Incomplete lineage sorting and reticulate evolution mask species relationships in Brunelliaceae, an Andean family with rapid, recent diversification.

PREMISE: To date, phylogenetic relationships within the monogeneric Brunelliaceae have been based on morphological evidence, which does not provide sufficient phylogenetic resolution. Here we use target-enriched nuclear data to improve our understanding of phylogenetic relationships in the family. METHODS: We used the Angiosperms353 toolkit for targeted recovery of exonic regions and supercontigs (exons + introns) from low copy nuclear genes from 53 of 70 species in Brunellia, and several outgroup taxa. We removed loci that indicated biased inference of relationships and applied concatenated and coalescent methods to infer Brunellia phylogeny. We identified conflicts among gene trees that may reflect hybridization or incomplete lineage sorting events and assessed their impact on phylogenetic inference. Finally, we performed ancestral-state reconstructions of morphological traits and assessed the homology of character states used to define sections and subsections in Brunellia. RESULTS: Brunellia comprises two major clades and several subclades. Most of these clades/subclades do not correspond to previous infrageneric taxa. There is high topological incongruence among the subclades across analyses. CONCLUSIONS: Phylogenetic reconstructions point to rapid species diversification in Brunelliaceae, reflected in very short branches between successive species splits. The removal of putatively biased loci slightly improves phylogenetic support for individual clades. Reticulate evolution due to hybridization and/or incomplete lineage sorting likely both contribute to gene-tree discordance. Morphological characters used to define taxa in current classification schemes are homoplastic in the ancestral character-state reconstructions. While target enrichment data allows us to broaden our understanding of diversification in Brunellia, the relationships among subclades remain incompletely understood.

-Complete plastid genome sequences of two species of the Neotropical genus Brunellia (Brunelliaceae).

Here we present the first two complete plastid genomes for Brunelliaceae, a Neotropical family with a single genus, Brunellia. We surveyed the entire plastid genome in order to find variable cpDNA regions for further phylogenetic analyses across the family. We sampled morphologically different species, B. antioquensis and B. trianae, and found that the plastid genomes are 157,685 and 157,775 bp in length and display the typical quadripartite structure found in angiosperms. Despite the clear morphological distinction between both species, the molecular data show a very low level of divergence. The amount of nucleotide substitutions per site is one of the lowest reported to date among published congeneric studies (π = 0.00025). The plastid genomes have gene order and content coincident with other COM (Celastrales, Oxalidales, Malpighiales) relatives. Phylogenetic analyses of selected superrosid representatives show high bootstrap support for the ((C,M)O) topology. The N-fixing clade appears as the sister group of the COM clade and Zygophyllales as the sister to the rest of the fabids group.

Effects of taxon sampling and tree reconstruction methods on phylodiversity metrics.

The amount and patterns of phylodiversity in a community are often used to draw inferences about the local and historical factors affecting community assembly and can be used to prioritize communities and locations for conservation. Because measures of phylodiversity are based on the topology and branch lengths of phylogenetic trees, which are affected by the number and diversity of taxa in the tree, these analyses may be sensitive to changes in taxon sampling and tree reconstruction methods.To investigate the effects of taxon sampling and tree reconstruction methods on measures of phylodiversity, we investigated the community phylogenetics of the Ordway-Swisher Biological Station (Florida), which is home to over 600 species of vascular plants. We studied the effects of (a) the number of taxa included in the regional phylogeny; (b) random versus targeted sampling of species to assemble the regional species pool; (c) including only species from specific clades rather than broad sampling; (d) using trees reconstructed directly for the taxa under study compared to trees pruned from a larger reconstructed tree; and (e) using phylograms compared to chronograms.We found that including more taxa in a study increases the likelihood of observing significantly nonrandom phylogenetic patterns. However, there were no consistent trends in the phylodiversity patterns based on random taxon sampling compared to targeted sampling, or within individual clades compared to the complete dataset. Using pruned and reconstructed phylogenies resulted in similar patterns of phylodiversity, while chronograms in some cases led to significantly different results from phylograms.The methods commonly used in community phylogenetic studies can significantly impact the results, potentially influencing both inferences of community assembly and conservation decisions. We highlight the need for both careful selection of methods in community phylogenetic studies and appropriate interpretation of results, depending on the specific questions to be addressed.

