Environmental DNA metabarcoding of wild flowers reveals diverse communities of terrestrial arthropods

Terrestrial arthropods comprise the most species‐rich communities on Earth, and grassland flowers provide resources for hundreds of thousands of arthropod species. Diverse grassland ecosystems worldwide are threatened by various types of environmental change, which has led to decline in arthropod diversity. At the same time, monitoring grassland arthropod diversity is time‐consuming and strictly dependent on declining taxonomic expertise. Environmental DNA (eDNA) metabarcoding of complex samples has demonstrated that information on species compositions can be efficiently and non‐invasively obtained. Here, we test the potential of wild flowers as a novel source of arthropod eDNA. We performed eDNA metabarcoding of flowers from several different plant species using two sets of generic primers, targeting the mitochondrial genes 16S rRNA and COI. Our results show that terrestrial arthropod species leave traces of DNA on the flowers that they interact with. We obtained eDNA from at least 135 arthropod species in 67 families and 14 orders, together representing diverse ecological groups including pollinators, parasitoids, gall inducers, predators, and phytophagous species. Arthropod communities clustered together according to plant species. Our data also indicate that this experiment was not exhaustive, and that an even higher arthropod richness could be obtained using this eDNA approach. Overall, our results demonstrate that it is possible to obtain information on diverse communities of insects and other terrestrial arthropods from eDNA metabarcoding of wild flowers. This novel source of eDNA represents a vast potential for addressing fundamental research questions in ecology, obtaining data on cryptic and unknown species of plant‐associated arthropods, as well as applied research on pest management or conservation of endangered species such as wild pollinators.     

Biodiversity,conservation and inventory: why insects matter

Western culture views insects and arachnids as pests and vermin that need to be controlled. They usually are not considered as something to be preserved. Accordingly, arthropods and other small organisms have not been taken seriously for conservation by policy makers and the conservation community at large. Having existed for more than 400 million years and after surviving the Permian and Cretaceous mass extinctions, arthropods have been the most successful of all living things and along with other invertebrates constitute more than three-quarters of today's global biodiversity. Arthropods are major components of diverse ecosystems and are the major players in functioning of ecosystem processes. Nevertheless, arthropods, which are the least known and, along with other plants and animals, are relentlessly vanishing before our eyes. Thus, aside from anthropocentric perception and societal prejudice, arthropods certainly are not pests in an ecological or evolutionary context and have an inherent biological right to exist in an evolutionary context, with ecological and instrumental values. They must be preserved because of their inherent values but also because we need them for human survival. Thus, arthropods must become an important and necessary part of the conservation strategy at all levels of environmental organization, from populations and species to ecosystems and landscapes.Insect conservation aims at saving both endangered species and ecosystem processes with a multitude of approaches targeted at different scales. Conservation efforts for arthropods are daunting because all the odds are against them: whereas species diversity, population size and biomass are so large, taxonomy and faunal information are inadequate; whereas the need for taxonomic and biodiversity information increases greatly, the shortage of taxonomic expertise worsens.Basic issues of biodiversity and the loss of species are reviewed. The goals and strategies for insect conservation are discussed with a focus on inventory and monitoring. Taxonomic and environmental surveys are compared, and the needs for biodiversity monitoring are discussed as ecological monitoring process based on inventory data. This monitoring focuses on the health of nested biodiversity (composition, structure and process) and the state of species, differing from other contemporary monitoring efforts.     

Molecular phylogenetics at the population/species interface in cave spiders of the southern Appalachians (Araneae:Nesticidae:Nesticus)

This paper focuses on the relationship between population genetic structure and speciation mechanisms in a monophyletic species group of Appalachian cave spiders (Nesticus). Using mtDNA sequence data gathered from 256 individuals, I analyzed patterns of genetic variation within and between populations for three pairs of closely related sister species. Each sister-pair comparison involves taxa with differing distributional and ecological attributes; if these ecological attributes are reflected in basic demographic differences, then speciation might proceed differently across these sister taxa comparisons. Both frequency-based and gene tree analyses reveal that the genetic structure of the Nesticus species studied is characterized by similar and essentially complete population subdivision, regardless of differences in general ecology. These findings contrast with results of prior genetic studies of cave-dwelling arthropods that have typically revealed variation in population structure corresponding to differences in general ecology. Species fragmentation through both extrinsic and intrinsic evolutionary forces has resulted in discrete, perhaps independent, populations within morphologically defined species. Large sequence divergence values observed between populations suggest that this independence may extend well into the past. These patterns of mtDNA genealogical structure and divergence imply that species as morphological lineages are currently more inclusive than basal evolutionary or phylogenetic units, a suggestion that has important implications for the study of speciation mechanisms.      

