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Habitat fragmentation remains a major focus of research by ecologists decades after being put forward as a threat to the integrity of ecosystems. While studies have documented myriad biotic changes in fragmented landscapes, including the local extinction of species from fragments, the demographic mechanisms underlying these extinctions are rarely known. However, many of them-especially in lowland tropical forests-are thought to be driven by one of two mechanisms: (1) reduced recruitment in fragments resulting from changes in the diversity or abundance of pollinators and seed dispersers or (2) increased rates of individual mortality in fragments due to dramatically altered abiotic conditions, especially near fragment edges. Unfortunately, there have been few tests of these potential mechanisms due to the paucity of long-term and comprehensive demographic data collected in both forest fragments and continuous forest sites. Here we report 11 years (1998-2009) of demographic data from populations of the Amazonian understory herb Heliconia acuminata (LC Rich.) found at Brazil's Biological Dynamics of Forest Fragments Project (BDFFP). The data set comprises >66,000 plant × year records of 8586 plants, including 3464 seedlings established after the first census. Seven populations were in experimentally isolated fragments (one in each of four 1-ha fragments and one in each of three 10-ha fragments), with the remaining six populations in continuous forest. Each population was in a 50 × 100 m permanent plot, with the distance between plots ranging from 500 m to 60 km. The plants in each plot were censused annually, at which time we recorded, identified, marked, and measured new seedlings, identified any previously marked plants that died, and recorded the size of surviving individuals. Each plot was also surveyed four to five times during the flowering season to identify reproductive plants and record the number of inflorescences each produced. These data have been used to investigate topics ranging from the way fragmentation-related reductions in germination influence population dynamics to statistical methods for analyzing reproductive rates. This breadth of prior use reflects the value of these data to future researchers. In addition to analyses of plant responses to habitat fragmentation, these data can be used to address fundamental questions in plant demography and the evolutionary ecology of tropical plants and to develop and test demographic models and tools. Though we welcome opportunities to collaborate with interested users, there are no restrictions on the use of this data set. However, we do request that those using the data for teaching or research purposes inform us of how they are doing so and cite this paper and the data archive when appropriate. Any publication using the data must also include a BDFFP Technical Series Number in the Acknowledgments. Authors can request this series number upon the acceptance of their article by contacting the BDFFP's Scientific Coordinator or E. M. Bruna., (© 2023 The Ecological Society of America.)

Premise: Hedychium J. Koenig (Zingiberaceae) is endemic to the Indo-Malayan Realm and is known for its colorful and fragrant flowers. Historically, two different pollination syndromes characterize Hedychium: diurnal or bird pollination, and nocturnal or moth pollination. In this study, we aim to understand the evolution of nocturnal and diurnal flowers, and to test its putative association with lineage diversification in Hedychium., Methods: A molecular tree of Hedychium was used as a scaffold upon which we estimated ancestral character states, phylogenetic signals, and correlations for certain categorical and continuous floral traits. Furthermore, we used phylomorphospace and trait-dependent diversification rate estimation analyses to understand phenotypic evolution and associated lineage diversification in Hedychium., Results: Although floral color and size lacked any association with specific pollinators, white or pale flowers were most common in the early branching clades when compared to bright-colored flowers, which were more widely represented in the most-derived clade IV. Five categorical and two continuous characters were identified to have informative evolutionary patterns, which also emphasized that ecology may have played a critical role in the diversification of Hedychium., Conclusions: From our phylogenetic analyses and ecological observations, we conclude that specializations in pollinator interactions are rare in the hyperdiverse clade IV, thus challenging the role of both moth-specialization and bird-specialization as central factors in the diversification of Hedychium. However, our results also suggest that clade III (predominantly island clade) may show specializations, and future studies should investigate ecological and pollinator interactions, along with inclusion of new traits such as floral fragrance and anthesis time., (© 2022 Botanical Society of America.)

The Indo-Malayan Realm is a biogeographic realm that extends from the Indian Subcontinent to the islands of Southeast Asia (Malay Archipelago). Despite being megadiverse, evolutionary hypotheses explaining taxonomic diversity in this region have been rare. Here, we investigate the role of geoclimatic events such as Himalayan orogeny and monsoon intensification in the diversification of the ginger-lilies (Hedychium J.Koenig: Zingiberaceae). We first built a comprehensive, time-calibrated phylogeny of Hedychium with 75% taxonomic and geographic sampling. We found that Hedychium is a very young lineage that originated in Northern Indo-Burma, in the Late Miocene (c. 10.6 Ma). This was followed by a late Neogene and early Quaternary diversification, with multiple dispersal events to Southern Indo-Burma, Himalayas, Peninsular India, and the Malay Archipelago. The most speciose clade IV i.e., the predominantly Indo-Burmese clade also showed a higher diversification rate, suggesting its recent rapid radiation. Our divergence dating and GeoHiSSE results demonstrate that the diversification of Hedychium was shaped by both the intensifications in the Himalayan uplift as well as the Asian monsoon. Ancestral character-state reconstructions identified the occurrence of vegetative dormancy in both clades I and II, whereas the strictly epiphytic growth behavior, island dwarfism, lack of dormancy, and a distinct environmental niche were observed only in the predominantly island clade i.e., clade III. Finally, we show that the occurrence of epiphytism in clade III corresponds with submergence due to sea-level changes, suggesting it to be an adaptive trait. Our study highlights the role of recent geoclimatic events and environmental factors in the diversification of plants within the Indo-Malayan Realm and the need for collaborative work to understand biogeographic patterns within this understudied region. This study opens new perspectives for future biogeographic studies in this region and provides a framework to explain the taxonomic hyperdiversity of the Indo-Malayan Realm., (Copyright © 2022. Published by Elsevier Inc.)

