Rangewide analysis of fungal associations in the fully mycoheterotrophic Corallorhiza striata complex (Orchidaceae) reveals extreme specificity on ectomycorrhizal Tomentella (Thelephoraceae) across North America

Fully mycoheterotrophic plants offer a fascinating system for studying phylogenetic associations and dynamics of symbiotic specificity between hosts and parasites. These plants frequently parasitize mutualistic mycorrhizal symbioses between fungi and trees. Corallorhiza striata is a fully mycoheterotrophic, North American orchid distributed from Mexico to Canada, but the full extent of its fungal associations and specificity is unknown. Plastid DNA (orchids) and ITS (fungi) were sequenced for 107 individuals from 42 populations across North America to identify C. striata mycobionts and test hypotheses on fungal host specificity. Four largely allopatric orchid plastid clades were recovered, and all fungal sequences were most similar to ectomycorrhizal Tomentella (Thelephoraceae), nearly all to T. fuscocinerea. Orchid-fungal gene trees were incongruent but nonindependent; orchid clades associated with divergent sets of fungi, with a clade of Californian orchids subspecialized toward a narrow Tomentella fuscocinerea clade. Both geography and orchid clades were important determinants of fungal association, following a geographic mosaic model of specificity on Tomentella fungi. These findings corroborate patterns described in other fully mycoheterotrophic orchids and monotropes, represent one of the most extensive plant-fungal genetic investigations of fully mycoheterotrophic plants, and have conservation implications for the >400 plant species engaging in this trophic strategy worldwide.     

Morphological and genomic evidence for a new species of Corallorhiza (Orchidaceae: Epidendroideae) from SW China

Corallorhiza sinensis, a new species of mycoheterotrophic orchid from western Sichuan, China, is described and illustrated based on molecular and morphological evidence. It is morphologically similar to Corallorhiza trifida, but can be distinguished by bigger flowers, both sepals and petals with 3 veins, and longer lateral lobes of lip. To distinguish the new Corallorhiza species and explore its phylogenetic position within subtribe Calypsoinae, this study employed sequences of the nuclear ribosomal DNA (nrDNA) and whole plastome assembled from the genome skimming approach. The plastome is 148,124 bp in length, including a pair of inverted repeats (IRs) of 26,165 bp, a large single-copy region (LSC) of 82,207 bp, and a small single-copy region (SSC) of 13,587 bp. Further, phylogenetic analyses were performed using nrDNA sequence and 79 coding sequences (CDSs) from 26 complete plastomes of subtribe Calypsoinae. The phylogenetic tree based on nrDNA sequence suggested that Corallorhiza is a monophyletic group, and strongly support C. sinensis as sister to the rest species of Corallorhiza. The plastid tree showed that 10 Corallorhiza species grouped into two clades and C. sinensis is most closely related to the North American C. striata and C. bentleyi instead of Oreorchis foliosa and O.angustata in the same clade. The plastid tree and nrDNA tree indicate Oreorchis is a paraphyletic. Although the topological conflicts are displayed between plastome and nrDNA phylogenies of C. sinensis, it is still the most closely related to Corallorhiza. Comparative analysis showed that C. sinensis populations are characteristic of the intermediate morphological traits between Corallorhiza and Oreorchis. The finding of this new species from China shed new light on the phylogeny of Oreorchis and Corallorhiza.     

Phylogenetic analysis of the grape family (Vitaceae) based on three chloroplast markers

