Innovation in teaching and learning invertebrate zoology in remote and online classrooms

The COVID‐19 global pandemic caused instructors to pivot to remote and online teaching, an especially challenging task in hands‐on classes such as invertebrate biology. In this special 25th anniversary issue of Invertebrate Biology, the authors present a variety of clever and effective ways to help invertebrate biology instructors adapt to teaching in an online environment. Student‐centered research and learning are essential in all biology classes, and we explore scientific writing, field trips, do‐it‐yourself laboratories, and more. These techniques will be useful for classes of varying sizes and types, from non‐major undergraduates to graduate students, even after the pandemic is over. Innovation for teaching invertebrate biology online may help facilitate more inclusive courses that serve diverse students more equitably. Ideas for how to best move traditionally hands‐on laboratories into online or remote formats are currently also being informally discussed in a collaborative online space for instructors.     

Student exam performance in flipped classroom sections is similar to that in active learning sections,and satisfaction with the flipped classroom hinges on attitudes toward learning from videos

ABSTRACT

The flipped classroom is a teaching innovation in which instructional content is delivered out of the classroom, often via videos, and the class period is used for application of the course material. While the popularity of the flipped classroom is growing exponentially, its benefits have not been extensively studied. In this study we compared three semesters of an undergraduate Genetics course that was non-flipped, but included a significant amount of active learning, to three semesters of a flipped course with the same content. Student exam performance was not statistically different between the active non-flipped and the flipped courses, suggesting that the benefits of the flipped environment may be similar to those achieved via active learning. We also examined student attitudes toward the flipped classroom, and found 56% of students were satisfied, 39% were dissatisfied, and 5% were neutral toward the flipped classroom. Our survey revealed that the clearest defining characteristic of dissatisfied students was not a dislike of classroom active learning activities. Rather, dissatisfied students disproportionately disliked, and had difficulty learning the course material, from videos.     

The Contribution of Agriculture,Forestry and other Land Use activities to Global Warming, 1990–2012

We refine the information available through the IPCC AR5 with regard to recent trends in global GHG emissions from agriculture, forestry and other land uses (AFOLU), including global emission updates to 2012. Using all three available AFOLU datasets employed for analysis in the IPCC AR5, rather than just one as done in the IPCC AR5 WGIII Summary for Policy Makers, our analyses point to a down‐revision of global AFOLU shares of total anthropogenic emissions, while providing important additional information on subsectoral trends. Our findings confirm that the share of AFOLU emissions to the anthropogenic total declined over time. They indicate a decadal average of 28.7 ± 1.5% in the 1990s and 23.6 ± 2.1% in the 2000s and an annual value of 21.2 ± 1.5% in 2010. The IPCC AR5 had indicated a 24% share in 2010. In contrast to previous decades, when emissions from land use (land use, land use change and forestry, including deforestation) were significantly larger than those from agriculture (crop and livestock production), in 2010 agriculture was the larger component, contributing 11.2 ± 0.4% of total GHG emissions, compared to 10.0 ± 1.2% of the land use sector. Deforestation was responsible for only 8% of total anthropogenic emissions in 2010, compared to 12% in the 1990s. Since 2010, the last year assessed by the IPCC AR5, new FAO estimates indicate that land use emissions have remained stable, at about 4.8 Gt CO₂ eq yr^?1 in 2012. Emissions minus removals have also remained stable, at 3.2 Gt CO₂ eq yr^?1 in 2012. By contrast, agriculture emissions have continued to grow, at roughly 1% annually, and remained larger than the land use sector, reaching 5.4 Gt CO₂ eq yr^?1 in 2012. These results are useful to further inform the current climate policy debate on land use, suggesting that more efforts and resources should be directed to further explore options for mitigation in agriculture, much in line with the large efforts devoted to REDD+ in the past decade.     

Effects of 28 days of beta-alanine and creatine monohydrate supplementation on aerobic power,ventilatory and lactate thresholds,and time to exhaustion

Summary. The effect of beta-alanine (β-Ala) alone or in combination with creatine monohydrate (Cr) on aerobic exercise performance is unknown. The purpose of this study was to examine the effects of 4 weeks of β-Ala and Cr supplementation on indices of endurance performance. Fifty-five men (24.5 ± 5.3 yrs) participated in a double-blind, placebo-controlled study and randomly assigned to one of 4 groups; placebo (PL, n = 13), creatine (Cr, n = 12), beta-alanine (β-Ala, n = 14), or beta-alanine plus creatine (CrBA, n = 16). Prior to and following supplementation, participants performed a graded exercise test on a cycle ergometer to determine VO_2peak, time to exhaustion (TTE), and power output, VO₂, and percent VO_2peak associated with VT and LT. No significant group effects were found. However, within groups, a significant time effect was observed for CrBa on 5 of the 8 parameters measured. These data suggest that CrBA may potentially enhance endurance performance.     