Diversity of agromyzidae and associated hymenopteran parasitoid species in the afrotropical region: implications for biological control

Agromyzidae (Diptera) is a family with many species of economic importance on agricultural plants. However, many species are attacked by hymenopteran parasitoids which are known to be habitat rather than species specific. In the Afrotropical region, information about agromyzid and parasitoid diversity in different habitats is scattered in literature. Our aim was to assemble this dispersed information and discuss future needs of biological control efforts against invasive agromyzids in the Afrotropics. From published information, 581 agromyzid records comprising 302 species from 20 genera were recorded from 48 plant families. Parasitoids (90 records) were associated only with 20 agromyzid species belonging to 10 different genera. The comparatively low parasitoid diversity in the Aftrotropical region is, however, most likely a result of poor sampling effort and lack of taxonomic expertise for parasitoid species in this area. More research on native parasitoids and associated Agromyzidae is therefore required before embarking on extensive biological control programmes.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Coccinellid morphospecies as an alternative method for differentiating management regimes in olive orchards

Morphospecies, also known as morphotypes, recognizable taxonomic units (RTUs) and parataxonomic units (PUs) have been used for rapid biodiversity assessment (RBA) in invertebrate diversity studies worldwide. Their utilization might lighten taxonomists’ workload when rapidly evaluating the richness and diversity of arthropods for conservation or biological assessment. To validate morphospecies, as opposed to taxonomic species, ladybird beetles (Coleoptera, Coccinellidae) were chosen in order to differentiate organic and non-organic management regimes (integrated and conventional) in olive orchards in southern Spain. Ladybird beetle specimens collected over two years (1999 and 2000) from three locations were sorted by morphospecies, and then identified by Coleopteran specialists according to taxonomic species. Thus, two different datasets were created, independently analyzed and compared to measure the accuracy at the morphospecies level. The comparison of morphospecies and species datasets showed an accuracy of 62.18% (one morphospecies to one taxonomic species), with the identifying error principally made when one species was identified as two different morphospecies (32.74%). Although two Coccinellid species (Scymnus mediterraneus Iablokoff-Khnzorian, 1972 and Coccinella septempunctata Linnaeus, 1758) showed significant differences among regimes during the June–August period in spite of small errors, we suggest that the most abundant morphospecies of Coccinellidae and the June–August period could be adopted as a rapid and useful tool for evaluating the impacts of non-organic vs. organic management regimes in olive orchards.     

Taxonomic Resolution and Quantification of Freshwater Macroinvertebrate Samples from an Australian Dryland River: The Benefits and Costs of Using Species Abundance Data

In studies using macroinvertebrates as indicators for monitoring rivers and streams, species level identifications in comparison with lower resolution identifications can have greater information content and result in more reliable site classifications and better capacity to discriminate between sites, yet many such programmes identify specimens to the resolution of family rather than species. This is often because it is cheaper to obtain family level data than species level data. Choice of appropriate taxonomic resolution is a compromise between the cost of obtaining data at high taxonomic resolutions and the loss of information at lower resolutions. Optimum taxonomic resolution should be determined by the information required to address programme objectives. Costs saved in identifying macroinvertebrates to family level may not be justified if family level data can not give the answers required and expending the extra cost to obtain species level data may not be warranted if cheaper family level data retains sufficient information to meet objectives. We investigated the influence of taxonomic resolution and sample quantification (abundance vs. presence/absence) on the representation of aquatic macroinvertebrate species assemblage patterns and species richness estimates. The study was conducted in a physically harsh dryland river system (Condamine-Balonne River system, located in south-western Queensland, Australia), characterised by low macroinvertebrate diversity. Our 29 study sites covered a wide geographic range and a diversity of lotic conditions and this was reflected by differences between sites in macroinvertebrate assemblage composition and richness. The usefulness of expending the extra cost necessary to identify macroinvertebrates to species was quantified via the benefits this higher resolution data offered in its capacity to discriminate between sites and give accurate estimates of site species richness. We found that very little information (<6%) was lost by identifying taxa to family (or genus), as opposed to species, and that quantifying the abundance of taxa provided greater resolution for pattern interpretation than simply noting their presence/absence. Species richness was very well represented by genus, family and order richness, so that each of these could be used as surrogates of species richness if, for example, surveying to identify diversity hot-spots. It is suggested that sharing of common ecological responses among species within higher taxonomic units is the most plausible mechanism for the results. Based on a cost/benefit analysis, family level abundance data is recommended as the best resolution for resolving patterns in macroinvertebrate assemblages in this system. The relevance of these findings are discussed in the context of other low diversity, harsh, dryland river systems.     

