Collective and harmonized high throughput barcoding of insular arthropod biodiversity: Toward a Genomic Observatories Network for islands.

Current understanding of ecological and evolutionary processes underlying island biodiversity is heavily shaped by empirical data from plants and birds, although arthropods comprise the overwhelming majority of known animal species, and as such can provide key insights into processes governing biodiversity. Novel high throughput sequencing (HTS) approaches are now emerging as powerful tools to overcome limitations in the availability of arthropod biodiversity data, and hence provide insights into these processes. Here, we explored how these tools might be most effectively exploited for comprehensive and comparable inventory and monitoring of insular arthropod biodiversity. We first reviewed the strengths, limitations and potential synergies among existing approaches of high throughput barcode sequencing. We considered how this could be complemented with deep learning approaches applied to image analysis to study arthropod biodiversity. We then explored how these approaches could be implemented within the framework of an island Genomic Observatories Network (iGON) for the advancement of fundamental and applied understanding of island biodiversity. To this end, we identified seven island biology themes at the interface of ecology, evolution and conservation biology, within which collective and harmonized efforts in HTS arthropod inventory could yield significant advances in island biodiversity research.

Artemisia annua, a Traditional Plant Brought to Light.

Traditional remedies have been used for thousand years for the prevention and treatment of infectious diseases, particularly in developing countries. Of growing interest, the plant Artemisia annua, known for its malarial properties, has been studied for its numerous biological activities including metabolic, anti-tumor, anti-microbial and immunomodulatory properties. Artemisia annua is very rich in secondary metabolites such as monoterpenes, sesquiterpenes and phenolic compounds, of which the biological properties have been extensively studied. The purpose of this review is to gather and describe the data concerning the main chemical components produced by Artemisia annua and to describe the state of the art about the biological activities reported for this plant and its compounds beyond malaria.

MtDNA metagenomics reveals large-scale invasion of belowground arthropod communities by introduced species.

Using a series of standardized sampling plots within forest ecosystems in remote oceanic islands, we reveal fundamental differences between the structuring of aboveground and belowground arthropod biodiversity that are likely due to large-scale species introductions by humans. Species of beetle and spider were sampled almost exclusively from single islands, while soil-dwelling Collembola exhibited more than tenfold higher species sharing among islands. Comparison of Collembola mitochondrial metagenomic data to a database of more than 80 000 Collembola barcode sequences revealed almost 30% of sampled island species are genetically identical, or near identical, to individuals sampled from often very distant geographic regions of the world. Patterns of mtDNA relatedness among Collembola implicate human-mediated species introductions, with minimum estimates for the proportion of introduced species on the sampled islands ranging from 45% to 88%. Our results call for more attention to soil mesofauna to understand the global extent and ecological consequences of species introductions.

Islands as model systems in ecology and evolution: prospects fifty years after MacArthur-Wilson.

The study of islands as model systems has played an important role in the development of evolutionary and ecological theory. The 50th anniversary of MacArthur and Wilson's (December 1963) article, 'An equilibrium theory of insular zoogeography', was a recent milestone for this theme. Since 1963, island systems have provided new insights into the formation of ecological communities. Here, building on such developments, we highlight prospects for research on islands to improve our understanding of the ecology and evolution of communities in general. Throughout, we emphasise how attributes of islands combine to provide unusual research opportunities, the implications of which stretch far beyond islands. Molecular tools and increasing data acquisition now permit re-assessment of some fundamental issues that interested MacArthur and Wilson. These include the formation of ecological networks, species abundance distributions, and the contribution of evolution to community assembly. We also extend our prospects to other fields of ecology and evolution - understanding ecosystem functioning, speciation and diversification - frequently employing assets of oceanic islands in inferring the geographic area within which evolution has occurred, and potential barriers to gene flow. Although island-based theory is continually being enriched, incorporating non-equilibrium dynamics is identified as a major challenge for the future.

Relatedness defies biogeography: the tale of two island endemics (Acacia heterophylla and A. koa).