Phylogenomics in Cactaceae: A case study using the chollas sensu lato (Cylindropuntieae, Opuntioideae) reveals a common pattern out of the Chihuahuan and Sonoran deserts.

PREMISE: Although numerous phylogenetic studies have been conducted in Cactaceae, whole-plastome datasets have not been employed. We used the chollas to develop a plastome dataset for phylogeny reconstruction to test species relationships, biogeography, clade age, and morphological evolution. METHODS: We developed a plastome dataset for most known diploid members of the chollas (42 taxa) as well as for other members of Cylindropuntieae. Paired-end, raw reads from genome skimming were reference-mapped onto a de novo plastome assembly of one species of cholla, Cylindropuntia bigelovii, and were used to build our plastome dataset, which was analyzed using various methods. RESULTS: Our plastome dataset resolved the phylogeny of the chollas, including most interspecific and intraspecific relationships. Tribe Cylindropuntieae arose ~18 mya, during the early Miocene in southern South America, and is supported as sister to the South American clade Tephrocacteae. The (Micropuntia (Cylindropuntia + Grusonia)) clade most likely originated in the Chihuahuan Desert region around 16 mya and then migrated into other North American desert regions. Key morphological characters for recognizing traditional taxonomic series in Cylindropuntia (e.g., spiny fruit) are mostly homoplasious. CONCLUSIONS: This study provides the first comprehensive plastome phylogeny for any clade within Cactaceae. Although the chollas s.l. are widespread throughout western North American deserts, their most recent common ancestor likely arose in the Chihuahuan Desert region during the mid-Miocene, with much of their species diversity arising in the early to mid-Pliocene, a pattern strikingly similar to those found in other western North American desert groups.

Spatial Phylogenetics of Florida Vascular Plants: The Effects of Calibration and Uncertainty on Diversity Estimates.

Recent availability of biodiversity data resources has enabled an unprecedented ability to estimate phylogenetically based biodiversity metrics over broad scales. Such approaches elucidate ecological and evolutionary processes yielding a biota and help guide conservation efforts. However, the choice of appropriate phylogenetic resources and underlying input data uncertainties may affect interpretation. Here, we address how differences among phylogenetic source trees and levels of phylogenetic uncertainty affect these metrics and test existing hypotheses regarding geographic biodiversity patterns across the diverse vascular plant flora of Florida, US. Ecological niche models for 1,490 Florida species were combined with a "purpose-built" phylogenetic tree (phylogram and chronogram), as well as with trees derived from community resources (Phylomatic and Open Tree of Life). There were only modest differences in phylodiversity metrics given the phylogenetic source tree and taking into account the level of phylogenetic uncertainty; we identify similar areas of conservation interest across Florida regardless of the method used.

Phylogenomic inference in extremis: A case study with mycoheterotroph plastomes.

PREMISE OF THE STUDY: Phylogenomic studies employing large numbers of genes, including those based on plastid genomes (plastomes), are becoming common. Nonphotosynthetic plants such as mycoheterotrophs (which rely on root-associated fungi for essential nutrients, including carbon) tend to have highly elevated rates of plastome evolution, substantial genome reduction, or both. Mycoheterotroph plastomes therefore provide excellent test cases for investigating how extreme conditions impact phylogenomic inference. METHODS: We used parsimony and likelihood analysis of protein-coding gene sets from published and newly completed plastomes to infer the phylogenetic placement of taxa from the 10 angiosperm families in which mycoheterotrophy evolved. KEY RESULTS: Despite multiple very long branches that reflect elevated substitution rates, and frequently patchy gene recovery due to genome reduction, inferred phylogenetic placements of most mycoheterotrophic lineages in DNA-based likelihood analyses are both well supported and congruent with other studies. Amino-acid-based likelihood placements are broadly consistent with DNA-based inferences, but extremely rate-elevated taxa can have unexpected placements-albeit with weak support. In contrast, parsimony analysis is strongly misled by long-branch attraction among many distantly related mycoheterotrophic monocots. CONCLUSIONS: Mycoheterotrophic plastomes provide challenging cases for phylogenomic inference, as substitutional rates can be elevated and genome reduction can lead to sparse gene recovery. Nonetheless, diverse likelihood frameworks provide generally well-supported and mutually concordant phylogenetic placements of mycoheterotrophs, consistent with recent phylogenetic studies and angiosperm-wide classifications. Previous predictions of parallel photosynthesis loss within families are supported for Burmanniaceae, Ericaceae, Gentianaceae, and Orchidaceae. Burmanniaceae and Thismiaceae should not be combined as a single family in Dioscoreales.