Phylogeny and temporal diversification of darters (Percidae: Etheostomatinae)

Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species-sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species-sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters.     

A role for molecular genetics in the recognition and conservation of endangered species

Taxonomies based on morphological traits alone sometimes provide inadequate or misleading guides to phylogenetic distinctions at the subspecies and species levels. Yet taxonomic assignments inevitably shape perceptions of biotic diversity, including recognition of endangered species. Case histories are discussed in which the data of molecular genetics revealed prior systematic errors of the two possible kinds: taxonomic recognition of groups showing little evolutionary differentiation, and lack of taxonomic recognition of phylogenetically distinct forms. In such cases, conservation efforts for 'endangered species' can be misdirected with respect to the goal of protecting biological diversity.     

The Costs of Evaluating Species Densities and Composition of Snakes to Assess Development Impacts in Amazonia

Studies leading to decision-making for environmental licensing often fail to provide accurate estimates of diversity. Measures of snake diversity are regularly obtained to assess development impacts in the rainforests of the Amazon Basin, but this taxonomic group may be subject to poor detection probabilities. Recently, the Brazilian government tried to standardize sampling designs by the implementation of a system (RAPELD) to quantify biological diversity using spatially-standardized sampling units. Consistency in sampling design allows the detection probabilities to be compared among taxa, and sampling effort and associated cost to be evaluated. The cost effectiveness of detecting snakes has received no attention in Amazonia. Here we tested the effects of reducing sampling effort on estimates of species densities and assemblage composition. We identified snakes in seven plot systems, each standardised with 14 plots. The 250 m long centre line of each plot followed an altitudinal contour. Surveys were repeated four times in each plot and detection probabilities were estimated for the 41 species encountered. Reducing the number of observations, or the size of the sampling modules, caused significant loss of information on species densities and local patterns of variation in assemblage composition. We estimated the cost to find a snake as $ 120 U.S., but general linear models indicated the possibility of identifying differences in assemblage composition for half the overall survey costs. Decisions to reduce sampling effort depend on the importance of lost information to target-issues, and may not be the preferred option if there is the potential for identifying individual snake species requiring specific conservation actions. However, in most studies of human disturbance on species assemblages, it is likely to be more cost-effective to focus on other groups of organisms with higher detection probabilities.     

Terrestrial invertebrate surveys and rapid biodiversity assessment in New Zealand: lessons from Australia

Although invertebrates play a key role in the environment, their conservation and use in environmental monitoring is often considered “too difficult” and consequently ignored. One of the main problems in dealing with invertebrates is that even limited sampling can yield large numbers of specimens and an enormous diversity of species. Other problems include the taxonomic impediment (i.e. high proportions of invertebrate taxa are undescribed and there are few specialists available to identify specimens), the lack of knowledge on species distribution, diversity and ecological roles, and the fact that invertebrates are undervalued by the general public. A number of rapid biodiversity assessment (RBA) approaches have been suggested to overcome these problems. RBA approaches generally fall into four categories: (1) restricted sampling in place of intensive sampling (sampling surrogacy); (2) the use of higher taxonomic levels than species (species surrogacy); (3) the use of recognisable taxonomic units (RTUs) identified by non-specialists (taxonomic surrogacy); and (4) the use of surrogate taxa in place of all taxa (taxon-focusing). Australia has a long history of using invertebrates in terrestrial ecological studies, and in developing and using RBA approaches. Therefore, New Zealand could benefit from the experienced gained in Australia. Potentially one of the most useful RBA approaches to take in New Zealand involves focusing resources and attention on a limited range of taxa. However, this requires substantial communication, discussion, and agreement over which taxa should be selected for conservation priorities and environmental monitoring in terrestrial ecosystems.     

