Ecological Experiments Research Articles

Long-term persistence of experimental populations beyond a species' natural range.

Ecological experiments usually infer long-term processes from short-term data, and the analysis of geographic range limits is a good example. Species' geographic ranges may be limited by low fitness due to niche constraints, a hypothesis most directly tested by comparing the fitness of populations transplanted within and beyond the range. Such studies often fail to find beyond-range fitness declines strong enough to conclude that geographic range limits are solely imposed by niche limits. However, almost all studies only follow transplants for a single generation, which will underestimate the importance of niche limitation because critical but infrequent range-limiting events may be missed and methodological issues may artificially boost the fitness of beyond-range transplants. Here, we present the first multi-generation beyond-range transplant experiment that involves adequate replication and proper experimental controls. In 2005, experimental populations of the coastal dune plant Camissoniopsis cheiranthifolia were planted at four sites within and one site beyond the northern limit. Fitness of initial transplants was high beyond the limit, suggesting that the range was limited by dispersal and not niche constraints. To better address the niche-limitation hypothesis, we quantified density and fitness of descendant C. cheiranthifolia populations 12-14 yr (˜10 generations) after transplant. Average annual fruit production and density of reproductive individuals were as high beyond the range as at four comparable experimental populations and eight natural populations within the range, and the beyond-range population had more than tripled in size since it was planted. This provides unprecedented support for the conclusion that northern range limit of C. cheiranthifolia results from something other than niche limitation, likely involving constraints on local dispersal.

Manipulative experiments networks on response and adaptation of terrestrial ecosystems to environmental changes: Building the research methods and technology system.

The feedback relationship between organisms or ecosystems and environment has been a key issue in ecological research. Manipulative experiments with changing biological or environmental factors and large-scale field experiment networks were regarded as effective approaches to understand and accurately quantify the process and mechanisms underlying ecosystem response and adaptation to environmental changes. In recent years, a few networks have been developed, including large-scale networks of field physics simulation experiment (i.e., ECOTRON), ecosystem analysis and experiment platform (AnaEE), international Drought Network, Nutrient Network, and experiment networks based on regional ecological observation stations (i.e., USA-ILTER) at global scale. The development of continent-scale experiment network platform is attracting more attention from the academic community, and will play a more important role in understanding the process and mechanism underlying ecosystem responses to environmental change. We reviewed the development of method and experiment system of ecosystem manipulative experiments, and clearly pointed out that different experiments should form a joint collaborative system to answer fundamental scientific questions about the response and adaptation of ecosystem to global environment change. Manipulative experiments could be classified into four types: 1) Physiological and ecological experiments in closed laboratory equipment; 2) semi-open experiments with changing environmental factors in the field; 3) near-natural field experiments; 4) experiment networks based on field ecological station. Furthermore, we discussed the trends in network design of manipulative experiments focusing on ecosystem response and adaptation to environmental changes and the advantages of large-scale experiments based on natural environmental gradients. We put forward a proposal of integrating the technical advantages of different types of experiments and developing a new generation of field experiment network system. The study discussed the research system based on field experiment network, demonstrated the possibility to understand the patterns and mechanisms of the ecosystem short-term response and long-term adaptation to environmental changes, and proposed some equations to quantify the environmental response of ecosystems. The application of the design plan of the manipulative experiments network proposed here will greatly promote the scientific research level of ecosystems and environmental changes in China and even over the whole world, which has important scientific significance for the national response to climate change and ecological environmental construction.

A successful method of transplanting adult Ecklonia radiata kelp, and relevance to other habitat‐forming macroalgae

The ability to successfully transplant adult kelp has applications not only for ecological experiments, but also for habitat conservation and restoration projects. However, approaches to the long‐term transplanting of adult kelp (especially for stalked or ‘stipitate’ species), and the communication of these methods, has been relatively haphazard, often due to poor results and excessive mortality. Here, we provide a brief communication to describe a method to effectively transplant the stipitate kelp Ecklonia radiata—the most widespread and abundant kelp in Australasia—and which allowed the transplanting of >1,000 adult kelp over 1.5 ha. We also discuss additional observations relevant to the success of transplanting kelp (such as donor age/size), and the applicability of this method to other habitat‐forming macroalgae. Our method involved securing the adult Ecklonia to the substratum using large bands made from recycled rubber, which held the holdfast firmly but gently against the substratum. Re‐attachment of the adult kelp typically occurred within 3–6 weeks, while rates of survivorship were approximately 75% and not affected by the density nor size of the transplanted patch. This method could also readily be adapted to suit different substratum types and other species of kelp and habitat‐forming algae. Ultimately, this transplanting method adds to the collection of effective techniques for restoring habitat‐forming macroalgae, especially for stipitate species where few methods have been communicated.

