No phylogenomic support for a Cenozoic origin of the "living fossil" Isoetes.

PREMISE: The isoetalean lineage has a rich fossil record that extends to the Devonian, but the age of the living clade is unclear. Recent results indicate that it is young, from the Cenozoic, whereas earlier work based on less data from a denser taxon sampling yielded Mesozoic median ages. METHODS: We analyzed node ages in Isoetes using two genomic data sets (plastome and nuclear ribosomal cistron), three clock models implemented in MrBayes (ILN, WN, and TK02 models), and a conservative approach to calibration. RESULTS: While topological results were consistently resolved in Isoetes estimated crown group ages range from the latest Paleozoic (mid-Permian) to the Mesozoic depending on data type and clock model. The oldest estimates were retrieved using the autocorrelated TK02 clock model. An (early) Cenozoic age was only obtained under one specific condition (plastome data analyzed with the uncorrelated ILN clock model). That same plastome data set also yielded the oldest (mid-Permian) age estimate when analyzed with the autocorrelated TK02 clock model. Adding the highly divergent, recently established sister species Isoetes wormaldii to the data set approximately doubled the average median node depth to the Isoetes crown group. CONCLUSIONS: There is no consistent support for a Cenozoic origin of the living clade Isoetes. We obtained seemingly well-founded, yet strongly deviating results depending on data type and clock model. The single most important future improvement is probably to add calibration points, which requires an improved understanding of the isoetalean fossil record or alternative bases for calibration.

Phylogeny of the Madagascar-centred tribe Danaideae (Rubiaceae) as a precursor to taxonomic revision: insights into its generic and species limits, affinities and distribution.

BACKGROUND AND AIMS: The tribe Danaideae (Rubiaceae) is almost exclusively endemic to the Western Indian Ocean Region (WIOR), and encompasses the genera Danais, Payera and Schismatoclada that occur in humid, sub-humid and mountain and mountain bio climate zones. Much of the species diversity is endemic to restricted, remote and/or mountainous areas of Madagascar and recent field work on the island indicates substantial unknown diversity of the Danaideae. Furthermore, the monophyly of the Malagasy genera Payera and Schismatoclada has been questioned in previous work, species delimitations and phylogenetic relationships within the genera are poorly understood, and the distribution and evolution of gross morphological features have not been assessed. METHODS: We conducted morphological investigations, and produced robust phylogenies of Danaideae based on nuclear and plastid sequence data from 193 terminals. Ample plant material has been newly collected in the WIOR for the purpose of the present study, including potentially new species unknown to science. We performed Bayesian non-clock and relaxed-clock analyses employing three alternative clock models of a dataset with a dense sample of taxa from the entire geographical ranges of Danaideae. Based on the results, we discuss species diversity and distribution, relationships, and morphology in Danaideae. KEY RESULTS: Our results demonstrate the monophyly of Danaideae, its three genera and 42 species. Nine species are resolved as non-monophyletic. Many geographically distinct but morphologically heterogeneous lineages were identified, and morphological features traditionally considered diagnostic of subgroups of the genera, used for example in species identification keys, are not clade-specific. CONCLUSIONS: Our results demonstrate that Madagascar contains ample previously undocumented morphological and species diversity of Danaideae. Our novel approach to molecular phylogenetic analyses as a precursor to taxonomic revisions provides numerous benefits for the latter. There are tentative indications of parallel northward diversification in Payera and Schismatoclada on Madagascar, and of geographical phylogenetic clustering despite the anemochorous condition of Danaideae.

Phylogenetic affinity of an enigmatic Rubiaceae from the Seychelles revealing a recent biogeographic link with Central Africa: gen. nov. Seychellea and trib. nov. Seychelleeae.

