Comprehensive phylogenomic time tree of bryophytes reveals deep relationships and uncovers gene incongruences in the last 500 million years of diversification.

PREMISE: Bryophytes form a major component of terrestrial plant biomass, structuring ecological communities in all biomes. Our understanding of the evolutionary history of hornworts, liverworts, and mosses has been significantly reshaped by inferences from molecular data, which have highlighted extensive homoplasy in various traits and repeated bursts of diversification. However, the timing of key events in the phylogeny, patterns, and processes of diversification across bryophytes remain unclear. METHODS: Using the GoFlag probe set, we sequenced 405 exons representing 228 nuclear genes for 531 species from 52 of the 54 orders of bryophytes. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed gene conflict, and estimated the timing of divergences based on 29 fossil calibrations. RESULTS: The phylogeny resolves many relationships across the bryophytes, enabling us to resurrect five liverwort orders and recognize three more and propose 10 new orders of mosses. Most orders originated in the Jurassic and diversified in the Cretaceous or later. The phylogenomic data also highlight topological conflict in parts of the tree, suggesting complex processes of diversification that cannot be adequately captured in a single gene-tree topology. CONCLUSIONS: We sampled hundreds of loci across a broad phylogenetic spectrum spanning at least 450 Ma of evolution; these data resolved many of the critical nodes of the diversification of bryophytes. The data also highlight the need to explore the mechanisms underlying the phylogenetic ambiguity at specific nodes. The phylogenomic data provide an expandable framework toward reconstructing a comprehensive phylogeny of this important group of plants.

Lethal and sublethal effects of essential oils fromPiper capitarianumYunck andPiper krukoffiiYunck onPlutella xylostellaL.

Plutella xylostella (L.) is responsible for considerable vegetable crop losses in the metropolitan region of Manaus, Brazil. In recent decades, essential oils have been investigated as an alternative to synthetic insecticides. The genusPiperis widely distributed in Amazonia and essential oils from these plants have insecticidal properties. This study describes the chemical composition of the essential oils fromPiper capiterianumandPiper krukoffiias well as the lethal and sublethal effects onP. xylostella. The phytotoxicity of the oils on the host plant was also evaluated. Globulol was the major constituent of theP. krukoffiioil ando-cymene was the major constituent of theP. capitarianumoil. The oil fromP. capiterianumexhibited greater toxicity to larvae and eggs. This oil also presented greater repellant action, feeding deterrence and mild phytotoxicity to the host plant (Brassicae oleraceae). The findings suggest that this oil can be used in the preparation of a formulated insecticide for the management ofP. xylostellain different development phases. However, further studies are needed to evaluate the effect of this oil on crops under field conditions as well as non-target organisms and determine the cost-benefit ratio of a product formulated withP. capitarianumoil.

The innovation of the symbiosome has enhanced the evolutionary stability of nitrogen fixation in legumes.

Nitrogen-fixing symbiosis is globally important in ecosystem functioning and agriculture, yet the evolutionary history of nodulation remains the focus of considerable debate. Recent evidence suggesting a single origin of nodulation followed by massive parallel evolutionary losses raises questions about why a few lineages in the N2 -fixing clade retained nodulation and diversified as stable nodulators, while most did not. Within legumes, nodulation is restricted to the two most diverse subfamilies, Papilionoideae and Caesalpinioideae, which show stable retention of nodulation across their core clades. We characterize two nodule anatomy types across 128 species in 56 of the 152 genera of the legume subfamily Caesalpinioideae: fixation thread nodules (FTs), where nitrogen-fixing bacteroids are retained within the apoplast in modified infection threads, and symbiosomes, where rhizobia are symplastically internalized in the host cell cytoplasm within membrane-bound symbiosomes (SYMs). Using a robust phylogenomic tree based on 997 genes from 147 Caesalpinioideae genera, we show that losses of nodulation are more prevalent in lineages with FTs than those with SYMs. We propose that evolution of the symbiosome allows for a more intimate and enduring symbiosis through tighter compartmentalization of their rhizobial microsymbionts, resulting in greater evolutionary stability of nodulation across this species-rich pantropical legume clade.

Chemical composition of essential oils from four Piper species, differentiation using multivariate analysis and antioxidant activity.