Seventy-nine species representing 12 genera of Vitaceae were sequenced for the trnL-F spacer, 37 of which were subsequently sequenced for the atpB-rbcL spacer and the rps16 intron. Phylogenetic analysis of the combined data provided a fairly robust phylogeny for Vitaceae. Cayratia, Tetrastigma, and Cyphostemma form a clade. Cyphostemma and Tetrastigma are each monophyletic, and Cayratia may be paraphyletic. Ampelopsis is paraphyletic with the African Rhoicissus and the South American Cissus striata nested within it. The pinnately leaved Ampelopsis form a subclade, and the simple and palmately leaved Ameplopsis constitutes another with both subclades containing Asian and American species. Species of Cissus from Asia and Central America are monophyletic, but the South American C. striata does not group with other Cissus species. The Asian endemic Nothocissus and Pterisanthes form a clade with Asian Ampelocissus, and A. javalensis from Central America is sister to this clade. Vitis is monophyletic and forms a larger clade with Ampelocissus, Pterisanthes, and Nothocissus. The eastern Asian and North American disjunct Parthenocissus forms a clade with Yua austro-orientalis, a species of a small newly recognized genus from China to eastern Himalaya. Vitaceae show complex multiple intercontinental relationships within the northern hemisphere and between northern and southern hemispheres.     

Angiosperm phylogeny based on matK sequence information

Plastid matK gene sequences for 374 genera representing all angiosperm orders and 12 genera of gymnosperms were analyzed using parsimony (MP) and Bayesian inference (BI) approaches. Traditionally, slowly evolving genomic regions have been preferred for deep-level phylogenetic inference in angiosperms. The matK gene evolves approximately three times faster than the widely used plastid genes rbcL and atpB. The MP and BI trees are highly congruent. The robustness of the strict consensus tree supercedes all individual gene analyses and is comparable only to multigene-based phylogenies. Of the 385 nodes resolved, 79% are supported by high jackknife values, averaging 88%. Amborella is sister to the remaining angiosperms, followed by a grade of Nymphaeaceae and Austrobaileyales. Bayesian inference resolves Amborella + Nymphaeaceae as sister to the rest, but with weak (0.42) posterior probability. The MP analysis shows a trichotomy sister to the Austrobaileyales representing eumagnoliids, monocots + Chloranthales, and Ceratophyllum + eudicots. The matK gene produces the highest internal support yet for basal eudicots and, within core eudicots, resolves a crown group comprising Berberidopsidaceae/Aextoxicaceae, Santalales, and Caryophyllales + asterids. Moreover, matK sequences provide good resolution within many angiosperm orders. Combined analyses of matK and other rapidly evolving DNA regions with available multigene data sets have strong potential to enhance resolution and internal support in deep level angiosperm phylogenetics and provide additional insights into angiosperm evolution.     

Character transformation and relationships in Corallorhiza (Orchidaceae: Epidendroideae). II. Morphological variation and phylogenetic analysis

Morphological and anatomical study of Corallorhiza, a genus of primarily New World leafless mycoparasitic orchids, was undertaken in order to produce a hypothesis of relationships among the species and to gain some understanding of character transformations. Cladistic analysis of the resulting data set gave two most parsimonious trees. Analysis of combined plastid DNA and morphological data yielded a single topology, identical to one of the two from the analysis of morphological data alone. Molecular data do not conflict with the morphological data set, and provide more resolution within the C. maculata complex. The combined data indicate that C. striata is the sister group to the remainder of the genus; the circumboreal C. trifida also occupies a basal position. Corallorhiza wisteriana and C. odontorhiza comprise the sister group to the C. maculata + C. mertensiana + C. bulbosa clade. Only two synapomorphies, presence of the coralloid rhizome and loss of leaves, unite the species of Corallorhiza. The coralloid rhizome appears to be a paedomorphic development, due to its similarity to a protocorm; if so, it too is a loss character and may be considered only weak support for monophyly of the genus. Predominant autogamy, seen in C. trifida and cleistogamous C. odontorhiza, has probably arisen independently in these taxa.     