The granitic islands of the Seychelles harbor about 268 native angiosperm species, with 28% being endemics there. The Seychelles biota contains a mix of ancient taxa with Gondwanan origins and young taxa that arrived there via dispersals. We investigate the phylogenetic position of an enigmatic, critically endangered, Seychellean endemic of the coffee family (Rubiaceae), Psathura/Psychotria sechellarum, and assess whether its presence on the granitic islands of the Seychelles is the result of vicariance or long-distance dispersal. Phylogenetic relationships of the family were reconstructed based on the Bayesian and maximum-likelihood phylogenetic analyses of sequence data from five plastid markers of 107 terminals. Divergence times were estimated using a Bayesian-based method. Psathura/Psychotria sechellarum is distantly related to Psychotria s.l. (including Psathura), and is strongly supported as sister to the Central African genus Colletoecema. Their striking morphological differences, coupled with their geographic separation and genetic distinctness, support the recognition of the new genus Seychellea and new tribe Seychelleeae to accommodate the Seychellean species. The Colletoecema-Seychellea clade constitutes an early-divergent lineage in the subfamily Rubioideae, with an old stem and a young crown ages estimated to be in the Late Cretaceous and late Oligocene-early Pliocene, respectively. Colletoecema diverged from Seychellea in the late Oligocene-early Pliocene and their respective crown ages are inferred to be late Miocene-middle Pleistocene and Pleistocene, respectively. The ancestor of the two genera was likely present in Africa, and reached the Seychelles via avian dispersal. Unlike Colletoecema with three species, Seychellea is monospecific, with very few individuals left in the wild. The species should be a top candidate for conservation priority, as its extinction would cause loss of genetic diversity of this entire lineage.

Conflicting results from mitochondrial genomic data challenge current views of Rubiaceae phylogeny.

PREMISE OF THE STUDY: Reconstruction of plant phylogeny has heavily relied on single-gene or multigene plastid data. New sequencing methods have led to an increasing number of studies based on data from the entire plastid, but the mitochondrion has rarely been used to infer plant phylogeny because of an assumed information poverty and demonstrated lateral transfer of mitochondrial gene regions between distantly related species. METHODS: We explored phylogenetic information from the plant mitochondrion using 57 representatives of the species-rich coffee family as study system and assessed consistency with previous results based (mostly) on plastid data. KEY RESULTS: We showed that the mitochondrial genome can provide structured and statistically significant information on plant phylogeny. While most of our results are consistent with those based on plastid data, some surprising and statistically significant conflicts emerge, and our study demonstrates with striking clarity that the phylogeny of Rubiaceae is far from resolved. CONCLUSIONS: It appears unlikely that conflicts between results retrieved from the different genomic compartments would be restricted to Rubiaceae. Rather, they are probably a general phenomenon and an important factor behind longstanding "difficult" phylogenetic questions. The biological processes responsible for the conflicting results detected here are unclear, but some conflicts are likely caused by hybridization events that occurred tens of millions of years ago. Whether such ancient events can be reconstructed based on molecular data from extant plants remains to be seen, but future studies of the nuclear genome may provide a way forward.

Aerodynamics and pollen ultrastructure in Ephedra.

UNLABELLED: • PREMISE OF THE STUDY: Pollen dispersal is affected by the terminal settling velocity (Ut) of the grains, which is determined by their size, bulk density, and by atmospheric conditions. The likelihood that wind-dispersed pollen is captured by ovulate organs is influenced by the aerodynamic environment created around and by ovulate organs. We investigated pollen ultrastructure and Ut of Ephedra foeminea (purported to be entomophilous), and simulated the capture efficiency of its ovules. Results were compared with those from previously studied anemophilous Ephedra species.• METHODS: Ut was determined using stroboscopic photography of pollen in free fall. The acceleration field around an "average" ovule was calculated, and inflight behavior of pollen grains was predicted using computer simulations. Pollen morphology and ultrastructure were investigated using SEM and STEM.• KEY RESULTS: Pollen wall ultrastructure was correlated with Ut in Ephedra. The relative proportion and amount of granules in the infratectum determine pollen bulk densities, and (together with overall size) determine Ut and thus dispersal capability. Computer simulations failed to reveal any functional traits favoring anemophilous pollen capture in E. foeminea.• CONCLUSION: The fast Ut and dense ultrastructure of E. foeminea pollen are consistent with functional traits that distinguish entomophilous species from anemophilous species. In anemophilous Ephedra species, ovulate organs create an aerodynamic microenvironment that directs airborne pollen to the pollination drops. In E. foeminea, no such microenvironment is created. Ephedroid palynomorphs from the Cretaceous share the ultrastructural characteristics of E. foeminea, and at least some may, therefore, have been produced by insect-pollinated plants.

Moonlight pollination in the gymnosperm Ephedra (Gnetales).