Many well-studied animal species use conspicuous, repetitive signals that attract both mates and predators. Orthopterans (crickets, katydids, and grasshoppers) are renowned for their acoustic signals. In Neotropical forests, however, many katydid species produce extremely short signals, totaling only a few seconds of sound per night, likely in response to predation by acoustically orienting predators. The rare signals of these katydid species raises the question of how they find conspecific mates in a structurally complex rainforest. While acoustic mechanisms, such as duetting, likely facilitate mate finding, we test the hypothesis that mate finding is further facilitated by colocalization on particular host plant species. DNA barcoding allows us to identify recently consumed plants from katydid stomach contents. We use DNA barcoding to test the prediction that katydids of the same species will have closely related plant species in their stomach. We do not find evidence for dietary specialization. Instead, katydids consumed a wide mix of plants within and across the flowering plants (27 species in 22 genera, 16 families, and 12 orders) with particular representation in the orders Fabales and Laurales. Some evidence indicates that katydids may gather on plants during a narrow window of rapid leaf out, but additional investigations are required to determine whether katydid mate finding is facilitated by gathering at transient food resources., Competing Interests: Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Life on Earth has evolved from initial simplicity to the astounding complexity we experience today. Bacteria and archaea have largely excelled in metabolic diversification, but eukaryotes additionally display abundant morphological innovation. How have these innovations come about and what constraints are there on the origins of novelty and the continuing maintenance of biodiversity on Earth? The history of life and the code for the working parts of cells and systems are written in the genome. The Earth BioGenome Project has proposed that the genomes of all extant, named eukaryotes-about 2 million species-should be sequenced to high quality to produce a digital library of life on Earth, beginning with strategic phylogenetic, ecological, and high-impact priorities. Here we discuss why we should sequence all eukaryotic species, not just a representative few scattered across the many branches of the tree of life. We suggest that many questions of evolutionary and ecological significance will only be addressable when whole-genome data representing divergences at all of the branchings in the tree of life or all species in natural ecosystems are available. We envisage that a genomic tree of life will foster understanding of the ongoing processes of speciation, adaptation, and organismal dependencies within entire ecosystems. These explorations will resolve long-standing problems in phylogenetics, evolution, ecology, conservation, agriculture, bioindustry, and medicine., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Competing Interests: The authors declare no competing interest.

Green plants play a fundamental role in ecosystems, human health, and agriculture. As de novo genomes are being generated for all known eukaryotic species as advocated by the Earth BioGenome Project, increasing genomic information on green land plants is essential. However, setting standards for the generation and storage of the complex set of genomes that characterize the green lineage of life is a major challenge for plant scientists. Such standards will need to accommodate the immense variation in green plant genome size, transposable element content, and structural complexity while enabling research into the molecular and evolutionary processes that have resulted in this enormous genomic variation. Here we provide an overview and assessment of the current state of knowledge of green plant genomes. To date fewer than 300 complete chromosome-scale genome assemblies representing fewer than 900 species have been generated across the estimated 450,000 to 500,000 species in the green plant clade. These genomes range in size from 12 Mb to 27.6 Gb and are biased toward agricultural crops with large branches of the green tree of life untouched by genomic-scale sequencing. Locating suitable tissue samples of most species of plants, especially those taxa from extreme environments, remains one of the biggest hurdles to increasing our genomic inventory. Furthermore, the annotation of plant genomes is at present undergoing intensive improvement. It is our hope that this fresh overview will help in the development of genomic quality standards for a cohesive and meaningful synthesis of green plant genomes as we scale up for the future., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

The Earth BioGenome Project (EBP) is an audacious endeavor to obtain whole-genome sequences of representatives from all eukaryotic species on Earth. In addition to the project's technical and organizational challenges, it also faces complicated ethical, legal, and social issues. This paper, from members of the EBP's Ethical, Legal, and Social Issues (ELSI) Committee, catalogs these ELSI concerns arising from EBP. These include legal issues, such as sample collection and permitting; the applicability of international treaties, such as the Convention on Biological Diversity and the Nagoya Protocol; intellectual property; sample accessioning; and biosecurity and ethical issues, such as sampling from the territories of Indigenous peoples and local communities, the protection of endangered species, and cross-border collections, among several others. We also comment on the intersection of digital sequence information and data rights. More broadly, this list of ethical, legal, and social issues for large-scale genomic sequencing projects may be useful in the consideration of ethical frameworks for future projects. While we do not-and cannot-provide simple, overarching solutions for all the issues raised here, we conclude our perspective by beginning to chart a path forward for EBP's work., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Ecology cannot yet fully explain why so many tree species coexist in natural communities such as tropical forests. A major difficulty is linking individual-level processes to community dynamics. We propose a combination of tree spatial data, spatial statistics and dynamical theory to reveal the relationship between spatial patterns and population-level interaction coefficients and their consequences for multispecies dynamics and coexistence. Here we show that the emerging population-level interaction coefficients have, for a broad range of circumstances, a simpler structure than their individual-level counterparts, which allows for an analytical treatment of equilibrium and stability conditions. Mechanisms such as animal seed dispersal, which result in clustering of recruits that is decoupled from parent locations, lead to a rare-species advantage and coexistence of otherwise neutral competitors. Linking spatial statistics with theories of community dynamics offers new avenues for explaining species coexistence and calls for rethinking community ecology through a spatial lens.