Despite the normally strong link between geographic proximity and relatedness of recently diverged taxa, truly puzzling biogeographic anomalies to this expectation exist in nature. Using a dated phylogeny, population genetic structure and estimates of ecological niche overlap, we tested the hypothesis that two geographically very disjunct, but morphologically very similar, island endemics (Acacia heterophylla from Réunion Island and A. koa from the Hawaiian archipelago) are the result of dispersal between these two island groups, rather than independent colonization events from Australia followed by convergent evolution. Our genetic results indicated that A. heterophylla renders A. koa paraphyletic and that the former colonized the Mascarene archipelago directly from the Hawaiian Islands ≤ 1.4 million yr ago. This colonization sequence was corroborated by similar ecological niches between the two island taxa, but not between A. melanoxylon from Australia (a sister, and presumed ancestral, taxon to A. koa and A. heterophylla) and Hawaiian A. koa. It is widely accepted that the long-distance dispersal of plants occurs more frequently than previously thought. Here, however, we document one of the most exceptional examples of such dispersal. Despite c. 18 000 km separating A. heterophylla and A. koa, these two island endemics from two different oceans probably represent a single taxon as a result of recent extreme long-distance dispersal.

Molecular characterization of trophic ecology within an island radiation of insect herbivores (Curculionidae: Entiminae: Cratopus).

The phytophagous beetle family Curculionidae is the most species-rich insect family known, with much of this diversity having been attributed to both co-evolution with food plants and host shifts at key points within the early evolutionary history of the group. Less well understood is the extent to which patterns of host use vary within or among related species, largely because of the technical difficulties associated with quantifying this. Here we develop a recently characterized molecular approach to quantify diet within and between two closely related species of weevil occurring primarily within dry forests on the island of Mauritius. Our aim is to quantify dietary variation across populations and assess adaptive and nonadaptive explanations for this and to characterize the nature of a trophic shift within an ecologically distinct population within one of the species. We find that our study species are polyphagous, consuming a much wider range of plants than would be suggested by the literature. Our data suggest that local diet variation is largely explained by food availability, and locally specialist populations consume food plants that are not phylogenetically novel, but do appear to represent a novel preference. Our results demonstrate the power of molecular methods to unambiguously quantify dietary variation across populations of insect herbivores, providing a valuable approach to understanding trophic interactions within and among local plant and insect herbivore communities.

Do Native and Alien Species Differ in Their Ecological Strategies? A Test with Woody Plants in Tropical Rainforests on Réunion Island (Mascarene Archipelago, Indian Ocean).

Understanding the mechanisms of biological invasions (e.g., competitive exclusion) is a key conservation challenge, especially on islands. Many mechanisms have been tested by comparing the characteristics of native and alien species, but few studies have considered ecological strategies. Here we aim at comparing the competitive ability, stress tolerance, and ruderalism (CSR) of native and alien trees in the tropical rainforests of Réunion Island. A total of sixteen 100 m2 plots (eight 'near-trail' and eight 'off-trail', at less disturbed sites) were established over a 2100 m elevational gradient. Three traits were measured in 1093 leaves from 237 trees: leaf area, leaf dry matter content and specific leaf area. They were converted into a CSR score assigned to each of the 80 surveyed tree species (70 native and 10 alien) using the 'Stratefy' ordination approach. C scores increased with basal area and S scores with elevation, but R scores were not higher along the trail, thus only partially validating Stratefy. Native and alien trees had similar CS strategies, thus challenging invasion hypotheses predicting a difference in ecological strategies and rather demonstrating the importance of environmental filtering. However, other differences falling outside the CSR theory may also explain the success of alien species on Réunion.

Soil filtration-sedimentation improves shelled protist recovery in eukaryotic eDNA surveys.