Essential oils from the leaves of four species of Piper obtained through hydrodistillation were analyzed using GC-MS andmultivariate data analysis. The chemical analysis enabled the identification of qualitative and quantitative differences among the oils. ß-selinene (32.44 ± 1.14%), (E)-nerolidol (44.23 ± 2.23%), ß-caryophyllene (19.11 ± 0.40%) and caryophyllene oxide (16.92 ± 0.21%) were identified as the major constituents of the P. mollipilosum, P. brachypetiolatum, P. glandulosissimum and P. madeiranum oils, respectively. The differences in the chemical profiles of the oils were confirmed by principal component analysis. All four species exhibited antioxidant activity. The oil from P. brachypetiolatum achieved the best results on the DPPH test (EC50 = 64.8 µg/ml) and with the ABTS radical (EC50 = 159.7 µg/ml).

A dated molecular phylogeny and biogeographical analysis reveals the evolutionary history of the trans-pacifically disjunct tropical tree genus Ormosia (Fabaceae).

The papilionoid legume genus Ormosia (Fabaceae) comprises about 150 species of trees and exhibits a striking disjunct geographical distribution between the New World- and Asian and Australasian wet tropics and subtropics. Modern classifications of Ormosia are not grounded on a well-substantiated phylogenetic hypothesis and have been limited to just portions of the geographical range of the genus. The lack of an evolutionarily-based foundation for systematic studies has hindered taxonomic work on the genus and prevented the testing of biogeographical hypotheses related to the origin of the Old World/New World disjunction and the individual dispersal histories within both areas. Here, we present the most comprehensively sampled molecular phylogeny of Ormosia to date, based on analysis of both nuclear (ITS) and plastid (matK and trnL-F) DNA sequences from 82 species of the genus. Phylogenetically-based divergence times and ancestral range estimations are employed to test hypotheses related to the biogeographical history of the genus. We find strong support for the monophyly of Ormosia and the grouping of all sampled Asian species of the genus into two comparably sized clades, one of which is sister to another large clade containing all sampled New World species. Within the New World clade, additional resolution supports the grouping of most species into three mutually exclusive subordinate clades. The remaining New World species form a fourth well-supported clade in the analyses of plastid sequences, but that result is contradicted by the analysis of ITS. With few exceptions the supported clades have not been previously recognized as taxonomic groups. The biogeographical analysis suggests that Ormosia originated in continental Asia and dispersed to the New World in the Oligocene or early Miocene via long-distance trans-oceanic dispersal. We reject the hypothesis that the inter-hemispheric disjunction in Ormosia resulted from fragmentation of a more continuous "Boreotropical" distribution since the dispersal post-dates Eocene climatic maxima. Both of the Old World clades appear to have originated in mainland Asia and subsequently dispersed into the Malay Archipelago and beyond, at least two lineages dispersing across Wallace's Line as far as the Solomon Islands and northeastern Australia. In the New World, the major clades all originated in Amazonia. Dispersal from Amazonia into peripheral areas in Central America, the Caribbean, and Extra-Amazonian Brazil occurred multiple times over varying time scales, the earliest beginning in the late Miocene. In a few cases, these dispersals were followed by local diversification, but not by reverse migration back to Amazonia. Within each of the two main areas of distribution, multiple modest bouts of oceanic dispersal were required to achieve the modern distributions.

What do tropical cryptogams reveal? Strong genetic structure in Amazonian bryophytes.

Lowland tropical bryophytes have been perceived as excellent dispersers. In such groups, the inverse isolation hypothesis proposes that spatial genetic structure is erased beyond the limits of short-distance dispersal. Here, we determine the influence of environmental variation and geographic barriers on the spatial genetic structure of a widely dispersed and phylogenetically independent sample of Amazonian bryophytes. Single nucleotide polymorphism data were produced from a restriction site-associated DNA sequencing protocol for 10 species and analyzed through F-statistics and Mantel tests. Neither isolation-by-environment nor the impact of geographic barriers were recovered from the analyses. However, significant isolation-by-distance patterns were observed for 8 out of the 10 investigated species beyond the scale of short-distance dispersal (> 1 km), offering evidence contrary to the inverse isolation hypothesis. Despite a cadre of life-history traits and distributional patterns suggesting that tropical bryophytes are highly vagile, our analyses reveal spatial genetic structures comparable to those documented for angiosperms, whose diaspores are orders of magnitude larger. Dispersal limitation for tropical bryophytes flies in the face of traditional assumptions regarding their dispersal potential, and suggests that the plight of this component of cryptic biodiversity is more dire than previously considered in light of accelerated forest fragmentation in the Amazon.

Insights on the systematics and morphology of Humiriaceae (Malpighiales): androecial and extrafloral nectary variation, two new combinations, and a new Sacoglottis from Guyana.