Most gymnosperms are wind-pollinated, but some are insect-pollinated, and in Ephedra (Gnetales), both wind pollination and insect pollination occur. Little is, however, known about mechanisms and evolution of pollination syndromes in gymnosperms. Based on four seasons of field studies, we show an unexpected correlation between pollination and the phases of the moon in one of our studied species, Ephedra foeminea. It is pollinated by dipterans and lepidopterans, most of them nocturnal, and its pollination coincides with the full moon of July. This may be adaptive in two ways. Many nocturnal insects navigate using the moon. Further, the spectacular reflection of the full-moonlight in the pollination drops is the only apparent means of nocturnal attraction of insects in these plants. In the sympatric but wind-pollinated Ephedra distachya, pollination is not correlated to the full moon but occurs at approximately the same dates every year. The lunar correlation has probably been lost in most species of Ephedra subsequent an evolutionary shift to wind pollination in the clade. When the services of insects are no longer needed for successful pollination, the adaptive value of correlating pollination with the full moon is lost, and conceivably also the trait.

Phylogenomics and topological conflicts in the tribe Anthospermeae (Rubiaceae).

Genome skimming (shallow whole-genome sequencing) offers time- and cost-efficient production of large amounts of DNA data that can be used to address unsolved evolutionary questions. Here we address phylogenetic relationships and topological incongruence in the tribe Anthospermeae (Rubiaceae), using phylogenomic data from the mitochondrion, the nuclear ribosomal cistron, and the plastome. All three genomic compartments resolve relationships in the Anthospermeae; the tribe is monophyletic and consists of three major subclades. Carpacoce Sond. is sister to the remaining clade, which comprises an African subclade and a Pacific subclade. Most results, from all three genomic compartments, are statistically well supported; however, not fully consistent. Intergenomic topological incongruence is most notable in the Pacific subclade but present also in the African subclade. Hybridization and introgression followed by organelle capture may explain these conflicts but other processes, such as incomplete lineage sorting (ILS), can yield similar patterns and cannot be ruled out based on the results. Whereas the null hypothesis of congruence among all sequenced loci in the individual genomes could not be rejected for nuclear and mitochondrial data, it was rejected for plastid data. Phylogenetic analyses of three subsets of plastid loci identified using the hierarchical likelihood ratio test demonstrated statistically supported intragenomic topological incongruence. Given that plastid genes are thought to be fully linked, this result is surprising and may suggest modeling or sampling error. However, biological processes such as biparental inheritance and inter-plastome recombination have been reported and may be responsible for the observed intragenomic incongruence. Mitochondrial insertions into the plastome are rarely documented in angiosperms. Our results indicate that a mitochondrial insertion event in the plastid trnS GGA - rps4 IGS region occurred in the common ancestor of the Pacific clade of Anthospermeae. Exclusion/inclusion of this locus in phylogenetic analyses had a strong impact on topological results in the Pacific clade.

Plastid phylogenomics and cytonuclear discordance in Rubioideae, Rubiaceae.

In this study of evolutionary relationships in the subfamily Rubioideae (Rubiaceae), we take advantage of the off-target proportion of reads generated via previous target capture sequencing projects based on nuclear genomic data to build a plastome phylogeny and investigate cytonuclear discordance. The assembly of off-target reads resulted in a comprehensive plastome dataset and robust inference of phylogenetic relationships, where most intratribal and intertribal relationships are resolved with strong support. While the phylogenetic results were mostly in agreement with previous studies based on plastome data, novel relationships in the plastid perspective were also detected. For example, our analyses of plastome data provide strong support for the SCOUT clade and its sister relationship to the remaining members of the subfamily, which differs from previous results based on plastid data but agrees with recent results based on nuclear genomic data. However, several instances of highly supported cytonuclear discordance were identified across the Rubioideae phylogeny. Coalescent simulation analysis indicates that while ILS could, by itself, explain the majority of the discordant relationships, plastome introgression may be the better explanation in some cases. Our study further indicates that plastomes across the Rubioideae are, with few exceptions, highly conserved and mainly conform to the structure, gene content, and gene order present in the majority of the flowering plants.

Phylogeny of Merlin's grass (Isoetaceae): revealing an "Amborella syndrome" and the importance of geographic distribution for understanding current and historical diversity.