A large part of the soil protist diversity is missed in metabarcoding studies based on 0.25 g of soil environmental DNA (eDNA) and universal primers due to ca. 80% co-amplification of non-target plants, animals and fungi. To overcome this problem, enrichment of the substrate used for eDNA extraction is an easily implemented option but its effect has not yet been tested. In this study, we evaluated the effect of a 150 µm mesh size filtration and sedimentation method to improve the recovery of protist eDNA, while reducing the co-extraction of plant, animal and fungal eDNA, using a set of contrasted forest and alpine soils from La Réunion, Japan, Spain and Switzerland. Total eukaryotic diversity was estimated by V4 18S rRNA metabarcoding and classical amplicon sequence variant calling. A 2- to 3-fold enrichment in shelled protists (Euglyphida, Arcellinida and Chrysophyceae) was observed at the sample level with the proposed method, with, at the same time, a 2-fold depletion of Fungi and a 3-fold depletion of Embryophyceae. Protist alpha diversity was slightly lower in filtered samples due to reduced coverage in Variosea and Sarcomonadea, but significant differences were observed in only one region. Beta diversity varied mostly between regions and habitats, which explained the same proportion of variance in bulk soil and filtered samples. The increased resolution in soil protist diversity estimates provided by the filtration-sedimentation method is a strong argument in favour of including it in the standard protocol for soil protist eDNA metabarcoding studies.

Structure and specialization of mycorrhizal networks in phylogenetically diverse tropical communities.

BACKGROUND: The root mycobiome plays a fundamental role in plant nutrition and protection against biotic and abiotic stresses. In temperate forests or meadows dominated by angiosperms, the numerous fungi involved in root symbioses are often shared between neighboring plants, thus forming complex plant-fungus interaction networks of weak specialization. Whether this weak specialization also holds in rich tropical communities with more phylogenetically diverse sets of plant lineages remains unknown. We collected roots of 30 plant species in semi-natural tropical communities including angiosperms, ferns, and lycophytes, in three different habitat types on La Réunion island: a recent lava flow, a wet thicket, and an ericoid shrubland. We identified root-inhabiting fungi by sequencing both the 18S rRNA and the ITS2 variable regions. We assessed the diversity of mycorrhizal fungal taxa according to plant species and lineages, as well as the structure and specialization of the resulting plant-fungus networks. RESULTS: The 18S and ITS2 datasets are highly complementary at revealing the root mycobiota. According to 18S, Glomeromycotina colonize all plant groups in all habitats forming the least specialized interactions, resulting in nested network structures, while Mucoromycotina (Endogonales) are more abundant in the wetland and show higher specialization and modularity compared to the former. According to ITS2, mycorrhizal fungi of Ericaceae and Orchidaceae, namely Helotiales, Sebacinales, and Cantharellales, also colonize the roots of most plant lineages, confirming that they are frequent endophytes. While Helotiales and Sebacinales present intermediate levels of specialization, Cantharellales are more specialized and more sporadic in their interactions with plants, resulting in highly modular networks. CONCLUSIONS: This study of the root mycobiome in tropical environments reinforces the idea that mycorrhizal fungal taxa are locally shared between co-occurring plants, including phylogenetically distant plants (e.g. lycophytes and angiosperms), where they may form functional mycorrhizae or establish endophytic colonization. Yet, we demonstrate that, irrespectively of the environmental variations, the level of specialization significantly varies according to the fungal lineages, probably reflecting the different evolutionary origins of these plant-fungus symbioses. Frequent fungal sharing between plants questions the roles of the different fungi in community functioning and highlights the importance of considering networks of interactions rather than isolated hosts.

Orthoptera, a new order of pollinator.