Humiriaceae have had little recent comparative morphological study except for their distinctive fruits. We surveyed the diversity of stamen structures in the family with consideration of dehiscence patterns and the evolutionary transitions between tetra- and disporangiate anthers. Novel interpretations of floral morphology support new combinations (Duckesialiesneri K.Wurdack & C.E.Zartman, comb. nov. and Vantaneaspiritu-sancti K.Wurdack & C.E.Zartman, comb. nov.) for two species formerly in Humiriastrum. We investigated all eleven species of Sacoglottis for diagnostic features that may contribute to better species delimitations, and describe Sacoglottisperryi K.Wurdack & C.E.Zartman, sp. nov. as an endemic of the Pakaraima Mountains in western Guyana. Finally, our survey across Humiriaceae for extrafloral nectaries (EFNs) revealed their presence on leaves of all extant species as adaxial basilaminar and/or abaxial embedded glands, in addition to the frequent occurrence of marginal glandular setae. The significance of inter-generic variation in gland position and anther morphology within the family are discussed.

Amazon plant diversity revealed by a taxonomically verified species list.

Recent debates on the number of plant species in the vast lowland rain forests of the Amazon have been based largely on model estimates, neglecting published checklists based on verified voucher data. Here we collate taxonomically verified checklists to present a list of seed plant species from lowland Amazon rain forests. Our list comprises 14,003 species, of which 6,727 are trees. These figures are similar to estimates derived from nonparametric ecological models, but they contrast strongly with predictions of much higher tree diversity derived from parametric models. Based on the known proportion of tree species in neotropical lowland rain forest communities as measured in complete plot censuses, and on overall estimates of seed plant diversity in Brazil and in the neotropics in general, it is more likely that tree diversity in the Amazon is closer to the lower estimates derived from nonparametric models. Much remains unknown about Amazonian plant diversity, but this taxonomically verified dataset provides a valid starting point for macroecological and evolutionary studies aimed at understanding the origin, evolution, and ecology of the exceptional biodiversity of Amazonian forests.

How does reproductive strategy influence demography? A case study in the tropical, unisexual epiphyllous moss Crossomitrium patrisiae.

PREMISE OF THE STUDY: Leaf-inhabiting organisms offer an experimentally tractable model system within which to investigate the influence of alternative reproductive strategies on plant metapopulation dynamics. We conducted a field study to determine whether (1) threshold colony sizes exist for the onset of sexual and asexual expression, and (2) alternative reproductive strategies differentially influence within-patch dynamics of the tropical pleurocarpous moss Crossomitrium patrisiae. METHODS: The growth, reproduction, and fate of 2101 colonies of C. patrisiae were followed over 2 years to investigate threshold size and age for sporophyte and brood branch formation and their influence on within-patch growth rates and longevity. KEY RESULTS: Asexual expression rather than sexual onset was limited by a minimal colony size. Age was uncoupled with threshold sizes. Colonies bearing brood branches survived nearly twice as long as sterile and solely sporophytic colonies. However, no effect of reproductive strategies on colony growth rates was found. CONCLUSIONS: This study is among the few attempts to correlate life history strategies with demographic parameters of terrestrial plants. Specifically, we provide evidence for differential influence of reproductive strategies on metapopulation survivorship.

Prezygotic resource-allocation dynamics and reproductive trade-offs in Calymperaceae (Bryophyta).

PREMISE OF THE STUDY: Resource allocation is difficult to characterize in plants because of the challenges of quantifying gametes and propagules. We surveyed six sympatric, unisexual species in the family Calymperaceae (Bryophyta) to test for trade-offs in prezygotic sexual and asexual expression and density-dependent survivorship of female gametangia. METHODS: We tallied gametangial and asexual propagule output for 1820 shoots from 17 populations of six species at monthly intervals during one year (2010-2011) in a central Amazonian forest. Generalized linear mixed models were used to test for trade-offs in sexual and asexual expression and density-dependent senescence probability of gametangia. Precipitation and microsite variables were also included in the model. KEY RESULTS: For all species, sexual and asexual expression were positively correlated with mean monthly precipitation. Asexually expressing shoots produced significantly fewer gametangia than nonexpressing ones, and the probability of senescence increased with shoot density. Archegonium density per shoot was also consistently lower than the modeled optimum to maximize the number of receptive archegonia. CONCLUSIONS: Trade-offs among reproductive strategies and positive density-dependent senescence of female gametangia suggest that prezygotic sexual and asexual expression come at a tangible investment. However, the apparently inefficient resource-allocation dynamics in the production of female gametangia makes the possible advantages of squandering such investments unclear. One possibility is that the study populations, like those of many dioicous mosses, are skewed toward expressing females with low sporophyte production, which would suggest that asexual reproduction predominates and upstages efficient resource allocation in prezygotic investment.

Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes.

Aldina (Leguminosae) is among the very few ecologically successful ectomycorrhizal lineages in a family largely marked by the evolution of nodulating symbiosis. The genus comprises 20 species predominantly distributed in Amazonia and has been traditionally classified in the tribe Swartzieae because of its radial flowers with an entire calyx and numerous free stamens. The taxonomy of Aldina is complicated due to its poor representation in herbaria and the lack of a robust phylogenetic hypothesis of relationship. Recent phylogenetic analyses of matK and trnL sequences confirmed the placement of Aldina in the 50-kb inversion clade, although the genus remained phylogenetically isolated or unresolved in the context of the evolutionary history of the main early-branching papilionoid lineages. We performed maximum likelihood and Bayesian analyses of combined chloroplast datasets (matK, rbcL, and trnL) and explored the effect of incomplete taxa or missing data in order to shed light on the enigmatic phylogenetic position of Aldina. Unexpectedly, a sister relationship of Aldina with the Andira clade (Andira and Hymenolobium) is revealed. We suggest that a new tribal phylogenetic classification of the papilionoid legumes should place Aldina along with Andira and Hymenolobium. These results highlight yet another example of the independent evolution of radial floral symmetry within the early-branching Papilionoideae, a large collection of florally heterogeneous lineages dominated by papilionate or bilaterally symmetric flower morphology.

Flowers of the early-branching papilionoid legume Petaladenium urceoliferum display unique morphological and ontogenetic features.

PREMISE OF THE STUDY: Floral development can help to shed light on puzzling features across flowering plants. The enigmatic Amazonian monospecific genus Petaladenium of the legume family (Leguminosae) had rarely been collected and only recently became available for ontogenetic studies. The fimbriate-glandular wing petals of P. urceoliferum are unique among the more than 19000 legume species. Ontogenetic data illuminate the systematic position of the genus and foster our understanding on floral evolution during the early diversification of the papilionoid legumes. METHODS: Flower buds were collected in the field, fixed in 70% ethanol, and investigated using scanning electron microscopy (SEM). Results were compared with existing material from early-diverging papilionoid legumes. KEY RESULTS: Formation of sepals and petals shows bidirectional tendencies. Stamens arise in two whorls, and the single carpel arises concomitantly with the outer stamen whorl. Gland formation starts early on the edges of the wing petals. The carpel reopens for a short time when the initiation of ovules is visible. Stomata at the base of the hypanthium indicate that the flower functions like other standard flag blossoms. CONCLUSIONS: The floral ontogeny confirms the close affinity of P. urceoliferum with the florally heterogeneous, early-diverging papilionoid Amburaneae clade. The results strengthen the theory of a distinct experimental phase among early-branching papilionoid legumes during which a wider range of floral morphologies arose. Polysymmetry, monosymmetry, variable organ numbers, and a wide range of ontogenetic patterns laid the foundation for a successful canalization toward the more restricted but well-adapted dorsiventral papilionoid flag blossom.

Monomethyl ethers of 4,5-dihydroxypipecolic acid from Petaladenium urceoliferum: Enigmatic chemistry of an enigmatic legume.

Leaves of Petaladenium (Leguminosae), an Amazonian monospecific genus recently revealed as a member of the Amburaneae clade among the earliest-diverging papilionoid legumes, were found to accumulate three monomethyl ethers of 4,5-dihydroxypipecolic acids. These were characterised by spectroscopic means as the (2S,4S,5R) and (2S,4R,5S) epimers of 5-hydroxy-4-methoxypipecolic acid and (2S,4R,5R)-4-hydroxy-5-methoxypipecolic acid. These compounds were not detected in any other genera in the Amburaneae clade or the wider Angylocalyceae-Dipterygeae-Amburaneae (ADA) clade of papilionoid legumes. Hydroxypipecolic acids, however, were detected in leaves of Myrocarpus and Myroxylon (sister genera in the Amburaneae clade), Angylocalyx and Xanthocercis (sister genera in the Angylocalyceae clade) and Monopteryx (Dipterygeae clade), and were also present in Petaladenium. Iminosugars, known to be accumulated by all four genera in the Angylocalyceae clade (Alexa, Angylocalyx, Castanospermum and Xanthocercis), were found to be characteristic of this group within the ADA clade.