Quantifying biodiversity: procedures and pitfalls in the measurement and comparison of species richness

Species richness is a fundamental measurement of community and regional diversity, and it underlies many ecological models and conservation strategies. In spite of its importance, ecologists have not always appreciated the effects of abundance and sampling effort on richness measures and comparisons. We survey a series of common pitfalls in quantifying and comparing taxon richness. These pitfalls can be largely avoided by using accumulation and rarefaction curves, which may be based on either individuals or samples. These taxon sampling curves contain the basic information for valid richness comparisons, including category–subcategory ratios (species-to-genus and species-to-individual ratios). Rarefaction methods – both sample-based and individual-based – allow for meaningful standardization and comparison of datasets. Standardizing data sets by area or sampling effort may produce very different results compared to standardizing by number of individuals collected, and it is not always clear which measure of diversity is more appropriate. Asymptotic richness estimators provide lower-bound estimates for taxon-rich groups such as tropical arthropods, in which observed richness rarely reaches an asymptote, despite intensive sampling. Recent examples of diversity studies of tropical trees, stream invertebrates, and herbaceous plants emphasize the importance of carefully quantifying species richness using taxon sampling curves.     

Ecological integrity assessment as a metric of biodiversity: are we measuring what we say we are?

As the recognition of the importance of biological diversity in biological conservation grows, an ongoing challenge is to develop metrics that can be used for effective conservation and management. The ecological integrity assessment has been proposed as such a metric. It is held by some to measure species composition, diversity, and habitat quality, as well as ecosystem structure, composition, and function. The methodology relies on proxy variables that include data on landscape characteristics such as patch size, abiotic factors such as hydrology, and some features of vegetation structure and composition. We suggest that the measure is flawed on four levels. First, its putative representation of general ecological form and function, and its lack of specific detail about how it actually represents those attributes, leaves the metric without the focus needed to be useful for measuring ecological features on the ground and testing associated hypotheses and predictions. Second, the proxy variables used to represent biological diversity, such as habitat (vegetation) metrics and vascular plant species diversity, are not empirically correlated with diversity of a range of taxa or of other components of the biota. Third, like other ecological indices that integrate many distinct features, the ecological integrity index is subject to the loss of information in its condensation of multi-dimensional variability into a one-dimensional index, and it may be subject to systematic bias from the conversion of raw data into categorical scores. Fourth, the sampling protocols are at risk of sampling bias, observer bias, and measurement error, any of which can confound the estimation of conservation value. In terms of biological diversity, the methodology produces an unreliable estimate of the number of vascular plant species and their relative percentages of occurrence, and an absence of any protocols for taxa other than plants. For these reasons we believe that ecological integrity assessment is currently of limited value as a measure of site-specific biological diversity and its change over time. A considerable amount of investigation is needed in order to have confidence in the results of an ecological integrity assessment, especially if it is to be used for regulatory purposes. We suggest further refinements and discuss alternative measures of biological diversity that provide reliable metrics for assessing change. A thoughtful choice among measures can help to identify the most appropriate assessment for conservation decisions.     

Toward accurate species‐level metabarcoding of arthropod communities from the tropical forest canopy

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Arthropod Distribution in a Tropical Rainforest: Tackling a Four Dimensional Puzzle

Quantifying the spatio-temporal distribution of arthropods in tropical rainforests represents a first step towards scrutinizing the global distribution of biodiversity on Earth. To date most studies have focused on narrow taxonomic groups or lack a design that allows partitioning of the components of diversity. Here, we consider an exceptionally large dataset (113,952 individuals representing 5,858 species), obtained from the San Lorenzo forest in Panama, where the phylogenetic breadth of arthropod taxa was surveyed using 14 protocols targeting the soil, litter, understory, lower and upper canopy habitats, replicated across seasons in 2003 and 2004. This dataset is used to explore the relative influence of horizontal, vertical and seasonal drivers of arthropod distribution in this forest. We considered arthropod abundance, observed and estimated species richness, additive decomposition of species richness, multiplicative partitioning of species diversity, variation in species composition, species turnover and guild structure as components of diversity. At the scale of our study (2km of distance, 40m in height and 400 days), the effects related to the vertical and seasonal dimensions were most important. Most adult arthropods were collected from the soil/litter or the upper canopy and species richness was highest in the canopy. We compared the distribution of arthropods and trees within our study system. Effects related to the seasonal dimension were stronger for arthropods than for trees. We conclude that: (1) models of beta diversity developed for tropical trees are unlikely to be applicable to tropical arthropods; (2) it is imperative that estimates of global biodiversity derived from mass collecting of arthropods in tropical rainforests embrace the strong vertical and seasonal partitioning observed here; and (3) given the high species turnover observed between seasons, global climate change may have severe consequences for rainforest arthropods.     