The first complete plastid genomes of Melastomataceae are highly structurally conserved.

BACKGROUND: In the past three decades, several studies have predominantly relied on a small sample of the plastome to infer deep phylogenetic relationships in the species-rich Melastomataceae. Here, we report the first full plastid sequences of this family, compare general features of the sampled plastomes to other sequenced Myrtales, and survey the plastomes for highly informative regions for phylogenetics. METHODS: Genome skimming was performed for 16 species spread across the Melastomataceae. Plastomes were assembled, annotated and compared to eight sequenced plastids in the Myrtales. Phylogenetic inference was performed using Maximum Likelihood on six different data sets, where putative biases were taken into account. Summary statistics were generated for all introns and intergenic spacers with suitable size for polymerase chain reaction (PCR) amplification and used to rank the markers by phylogenetic information. RESULTS: The majority of the plastomes sampled are conserved in gene content and order, as well as in sequence length and GC content within plastid regions and sequence classes. Departures include the putative presence of rps16 and rpl2 pseudogenes in some plastomes. Phylogenetic analyses of the majority of the schemes analyzed resulted in the same topology with high values of bootstrap support. Although there is still uncertainty in some relationships, in the highest supported topologies only two nodes received bootstrap values lower than 95%. DISCUSSION: Melastomataceae plastomes are no exception for the general patterns observed in the genomic structure of land plant chloroplasts, being highly conserved and structurally similar to most other Myrtales. Despite the fact that the full plastome phylogeny shares most of the clades with the previously widely used and reduced data set, some changes are still observed and bootstrap support is higher. The plastome data set presented here is a step towards phylogenomic analyses in the Melastomataceae and will be a useful resource for future studies.

Seven New Complete Plastome Sequences Reveal Rampant Independent Loss of the ndh Gene Family across Orchids and Associated Instability of the Inverted Repeat/Small Single-Copy Region Boundaries.

Earlier research has revealed that the ndh loci have been pseudogenized, truncated, or deleted from most orchid plastomes sequenced to date, including in all available plastomes of the two most species-rich subfamilies, Orchidoideae and Epidendroideae. This study sought to resolve deeper-level phylogenetic relationships among major orchid groups and to refine the history of gene loss in the ndh loci across orchids. The complete plastomes of seven orchids, Oncidium sphacelatum (Epidendroideae), Masdevallia coccinea (Epidendroideae), Sobralia callosa (Epidendroideae), Sobralia aff. bouchei (Epidendroideae), Elleanthus sodiroi (Epidendroideae), Paphiopedilum armeniacum (Cypripedioideae), and Phragmipedium longifolium (Cypripedioideae) were sequenced and analyzed in conjunction with all other available orchid and monocot plastomes. Most ndh loci were found to be pseudogenized or lost in Oncidium, Paphiopedilum and Phragmipedium, but surprisingly, all ndh loci were found to retain full, intact reading frames in Sobralia, Elleanthus and Masdevallia. Character mapping suggests that the ndh genes were present in the common ancestor of orchids but have experienced independent, significant losses at least eight times across four subfamilies. In addition, ndhF gene loss was correlated with shifts in the position of the junction of the inverted repeat (IR) and small single-copy (SSC) regions. The Orchidaceae have unprecedented levels of homoplasy in ndh gene presence/absence, which may be correlated in part with the unusual life history of orchids. These results also suggest that ndhF plays a role in IR/SSC junction stability.