Cyberneticisation as a Theory and Practice of Matter

The ecological implementation of cybernetic ideas in architecture requires a material theory and practice that enables their propositions to be tested. The need for approaches that move from simulation to cybernetic reality is a documented limitation of cybernetics recognised by Stafford Beer with his pond ecology experiments and Gordon Pask through electrochemical devices. While both experimented with adaptive material platforms as embodiments of designed cybernetic systems, their approaches were limited by the available toolsets. This article considers an ecological trajectory of cybernetisation by revisiting notions of biological computation as a generative material practice. In particular, the growing fields of biodesign and living architecture go beyond notions of biological analogues that inform modern architecture by directly incorporating living systems into the very fabric of buildings as designed expressions of ecology.

Ethical considerations of urban ecological design and planning experiments

Societal Impact StatementIt is increasingly common for plant scientists and urban planning and design professionals to collaborate on interdisciplinary teams that integrate scientific experiments into public and social urban spaces. However, neither the procedural ethics that govern scientific experimentation, nor the professional ethics of urban design and planning practice, fully account for the possible impacts of urban ecological experiments on local residents and communities. Scientists that participate in design and planning teams act as decision‐makers, and must expand their domain of ethical consideration accordingly. Conversely, practitioners who engage in ecological experiments take on the moral responsibilities inherent in generation of knowledge. To avoid potential harm to human and non‐human inhabitants of cities while maintaining scientific and professional integrity in research and practice, an integrated ethical framework is needed for urban ecological planning and design.SummaryWhile there are many ethical and procedural guidelines for scientists who wish to inform decision‐making and public policy, urban ecologists are increasingly embedded in planning and design teams to integrate scientific measurements and experiments into urban landscapes. These scientists are not just informing decision‐making – they are themselves acting as decision‐makers. As such, researchers take on additional moral obligations beyond scientific procedural ethics when designing and conducting ecological design and planning experiments. We describe the growing field of urban ecological design and planning and present a framework for expanding the ethical considerations of socioecological researchers and urban practitioners who collaborate on interdisciplinary teams. Drawing on existing ethical frameworks from a range of disciplines, we outline possible ways in which ecologists, social scientists, and practitioners should expand the traditional ethical considerations of their work to ensure that urban residents, communities, and non‐human entities are not harmed as researchers and practitioners carry out their individual obligations to clients, municipalities, and scientific practice. We present an integrated framework to aid in the development of ethical codes for research, practice, and education in integrated urban ecology, socioenvironmental sciences, and design and planning.

Performing Through Privatization: An Ecological Natural Experiment of the Impact of the Swedish Free Choice Reform on Ambulatory Care Sensitive Conditions.

Background: In 2010, Sweden opened up for establishment of privately owned primary health care providers, as part of a national Free Choice in Primary Health Care reform. The reform has been highly debated, and evidence on its effects is scarce. The present study therefore sought to evaluate whether the reform have impacted on primary health care service performance.Methods: This ecological register-based study used a natural experimental approach through an interrupted time series design. Data comprised the total adult population of the 21 counties of Sweden 2001–2009 (pre-intervention period) and 2010–2016 (post-intervention period). Hospitalizations and emergency department visits for ambulatory care sensitive conditions (ACSC) were used as indicators of primary health care performance. Segmented regression analysis was used to assess the effects of the reform, in Sweden as a whole, as well as compared between counties grouped by (i) change in private provision pre- to post reform; (ii) the timing of the implementation; and (iii) sustained presence of private providers both pre- and post-reform.Results: The results suggest that, following the introduction of the reform in Sweden as a whole, the trends in total hospitalizations rates were slowed down by 1.0% albeit acute emergency visits increased 1.1% more rapidly after the introduction of the reform. However, we found no evidence of more beneficial effects in counties where the reform had been implemented more ambitiously, specifically those with a larger increase in private primary care providers, or where the reform was introduced early and thus had longer time effects to emerge. Lastly, counties with a sustained high presence of private primary care providers displayed the least favorable development when it comes to ACSC.Conclusion: Taken together, the present study does not support that the Swedish Free Choice reform has improved performance of the primary care delivery system in Sweden, and suggests that high degree of private provision may involve worse performance and higher care burden for specialized health care. Further evaluations of the consequences of the reform are dire needed to provide a comprehensive picture of its intended and unintended impact on health care provision, delivery and results.