BACKGROUND AND AIMS: Pollinator-mediated selection and evolution of floral traits have long fascinated evolutionary ecologists. No other plant family shows as wide a range of pollinator-linked floral forms as Orchidaceae. In spite of the large size of this model family and a long history of orchid pollination biology, the identity and specificity of most orchid pollinators remains inadequately studied, especially in the tropics where the family has undergone extensive diversification. Angraecum (Vandeae, Epidendroideae), a large genus of tropical Old World orchids renowned for their floral morphology specialized for hawkmoth pollination, has been a model system since the time of Darwin. METHODS: The pollination biology of A. cadetii, an endemic species of the islands of Mauritius and Reunion (Mascarene Islands, Indian Ocean) displaying atypical flowers for the genus (white and medium-size, but short-spurred) was investigated. Natural pollinators were observed by means of hard-disk camcorders. Pollinator-linked floral traits, namely spur length, nectar volume and concentration and scent production were also investigated. Pollinator efficiency (pollen removal and deposition) and reproductive success (fruit set) were quantified in natural field conditions weekly during the 2003, 2004 and 2005 flowering seasons (January to March). KEY RESULTS: Angraecum cadetii is self-compatible but requires a pollinator to achieve fruit set. Only one pollinator species was observed, an undescribed species of raspy cricket (Gryllacrididae, Orthoptera). These crickets, which are nocturnal foragers, reached flowers by climbing up leaves of the orchid or jumping across from neighbouring plants and probed the most 'fresh-looking' flowers on each plant. Visits to flowers were relatively long (if compared with the behaviour of birds or hawkmoths), averaging 16.5 s with a maximum of 41.0 s. At the study site of La Plaine des Palmistes (Pandanus forest), 46.5 % of flowers had pollen removed and 27.5 % had pollinia deposited on stigmas. The proportion of flowers that set fruit ranged from 11.9 % to 43.4 %, depending of the sites sampled across the island. CONCLUSIONS: Although orthopterans are well known for herbivory, this represents the first clearly supported case of orthopteran-mediated pollination in flowering plants.

Why does the biota of the Madagascar region have such a strong Asiatic flavour?

A corollary of island biogeographical theory is that islands are largely colonized from their nearest mainland source. Despite Madagascar's extreme isolation from India and proximity to Africa, a high proportion of the biota of the Madagascar region has Asian affinities. This pattern has rarely been viewed as surprising, as it is consistent with Gondwanan vicariance. Molecular phylogenetic data provide strong support for such Asian affinities, but often not for their vicariant origin; most divergences between lineages in Asia and the Madagascar region post-date the separation of India and Madagascar considerably (up to 87 Myr), implying a high frequency of dispersal that mirrors colonization of the Hawaiian archipelago in distance. Indian Ocean bathymetry and the magnitude of recent sea-level lowstands support the repeated existence of sizeable islands across the western Indian Ocean, greatly reducing the isolation of Madagascar from Asia. We put forward predictions to test the role of this historical factor in the assembly of the regional biota. © The Willi Hennig Society 2009.

Fragrant volatile compounds in the liverwort Drepanolejeunea madagascariensis (Steph.) Grolle: approach by the HS-SPME technique.

Three populations of the epiphyllous liverwort Drepanolejeunea madagascariensis collected in the cloud forests of Reunion Island (Mascarene Archipelago) were investigated for their volatile compounds, because of the pleasant, sweet, warm, woody-spicy, and herbaceous fragrance, slightly reminiscent of dill, of this species. By applying the headspace solid-phase microextraction (HS-SPME) technique coupled to GC/MS analysis, 34 compounds were detected in total, with p-menth-1-en-9-ol (28.8-43.5%), limonene (10.5-14.7%), beta-phellandrene (8.8-11.6%), and the so-called dill ether (8.5-16.6%) as the main components. The presence of 1-epi-alpha-pinguisene confirms the possible use of pinguisane-type sesquiterpenoids as a characteristic chemical marker for the order Jungermanniales.

Volatile compounds from Melicope obscura.

To evaluate the interpopulation variability of volatile compounds in Melicope obscura, four samples representing four populations were collected all over the distribution area of the species in Reunion Island (Indian Ocean). The samples were extracted by hydrodistillation, and analyzed using GC/FID and GC/MS techniques. The study revealed that, in the four essential oils obtained, oxygenated sesquiterpenes were one of the major chemical classes (9.2-35.2%), mainly consisting of a new compound, (+)-6-ethenyl-2-hydroxy-6,10-dimethylundeca-2,9-dien-4-one (1), called melicopenol (8.6-30.1%). The compound was isolated by column chromatography and identified by spectral analyses including 1D- and 2D-NMR.