The circumscription and phylogenetic relationships of Callitropsis and the newly described genus Xanthocyparis (Cupressaceae)

A new species of conifer was recently discovered in northern Vietnam. In a preliminary phylogenetic analysis of morphological data a possible sister species, Chamaecyparis nootkatensis (D. Don) Spach, was identified; however, because of the presumed phylogenetic remoteness of these two species to the remainder of the Cupressaceae, a new genus-Xanthocyparis-was described to accommodate both species. Here an analysis of ITS (nrDNA), matK, and rbcL sequence data in combination with 58 informative morphological characters was aimed at testing the monophyly of the remainder of Chamaecyparis and evaluating the placement and monophyly of Xanthocyparis. Chamaecyparis, minus C. nootkatensis, was resolved as a monophyletic group, remote from Cupressus and Xanthocyparis. Cupressus, Juniperus, and Xanthocyparis formed a very highly supported monophyletic group. However, Cupressus was not monophyletic. Instead the Old World species sampled were resolved sister to a clade containing a monophyletic Juniperus, a monophyletic Xanthocyparis, and a clade of New World Cupressus species. If both species of Xanthocyparis are to be treated as members of the same genus, then due to the principal of priority they will have to be recognized in the genus Callitropsis. Research is continuing to resolve the status of New World and Old World Cupressus.     

DNA barcoding of European Herbertus (Marchantiopsida, Herbertaceae) and the discovery and description of a new species

DNA barcoding of a group of European liverwort species from the genus Herbertus was undertaken using three plastid (matK, rbcL and trnH-psbA) and one nuclear (ITS) marker. The DNA barcode data were effective in discriminating among the sampled species of Herbertus and contributed towards the detection of a previously overlooked European Herbertus species, described here as H. norenus sp. nov. This species shows clear-cut differences in DNA sequence for multiple barcode regions and is also morphologically distinct. The DNA barcode data were also useful in clarifying taxonomic relationships of the European species with some species from Asia and North America. In terms of the discriminatory power of the different barcode markers, ITS was the most informative region, followed closely by matK. All species were distinguishable by ITS alone, rbcL + matK and various other multimarker combinations.     

Resolving the evolutionary history of Campanula (Campanulaceae) in western North America

Recent phylogenetic works have begun to address long-standing questions regarding the systematics of Campanula (Campanulaceae). Yet, aspects of the evolutionary history, particularly in northwestern North America, remain unresolved. Thus, our primary goal in this study was to infer the phylogenetic positions of northwestern Campanula species within the greater Campanuloideae tree. We combined new sequence data from 5 markers (atpB, rbcL, matK, and trnL-F regions of the chloroplast and the nuclear ITS) representing 12 species of Campanula with previously published datasets for worldwide campanuloids, allowing us to include approximately 75% of North American Campanuleae in a phylogenetic analysis of the Campanuloideae. Because all but one of North American Campanula species are nested within a single campanuloid subclade (the Rapunculus clade), we conducted a separate set of analyses focused specifically on this group. Our findings show that i) the campanuloids have colonized North America at least 6 times, 4 of which led to radiations, ii) all but one North American campanuloid are nested within the Rapunculus clade, iii) in northwestern North America, a C. piperi-C. lasiocarpa ancestor gave rise to a monophyletic Cordilleran clade that is sister to a clade containing C. rotundifolia, iv) within the Cordilleran clade, C. parryi var. parryi and C. parryi var. idahoensis exhibit a deep, species-level genetic divergence, and v) C. rotundifolia is genetically diverse across its range and polyphyletic. Potential causes of diversification and endemism in northwestern North America are discussed.     

Phylogeny of Eastern North American Coreopsis (Asteraceae-Coreopsideae): insights from nuclear and plastid sequences, and comments on character evolution

A molecular phylogenetic study of eastern North American Coreopsis and representatives of other genera of tribe Coreopsideae was conducted using combined sequences from nuclear ITS and two plastid regions (matK, rpl16). A total of 25-30 species has been recognized in five sections of Coreopsis in eastern North America. Based on morphological characters, these taxa have generally been considered a monophyletic group. Our well-resolved phylogeny supports the monophyly of sections that have been recognized in Coreopsis, but the sections collectively do not comprise a monophyletic group because species of north temperate Bidens occur within one of the two major Coreopsis clades. The most notable departure of present results from prior views of relationships among sections is the lack of a sister group relationship between sections Calliopsis and Eublepharis; the shared presence of four-lobed disk floret corollas had been used to support a close relationship between these two sections. Relationships within sections show both similarities and differences with the results of previous studies based primarily on morphological characters. Mapping of morphological characters used taxonomically in Coreopsis and related genera onto the phylogeny indicates that the evolution of these characters has been complex, and this compromises their value for defining monophyletic groups. Examples include the annual habit, alternate leaves, winged fruits, red or brown basal spots on the yellow ligules, and four-lobed disk floret corollas.     