Bracon wasps for ecological pest control-a laboratory experiment.

Biological control of pest insects by natural enemies may be an effective, cheap and environmentally friendly alternative to synthetic pesticides. The cosmopolitan parasitoid wasp species Bracon brevicornis Wesmael and B. hebetor Say (Hymenoptera: Braconidae) use lepidopteran species as hosts, including insect pests like Ephestia kuehniella or Ostrinia nubilalis. Here, we compare the reproductive success of both Bracon species on E. kuehniella in a laboratory experiment. We asked (1) how the reproductive success on a single host larva changes with temperature, (2) how it changes with temperature when more host larvae are present and (3) how temperature and availability of host larvae influence the efficacy of Bracon species as biological control agents. In general, differences between B. brevicornis and B. hebetor have been small. For rearing both Bracon species in the laboratory on one host larva, a temperature between 20–27 °C seems appropriate to obtain the highest number of offspring with a female-biased sex ratio. Rearing the braconid wasps on more than one host larva revealed a higher number of total offspring but less offspring per host larva on average. Again, highest numbers of offspring hatched at 27 °C and the sex ratio was independent from temperature. Although no parasitoids hatched at 12 °C and only few at 36 °C, host larvae were still paralyzed. The efficacy of B. brevicornis was higher than 80% at all numbers of host larvae presented at all temperatures while the efficacy of B. hebetor was less than 80% at 12 °C and 27 °C at low numbers of host larvae presented. In conclusion, practitioners can use either B. brevicornis or B. hebetor at low and high temperatures and at varying host densities to achieve high pest control efficacy.

Mahalanobis distances for ecological niche modelling and outlier detection: implications of sample size, error, and bias for selecting and parameterising a multivariate location and scatter method.

The Mahalanobis distance is a statistical technique that has been used in statistics and data science for data classification and outlier detection, and in ecology to quantify species-environment relationships in habitat and ecological niche models. Mahalanobis distances are based on the location and scatter of a multivariate normal distribution, and can measure how distant any point in space is from the centre of this kind of distribution. Three different methods for calculating the multivariate location and scatter are commonly used: the sample mean and variance-covariance, the minimum covariance determinant, and the minimum volume ellipsoid. The minimum covariance determinant and minimum volume ellipsoid were developed to be robust to outliers by minimising the multivariate location and scatter for a subset of the full sample, with the proportion of the full sample forming the subset being controlled by a user-defined parameter. This outlier robustness means the minimum covariance determinant and the minimum volume ellipsoid are highly relevant for ecological niche analyses, which are usually based on natural history observations that are likely to contain errors. However, natural history observations will also contain extreme bias, to which the minimum covariance determinant and the minimum volume ellipsoid will also be sensitive. To provide guidance for selecting and parameterising a multivariate location and scatter method, a series of virtual ecological niche modelling experiments were conducted to demonstrate the performance of each multivariate location and scatter method under different levels of sample size, errors, and bias. The results show that there is no optimal modelling approach, and that choices need to be made based on the individual data and question. The sample mean and variance-covariance method will perform best on very small sample sizes if the data are free of error and bias. At larger sample sizes the minimum covariance determinant and minimum volume ellipsoid methods perform as well or better, but only if they are appropriately parameterised. Modellers who are more concerned about the prevalence of errors should retain a smaller proportion of the full data set, while modellers more concerned about the prevalence of bias should retain a larger proportion of the full data set. I conclude that Mahalanobis distances are a useful niche modelling technique, but only for questions relating to the fundamental niche of a species where the assumption of multivariate normality is reasonable. Users of the minimum covariance determinant and minimum volume ellipsoid methods must also clearly report their parameterisations so that the results can be interpreted correctly.