DNA barcoding is an emerging molecular identification and classification technology that has been applied to medicinal plants since 2008. The application of this technique has greatly ensured the safety and effectiveness of medicinal materials. In this paper, we review the application of DNA barcoding and some related technologies over the past 10 years with respect to improving our knowledge of medicinal plant identification and authentication. From single locus-based DNA barcodes to combined markers to genome-scale levels, DNA barcodes contribute more and more genetic information. At the same time, other technologies, such as high-resolution melting (HRM), have been combined with DNA barcoding. With the development of next-generation sequencing (NGS), metabarcoding technology has also been shown to identify species in mixed samples successfully. As a widely used and effective tool, DNA barcoding will become more useful over time in the field of medicinal plants., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: The authors declare no competing interest.

DNA barcoding is a valuable tool to support species identification with broad applications from traditional taxonomy, ecology, forensics, food analysis, and environmental science. We introduce Microfluidic Enrichment Barcoding (MEBarcoding) for plant DNA Barcoding, a cost-effective method for high-throughput DNA barcoding. MEBarcoding uses the Fluidigm Access Array to simultaneously amplify targeted regions for 48 DNA samples and hundreds of PCR primer pairs (producing up to 23,040 PCR products) during a single thermal cycling protocol. As a proof of concept, we developed a microfluidic PCR workflow using the Fluidigm Access Array and Illumina MiSeq. We tested 96 samples for each of the four primary DNA barcode loci in plants: rbcL, matK, trnH-psbA, and ITS. This workflow was used to build a reference library for 78 families and 96 genera from all major plant lineages - many currently lacking in public databases. Our results show that this technique is an efficient alternative to traditional PCR and Sanger sequencing to generate large amounts of plant DNA barcodes and build more comprehensive barcode databases.

Applications of DNA barcoding include identifying species, inferring ecological and evolutionary relationships between species, and DNA metabarcoding. These applications require reference libraries that are not yet available for many taxa and geographic regions. We collected, identified, and vouchered plant specimens from Mpala Research Center in Laikipia, Kenya, to develop an extensive DNA-barcode library for a savanna ecosystem in equatorial East Africa. We amassed up to five DNA barcode markers (rbcL, matK, trnL-F, trnH-psbA, and ITS) for 1,781 specimens representing up to 460 species (~92% of the known flora), increasing the number of plant DNA barcode records for Africa by ~9%. We evaluated the ability of these markers, singly and in combination, to delimit species by calculating intra- and interspecific genetic distances. We further estimated a plant community phylogeny and demonstrated its utility by testing if evolutionary relatedness could predict the tendency of members of the Mpala plant community to have or lack "barcode gaps", defined as disparities between the maximum intra- and minimum interspecific genetic distances. We found barcode gaps for 72%-89% of taxa depending on the marker or markers used. With the exception of the markers rbcL and ITS, we found that evolutionary relatedness was an important predictor of barcode-gap presence or absence for all of the markers in combination and for matK, trnL-F, and trnH-psbA individually. This plant DNA barcode library and community phylogeny will be a valuable resource for future investigations., (© 2019 John Wiley & Sons Ltd.)

Many cases of rapid evolutionary radiations in plant and animal lineages are known; however phylogenetic relationships among these lineages have been difficult to resolve by systematists. Increasing amounts of genomic data have been sequentially applied in an attempt to resolve these radiations, dissecting their evolutionary patterns into a series of bifurcating events. Here we explore one such rapid radiation in the tropical plant order Zingiberales (the bananas and relatives) which includes eight families, approximately 110 genera, and more than 2600 species. One clade, the "Ginger families", including (Costaceae + Zingiberaceae) (Marantaceae + Cannaceae), has been well-resolved and well-supported in all previous studies. However, well-supported reconstructions among the "Banana families" (Musaceae, Heliconiaceae, Lowiaceae, Strelitziaceae), which most likely diverged about 90 Mya, have been difficult to confirm. Supported with anatomical, morphological, single locus, and genome-wide data, nearly every possible phylogenetic placement has been proposed for these families. In an attempt to resolve this complex evolutionary event, hybridization-based target enrichment was used to obtain sequences from up to 378 putatively orthologous low-copy nuclear genes (all ≥ 960 bp). Individual gene trees recovered multiple topologies among the early divergent lineages, with varying levels of support for these relationships. One topology of the "Banana families" (Musaceae (Heliconiaceae (Lowiaceae + Strelitziaceae))), which has not been suggested until now, was almost consistently recovered in all multilocus analyses of the nuclear dataset (concatenated - ExaML, coalescent - ASTRAL and ASTRID, supertree - MRL, and Bayesian concordance - BUCKy). Nevertheless, the multiple topologies recovered among these lineages suggest that even large amounts of genomic data might not be able to fully resolve relationships at this phylogenetic depth. This lack of well-supported resolution could suggest methodological problems (i.e., violation of model assumptions in both concatenated and coalescent analyses) or more likely reflect an evolutionary history shaped by an explosive, rapid, and nearly simultaneous polychotomous radiation in this group of plants towards the end of the Cretaceous, perhaps driven by vertebrate pollinator selection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Increasing our understanding of Earth's biodiversity and responsibly stewarding its resources are among the most crucial scientific and social challenges of the new millennium. These challenges require fundamental new knowledge of the organization, evolution, functions, and interactions among millions of the planet's organisms. Herein, we present a perspective on the Earth BioGenome Project (EBP), a moonshot for biology that aims to sequence, catalog, and characterize the genomes of all of Earth's eukaryotic biodiversity over a period of 10 years. The outcomes of the EBP will inform a broad range of major issues facing humanity, such as the impact of climate change on biodiversity, the conservation of endangered species and ecosystems, and the preservation and enhancement of ecosystem services. We describe hurdles that the project faces, including data-sharing policies that ensure a permanent, freely available resource for future scientific discovery while respecting access and benefit sharing guidelines of the Nagoya Protocol. We also describe scientific and organizational challenges in executing such an ambitious project, and the structure proposed to achieve the project's goals. The far-reaching potential benefits of creating an open digital repository of genomic information for life on Earth can be realized only by a coordinated international effort., Competing Interests: The authors declare no conflict of interest.