Orchid phylogenomics and multiple drivers of their extraordinary diversification.

Orchids are the most diverse family of angiosperms, with over 25 000 species,more than mammals, birds and reptiles combined. Tests of hypotheses to account for such diversity have been stymied by the lack of a fully resolved broad-scale phylogeny. Here,we provide such a phylogeny, based on 75 chloroplast genes for 39 species representing all orchid subfamilies and 16 of 17 tribes, time-calibrated against 17 angiosperm fossils. Asupermatrix analysis places an additional 144 species based on three plastid genes. Orchids appear to have arisen roughly 112 million years ago (Mya); the subfamilies Orchidoideae and Epidendroideae diverged from each other at the end of the Cretaceous; and the eight tribes and three previously unplaced subtribes of the upper epidendroids diverged rapidly from each other between 37.9 and 30.8 Mya. Orchids appear to have undergone one significant acceleration of net species diversification in the orchidoids, and two accelerations and one deceleration in the upper epidendroids. Consistent with theory, such accelerations were correlated with the evolution of pollinia, the epiphytic habit, CAM photosynthesis, tropical distribution (especially in extensive cordilleras),and pollination via Lepidoptera or euglossine bees. Deceit pollination appears to have elevated the number of orchid species by one-half but not via acceleration of the rate of net diversification. The highest rate of net species diversification within the orchids (0.382 sp sp(-1) My(-1)) is 6.8 times that at the Asparagales crown.

Recent gene-capture on the UV sex chromosomes of the moss Ceratodon purpureus.

Sex chromosomes evolve from ordinary autosomes through the expansion and subsequent degeneration of a region of suppressed recombination that is inherited through one sex. Here we investigate the relative timing of these processes in the UV sex chromosomes of the moss Ceratodon purpureus using molecular population genetic analyses of eight newly discovered sex-linked loci. In this system, recombination is suppressed on both the female-transmitted (U) sex chromosome and the male-transmitted (V) chromosome. Genes on both chromosomes therefore should show the deleterious effects of suppressed recombination and sex-limited transmission, while purifying selection should maintain homologs of genes essential for both sexes on both sex chromosomes. Based on analyses of eight sex-linked loci, we show that the nonrecombining portions of the U and V chromosomes expanded in at least two events (~0.6-1.3 MYA and ~2.8-3.5 MYA), after the divergence of C. purpureus from its dioecious sister species, Trichodon cylindricus and Cheilothela chloropus. Both U- and V-linked copies showed reduced nucleotide diversity and limited population structure, compared to autosomal loci, suggesting that the sex chromosomes experienced more recent selective sweeps that the autosomes. Collectively these results highlight the dynamic nature of gene composition and molecular evolution on nonrecombining portions of the U and V sex chromosomes.

Primer development for the plastid region ycf1 in Annonaceae and other magnoliids.

PREMISE OF THE STUDY: Primers were developed for a portion of the ycf1 plastid gene in magnoliid taxa to investigate the utility of ycf1 in phylogenetic analyses. • METHODS AND RESULTS: Twenty-six species across six families within the magnoliid group (Canellales, Piperales, Laurales, and Magnoliales) were sampled to examine the ability to amplify ycf1. Additionally, 29 accessions of Asimina and Deeringothamnus (Annonaceae) were sequenced to assess levels of variation in ycf1 compared to matK and trnL-F. • CONCLUSIONS: Results indicate that ycf1 is easily amplified and sequenced. In Annonaceae, ycf1 provides more informative phylogenetic characters than commonly used markers such as matK and trnL-F.

Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia.