Molecular systematics of Trilliaceae II. Phylogenetic analyses of Trillium and its allies using sequences of rbcL and matK genes of cpDNA and internal transcribed spacers of 18S–26S nrDNA

Coding regions of the rbcL and matK genes of cp DNA and internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced to study phylogenetic relationships within and among all four genera of Trilliaceae: Trillium, Paris, Daiswa and Kinugasa . The rbcL gene has evolved much slower than matK and in particular ITS; hence the phylogenetic trees based on the rbcL gene show a much lower resolution than trees based on either matK or ITS. The general topology of phylogenetic trees resulting from separate parsimony analyses of the matK and ITS sequences are relatively congruent, with the exception of the placement of T. pusillum . Both matK and ITS phylogenies reveal that T. rivale diverges at the base of the trees. In both trees, Paris, Daiswa and Kinugasa form a relatively weakly supported group. Within this group, the allo-octaploid Kinugasa japonica is the sister group of Daiswa species. The Paris–Daiswa – Kinugasa group, the major Trillium group, and T. undulatum and T. govanianum showed a loosely related topology, but their affinities are not evident according to these two molecular markers. However, phylogenetic analysis of amino acid sequences derived from matK shows that T. rivale together with clades T. undulatum–T. govanianum, Daiswa–Kinugasa and Paris is basally diverged as a sister group to the remainder of Trillium .     

Molecular evolution of rbcL in the mycoheterotrophic coralroot orchids (Corallorhiza Gagnebin, Orchidaceae)

The RuBisCO large subunit gene (rbcL) has been the focus of numerous plant phylogenetic studies and studies on molecular evolution in parasitic plants. However, there has been a lack of investigation of photosynthesis gene molecular evolution in fully mycoheterotrophic plants. These plants invade pre-existing mutualistic associations between ectomycorrhizal trees and fungi, from which they obtain fixed carbon and nutrients. The mycoheterotrophic orchid Corallorhiza contains both green (photosynthetic) and non-green (putatively nonphotosynthetic) species. We sequenced rbcL from 31 accessions of eight species of Corallorhiza and hypothesized that some lineages would have pseudogenes resulting from relaxation of purifying selection on RuBisCO's carboxylase function. Phylogenetic analysis of rbcL+ITS gave high jackknife support for relationships among species. We found evidence of pseudogene formation in all lineages of the Corallorhiza striata complex and in some lineages of the C. maculata complex. Evidence includes: stop codons, frameshifts, decreased d(S)/d(N) ratios, replacements not observed in photosynthetic species, rate heterogeneity, and high likelihood of neutral evolution. The evolution of rbcL in Corallorhiza may serve as an exemplary system in which to study the effects of relaxed evolutionary constraints on photosynthesis genes for >400 documented fully mycoheterotrophic plant species.     

Northern hemisphere biogeography of Cerastium (Caryophyllaceae): insights from phylogenetic analysis of noncoding plastidnucleotide sequences

Phylogenetic relationships and biogeography of the genus Cerastium were studied using sequences of three noncoding plastid DNA regions (trnL intron, trnL-trnF spacer, and psbA-trnH spacer). A total of 57 Cerastium taxa was analyzed using two species of the putative sister genus Stellaria as outgroups. Maximum parsimony analyses identified four clades that largely corresponded to previously recognized infrageneric groups. The results suggest an Old World origin and at least two migration events into North America from the Old World. The first event possibly took place across the Bering land bridge during the Miocene. Subsequent colonization of South America occurred after the North and South American continents joined during the Pliocene. A more recent migration event into North America probably across the northern Atlantic took place during the Quaternary, resulting in the current circumpolar distribution of the Arctic species. Molecular clock dating of major biogeographic events was internally consistent on the phylogenetic trees. The arctic high-polyploid species form a polytomy together with some boreal and temperate species of the C. tomentosum group and the C. arvense group. Lack of genetic variation among the arctic species probably indicates a recent origin. The annual life form is shown to be of polyphyletic origin.     