BACKGROUND: Merlin's grass (Isoetes, Isoetaceae, Lycopsida), is the extant remnant of the isoetalean wood-producing lycopsids that originated during the Paleozoic, possibly in aquatic or boggy habitats. Modern day species are aquatic, semi-aquatic or terrestrial and occur almost worldwide. They display little morphological variation; the lobed corm has helically arranged leaves with internal air channels and basal sporangia. Genetic variation has also proven limited, which has hampered phylogenetic inference. We investigate evolutionary relationships in Isoetes, using molecular data and an extended sample of species compared to previous work, adding species that have never before been included in a phylogenetic study. RESULTS: Our results reveal an unexpected discovery of an "Amborella syndrome" in Isoetaceae: a single poorly known species is sister to the remaining family. The species, Isoetes wormaldii, is a rare endemic to the Eastern Cape of South Africa. Its leaves are flattened with a rounded point, which sharply contrasts with the awl-shaped leaves of most other species of Isoetes. The remaining species of Isoetes are resolved in five major clades, also indicated in previous work. While the phylogeny shows geographic structure, the patterns are complex. For example, tropical-southern African species occur in at least five clades, and Indian, Australian and Mediterranean species in at least three clades each. CONCLUSION: The evolutionary and biogeographical history of Isoetes is not easily explained, and may conceivably include ample extinction and a mixture of ancient and more recent processes. Previously shown difficulties with node age estimation increase the problem. The here demonstrated sister-relationship between the phylogenetically, morphologically and genetically distinct Isoetes wormaldii and the remaining family appears to bridge the morphological gap between Isoetes and its extinct relatives, although further studies are needed. Moreover, it shortens the branch length to its living sister genus Selaginella, and may enhance node age estimation in future studies. Isoetes wormaldii is critically endangered, known only from one (to a few) minor populations. Immediate actions need to be taken if we want to prevent this unique species from going extinct.

Target capture data resolve recalcitrant relationships in the coffee family (Rubioideae, Rubiaceae).

Subfamily Rubioideae is the largest of the main lineages in the coffee family (Rubiaceae), with over 8,000 species and 29 tribes. Phylogenetic relationships among tribes and other major clades within this group of plants are still only partly resolved despite considerable efforts. While previous studies have mainly utilized data from the organellar genomes and nuclear ribosomal DNA, we here use a large number of low-copy nuclear genes obtained via a target capture approach to infer phylogenetic relationships within Rubioideae. We included 101 Rubioideae species representing all but two (the monogeneric tribes Foonchewieae and Aitchinsonieae) of the currently recognized tribes, and all but one non-monogeneric tribe were represented by more than one genus. Using data from the 353 genes targeted with the universal Angiosperms353 probe set we investigated the impact of data type, analytical approach, and potential paralogs on phylogenetic reconstruction. We inferred a robust phylogenetic hypothesis of Rubioideae with the vast majority (or all) nodes being highly supported across all analyses and datasets and few incongruences between the inferred topologies. The results were similar to those of previous studies but novel relationships were also identified. We found that supercontigs [coding sequence (CDS) + non-coding sequence] clearly outperformed CDS data in levels of support and gene tree congruence. The full datasets (353 genes) outperformed the datasets with potentially paralogous genes removed (186 genes) in levels of support but increased gene tree incongruence slightly. The pattern of gene tree conflict at short internal branches were often consistent with high levels of incomplete lineage sorting (ILS) due to rapid speciation in the group. While concatenation- and coalescence-based trees mainly agreed, the observed phylogenetic discordance between the two approaches may be best explained by their differences in accounting for ILS. The use of target capture data greatly improved our confidence and understanding of the Rubioideae phylogeny, highlighted by the increased support for previously uncertain relationships and the increased possibility to explore sources of underlying phylogenetic discordance.

A new early Cretaceous relative of Gnetales: Siphonospermum simplex gen. et sp. nov. from the Yixian formation of northeast China.