Boreal forest fertilization leads to functional homogenization of ground beetle assemblages

Abstract Intensive fertilization of young spruce forest plantations (i.e. ‘nutrient optimization’) has the potential to meet increasing demands for carbon sequestration and biomass production from boreal forests. However, its effects on biodiversity, other than the homogenization of ground‐layer plant communities, are widely unknown. We sampled ground beetles (Coleoptera: Carabidae) in young spruce forest plantations of southern Sweden, within a large‐scale, replicated ecological experiment initiated in 2012, where half of the forest stands were fertilized every second year. We assessed multi‐scale effects of forest fertilization on ground beetle diversity and community assembly, 4 years after commencement of the experiment. We found that nutrient optimization had negative effects on ground beetle diversity at multiple spatial scales, despite having negligible effects on species richness. At the local scale, ground beetle species had lower variation in body size at fertilized sites, resulting in within‐site functional homogenization. At the landscape scale, fertilized sites, with higher basal area and lower bilberry cover, filtered carabid traits composition to larger body sizes, generalist predators and summer breeding species. Synthesis and applications. Fertilization of young spruce plantations is a strong filter for ground beetle assemblages, leading to functionally homogeneous communities in the short term, without changes in species richness. The large‐scale functional impoverishment of carabid communities because of fertilization may have negative consequences on system resilience and on ecosystem service provision by this functionally diverse group. Large‐scale establishment of nutrient optimization threatens ground beetle diversity in young conifer plantations, underlining the risks of introducing more intensive management schemes in already heavily managed forest landscapes.

Fungus-bacterium associations are widespread in fungal cultures isolated from a semi-arid natural grassland in Germany.

We report on a study that aimed at establishing a large soil-fungal culture collection spanning a wide taxonomic diversity and systematically screening the collection for bacterial associations. Fungal cultures were isolated from soil samples obtained from a natural grassland in eastern Germany and bacterial associations were assessed by PCR-amplification and sequencing of bacterial 16S rRNA. In addition, intraspecies genetic diversities of a subset of the isolated species were estimated by double-digest restriction associated DNA sequencing. A total of 688 fungal cultures, representing at least 106 fungal species from 36 different families, were obtained and even though clonal isolates were identified in almost all fungal species subjected to ddRAD-seq, relatively high genetic diversities could be observed in some of the isolated species. A total of 69% of the fungal isolates in our collection were found to be associated with bacteria and the most commonly identified bacterial genera were Pelomonas, Enterobacter and Burkholderia. Our results indicate that bacterial associations commonly occur in soil fungi, even if antibiotics are being applied during the isolation process, and provide a basis for the use of our culture collection in ecological experiments that want to acknowledge the importance of intraspecies genetic diversity.

Instrumental variable methods in structural equation models

Abstract Instrumental variable regression (RegIV) provides a means for detecting and correcting parameter bias in causal models. Widely used in economics, recently several papers have highlighted its potential utility for ecological applications. Little attention has thus far been paid to the fact that IV methods can also be implemented within structural equation models (SEMIV). In this paper I present the motivations, requirements and basic procedures for using SEMIV. I first consider causal inference and IVs from the perspective of a randomized experiment with partial control of the cause of interest. I consider common sources of bias, the role of randomization and limits to its capacity to exclude bias. Sources of bias include omitted confounders, reciprocal causation, reverse causation and measurement error, all of which can all be seen as a single problem—endogeneity. The approach to estimating IV models most commonly used in econometric practice, two‐stage least squares regression (2SLS), is explained, followed by a brief exposition of the covariance modelling approach used in SEM. Using data from an ecological field experiment, I illustrate the use of the treatment variable as an IV and then illustrate procedures for evaluating candidate variables that might serve as additional IVs. IV methods are shown to be useful for both detecting endogeneity and removing its influences. I illustrate some of the ways that bias can be generated, as well as diagnostic capabilities and means for remedy embedded within SEM. Procedures for screening and evaluating additional IVs reveal valuable lessons regarding the theoretical requirements and empirical standards for IVs. SEMIV provides a useful way to detect and control for bias. I suggest that the use of IVs within the SEM framework can support the simultaneous pursuit of causal inference and explanatory modelling, a common pair of aspirations for ecologists. Moving forward, there is a need for a better understanding of the capabilities of SEMIV and requirements for successful application.