Phylogenetic relationships in Nicotiana (Solanaceae) inferred from multiple plastid DNA regions

For Nicotiana, with 75 naturally occurring species (40 diploids and 35 allopolyploids), we produced 4656bp of plastid DNA sequence for 87 accessions and various outgroups. The loci sequenced were trnL intron and trnL-F spacer, trnS-G spacer and two genes, ndhF and matK. Parsimony and Bayesian analyses yielded identical relationships for the diploids, and these are consistent with other data, producing the best-supported phylogenetic assessment currently available for the genus. For the allopolyploids, the line of maternal inheritance is traced via the plastid tree. Nicotiana and the Australian endemic tribe Anthocercideae form a sister pair. Symonanthus is sister to the rest of Anthocercideae. Nicotiana sect. Tomentosae is sister to the rest of the genus. The maternal parent of the allopolyploid species of N. sect. Polydicliae were ancestors of the same species, but the allopolyploids were produced at different times, thus making such sections paraphyletic to their extant diploid relatives. Nicotiana is likely to have evolved in southern South America east of the Andes and later dispersed to Africa, Australia, and southwestern North America.     

Phylogenetic relationships of Malvatheca (Bombacoideae and Malvoideae; Malvaceae sensu lato) as inferred from plastid DNA sequences

Previous molecular phylogenetic analyses have revealed that elements of the former families Malvaceae sensu stricto and Bombacaceae together form a well-supported clade that has been named Malvatheca. Within Malvatheca, two major lineages have been observed; one, Bombacoideae, corresponds approximately to the palmate-leaved Bombacaceae, and the other, Malvoideae, includes the traditional Malvaceae (the mallows or Eumalvoideae). However, the composition of these two groups and their relationships to other elements of Malvatheca remain a source of uncertainty. Sequence data from two plastid regions, ndhF and trnK/matK, from 34 exemplars of Malvatheca and six outgroups were analyzed. Parsimony, likelihood, and Bayesian analyses of the sequence data provided a well-resolved phylogeny except that relationships among five lineages at the base of Malvatheca are poorly resolved. Nonetheless, a 6-bp insertion in matK suggests that Fremontodendreae is sister to the remainder of Malvatheca. Our results suggest that the Malvoideae originated in the Neotropics and that a mangrove taxon dispersed across the Pacific from South America to Australasia and later radiated out of Australasia to give rise to the ca. 1700 living species of Eumalvoideae. Local clock analyses imply that the plastid genome underwent accelerated molecular evolution coincident with the dispersal out of the Americas and again with the radiation into the three major clades of Eumalvoideae.     

An integrative approach to delimiting species in a rare but widespread mycoheterotrophic orchid

In the spirit of recent calls for species delimitation studies to become more pluralistic, incorporating multiple sources of evidence, we adopted an integrative, phylogeographic approach to delimiting species and evolutionarily significant units (ESUs) in the Corallorhiza striata species complex. This rare, North American, mycoheterotrophic orchid has been a taxonomic challenge regarding species boundaries, displaying complex patterns of variation and reduced vegetative morphology. We employed plastid DNA, nuclear DNA and morphometrics, treating the C. striata complex as a case study for integrative species delimitation. We found evidence for the differentiation of the endangered C. bentleyi (eastern USA) + C. striata var. involuta (Mexico) from the remaining C. striata (= C. striata s.s.; USA, Canada, Mexico). Corallorhiza striata involuta and C. bentleyi, disjunct by thousands of kilometres (Mexico‐Appalachia), were genetically identical but morphologically distinct. Evidence suggests the C. striata complex represents three species: C. bentleyi, C. involuta and a widespread C. striata s.s under operational criteria of diagnosability and common allele pools. In contrast, Bayesian coalescent estimation delimited four species, but more informative loci and a resultant species tree will be needed to place higher confidence in future analyses. Three distinct groupings were identified within C. striata s.s., corresponding to C. striata striata, C. striata vreelandii, and Californian accessions, but these were not delimited as species because of occupying a common allele pool. Each comprises an ESU, warranting conservation considerations. This study represents perhaps the most geographically comprehensive example of integrative species delimitation for any orchid and any mycoheterotroph.     