BACKGROUND: Floral morphology, particularly the angle of lip attachment to the column, has historically been the fundamental character used in establishing generic limits in subtribe Oncidiinae (Orchidaceae), but it has also been long recognized that reliance on this character alone has produced a highly artificial set of genera. In essence, lip/column relationships reflect syndromes associated with pollinator preferences; most genera of Oncidiinae as previously defined have consisted of a single floral type. Here, the degree to which this has influenced generic delimitation in Brazilian members of the largest genus of Oncidiinae, Oncidium, which previous molecular (DNA) studies have demonstrated to be polyphyletic, is evaluated. METHODS: Phylogenetic analyses of the following multiple DNA regions were used: the plastid psbA-trnH intergenic spacer, matK exon and two regions of ycf1 exon and nuclear ribosomal DNA, comprised of the two internal transcribed spacers, ITS1 and ITS2, and the 5.8S gene. Results from all regions analysed separately indicated highly similar relationships, so a combined matrix was analysed. KEY RESULTS: Nearly all species groups of Brazilian Oncidium are only distantly related to the type species of the genus, O. altissimum, from the Caribbean. There are two exceptions to this geographical rule: O. baueri is related to the type group and O. orthostates, an isolated species that lacks the defining tabula infrastigmata of Oncidium, is not exclusively related to any previously described genus in the subtribe. Several well-supported subclades can be observed in these results, but they do not correspond well to sections of Oncidium as previously circumscribed or to segregate genera as defined by several recent authors. In spite of their floral differences, these groups of Oncidium, formerly treated as O. sections Barbata, Concoloria pro parte, Crispa, Ranifera, Rhinocerotes, Rostrata (only O. venustum), Synsepala, Verrucituberculata pro parte and Waluewa, form a well-supported clade with Gomesa (including Rodrigueziella and Rodrigueziopsis) embedded in it. Two often recognized segregate genera, Baptistonia and Ornithophora, and the recently described Carriella are also embedded within the Brazilian clade. The level of variation within major subclades of the Gomesa clade is low and similar to that observed within other genera of Oncidiinae. CONCLUSIONS: Convergence on a stereotypical syndrome of floral traits associated with pollination by oil-collecting bees has resulted in these characters not being reliable for producing monophyletic taxa, and the genus Oncidium, defined by these characters, is grossly polyphyletic. Vegetative and a few floral/inflorescence characters link these taxa with a mainly Brazilian distribution, and they were all transferred to Gomesa on this basis rather than separated from Gomesa based on their floral differences, which we hypothesize to be simple shifts in pollination strategies. Other authors have described a large number of new genera for these former members of Oncidium, but most of these are not supported by the results presented here (i.e. they are not monophyletic). A new genus, Nohawilliamsia, is described for O. orthostates because it does not fit in any currently recognized genus and is only distantly related to any other member of Oncidiinae.

Molecular phylogenetics and the evolution of fruit and leaf morphology of Dichaea (Orchidaceae: Zygopetalinae).

BACKGROUND AND AIMS: The orchid genus Dichaea, with over 100 species found throughout the neotropics, is easily recognized by distichous leaves on long stems without pseudobulbs and flowers with infrastigmatic ligules. The genus has previously been divided into four sections based primarily on presence of ovary bristles and a foliar abscission layer. The aim of this work is to use DNA sequence data to estimate phylogenetic relationships within Dichaea and map the distribution of major morphological characters that have been used to delimit subgenera/sections. METHODS: Sequence data for the nuclear ribosomal internal transcribed spacers and plastid matK, trnL intron, trnL-F spacer and ycf1 for 67 ingroup and seven outgroup operational taxonomic units were used to estimate phylogenetic relationships within Dichaea. Taxa from each of the four sections were sampled, with the greatest representation from section Dichaea, the most diverse and taxonomically puzzling group. KEY RESULTS: Molecular data and morphology support monophyly of Dichaea. Results indicate that section Dichaeopsis is polyphyletic and based on symplesiomorphies, including deciduous leaves and smooth ovaries that are widespread in Zygopetalinae. There are at least three well-supported clades within section Dichaeopsis. Section Pseudodichaea is monophyletic and defined by setose ovaries and leaves with an abscission layer. Sections Dichaea and Dichaeastrum are monophyletic and defined by pendent habit and persistent leaves. Section Dichaeastrum, distinguished from section Dichaea primarily by a glabrous ovary, is potentially polyphyletic. CONCLUSIONS: The leaf abscission layer was lost once, occurring only in the derived sections Dichaea and Dichaeastrum. The setose fruit is a more homoplasious character with several losses and gains within the genus. We propose an informal division of the genus based upon five well-supported clades.