Effectively integrating experiments into conservation practice

AbstractMaking effective decisions in conservation requires a broad and robust evidence base describing the likely outcomes of potential actions to draw on. Such evidence is typically generated from experiments or trials that evaluate the effectiveness of actions, but for many actions evidence is missing or incomplete.We discuss how evidence can be generated by incorporating experiments into conservation practice. This is likely to be most efficient if opportunities for carrying out informative, well‐designed experiments are identified at an early stage during conservation management planning.We consider how to navigate a way between the stringent requirements of statistical textbooks and the complexities of carrying out ecological experiments in the real world by considering practical approaches to the key issues of replication, controls and randomization.We suggest that routinely sharing the results of experiments could increase both the value for money and effectiveness of conservation practice.We argue that with early planning and a small additional input of effort, important new learning can be gained during the implementation of many conservation actions.

Assessing long‐term effects of artificial light at night on insects: what is missing and how to get there

Abstract Widespread and significant declines of insect population abundances and biomass are currently one of the most pressing issues in entomology, ecology and conservation biology. It has been suggested that artificial light at night is one major driver behind this trend. Recent advances in the gathering and analysis of long‐term data sets of insect population and biomass trends, however, have mostly focused on the effects of climate change and agricultural intensification. We posit here that adequate assessment of artificial night at light that would be required to evaluate its role as a driver of insect declines is far from trivial. Currently its implementation into entomological monitoring programmes and long‐running ecological experiments is hampered by several challenges that arise due to (i) its relatively late appearance as a biodiversity threat on the research agenda and (ii) the interdisciplinary nature of the research field where biologists, physicists and engineers still need to develop a set of standardised assessment methods that are both biologically meaningful and easy to implement. As more studies that address these challenges are urgently needed, this article aims to provide a short overview of the few existing studies that have attempted to investigate longer‐term effects of artificial light at night on insect populations. To improve the quality and relevance of studies addressing artificial light at night and its effect on insects, we present a set of best practise recommendations where this field needs to be heading in the coming years and how to achieve it.

Interaction analyses based on growth parameters of GWAS between Escherichia coli and Staphylococcus aureus

To accurately explore the interaction mechanism between Escherichia coli and Staphylococcus aureus, we designed an ecological experiment to monoculture and co-culture E. coli and S. aureus. We co-cultured 45 strains of E. coli and S. aureus, as well as each species individually to measure growth over 36 h. We implemented a genome wide association study (GWAS) based on growth parameters (λ, R, A and s) to identify significant single nucleotide polymorphisms (SNPs) of the bacteria. Three commonly used growth regression equations, Logistic, Gompertz, and Richards, were used to fit the bacteria growth data of each strain. Then each equation’s Akaike’s information criterion (AIC) value was calculated as a commonly used information criterion. We used the optimal growth equation to estimate the four parameters above for strains in co-culture. By plotting the estimates for each parameter across two strains, we can visualize how growth parameters respond ecologically to environment stimuli. We verified that different genotypes of bacteria had different growth trajectories, although they were the same species. We reported 85 and 52 significant SNPs that were associated with interaction in E. coli and S. aureus, respectively. Many significant genes might play key roles in interaction, such as yjjW, dnaK, aceE, tatD, ftsA, rclR, ftsK, fepA in E. coli, and scdA, trpD, sdrD, SAOUHSC_01219 in S. aureus. Our study illustrated that there were multiple genes working together to affect bacterial interaction, and laid a solid foundation for the later study of more complex inter-bacterial interaction mechanisms.

Evaluating natural experiments in ecology: using synthetic controls in assessments of remotely sensed land treatments.