BACKGROUND: Knowledge on fossil and evolutionary history of the Gnetales has expanded rapidly; Ephedra and ephedroids as well as the Gnetum-Welwitschia clade are now well documented in the Early Cretaceous. However, hypotheses on evolutionary relationships among living and fossil species are hampered by restricted knowledge of morphological variation in living groups and recent studies indicate that gnetalean diversity and character evolution may be more complex than previously assumed and involve additional extinct groups (Bennettitales, Erdtmanithecales and unassigned fossil taxa). RESULTS: Here we describe a new fossil related to Gnetales, Siphonospermum simplex from the Early Cretaceous Yixian Formation, an impression/compression of a reproductive shoot. The slender main axis bears one pair of opposite and linear leaves with primary parallel venation. The reproductive units are ovoid, without supporting bracts and borne on one median and two lateral branches. The most conspicuous feature of the fossil is the long, thread-like micropylar tube formed by the integument. Each ovule is surrounded by two different layers representing one or two seed envelopes; an inner sclerenchymatous layer and an outer probably parenchymatous layer. CONCLUSIONS: The vegetative and reproductive features of Siphonospermum simplex exclude a relationship to any other group than the Gnetales. A combination of opposite phyllotaxis, linear leaves and ovules surrounded by seed envelope(s) and with a long exposed micropylar tube are known only for extant and extinct Gnetales. Siphonospermum simplex constitutes a new lineage within the Gnetales. Its morphology cannot be directly linked to any previously known plant, but the organization of the reproductive units indicates that it belongs to the Gnetum-Welwitschia clade. Based on the absence of cone bracts and the inferred histology of the seed envelope(s) it could be related to Gnetum, however, there are also affinities with the ephedran lineage, some of which are likely plesiomorphic features, others perhaps not. Phylogeny and character evolution in the Bennettitales, Erdtmanithecales and Gnetales are currently only partly understood and under debate; the exact systematic position of Siphonospermum simplex, i.e., its position within the Gnetales, cannot be resolved with certainty.

Rare and enigmatic genera (Dunnia, Schizocolea, Colletoecema), sisters to species-rich clades: phylogeny and aspects of conservation biology in the coffee family.

Despite extensive efforts, parts of the phylogeny of the angiosperm family Rubiaceae has not been resolved and consequently, character evolution, ancestral areas and divergence times of major radiations are difficult to estimate. Here, phylogenetic analyses of 149 taxa and five plastid gene regions show that three enigmatic genera are sisters to considerably species rich clades. The rare and endangered species Dunnia, endemic to southern Guangdong, China, is sister to a large clade in the Spermacoceae alliance; the rarely collected Schizocolea from western tropical Africa is sister to the Psychotrieae alliance; and Colletoecema from central tropical Africa is sister to remaining Rubioideae. The morphology of these taxa has been considered "puzzling". In combination with further morphological studies, our results may help understanding the apparently confusing traits of these plants. Phylogenetic, morphological, and geographical isolation of Dunnia, Schizocolea and Colletocema may indicate high genetic diversity. They are lone representatives of unique lineages and if extinct, the loss would not only mean loss of genetic diversity of a single species but of an entire lineage.

Evolution and trends in the Psychotrieae alliance (Rubiaceae)--a rarely reported evolutionary change of many-seeded carpels from one-seeded carpels.

Bayesian and parsimony analyses of five plastid gene and nrITS regions from 58 Rubioideae (Rubiaceae) taxa further support the sister-group relationship between the African monotypic genus Schizocolea and the Psychotrieae alliance sensu Bremer & Manen. Our analyses show that the Psychotrieae alliance can be subdivided into in four well-supported clades: Schizocolea, (Schradereae(Gaertnereae(Mitchelleae-Morindeae s.s.))), Palicoureeae-Psychotrieae s.s., and Craterispermeae-Prismatomerideae. The relationships between the latter three clades remain unsettled. Our study further reveals much higher numbers of molecular autapomorphies of the tribes compared with those of molecular synapomorphies of two sister tribes or groups of tribes. Within the newly delimited Psychotrieae alliance a one-seeded carpel was inferred as ancestral and many- and two-seeded carpels evolved once each. We describe Mitchelleae to accommodate Damnacanthus and Mitchella and restrict Morindeae to include only Appunia, Coelospermum, Gynochthodes, Morinda, Pogonolobus, and Syphonandrium. Mitchelleae is characterized e.g., by placentae inserted near the top of the septum and a single campylotropous ovule per carpel, while Morindeae s.s. has massive and T-shaped placentae inserted in the middle of the septum and two anatropous ovules per carpel.

Phylogeny and generic limits in the Niemeyera complex of New Caledonian Sapotaceae: evidence of multiple origins of the anisomerous flower.