Filling in the gaps of the papilionoid legume phylogeny: the enigmatic Amazonian genus Petaladenium is a new branch of the early-diverging Amburaneae clade.

Recent deep-level phylogenies of the basal papilionoid legumes (Leguminosae, Papilionoideae) have resolved many clades, yet left the phylogenetic placement of several genera unassessed. The phylogenetically enigmatic Amazonian monospecific genus Petaladenium had been believed to be close to the genera of the Genistoid Ormosieae clade. In this paper we provide the first DNA phylogenetic study of Petaladenium and show it is not part of the large Genistoid clade, but is a new branch of the Amburaneae clade, one of the first-diverging lineages of the Papilionoideae phylogeny. This result is supported by the chemical observation that the quinolizidine alkaloids, a chemical synapomorphy of the Genistoids, are absent in Petaladenium. Parsimony and Bayesian phylogenetic analysis of nuclear ITS/5.8S and plastid matK and trnL intron agree with a new interpretation of morphology that Petaladenium is sister to Dussia, a genus comprising ∼18 species of trees largely confined to rainforests in Central America and northern South America. Petaladenium, Dussia, and Myrospermum have papilionate flowers in a clade otherwise with radial floral symmetry, loss of petals or incompletely differentiated petals. Our phylogenetic analyses also revealed well-supported resolution within the three main lineages of the ADA clade (Angylocalyceae, Dipterygeae, and Amburaneae). We also discuss further molecular phylogenetic evidence for the undersampled Amazonian genera Aldina and Monopteryx, and the tropical African Amphimas, Cordyla, Leucomphalos, and Mildbraediodendron.

Experimental habitat fragmentation increases linkage disequilibrium but does not affect genetic diversity or population structure in the Amazonian liverwort Radula flaccida.

Habitat fragmentation increases the migration distances among remnant populations, and is predicted to play a significant role in altering both demographic and genetic processes. Nevertheless, few studies have evaluated the genetic consequences of habitat fragmentation in light of information about population dynamics in the same set of organisms. In a 10,000-km(2) experimentally fragmented landscape of rainforest reserves in central Amazonia, we examine patterns of genetic variation (amplified fragment length polymorphisms, AFLPs) in the epiphyllous (e.g. leaf-inhabiting) liverwort Radula flaccida Gott. Previous demographic work indicates that colonization rates in this species are significantly reduced in small forest reserves. We scored 113 polymorphic loci in 86 individuals representing five fragmented and five experimentally unmanipulated populations. Most of the variation (82%) in all populations was harboured at the smallest (400 m(2)) sampling unit. The mean ((+/-) SD) within-population genetic diversity (Nei's), of forest remnants (0.412 +/- 0.2) was indistinguishable from continuous (0.413 +/- 0.2) forests. Similarly, F(ST) was identical among small (1- and 10-ha) and large (> or = 100-ha) reserves (0.19 and 0.18, respectively), but linkage disequilibrium between pairs of loci was significantly elevated in fragmented populations relative to those in continuous forests. These results illustrate that inferences regarding the long-term viability of fragmented populations based on neutral marker data alone must be viewed with caution, and underscore the importance of jointly evaluating information on both genetic structure and demography. Second, multilocus analyses may be more sensitive to the effects of fragmentation in the short term, although the effects of increasing linkage disequilibrium on population viability remain uncertain.

Metapopulation extinction thresholds in rain forest remnants.

Although habitat fragmentation is a major threat to global biodiversity, the demographic mechanisms underlying species loss from tropical forest remnants remain largely unexplored. In particular, no studies at the landscape scale have quantified fragmentation's impacts on colonization, extinction, and local population growth simultaneously. In central Amazonia, we conducted a multiyear demographic census of 292 populations of two leaf-inhabiting (i.e., epiphyllous) bryophyte species transplanted from continuous forest into a network of 10 study sites ranging from 1, 10, and 100 to > 10,000 ha in size. All populations experienced significantly positive local growth (lambda > 1) and a nearly constant per-generational extinction probability (15%). However, experimental leaf patches in reserves of > or = 100 ha experienced nearly double (48%) the colonization probability observed in small reserves (27%), suggesting that the proximate cause of epiphyll species loss in small fragments (< or = 10 ha) is reduced colonization. Nonetheless, populations of small fragments exhibit rates of colonization above patch extinction, positive local growth, and low temporal variation, which are features that should theoretically reduce the probability of extinction. This result suggests that for habitat-tracking metapopulations subject to frequent and stochastic turnover events, including epiphylls, colonization/extinction ratios must be maintained well above unity to ensure metapopulation persistence.