Trade-offs among plant functional traits indicate diversity in plant strategies of growth and survival. The leaf economics spectrum (LES) reflects a trade-off between short-term carbon gain and long-term leaf persistence. A related trade-off, between foliar growth and anti-herbivore defense, occurs among plants growing in contrasting resource regimes, but it is unclear whether this trade-off is maintained within plant communities, where resource gradients are minimized. The LES and the growth-defense trade-off involve related traits, but the extent to which these trade-off dimensions are correlated is poorly understood. We assessed the relationship between leaf economic and anti-herbivore defense traits among sunlit foliage of 345 canopy trees in 83 species on Barro Colorado Island, Panama. We quantified ten traits related to resource allocation and defense, and identified patterns of trait co-variation using multivariate ordination. We tested whether traits and ordination axes were correlated with patterns of phylogenetic relatedness, juvenile demographic trade-offs, or topo-edaphic variation. Two independent axes described ~ 60% of the variation among canopy trees. Axis 1 revealed a trade-off between leaf nutritional and structural investment, consistent with the LES. Physical defense traits were largely oriented along this axis. Axis 2 revealed a trade-off between investments in phenolic defenses versus other foliar defenses, which we term the leaf defense spectrum. Phylogenetic relationships and topo-edaphic variation largely did not explain trait co-variation. Our results suggest that some trade-offs among the growth and defense traits of outer-canopy trees may be captured by the LES, while others may occur along additional resource allocation dimensions.

Geographic isolation is the first step in insect herbivore diet specialization. Such specialization is postulated to increase insect fitness, but may simultaneously reduce insect ability to colonize novel hosts. During the Paleocene-Eocene, plants from the order Zingiberales became isolated either in the Paleotropics or in the Neotropics. During the Cretaceous, rolled-leaf beetles diversified in the Neotropics concurrently with Neotropical Zingiberales. Using a community of Costa Rican rolled-leaf beetles and their Zingiberales host plants as study system, we explored if previous geographic isolation precludes insects to expand their diets to exotic hosts. We recorded interactions between rolled-leaf beetles and native Zingiberales by combining DNA barcodes and field records for 7450 beetles feeding on 3202 host plants. To determine phylogenetic patterns of diet expansions, we set 20 field plots including five exotic Zingiberales, recording beetles feeding on these exotic hosts. In the laboratory, using both native and exotic host plants, we reared a subset of insect species that had expanded their diets to the exotic plants. The original plant-herbivore community comprised 24 beetle species feeding on 35 native hosts, representing 103 plant-herbivore interactions. After exotic host plant introduction, 20% of the beetle species expanded their diets to exotic Zingiberales. Insects only established on exotic hosts that belong to the same plant family as their native hosts. Laboratory experiments show that beetles are able to complete development on these novel hosts. In conclusion, rolled-leaf beetles are pre-adapted to expand their diets to novel host plants even after millions of years of geographic isolation.

DNA barcoding is a commonly used bio-technology in multiple disciplines including biology, environmental science, forensics and inspection, etc. Forest dynamic plots provide a unique opportunity to carry out large-scale, comparative, and multidisciplinary research for plant DNA barcoding. The paper concisely reviewed four previous progresses in China; specifically, species discrimination, community phylogenetic reconstruction, phylogenetic community structure exploration, and biodiversity index evaluation. Further, we demonstrated three major challenges; specifically, building the impetus to generate DNA barcodes using multiple plant DNA markers for all woody species at forest community levels, analyzing massive DNA barcoding sequence data, and promoting theoretical innovation. Lastly, we raised five possible directions; specifically, proposing a "purpose-driven barcode" fit for multi-level applications, developing new integrative sequencing strategies, pushing DNA barcoding beyond terrestrial ecosystem, constructing national-level DNA barcode sequence libraries for special plant groups, and establishing intelligent identification systems or online server platforms. These efforts will be potentially valuable to explore large-scale biodiversity patterns, the origin and evolution of life, and will also facilitate preservation and utilization of biodiversity resources.

The taxonomic status of a single island, narrow range endemic plant species from Palau, Micronesia (Timonius salsedoi) was assessed using DNA barcode markers, additional plastid loci, and morphology in order to verify its conservation status. DNA barcode loci distinguished T. salsedoi from all other Timonius species sampled from Palau, and were supported by sequence data from the atpB-rbcL intergenic spacer region. Timonius salsedoi was only known from two mature individual trees in 2012. Due to its extremely narrow range and population size, it had previously been recommended to be listed as Critically Endangered Status under three separate IUCN Criteria. In 2014 a second survey of the population following a typhoon revealed that the only two known trees had died suggesting that this species may now be extinct. Comprehensive follow up surveys of suitable habitat for this species are urgently required.