Traditional generic limits within the "Niemeyera complex" (Sapotaceae: Chrysophylloideae) in Australia and New Caledonia do not correspond to natural groups. We analyzed nuclear (ETS, ITS) and chloroplast (trnH-psbA, trnS-G) sequence data, and 42 morphological characters, using a near-complete taxon sampling. The resulting phylogeny provides a new generic framework where Leptostylis and Sebertia are monophyletic, Niemeyera is recognized as a small genus confined to Australia, and the circumscriptions of Achradotypus and Pycnandra require significant modification. This framework allows about 20 recently discovered species to be described in appropriate genera and assessed for their conservation status. Evolutionary changes in two widely used characters, anisomerous flowers and the number of stamens inserted opposite each corolla lobe, have occurred multiple times. There is no evidence that anisomery originated through hybridization as suggested in other groups. Instead, the two characters are closely coupled and often mutually exclusive. The diagnostic value of morphological characters varies considerably; for example, the presence, absence, and type of malpighiaceous hairs convey more phylogenetic information than many traditionally used features. Criteria and options for a new generic classification are discussed, and a reconstruction of the hypothesized ancestor that likely colonized New Caledonia in the Oligocene is presented.

Gymnosperms on the EDGE.

Driven by limited resources and a sense of urgency, the prioritization of species for conservation has been a persistent concern in conservation science. Gymnosperms (comprising ginkgo, conifers, cycads, and gnetophytes) are one of the most threatened groups of living organisms, with 40% of the species at high risk of extinction, about twice as many as the most recent estimates for all plants (i.e. 21.4%). This high proportion of species facing extinction highlights the urgent action required to secure their future through an objective prioritization approach. The Evolutionary Distinct and Globally Endangered (EDGE) method rapidly ranks species based on their evolutionary distinctiveness and the extinction risks they face. EDGE is applied to gymnosperms using a phylogenetic tree comprising DNA sequence data for 85% of gymnosperm species (923 out of 1090 species), to which the 167 missing species were added, and IUCN Red List assessments available for 92% of species. The effect of different extinction probability transformations and the handling of IUCN data deficient species on the resulting rankings is investigated. Although top entries in our ranking comprise species that were expected to score well (e.g. Wollemia nobilis, Ginkgo biloba), many were unexpected (e.g. Araucaria araucana). These results highlight the necessity of using approaches that integrate evolutionary information in conservation science.

The chloroplast genome of Ephedra foeminea (Ephedraceae, Gnetales), an entomophilous gymnosperm endemic to the Mediterranean area.

This study presents the chloroplast genome of Ephedra foeminea, an entomophilous gymnosperm, sister to the remaining (wind-pollinated) species of Ephedra (Ephedraceae, Gnetales). Based on the reference-guided assembly, the length of the chloroplast genome was estimated to be 109 584 bp, comprising a large single copy region of 60 027 bp, a small single copy 8079 bp, and inverted repeat regions of 20 739 bp. In total, 118 genes were detected, including 73 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The gene density is 1.076 (genes/kb) and the GC content is 36.7%. The genomic sequence of the entomophilous, Mediterranean species E. foeminea, differs from that of the anemophilous, Asian species E. equisetina by 1018 point mutations and 1334 indels. The detected variation is useful for future development of new plastid markers for phylogenetic purposes. Our phylogenetic analysis based on 55 protein-coding chloroplast genes resolve Ephedra as monophyletic and sister to a Gnetum-Welwitschia clade. The Gnetales are sister to Cupressophytes.

The Huperzia selago Shoot Tip Transcriptome Sheds New Light on the Evolution of Leaves.

Lycopodiophyta-consisting of three orders, Lycopodiales, Isoetales and Selaginellales, with different types of shoot apical meristems (SAMs)-form the earliest branch among the extant vascular plants. They represent a sister group to all other vascular plants, from which they differ in that their leaves are microphylls-that is, leaves with a single, unbranched vein, emerging from the protostele without a leaf gap-not megaphylls. All leaves represent determinate organs originating on the flanks of indeterminate SAMs. Thus, leaf formation requires the suppression of indeterminacy, that is, of KNOX transcription factors. In seed plants, this is mediated by different groups of transcription factors including ARP and YABBY.We generated a shoot tip transcriptome of Huperzia selago (Lycopodiales) to examine the genes involved in leaf formation. Our H. selago transcriptome does not contain any ARP homolog, although transcriptomes of Selaginella spp. do. Surprisingly, we discovered a YABBY homolog, although these transcription factors were assumed to have evolved only in seed plants.The existence of a YABBY homolog in H. selago suggests that YABBY evolved already in the common ancestor of the vascular plants, and subsequently was lost in some lineages like Selaginellales, whereas ARP may have been lost in Lycopodiales. The presence of YABBY in the common ancestor of vascular plants would also support the hypothesis that this common ancestor had a simplex SAM. Furthermore, a comparison of the expression patterns of ARP in shoot tips of Selaginella kraussiana (Harrison CJ, etal. 2005. Independent recruitment of a conserved developmental mechanism during leaf evolution. Nature 434(7032):509-514.) and YABBY in shoot tips of H. selago implies that the development of microphylls, unlike megaphylls, does not seem to depend on the combined activities of ARP and YABBY. Altogether, our data show that Lycopodiophyta are a diverse group; so, in order to understand the role of Lycopodiophyta in evolution, representatives of Lycopodiales, Selaginellales, as well as of Isoetales, have to be examined.