Molecular phylogeny of the neotropical genus Christensonella (Orchidaceae, Maxillariinae): species delimitation and insights into chromosome evolution.

BACKGROUND AND AIMS: Species' boundaries applied within Christensonella have varied due to the continuous pattern of variation and mosaic distribution of diagnostic characters. The main goals of this study were to revise the species' delimitation and propose a more stable classification for this genus. In order to achieve these aims phylogenetic relationships were inferred using DNA sequence data and cytological diversity within Christensonella was examined based on chromosome counts and heterochromatin patterns. The results presented describe sets of diagnostic morphological characters that can be used for species' identification. METHODS: Phylogenetic studies were based on sequence data of nuclear and plastid regions, analysed using maximum parsimony and maximum likelihood criteria. Cytogenetic observations of mitotic cells were conducted using CMA and DAPI fluorochromes. KEY RESULTS: Six of 21 currently accepted species were recovered. The results also support recognition of the 'C. pumila' clade as a single species. Molecular phylogenetic relationships within the 'C. acicularis-C. madida' and 'C. ferdinandiana-C. neowiedii' species' complexes were not resolved and require further study. Deeper relationships were incongruent between plastid and nuclear trees, but with no strong bootstrap support for either, except for the position of C. vernicosa. Cytogenetic data indicated chromosome numbers of 2n = 36, 38 and 76, and with substantial variation in the presence and location of CMA/DAPI heterochromatin bands. CONCLUSIONS: The recognition of ten species of Christensonella is proposed according to the molecular and cytogenetic patterns observed. In addition, diagnostic morphological characters are presented for each recognized species. Banding patterns and chromosome counts suggest the occurrence of centric fusion/fission events, especially for C. ferdinandiana. The results suggest that 2n = 36 karyotypes evolved from 2n = 38 through descendent dysploidy. Patterns of heterochromatin distribution and other karyotypic data proved to be a valuable source of information to understand evolutionary patterns within Maxillariinae orchids.

Molecular phylogenetics of Maxillaria and related genera (Orchidaceae: Cymbidieae) based on combined molecular data sets.

The orchid genus Maxillaria is one of the largest and most common of neotropical orchid genera, but its current generic boundaries and relationships have long been regarded as artificial. Phylogenetic relationships within subtribe Maxillariinae sensu Dressler (1993) with emphasis on Maxillaria s.l. were inferred using parsimony analyses of individual and combined DNA sequence data. We analyzed a combined matrix of nrITS DNA, the plastid matK gene and flanking trnK intron, and the plastid atpB-rbcL intergenic spacer for 619 individuals representing ca. 354 species. The plastid rpoC1 gene (ca. 2600 bp) was sequenced for 84 selected species and combined in a more limited analysis with the other data sets to provide greater resolution. In a well-resolved, supported consensus, most clades were present in more than one individual analysis. All the currently recognized minor genera of "core" Maxillariinae (Anthosiphon, Chrysocycnis, Cryptocentrum, Cyrtidiorchis, Mormolyca, Pityphyllum, and Trigonidium) are embedded within a polyphyletic Maxillaria s.l. Our results support the recognition of a more restricted Maxillaria, of some previously published segregate genera (Brasiliorchis, Camaridium, Christensonella, Heterotaxis, Ornithidium, Sauvetrea), and of several novel clades at the generic level. These revised monophyletic generic concepts should minimize further nomenclatural changes, encourage monographic studies, and facilitate more focused analyses of character evolution within Maxillariinae.