Studies of forest dynamics plots (FDPs) have revealed a variety of negative density-dependent (NDD) demographic interactions, especially among conspecific trees. These interactions can affect growth rate, recruitment and mortality, and they play a central role in the maintenance of species diversity in these complex ecosystems. Here we use an equal area annulus (EAA) point-pattern method to comprehensively analyze data from two tropical FDPs, Barro Colorado Island in Panama and Sinharaja in Sri Lanka. We show that these NDD interactions also influence the continued evolutionary diversification of even distantly related tree species in these FDPs. We examine the details of a wide range of these interactions between individual trees and the trees that surround them. All these interactions, and their cumulative effects, are strongest among conspecific focal and surrounding tree species in both FDPs. They diminish in magnitude with increasing phylogenetic distance between heterospecific focal and surrounding trees, but do not disappear or change the pattern of their dependence on size, density, frequency or physical distance even among the most distantly related trees. The phylogenetic persistence of all these effects provides evidence that interactions between tree species that share an ecosystem may continue to promote adaptive divergence even after the species' gene pools have become separated. Adaptive divergence among taxa would operate in stark contrast to an alternative possibility that has previously been suggested, that distantly related species with dispersal-limited distributions and confronted with unpredictable neighbors will tend to converge on common strategies of resource use. In addition, we have also uncovered a positive density-dependent effect: growth rates of large trees are boosted in the presence of a smaller basal area of surrounding trees. We also show that many of the NDD interactions switch sign rapidly as focal trees grow in size, and that their cumulative effect can strongly influence the distributions and species composition of the trees that surround the focal trees during the focal trees' lifetimes., Competing Interests: The authors have declared that no competing interests exist.

Australia's Great Sandy Region is of international significance containing two World Heritage areas and patches of rainforest growing on white sand. Previous broad-scale analysis found the Great Sandy biogeographic subregion contained a significantly more phylogenetically even subset of species than expected by chance contrasting with rainforest on white sand in Peru. This study aimed to test the patterns of rainforest diversity and relatedness at a finer scale and to investigate why we may find different patterns of phylogenetic evenness compared with rainforests on white sands in other parts of the world. This study focussed on rainforest sites within the Great Sandy and surrounding areas in South East Queensland (SEQ), Australia. We undertook field collections, expanded our three-marker DNA barcode library of SEQ rainforest plants and updated the phylogeny to 95% of the SEQ rainforest flora. We sampled species composition of rainforest in fixed area plots from 100 sites. We calculated phylogenetic diversity (PD) measures as well as species richness (SR) for each rainforest community. These combined with site variables such as geology, were used to evaluate patterns and relatedness. We found that many rainforest communities in the Great Sandy area were significantly phylogenetically even at the individual site level consistent with a broader subregion analysis. Sites from adjacent areas were either not significant or were significantly phylogenetically clustered. Some results in the neighbouring areas were consistent with historic range expansions. In contrast with expectations, sites located on the oldest substrates had significantly lower phylogenetic diversity (PD). Fraser Island was once connected to mainland Australia, our results are consistent with a region geologically old enough to have continuously supported rainforest in refugia. The interface of tropical and temperate floras in part also explains the significant phylogenetic evenness and higher than expected phylogenetic diversity.

Optimal foraging models of floral divergence predict that competition between two different types of pollinators will result in partitioning, increased assortative mating, and divergence of two floral phenotypes. We tested these predictions in a tropical plant-pollinator system using sexes of purple-throated carib hummingbirds (Anthracothorax jugularis) as the pollinators, red and yellow inflorescence morphs of Heliconia caribaea as the plants, and fluorescent dyes as pollen analogs in an enclosed outdoor garden. When foraging alone, males exhibited a significant preference for the yellow morph of H. caribaea, whereas females exhibited no preference. In competition, males maintained their preference for the yellow morph and through aggression caused females to over-visit the red morph, resulting in resource partitioning. Competition significantly increased within-morph dye transfer (assortative mating) relative to non-competitive environments. Competition and partitioning of color morphs by sexes of purple-throated caribs also resulted in selection for floral divergence as measured by dye deposition on stigmas. Red and yellow morphs did not differ significantly in dye deposition in the competition trials, but differences in dye deposition and preferences for morphs when sexes of purple-throated caribs foraged alone implied fixation of one or the other color morph in the absence of competition. Competition also resulted in selection for divergence in corolla length, with the red morph experiencing directional selection for longer corollas and the yellow morph experiencing stabilizing selection on corolla length. Our results thus support predictions of foraging models of floral divergence and indicate that pollinator competition is a viable mechanism for divergence in floral traits of plants.

The critical thermal maximum (CTmax), the temperature at which motor control is lost in animals, has the potential to determine if species will tolerate global warming. For insects, tolerance to high temperatures decreases with latitude, suggesting that similar patterns may exist along elevational gradients as well. This study explored how CTmax varies among species and populations of a group of diverse tropical insect herbivores, the rolled-leaf beetles, across both broad and narrow elevational gradients. Data from 6,948 field observations and 8,700 museum specimens were used to map the elevational distributions of rolled-leaf beetles on two mountains in Costa Rica. CTmax was determined for 1,252 individual beetles representing all populations across the gradients. Initial morphological identifications suggested a total of 26 species with populations at different elevations displaying contrasting upper thermal limits. However, compared with morphological identifications, DNA barcodes (cytochrome oxidase I) revealed significant cryptic species diversity. DNA barcodes identified 42 species and haplotypes across 11 species complexes. These 42 species displayed much narrower elevational distributions and values of CTmax than the 26 morphologically defined species. In general, species found at middle elevations and on mountaintops are less tolerant to high temperatures than species restricted to lowland habitats. Species with broad elevational distributions display high CTmax throughout their ranges. We found no significant phylogenetic signal in CTmax, geography, or elevational range. The narrow variance in CTmax values for most rolled-leaf beetles, especially high-elevation species, suggests that the risk of extinction of insects may be substantial under some projected rates of global warming.