Cratonia cotyledon gen. et sp. nov: a unique Cretaceous seedling related to Welwitschia.

The fossil history of most extant seed plant groups is relatively well documented. Cycads, conifers and Ginkgo all have an extensive fossil record, and the understanding of early angiosperm diversity is increasing. The Gnetales are an exception. Few macrofossils have been described, and character evolution within the group is poorly known. Cratonia cotyledon is a new gnetalean fossil from the Early Cretaceous Crato Formation of Brazil. This well-preserved seedling consists of two cotyledons, a feeder and a root. The leaf surface shows polygonal epidermal cells and apparently paracytic or actinocytic stomata. The cotyledons have a very specific venation pattern, shared only by Cratonia and Welwitschia, with parallel primary veins and secondary veins fusing to form inverted 'Y's between the main veins. Based on the 'Y'-venation and the presence of a feeder, we assign Cratonia to the Gnetum-Welwitschia clade. Fossil seedlings are unusual and this complete specimen with unambiguously welwitschioid characters is spectacular. Cratonia indicates that the evolutionary split between Gnetum and Welwitschia had occurred in the Early Cretaceous. Further, the close relationship between a West African plant and an east South American Early Cretaceous fossil is consistent with a major geological event: the rifting of the Gondwana continent.

Taxon sampling and seed plant phylogeny.

We investigated the effects of taxon sampling on phylogenetic inference by exchanging terminals in two sizes of rbcL matrices for seed plants, applying parsimony and bayesian analyses to ten 38-taxon matrices and ten 80-taxon matrices. In comparing tree topologies we concentrated on the position of the Gnetales, an important group whose placement has long been disputed. With either method, trees obtained from different taxon samples could be mutually contradictory and even disagree on groups that seemed strongly supported. Adding terminals improved the consistency of results for unweighted parsimony, but not for parsimony with third positions excluded and not for bayesian analysis, particularly when the general time-reversible model was employed. This suggests that attempting to resolve deep relationships using only a few taxa can lead to spurious conclusions, groupings unlikely to be repeatable with different taxon samplings or larger data sets. The effect of taxon sampling has not generally been recognized, and phylogenetic studies of seed plants have often been based on few taxa. Such insufficient sampling may help explain the variety of phylogenetic hypotheses for seed plants proposed in recent years. We recommend that restricted data sets such as single-gene subsets of multigene studies should be reanalyzed with alternative selections of terminals to assess topological consistency.

The female reproductive unit of ephedra (Gnetales): comparative morphology and evolutionary perspectives.

Morphological variation in Ephedra (Gnetales) is limited and confusing from an evolutionary perspective, with parallelisms and intraspecific variation. However, recent analyses of molecular data provide a phylogenetic framework for investigations of morphological traits, albeit with few informative characters in the investigated gene regions. We document morphological, anatomical and histological variation patterns in the female reproductive unit and test the hypothesis that some Early Cretaceous fossils, which share synapomorphies with Ephedra, are members of the extant clade. Results indicate that some morphological features are evolutionarily informative although intraspecific variation is evident. Histology and anatomy of cone bracts and seed envelopes show clade-specific variation patterns. There is little evidence for an inclusion of the Cretaceous fossils in the extant clade. Rather, a hypothesized general pattern of reduction of the vasculature in the ephedran seed envelope, probably from four vascular bundles in the fossils, to ancestrally three in the living clade, and later to two, is consistent with phylogenetic and temporal analyses, which indicate that extant diversity evolved after the Cretaceous-Tertiary boundary. Notwithstanding striking similarities between living and Cretaceous Ephedra, available data indicate that the Mesozoic diversity went almost entirely extinct in the late Cretaceous causing a bottleneck effect in Ephedra, still reflected today by an extraordinarily low level of genetic and structural diversity.