Phylogeny of Castanea (Fagaceae) based on chloroplast trnT-L-F sequence data

Species in the genus Castanea are widely distributed in the deciduous forests of the Northern Hemisphere from Asia to Europe and North America. They show floristic similarity but differences in chestnut blight resistance especially among eastern Asian and eastern North American species. Phylogenetic analyses were conducted in this study using sequences of three chloroplast noncoding trnT-L-F regions. The trnT-L region was found to be the most variable and informative region. The highest proportion of parsimony informative sites, more and larger indels, and higher pairwise distances between taxa were obtained at trnT-L than at the other two regions. The high A+T values (74.5%) in the Castanea trnT-L region may explain the high proportion of transversions found in this region where as comparatively lower A+T values were found in the trnL intron (68.35%) and trnL-F spacer (70.07%) with relatively balanced numbers of transitions and transversions. The genus Castanea is supported as a monophyletic clade, while the section Eucastanon is paraphyletic. C. crenata is the most basal clade and sister to the remainder of the genus. The three Chinese species of Castanea are supported as a single monophyletic clade, whose sister group contains the North American and European species. There is consistent but weak support for a sister–group relationship between the North American species and European species.     

Phylogeny, biogeography, and molecular dating of cornelian cherries (Cornus, Cornaceae): tracking Tertiary plant migration

Data from four DNA regions (rbcL, matK, 26S rDNA, and ITS) as well as extant and fossil morphology were used to reconstruct the phylogeny and biogeographic history of an intercontinentally disjunct plant group, the cornelian cherries of Cornus (dogwoods). The study tests previous hypotheses on the relative roles of two Tertiary land bridges, the North Atlantic land bridge (NALB) and the Bering land bridge (BLB), in plant migration across continents. Three approaches, the Bayesian, nonparametric rate smoothing (NPRS), and penalized likelihood (PL) methods, were employed to estimate the times of geographic isolations of species. Dispersal and vicariance analysis (DIVA) was performed to infer the sequence and directionality of biogeographic pathways. Results of phylogenetic analyses suggest that among the six living species, C. sessilis from western North America represents the oldest lineage, followed by C. volkensii from Africa. The four Eurasian species form a clade consisting of two sister pairs, C. mas-C. officinalis and C. chinensis-C. eydeana. Results of DIVA and data from fossils and molecular dating indicate that the cornelian cherry subgroup arose in Europe as early as the Paleocene. Fossils confirm that the group was present in North America by the late Paleocene, consistent with the DIVA predictions that, by the end of the Eocene, it had diversified into several species and expanded its distribution to North America via the NALB and to Africa via the last direct connection between Eurasia and Africa prior to the Miocene, or via long-distance dispersal. The cornelian cherries in eastern Asia appear to be derived from two independent dispersal events from Europe. These events are inferred to have occurred during the Oligocene and Miocene. This study supports the hypothesis that the NALB served as an important land bridge connecting the North American and European floras, as well as connecting American and African floras via Europe during the early Tertiary.     