Background and Aims: Secondary pollen presentation (SPP), a floral mechanism of reproductive adaptation, has been described for more than 200 years, with nine types SPP recorded. However, few studies have been done experimentally to link the floral mechanism of SPP to its functional roles in pollination process. This study aims to describe a new SPP mechanism from a wild ginger (Zingiber densissimum, Zingiberaceae) and explore how the pollen arrangement of SPP affects pollen removal during the interaction with different pollinators., Methodology/principal Findings: Field observations and experiments revealed that flowers lasted for less than one day. The breeding system was partially self-incompatible. Two bee species, Macropis hedini (which carried pollen dorsally) and Amegilla zonata (which carried pollen ventrally) were the primary pollinators. About a third of pollen grains were relocated from the anther to the labellum staminode of flowers through the adherence of aggregated pollen chains, while other grains were presented on the anther. In a single visit, each bee species removed pollen grains from both the labellum staminode and the anther. Macropis hedini was more effective than Amegilla zonata., Conclusions/significance: Our study describes a new SPP mechanism in angiosperms. The new SPP mode enables pollen grains presented on the anther and the labellum staminode simultaneously via the adherence of aggregated pollen chains, thus promoting pollen to be taken away by different pollinators. This SPP mechanism plays a key role during pollen removal and may have evolved under the pressure to improve male fitness.

To determine how well DNA barcodes from the chloroplast region perform in forest dynamics plots (FDPs) from global CTFS-ForestGEO network, we analyzed DNA barcoding sequences of 1277 plant species from a wide phylogenetic range (3 FDPs in tropics, 5 in subtropics and 5 in temperate zone) and compared the rates of species discrimination (RSD). We quantified RSD by two DNA barcode combinations (rbcL + matK and rbcL + matK + trnH-psbA) using a monophyly-based method (GARLI). We defined two indexes of closely-related taxa (Gm/Gt and S/G ratios) and correlated these ratios with RSD. The combination of rbcL + matK averagely discriminated 88.65%, 83.84% and 72.51% at the local, regional and global scales, respectively. An additional locus trnH-psbA increased RSD by 2.87%, 1.49% and 3.58% correspondingly. RSD varied along a latitudinal gradient and were negatively correlated with ratios of closely-related taxa. Successes of species discrimination generally depend on scales in global FDPs. We suggested that the combination of rbcL + matK + trnH-psbA is currently applicable for DNA barcoding-based phylogenetic studies on forest communities.

Considerable debate focuses on whether invasive species establish and become abundant by being functionally and phylogenetically distinct from native species, leading to a host of invasion-specific hypotheses of community assembly. Few studies, however, have quantitatively assessed whether similar patterns of phylogenetic and functional similarity explain local abundance of both native and introduced species, which would suggest similar assembly mechanisms regardless of origin. Using a chronosequence of invaded temperate forest stands, we tested whether the occurrence and abundance of both introduced and native species were predicted by phylogenetic relatedness, functional overlap, and key environmental characteristics including forest age. Environmental filtering against functionally and phylogenetically distinct species strongly dictated the occurrence and abundance of both introduced and native species, with slight modifications of these patterns according to forest age. Thus, once functional and evolutionary novelty were quantified, introduced status provided little information about species' presence or abundance, indicating largely similar sorting mechanisms for both native and introduced species.

Niche partitioning facilitates species coexistence in a world of limited resources, thereby enriching biodiversity. For decades, biologists have sought to understand how diverse assemblages of large mammalian herbivores (LMH) partition food resources. Several complementary mechanisms have been identified, including differential consumption of grasses versus nongrasses and spatiotemporal stratification in use of different parts of the same plant. However, the extent to which LMH partition food-plant species is largely unknown because comprehensive species-level identification is prohibitively difficult with traditional methods. We used DNA metabarcoding to quantify diet breadth, composition, and overlap for seven abundant LMH species (six wild, one domestic) in semiarid African savanna. These species ranged from almost-exclusive grazers to almost-exclusive browsers: Grass consumption inferred from mean sequence relative read abundance (RRA) ranged from >99% (plains zebra) to <1% (dik-dik). Grass RRA was highly correlated with isotopic estimates of % grass consumption, indicating that RRA conveys reliable quantitative information about consumption. Dietary overlap was greatest between species that were similar in body size and proportional grass consumption. Nonetheless, diet composition differed between all species-even pairs of grazers matched in size, digestive physiology, and location-and dietary similarity was sometimes greater across grazing and browsing guilds than within them. Such taxonomically fine-grained diet partitioning suggests that coarse trophic categorizations may generate misleading conclusions about competition and coexistence in LMH assemblages, and that LMH diversity may be more tightly linked to plant diversity than is currently recognized.