DNA Barcoding is not enough: mismatch of taxonomy and genealogy in New Zealand grasshoppers (Orthoptera: Acrididae).

DNA barcoding has been touted as a program that will efficiently and relatively cheaply inform on biological diversity; yet many exemplars purporting to demonstrate the efficacy of the method have been undertaken by its principal proponents. Critics of DNA barcoding identify insufficient within-taxon sampling coupled with the knowledge that levels of haplotypic paraphyly are rather high as key reasons to be sceptical of the value of an exclusively DNA-based taxonomic. Here I applied a DNA barcoding approach using mtDNA sequences from the cytochrome oxidase I gene to examine diversity in a group of endemic New Zealand grasshoppers belonging to the genus Sigaus . The mtDNA data revealed high genetic distances among individuals of a single morpho-species, but this diversity was geographically partitioned. Phylogenetic analysis supported at least four haplogroups within one species ( Sigaus australis ) but paraphyly of this species with respect to several others. In some instances two morphologically and ecologically distinct species shared identical mtDNA haplotypes. The mismatch of genealogy and taxonomy revealed in the Sigaus australis complex indicates that, if used in isolation, DNA barcoding data can be highly misleading about biodiversity. Furthermore, failure to take into account evidence from natural history and morphology when utilizing DNA barcoding will tend to conceal the underlying evolutionary processes associated with speciation.
© The Willi Hennig Society 2007.     

Database records as a surrogate for sampling effort provide higher species richness estimations

The compilation of all the available taxonomic and distributional information on the species present in a territory frequently generates a biased picture of the distribution of biodiversity due to the uneven distribution of the sampling effort performed. Thus, quality protocol assessments such as those proposed by Hortal et al. (Conservation Biology 21:853–863, 2007) must be done before using this kind of information for basic and applied purposes. The discrimination of localities that can be considered relatively well-surveyed from those not surveyed enough is a key first step in this protocol and can be attained by the previous definition of a sampling effort surrogate and the calculation of survey completeness using different estimators. Recently it has been suggested that records from exhaustive databases can be used as a sampling-effort surrogate to recognize probable well-surveyed localities. In this paper, we use an Iberian dung beetle database to identify the 50 × 50 km UTM cells that appear to be reliably inventoried, using both data derived from standardized sampling protocols and database records as a surrogate for sampling effort. Observed and predicted species richness values in the shared cells defined as well-surveyed by both methods suggest that the use of database records provides higher species richness values, which are proportionally greater in the richest localities by the inclusion of rare species.     

Flyway structure in the circumpolar greater white‐fronted goose

Dispersal and migratory behavior are influential factors in determining how genetic diversity is distributed across the landscape. In migratory species, genetic structure can be promoted via several mechanisms including fidelity to distinct migratory routes. Particularly within North America, waterfowl management units have been delineated according to distinct longitudinal migratory flyways supported by banding data and other direct evidence. The greater white‐fronted goose (Anser albifrons) is a migratory waterfowl species with a largely circumpolar distribution consisting of up to six subspecies roughly corresponding to phenotypic variation. We examined the rangewide population genetic structure of greater white‐fronted geese using mtDNA control region sequence data and microsatellite loci from 23 locales across North America and Eurasia. We found significant differentiation in mtDNA between sampling locales with flyway delineation explaining a significant portion of the observed genetic variation (~12%). This is concordant with band recovery data which shows little interflyway or intercontinental movements. However, microsatellite loci revealed little genetic structure suggesting a panmictic population across most of the Arctic. As with many high‐latitude species, Beringia appears to have played a role in the diversification of this species. A common Beringian origin of North America and Asian populations and a recent divergence could at least partly explain the general lack of structure at nuclear markers. Further, our results do not provide strong support for the various taxonomic proposals for this species except for supporting the distinctness of two isolated breeding populations within Cook Inlet, Alaska (A. a. elgasi) and Greenland (A. a. flavirostris), consistent with their subspecies status.