Many important ecological phenomena occur on large spatial scales and/or are unplanned and thus do not easily fit within analytical frameworks that rely on randomization, replication, and interspersed a priori controls for statistical comparison. Analyses of such large-scale, natural experiments are common in the health and econometrics literature, where techniques have been developed to derive insight from large, noisy observational data sets. Here, we apply a technique from this literature, synthetic control, to assess landscape change with remote sensing data. The basic data requirements for synthetic control include (1) a discrete set of treated and untreated units, (2) a known date of treatment intervention, and (3) time series response data that include both pre- and post-treatment outcomes for all units. Synthetic control generates a response metric for treated units relative to a no-action alternative based on prior relationships between treated and unexposed groups. Using simulations and a case study involving a large-scale brush-clearing management event, we show how synthetic control can intuitively infer treatment effect sizes from satellite data, even in the presence of confounding noise from climate anomalies, long-term vegetation dynamics, or sensor errors. We find that accuracy depends on the number and quality of potential control units, highlighting the importance of selecting appropriate control populations. Although we consider the synthetic control approach in the context of natural experiments with remote sensing data, we expect the methodology to have wider utility in ecology, particularly for systems with large, complex, and poorly replicated experimental units.

Preferential snake detection in a simulated ecological experiment

According to Isbell's snake detection theory (SDT), the need to rapidly detect and thus avoid snakes had a major impact on the evolution of the primate visual system, and thus the origin and evolution of the primate lineage, as expansion of the visual sense is a key characteristic of primates. The SDT rests on the assumption that there are both cortical (conscious) and subcortical (unconscious) brain structures and pathways that are responsible for rapid visual detection of and quick avoidance reactions to snakes. Behavioral evidence for the SDT primarily comes from visual search tasks and presentations of images on a computer screen; our aim was to evaluate the SDT under more ecologically valid circumstances. We asked participants to take a virtual hike in which a realistic model of a snake, rabbit, or bottle had been placed on the trail. Subjects were instructed simply to imagine themselves as the hiker while watching the video. We measured heart rate and skin conductance reactions while the participants viewed the video. After the video, the participants were shown pictures of the three stimuli and asked if they had seen any of them. We found that snakes were detected more often than rabbits or bottles, and that participants showed greater changes in heart rate and greater skin conductance responses in the snake condition than in the other two conditions, even when the participant did not report having seen the snake. A critical component of the SDT is that primates must be able to quickly detect snakes even when their attention is directed elsewhere. Using a novel experimental context-a simulated hike-we assessed arousal and detection without directing participants to attend to any particular stimulus or event. Our data support the SDT by providing evidence of enhanced detection and autonomic arousal even in the absence of detection. Replication of these results using additional controls and experimental contexts will help refine our understanding of snake avoidance by primates.

Bracon wasps for ecological pest control-a laboratory experiment.

Biological control of pest insects by natural enemies may be an effective, cheap and environmentally friendly alternative to synthetic pesticides. The cosmopolitan parasitoid wasp species Bracon brevicornis Wesmael and B. hebetor Say (Hymenoptera: Braconidae) use lepidopteran species as hosts, including insect pests like Ephestia kuehniella or Ostrinia nubilalis. Here, we compare the reproductive success of both Bracon species on E. kuehniella in a laboratory experiment. We asked (1) how the reproductive success on a single host larva changes with temperature, (2) how it changes with temperature when more host larvae are present and (3) how temperature and availability of host larvae influence the efficacy of Bracon species as biological control agents. In general, differences between B. brevicornis and B. hebetor have been small. For rearing both Bracon species in the laboratory on one host larva, a temperature between 20–27 °C seems appropriate to obtain the highest number of offspring with a female-biased sex ratio. Rearing the braconid wasps on more than one host larva revealed a higher number of total offspring but less offspring per host larva on average. Again, highest numbers of offspring hatched at 27 °C and the sex ratio was independent from temperature. Although no parasitoids hatched at 12 °C and only few at 36 °C, host larvae were still paralyzed. The efficacy of B. brevicornis was higher than 80% at all numbers of host larvae presented at all temperatures while the efficacy of B. hebetor was less than 80% at 12 °C and 27 °C at low numbers of host larvae presented. In conclusion, practitioners can use either B. brevicornis or B. hebetor at low and high temperatures and at varying host densities to achieve high pest control efficacy.

