–Herreriaceae–Agavaceae (clades 2–5 collectively Asparagaceae s.l. sensu APG III). The position of Aphyllanthes is labile, but it is sister to Themidaceae in the combined maximum-parsimony tree and sister to Anemarrhenaceae in the Bayesian analysis. The highly supported clade of Xanthorrhoeaceae s.l. (sensu APG III, including Asphodelaceae and Hemerocallidaceae) is sister to the core asparagoids. Ruscaceae s.l. are a well-supported group. Asparagaceae s.s. are sister to Ruscaceae s.l., even though the clade of the two families is weakly supported; Laxmanniaceae are strongly supported as sister to Ruscaceae s.l. and Asparagaceae. Ruscaceae s.l. include six principal clades that often reflect previously named groups: (1) tribe Polygonateae (excluding Disporopsis); (2) tribe Ophiopogoneae; (3) tribe Convallarieae (excluding Theropogon); (4) Ruscaceae s.s. + Dracaenaceae + Theropogon + Disporopsis + Comospermum; (5) Nolinaceae, (6) Eriospermum.

Conclusions

The analyses here were largely conducted with new data collected for the same loci as in previous studies, but in this case from different species/DNA accessions and greater sampling in many cases than in previously published analyses; nonetheless, the results largely mirror those of previously conducted studies. This demonstrates the robustness of these results and answers questions often raised about reproducibility of DNA results, given the often sparse sampling of taxa in some studies, particularly the earliest ones. The results also provide a clear set of patterns on which to base a new classification of the subfamilies of Asparagaceae s.l., particularly Ruscaceae s.l. (= Nolinoideae of Asparagaceae s.l.), and examine other putatively important characters of Asparagales.     

Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences

To evaluate the monophyly of subtribe Pleurothallidinae (Epidendreae: Orchidaceae) and the component genera and to reveal evolutionary relationships and trends, we sequenced the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and 5.8S gene for 185 taxa. In addition, to improve the overall assessments along the spine of the topology, we added plastid sequences from matK, the trnL intron, and the trnL-F intergenic spacer for a representative subset of those taxa in the ITS study. All results were highly congruent, and so we then combined the sequence data from all three data sets in a separate analysis of 58 representative taxa. There is strong support in most analyses for the monophyly of Pleurothallidinae and in some for inclusion of Dilomilis and Neocognauxia of Laeliinae. Although most genera in the nine clades identified in the analyses are monophyletic, all data sets are highly congruent in revealing the polyphyly of Pleurothallis and its constitutent subgenera as presently understood. The high degree of homoplasy in morphological characters, especially floral characters, limits their usefulness in phylogenetic reconstruction of the subtribe.