Australian rainforests have been fragmented due to past climatic changes and more recently landscape change as a result of clearing for agriculture and urban spread. The subtropical rainforests of South Eastern Queensland are significantly more fragmented than the tropical World Heritage listed northern rainforests and are subject to much greater human population pressures. The Australian rainforest flora is relatively taxonomically rich at the family level, but less so at the species level. Current methods to assess biodiversity based on species numbers fail to adequately capture this richness at higher taxonomic levels. We developed a DNA barcode library for the SE Queensland rainforest flora to support a methodology for biodiversity assessment that incorporates both taxonomic diversity and phylogenetic relationships. We placed our SE Queensland phylogeny based on a three marker DNA barcode within a larger international rainforest barcode library and used this to calculate phylogenetic diversity (PD). We compared phylo- diversity measures, species composition and richness and ecosystem diversity of the SE Queensland rainforest estate to identify which bio subregions contain the greatest rainforest biodiversity, subregion relationships and their level of protection. We identified areas of highest conservation priority. Diversity was not correlated with rainforest area in SE Queensland subregions but PD was correlated with both the percent of the subregion occupied by rainforest and the diversity of regional ecosystems (RE) present. The patterns of species diversity and phylogenetic diversity suggest a strong influence of historical biogeography. Some subregions contain significantly more PD than expected by chance, consistent with the concept of refugia, while others were significantly phylogenetically clustered, consistent with recent range expansions.

Understanding the mechanisms enabling coevolution in complex mutualistic networks remains a central challenge in evolutionary biology. We show for the first time, to our knowledge, that a tropical plant species has the capacity to discriminate among floral visitors, investing in reproduction differentially across the pollinator community. After we standardized pollen quality in 223 aviary experiments, successful pollination of Heliconia tortuosa (measured as pollen tube abundance) occurred frequently when plants were visited by long-distance traplining hummingbird species with specialized bills (mean pollen tubes = 1.21 ± 0.12 SE) but was reduced 5.7 times when visited by straight-billed territorial birds (mean pollen tubes = 0.20 ± 0.074 SE) or insects. Our subsequent experiments revealed that plants use the nectar extraction capacity of tropical hummingbirds, a positive function of bill length, as a cue to turn on reproductively. Furthermore, we show that hummingbirds with long bills and high nectar extraction efficiency engaged in daily movements at broad spatial scales (∼1 km), but that territorial species moved only short distances (<100 m). Such pollinator recognition may therefore affect mate selection and maximize receipt of high-quality pollen from multiple parents. Although a diffuse pollinator network is implied, because all six species of hummingbirds carry pollen of H. tortuosa, only two species with specialized bills contribute meaningfully to its reproduction. We hypothesize that this pollinator filtering behavior constitutes a crucial mechanism facilitating coevolution in multispecies plant-pollinator networks. However, pollinator recognition also greatly reduces the number of realized pollinators, thereby rendering mutualistic networks more vulnerable to environmental change.

The genus Curcuma L. is commonly used as spices, medicines, dyes and ornamentals. Owing to its economic significance and lack of clear-cut morphological differences between species, this genus is an ideal case for developing DNA barcodes. In this study, four chloroplast DNA regions (matK, rbcL, trnH-psbA and trnL-F) and one nuclear region (ITS2) were generated for 44 Curcuma species and five species from closely related genera, represented by 96 samples. PCR amplification success rate, intra- and inter-specific genetic distance variation and the correct identification percentage were taken into account to assess candidate barcode regions. PCR and sequence success rate were high in matK (89.7%), rbcL (100%), trnH-psbA (100%), trnL-F (95.7%) and ITS2 (82.6%) regions. The results further showed that four candidate chloroplast barcoding regions (matK, rbcL, trnH-psbA and trnL-F) yield no barcode gaps, indicating that the genus Curcuma represents a challenging group for DNA barcoding. The ITS2 region presented large interspecific variation and provided the highest correct identification rates (46.7%) based on BLASTClust method among the five regions. However, the ITS2 only provided 7.9% based on NJ tree method. An increase in discriminatory power needs the development of more variable markers., (© 2014 John Wiley & Sons Ltd.)

The Neotropical genus Cephaloleia Chevrolat (Coleoptera: Chrysomelidae: Cassidinae) includes 214 species distributed from the south of Mexico to Argentina. Cephaloleia beetles feed mostly on plants from the order Zingiberales. The interactions between Cephaloleia beetles and their Zingiberales host plants is proposed as one of the oldest and most conservative associations. Here we describe a new species of Cephaloleia (Cephaloleiakuprewiczae sp. n.) that feeds on two species of bromeliads (Pitcairniaarcuata and Pitcairniabrittoniana, Bromeliaceae: Pitcairnioideae). Cephaloleiakuprewiczae was previously described as Cephaloleiahistrionica. This study includes evidence from DNA barcodes (COI), larval and adult morphology and insect diets that separates Cephaloleiakuprewiczae from Cephaloleiahistrionica as a new species.

The use of DNA barcodes, which are short gene sequences taken from a standardized portion of the genome and used to identify species, is entering a new phase of application as more and more investigations employ these genetic markers to address questions relating to the ecology and evolution of natural systems. The suite of DNA barcode markers now applied to specific taxonomic groups of organisms are proving invaluable for understanding species boundaries, community ecology, functional trait evolution, trophic interactions, and the conservation of biodiversity. The application of next-generation sequencing (NGS) technology will greatly expand the versatility of DNA barcodes across the Tree of Life, habitats, and geographies as new methodologies are explored and developed., (Published by Elsevier Ltd.)