Cyberneticisation as a Theory and Practice of Matter

The ecological implementation of cybernetic ideas in architecture requires a material theory and practice that enables their propositions to be tested. The need for approaches that move from simulation to cybernetic reality is a documented limitation of cybernetics recognised by Stafford Beer with his pond ecology experiments and Gordon Pask through electrochemical devices. While both experimented with adaptive material platforms as embodiments of designed cybernetic systems, their approaches were limited by the available toolsets. This article considers an ecological trajectory of cybernetisation by revisiting notions of biological computation as a generative material practice. In particular, the growing fields of biodesign and living architecture go beyond notions of biological analogues that inform modern architecture by directly incorporating living systems into the very fabric of buildings as designed expressions of ecology.

Identifying the unidentifiable: A PCR multiplex protocol for the diagnosis of invasive pheretimoid earthworm species, verified by morphological and barcode identification

Identification of taxa that are morphologically very similar is a standing problem in ecology and is particularly important for invasive species that may differ in their dispersal rates and environmental and economic costs. Three similar pheretimoid earthworms (genera of Amynthas, Metaphire) are important invasive species in North American forests. They often lack the diagnostic morphological characters and their hatchlings, juveniles, and cocoons are impossible to identify to species. We present a multiplex PCR protocol that accurately scores the three species and is inexpensive compared to other molecular methods. Multiplex PCR identification was as accurate as mitochondrial COI barcoding and better than morphological scoring. The method uses unique PCR fragments of different lengths for each species from the COI gene. The multiplex PCR correctly identified embryos within cocoons, juveniles, and adults of Amynthas agrestis, Amynthas tokioensis and Metaphire hilgendorfi, with 100% accuracy. Comparisons of COI sequences (GenBank) with other populations of the same species (including specimens from their native range in Japan), and many other species of earthworms showed the primers always amplify only the three target species. The multiplex PCR method is rapid and costs a fraction of standard COI barcoding. Also, tiny scratches of cells from living specimens can be entered directly into the PCR for easy identification, reducing costs even further by avoiding DNA extraction, and allows the earthworms to be conserved for ecological experiments. To show the utility of the method we present the hatching phenology of the three species which could not be done by morphology. The three species begin hatching at the same time with A. tokioensis producing the most abundant juveniles early in the season. The method facilitates studies on biogeography, phenology, life histories, and resource partitioning among the three co-occurring earthworm species.

Food Reward Chemistry Explains a Novel Pollinator Shift and Vestigialization of Long Floral Spurs in an Orchid

During the evolutionary history of flowering plants, transitions between pollinator groups (pollinator shifts) have been frequent,1 and contributed to the spectacular radiation of angiosperms.2 Although the evolution of floral traits during pollinator shifts has been studied in real time under controlled laboratory conditions,3 it is challenging to study in nature and therefore poorly understood.4-7 Using a comparative, multidisciplinary approach, we dissect the evolution of floral traits during a pollinator shift in the long-spurred African orchid Satyrium longicauda. Phylogenetic analysis and ecological experiments revealed a shift from moth- to oil-collecting bee pollination. Remarkably, flowers of the bee-pollinated form are similar in morphology, color, and overall volatile chemistry to those of moth-pollinated forms, but differ in having spurs that are mostly devoid of nectar, and have an elevated presence of the oil-derived compound diacetin, which oil-collecting bees use as a cue for oil presence.8 Experiments demonstrated that long spurs are critical for pollination of a moth-pollinated form, but are not needed for pollination of the bee-pollinated form. We conclude that the pollinator shift in Satyrium was mediated by a switch in chemistry of the pollinator reward. The ancestral presence of diacetin might have served as a pre-adaptation for bee pollination, whereas the current mismatch between flower morphology and bees is due to the retention of vestigial floral spurs. These results elucidate the sequence of floral evolution in the early stages of pollinator shifts and help to explain the assembly of suites of co-varying traits through pre-adaptation and vestigialization.9-12.