Phylogeny and biogeography of the carnivorous plant family Sarraceniaceae

The carnivorous plant family Sarraceniaceae comprises three genera of wetland-inhabiting pitcher plants: Darlingtonia in the northwestern United States, Sarracenia in eastern North America, and Heliamphora in northern South America. Hypotheses concerning the biogeographic history leading to this unusual disjunct distribution are controversial, in part because genus- and species-level phylogenies have not been clearly resolved. Here, we present a robust, species-rich phylogeny of Sarraceniaceae based on seven mitochondrial, nuclear, and plastid loci, which we use to illuminate this family's phylogenetic and biogeographic history. The family and genera are monophyletic: Darlingtonia is sister to a clade consisting of Heliamphora+Sarracenia. Within Sarracenia, two clades were strongly supported: one consisting of S. purpurea, its subspecies, and S. rosea; the other consisting of nine species endemic to the southeastern United States. Divergence time estimates revealed that stem group Sarraceniaceae likely originated in South America 44-53 million years ago (Mya) (highest posterior density [HPD] estimate = 47 Mya). By 25-44 (HPD = 35) Mya, crown-group Sarraceniaceae appears to have been widespread across North and South America, and Darlingtonia (western North America) had diverged from Heliamphora+Sarracenia (eastern North America+South America). This disjunction and apparent range contraction is consistent with late Eocene cooling and aridification, which may have severed the continuity of Sarraceniaceae across much of North America. Sarracenia and Heliamphora subsequently diverged in the late Oligocene, 14-32 (HPD = 23) Mya, perhaps when direct overland continuity between North and South America became reduced. Initial diversification of South American Heliamphora began at least 8 Mya, but diversification of Sarracenia was more recent (2-7, HPD = 4 Mya); the bulk of southeastern United States Sarracenia originated co-incident with Pleistocene glaciation, <3 Mya. Overall, these results suggest climatic change at different temporal and spatial scales in part shaped the distribution and diversity of this carnivorous plant clade.     

Molecular and cytological examination of Calopogon (Orchidaceae, Epidendroideae): circumscription, phylogeny, polyploidy, and possible hybrid speciation

The orchid genus Calopogon R.Br. (Orchidaceae), native to eastern North America and the northern Caribbean, currently contains five species and up to three varieties. Using nuclear internal transcribed spacer (ITS) ribosomal DNA sequences, amplified fragment length polymorphisms (AFLPs), chloroplast DNA restriction fragments, and chromosome counts, we present a phylogenetic and taxonomic study of the genus. Calopogon multiflorus and C. pallidus are consistently sister species, but the relationships of C. barbatus, C. oklahomensis, and C. tuberosus are not as clear. In the ITS analysis C. oklahomensis is sister to C. barbatus, whereas it is sister to C. tuberosus in the plastid restriction fragment analysis. Furthermore, all species were found to have chromosome numbers of 2n = 38 and 40, with the exception of the putatively hybrid-derived C. oklahomensis with 2n = 114 and 120. The hexaploidy of the latter, plus the discrepancy in its position between the ITS and plastid restriction fragment trees, could suggest that it is of hybrid origin. However, the presence of unique morphological and molecular characters might indicate that it is either an ancient hybrid or not of hybrid derivation at all. Finally, using these molecular methods all taxa appear to generally be discrete groups, with the exception of C. tuberosus vars. latifolius and tuberosus, the former of which is best combined with the latter.     

Occurrence of matK in a trnK group II intron in charophyte green algae and phylogeny of the Characeae

A group II intron containing the matK gene, which encodes a splicing-associated maturase, was found in the trnK (lysine tRNA) exon in the chloroplast genome of the six extant genera of green algae in the family Characeae, which among green algae are the sister group to embryophytes (land plants). The characean trnK intron (～2.5 kilobases [kb]) and matK ORF (～1.5 kb) are comparable in size to the intron and ORF of land plants, in which they are similarly found inserted in the trnK exon. Domain X, a sequence of conserved amino acid residues within matK, occurs in the Characeae. Phylogenetic analysis using maximum likelihood (GTR + I + gamma likelihood model) and parsimony (branch and bound search) yielded one tree with high bootstrap support for all branches. The matK tree was congruent with the rbcL tree for the same taxa. The number and proportion of informative sites was higher in matK (501, 31% of matK sequence) compared to rbcL (122, 10%). Characeae branch lengths were on average more than five times longer for matK compared to rbcL and provided better resolution within the Characeae. These findings along with recent genomic analyses demonstrate that the intron and matK invaded the chloroplast genome of green algae prior to the evolution of land plants.