A combined field survey and molecular identification protocol for comparing forest arthropod biodiversity across spatial scales.

Obtaining fundamental biodiversity metrics such as alpha, beta and gamma diversity for arthropods is often complicated by a lack of prior taxonomic information and/or taxonomic expertise, which can result in unreliable morphologically based estimates. We provide a set of standardized ecological and molecular sampling protocols that can be employed by researchers whose taxonomic skills may be limited, and where there may be a lack of robust a priori information regarding the regional pool of species. These protocols combine mass sampling of arthropods, classification of samples into parataxonomic units (PUs) and selective sampling of individuals for mtDNA sequencing to infer biological species. We sampled ten lowland rainforest plots located on the volcanic oceanic island of Réunion (Mascarene archipelago) for spiders, a group with limited taxonomic and distributional data for this region. We classified adults and juveniles into PUs and then demonstrated the reconciliation of these units with presumed biological species using mtDNA sequence data, ecological data and distributional data. Because our species assignment protocol is not reliant upon prior taxonomic information, or taxonomic expertise, it minimizes the problem of the Linnean shortfall to yield diversity estimates that can be directly compared across independent studies. Field sampling can be extended to other arthropod groups and habitats by adapting our field sampling protocol accordingly.

Predicting Where a Radiation Will Occur: Acoustic and Molecular Surveys Reveal Overlooked Diversity in Indian Ocean Island Crickets (Mogoplistinae: Ornebius).

Recent theory suggests that the geographic location of island radiations (local accumulation of species diversity due to cladogenesis) can be predicted based on island area and isolation. Crickets are a suitable group for testing these predictions, as they show both the ability to reach some of the most isolated islands in the world, and to speciate at small spatial scales. Despite substantial song variation between closely related species in many island cricket lineages worldwide, to date this characteristic has not received attention in the western Indian Ocean islands; existing species descriptions are based on morphology alone. Here we use a combination of acoustics and DNA sequencing to survey these islands for Ornebius crickets. We uncover a small but previously unknown radiation in the Mascarenes, constituting a three-fold increase in the Ornebius species diversity of this archipelago (from two to six species). A further new species is detected in the Comoros. Although double archipelago colonisation is the best explanation for species diversity in the Seychelles, in situ cladogenesis is the best explanation for the six species in the Mascarenes and two species of the Comoros. Whether the radiation of Mascarene Ornebius results from intra- or purely inter- island speciation cannot be determined on the basis of the phylogenetic data alone. However, the existence of genetic, song and ecological divergence at the intra-island scale is suggestive of an intra-island speciation scenario in which ecological and mating traits diverge hand-in-hand. Our results suggest that the geographic location of Ornebius radiations is partially but not fully explained by island area and isolation. A notable anomaly is Madagascar, where our surveys are consistent with existing accounts in finding no Ornebius species present. Possible explanations are discussed, invoking ecological differences between species and differences in environmental history between islands.

Methylenedioxy- and methoxyflavones from Melicope coodeana syn. Euodia simplex.

Three new natural products, 3,8-dimethoxy-5,7-dihydroxy-3',4'-methylenedioxyflavone, 3,6,8-trimethoxy-5,7-dihydroxy-3',4'-methylenedioxyflavone and 3,6,8,3',4'-pentamethoxy-5,7-dihydroxyflavone were isolated from Melicope coodeana syn. Euodia simplex (Rutaceae) along with 3,6,3'-trimethoxy-5,7,4'-trihydroxyflavone and 3,3'-dimethoxy-5,7,4'-trihydroxyflavone. The structural assignments are based on (1)H and (13)C NMR data, including discussion of the chemical shifts of C-2 in 3,5-dihydroxy- and 3-methoxy-5-hydroxyflavones. The presence of highly methoxylated and methylenedioxyflavones is characteristic of the genus Melicope, and the present findings support the recent transfer of Euodia simplex to Melicope.

Isolation and Characterization of Microsatellite Markers for Mimusops balata (Sapotaceae) and Cross-Amplification in Other Mimusops Species.