Background: The use of phylogenetic information in community ecology and conservation has grown in recent years. Two key issues for community phylogenetics studies, however, are (i) low terminal phylogenetic resolution and (ii) arbitrarily defined species pools., Methodology/principal Findings: We used three DNA barcodes (plastid DNA regions rbcL, matK, and trnH-psbA) to infer a phylogeny for 527 native and naturalized trees of Puerto Rico, representing the vast majority of the entire tree flora of the island (89%). We used a maximum likelihood (ML) approach with and without a constraint tree that enforced monophyly of recognized plant orders. Based on 50% consensus trees, the ML analyses improved phylogenetic resolution relative to a comparable phylogeny generated with Phylomatic (proportion of internal nodes resolved: constrained ML = 74%, unconstrained ML = 68%, Phylomatic = 52%). We quantified the phylogenetic composition of 15 protected forests in Puerto Rico using the constrained ML and Phylomatic phylogenies. We found some evidence that tree communities in areas of high water stress were relatively phylogenetically clustered. Reducing the scale at which the species pool was defined (from island to soil types) changed some of our results depending on which phylogeny (ML vs. Phylomatic) was used. Overall, the increased terminal resolution provided by the ML phylogeny revealed additional patterns that were not observed with a less-resolved phylogeny., Conclusions/significance: With the DNA barcode phylogeny presented here (based on an island-wide species pool), we show that a more fully resolved phylogeny increases power to detect nonrandom patterns of community composition in several Puerto Rican tree communities. Especially if combined with additional information on species functional traits and geographic distributions, this phylogeny will (i) facilitate stronger inferences about the role of historical processes in governing the assembly and composition of Puerto Rican forests, (ii) provide insight into Caribbean biogeography, and (iii) aid in incorporating evolutionary history into conservation planning.

Forest dynamics plots, which now span longitudes, latitudes, and habitat types across the globe, offer unparalleled insights into the ecological and evolutionary processes that determine how species are assembled into communities. Understanding phylogenetic relationships among species in a community has become an important component of assessing assembly processes. However, the application of evolutionary information to questions in community ecology has been limited in large part by the lack of accurate estimates of phylogenetic relationships among individual species found within communities, and is particularly limiting in comparisons between communities. Therefore, streamlining and maximizing the information content of these community phylogenies is a priority. To test the viability and advantage of a multi-community phylogeny, we constructed a multi-plot mega-phylogeny of 1347 species of trees across 15 forest dynamics plots in the ForestGEO network using DNA barcode sequence data (rbcL, matK, and psbA-trnH) and compared community phylogenies for each individual plot with respect to support for topology and branch lengths, which affect evolutionary inference of community processes. The levels of taxonomic differentiation across the phylogeny were examined by quantifying the frequency of resolved nodes throughout. In addition, three phylogenetic distance (PD) metrics that are commonly used to infer assembly processes were estimated for each plot [PD, Mean Phylogenetic Distance (MPD), and Mean Nearest Taxon Distance (MNTD)]. Lastly, we examine the partitioning of phylogenetic diversity among community plots through quantification of inter-community MPD and MNTD. Overall, evolutionary relationships were highly resolved across the DNA barcode-based mega-phylogeny, and phylogenetic resolution for each community plot was improved when estimated within the context of the mega-phylogeny. Likewise, when compared with phylogenies for individual plots, estimates of phylogenetic diversity in the mega-phylogeny were more consistent, thereby removing a potential source of bias at the plot-level, and demonstrating the value of assessing phylogenetic relationships simultaneously within a mega-phylogeny. An unexpected result of the comparisons among plots based on the mega-phylogeny was that the communities in the ForestGEO plots in general appear to be assemblages of more closely related species than expected by chance, and that differentiation among communities is very low, suggesting deep floristic connections among communities and new avenues for future analyses in community ecology.

In tropical and temperate trees, wood chemical traits are hypothesized to covary with species' life-history strategy along a 'wood economics spectrum' (WES), but evidence supporting these expected patterns remains scarce. Due to its role in nutrient storage, we hypothesize that wood nitrogen (N) concentration will covary along the WES, being higher in slow-growing species with high wood density (WD), and lower in fast-growing species with low WD. In order to test this hypothesis we quantified wood N concentrations in 59 Panamanian hardwood species, and used this dataset to examine ecological correlates and phylogenetic patterns of wood N. Wood N varied > 14-fold among species between 0.04 and 0.59%; closely related species were more similar in wood N than expected by chance. Wood N was positively correlated with WD, and negatively correlated with log-transformed relative growth rates, although these relationships were relatively weak. We found evidence for co-evolution between wood N and both WD and log-transformed mortality rates. Our study provides evidence that wood N covaries with tree life-history parameters, and that these patterns consistently co-evolve in tropical hardwoods. These results provide some support for the hypothesized WES, and suggest that wood is an increasingly important N pool through tropical forest succession., (© 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.)

Loss of native vegetation cover is thought to be a major driver of declines in pollination success worldwide. However, it is not well known whether reducing the fragmentation of remaining vegetation can ameliorate these negative effects. We tested the independent effects of composition vs. configuration on the reproductive success of a keystone tropical forest herb (Heliconia tortuosa). To do this we designed a large-scale mensurative experiment that independently varied connected forest-patch size (configuration) and surrounding amount of forest (composition). In each patch, we tested whether pollen tubes, fruit, and seed set were associated with these landscape variables. We also captured hummingbirds as an indication of pollinator availability in a subset of patches according to the same design. We found evidence for an effect of configuration on seed set of H. tortuosa, but not on other aspects of plant reproduction; proportion of seeds produced increased 40% across the gradient in patch size we observed (0.64 to > 1300 ha), independent of the amount of forest in the surrounding landscape at both local and landscape scales. We also found that the availability of pollinators was dependent upon forest configuration; hummingbird capture rates increased three and one-half times across the patch size gradient, independent of forest amount. Finally, pollinator availability was strongly positively correlated with seed set. We hypothesize that the effects of configuration on plant fitness that we observed are due to reduced pollen quality resulting from altered hummingbird availability and/or movement behavior. Our results suggest that prioritizing larger patches of tropical forest may be particularly important for conservation of this species.