Mimusops balata (Sapotaceae) is an endemic tree species from La Réunion and Mauritius. Like many species growing in lowland forests in La Réunion, it has suffered from human disturbances. We developed twelve microsatellite markers for M. balata and tested cross-amplification in five other Mimusops species to have powerful tools for genetic diversity studies. Genotyping peaks were of very low quality for two loci and were consequently abandoned for the genetic diversity analyses. Ten microsatellite loci were tested on 34 individuals of M. balata from two natural populations. The number of alleles per locus ranged from one to seven. The observed and expected heterozygosity levels varied from 0.000 to 0.823, and from 0.000 to 0.812 respectively. Two loci deviated from the Hardy-Weinberg equilibrium. The presence of null alleles was detected for one of these two loci. Nine to ten loci cross-amplified reliably in Mauritian species, for the other three species, four to six loci show successful amplifications. These polymorphic microsatellite markers are now available for population genetic investigations in Mimusops species aiming to establish accurate guidelines for conservation managers.

Hybridization and barriers to gene flow in an island bird radiation.

While reinforcement may play a role in all major modes of speciation, relatively little is known about the timescale over which species hybridize without evolving complete reproductive isolation. Birds have high potential for hybridization, and islands provide simple settings for uncovering speciation and hybridization patterns. Here we develop a phylogenetic hypothesis for a phenotypically diverse radiation of finch-like weaver-birds (Foudia) endemic to the western Indian Ocean islands. We find that unlike Darwin's finches, each island-endemic Foudia population is a monophyletic entity for which speciation can be considered complete. In explaining the only exceptions-mismatches between taxonomy, mitochondrial, and nuclear data-phylogenetic and coalescent methods support introgressive hybridization rather than incomplete lineage sorting. Human introductions of known timing of one island-endemic species, to all surrounding archipelagos provide two fortuitous experiments; (1) population sampling at known times in recent evolutionary history, (2) bringing allopatric lineages of an island radiation into secondary contact. Our results put a minimum time bound on introgression (235 years), and support hybridization between species in natural close contact (parapatry), but not between those in natural allopatry brought into contact by human introduction. Time in allopatry, rather than in sympatry, appears key in the reproductive isolation of Foudia species.

In and out of Madagascar: dispersal to peripheral islands, insular speciation and diversification of Indian Ocean daisy trees (Psiadia, Asteraceae).

Madagascar is surrounded by archipelagos varying widely in origin, age and structure. Although small and geologically young, these archipelagos have accumulated disproportionate numbers of unique lineages in comparison to Madagascar, highlighting the role of waif-dispersal and rapid in situ diversification processes in generating endemic biodiversity. We reconstruct the evolutionary and biogeographical history of the genus Psiadia (Asteraceae), a plant genus with near equal numbers of species in Madagascar and surrounding islands. Analyzing patterns and processes of diversification, we explain species accumulation on peripheral islands and aim to offer new insights on the origin and potential causes for diversification in the Madagascar and Indian Ocean Islands biodiversity hotspot. Our results provide support for an African origin of the group, with strong support for non-monophyly. Colonization of the Mascarenes took place by two evolutionary distinct lineages from Madagascar, via two independent dispersal events, each unique for their spatial and temporal properties. Significant shifts in diversification rate followed regional expansion, resulting in co-occurring and phenotypically convergent species on high-elevation volcanic slopes. Like other endemic island lineages, Psiadia have been highly successful in dispersing to and radiating on isolated oceanic islands, typified by high habitat diversity and dynamic ecosystems fuelled by continued geological activity. Results stress the important biogeographical role for Rodrigues in serving as an outlying stepping stone from which regional colonization took place. We discuss how isolated volcanic islands contribute to regional diversity by generating substantial numbers of endemic species on short temporal scales. Factors pertaining to the mode and tempo of archipelago formation and its geographical isolation strongly govern evolutionary pathways available for species diversification, and the potential for successful diversification of dispersed lineages, therefore, appears highly dependent on the timing of arrival, as habitat and resource properties change dramatically over the course of oceanic island evolution.