Nectar sugar composition of European Caryophylloideae (Caryophyllaceae) in relation to flower length,pollination biology and phylogeny

Floral nectar composition has been explained as an adaptation to factors that are either directly or indirectly related to pollinator attraction. However, it is often unclear whether the sugar composition is a direct adaptation to pollinator preferences. Firstly, the lower osmolality of sucrose solutions means that they evaporate more rapidly than hexose solutions, which might be one reason why sucrose‐rich nectar is typically found in flowers with long tubes (adapted to long‐tongued pollinators), where it is better protected from evaporation than in open or short‐tubed flowers. Secondly, it can be assumed that temperature‐dependent evaporation is generally lower during the night than during the day so that selection pressure to secrete nectar with high osmolality (i.e. hexose‐rich solutions) is relaxed for night‐active flowers pollinated at night. Thirdly, the breeding system may affect selection pressure on nectar traits; that is, for pollinator‐independent, self‐pollinated plants, a lower selective pressure on nectar traits can be assumed, leading to a higher variability of nectar sugar composition independent of pollinator preferences, nectar accessibility and nectar protection. To analyse the relations between flower tube length, day vs. night pollination and self‐pollination, the nectar sugar composition was investigated in 78 European Caryophylloideae (Caryophyllaceae) with different pollination modes (diurnal, nocturnal, self‐pollination) using high‐performance liquid chromatography (HPLC). All Caryophylleae species (Dianthus and relatives) were found to have nectar with more than 50% sucrose, whereas the sugar composition of Sileneae species (Silene and relatives) ranged from 0% to 98.2%. In the genus Silene, a clear dichotomous distribution of sucrose‐ and hexose‐dominant nectars is evident. We found a positive correlation between the flower tube length and sucrose content in Caryophylloideae, particularly in day‐flowering species, using both conventional analyses and phylogenetically independent contrasts.     

Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains.

Plastic embedding preserves tissue structure much more faithfully than does paraffin. Acrylic polymerization is innocuous to dye-binding groups in sections. The water solubility of glycol methacrylate monomer and the hydrophilic properties of the polymer allow for convenience in dehydration and for versatility in staining sections. Five years of experience with glycol methacrylate (GMA) embedding for light microscopy is summarized. Methods for purifying GMA monomer are cited. Procedures for fixing, dehydrating, embedding, polymerizing, sectioning and staining, using GMA, are explained. A method is provided for making glass knives long enough to cut large blocks. Simple, reliable, quick staining methods are outlined. When compared with paraffin, GMA offers opportunities for simpler, quicker procedures and yields sections of superior quality, greater information content, and less distortion.     

Floral biology,reciprocal herkogamy and breeding system in four Psychotria species (Rubiaceae) in Brazil

Distylous species have two floral morphs with reciprocal positions of sexual whorls, functioning to promote cross‐pollination. Additionally, most distylous species have an incompatibility system linked to the discrete variation of anthers and stigmas. Here we provide new data on heterostyly, reproductive biology and pollination in four Psychotria spp. from the Cerrado Region (Brazil). Psychotria deflexa, P. nitidula and P. trichophoroides fitted the distylous morphological syndrome, whereas P. prunifolia was monomorphic and self‐compatible. Reciprocity varied across species, with the upper whorl of anthers and stigmas being more reciprocal than the lower whorl. Psychotria nitidula has a heteromorphic incompatibility system, whereas P. deflexa and P. trichophoroides showed partial self‐compatibility. Psychotria prunifolia and P. trichophoroides were visited by large bees and exhibited lower reproductive efficacy than P. nitidula and P. deflexa, which are visited by wasps and small bees. Our results provide new evidence that the morphological distylous syndrome does not always co‐occur with heteromorphic incompatibility and additional mechanisms (e.g. disassortative pollen transfer) may operate to maintain the polymorphism and isoplethy of species with different incompatibility system.     

Artificial light and sun compass orientation in the sandhopper Talitrus saltator (Crustacea, Amphipoda)

Experiments on compass orientation under artificial light were conducted with adult individuals of Talitrus saltator. The aim was to reproduce in the laboratory an orientation based on the sun compass corresponding to that recorded in conditions of the true sun and sky. This was obtained by the creation within an opaque Plexiglas dome of a scenario that permitted variation of the brightness of the artificial sky and sun. The results show that it is possible to obtain sun compass orientation corresponding to the natural situation even in an artificial environment. It can be concluded that sandhoppers identify an artificial light source as the sun if the artificial sky is also illuminated and if the intensities of the artificial sun and sky exceed certain threshold values (1.13 and 10 μW cm⁻², respectively). The results of other experiments under the natural blue sky with an artificial sun and with the real sun attenuated are discussed. Accepted: 23 May 1997     

Responses to a warming world: Integrating life history,immune investment,and pathogen resistance in a model insect species

Environmental temperature has important effects on the physiology and life history of ectothermic animals, including investment in the immune system and the infectious capacity of pathogens. Numerous studies have examined individual components of these complex systems, but little is known about how they integrate when animals are exposed to different temperatures. Here, we use the Indian meal moth (Plodia interpunctella) to understand how immune investment and disease resistance react and potentially trade‐off with other life‐history traits. We recorded life‐history (development time, survival, fecundity, and body size) and immunity (hemocyte counts, phenoloxidase activity) measures and tested resistance to bacterial (E. coli) and viral (Plodia interpunctella granulosis virus) infection at five temperatures (20–30°C). While development time, lifespan, and size decreased with temperature as expected, moths exhibited different reproductive strategies in response to small changes in temperature. At cooler temperatures, oviposition rates were low but tended to increase toward the end of life, whereas warmer temperatures promoted initially high oviposition rates that rapidly declined after the first few days of adult life. Although warmer temperatures were associated with strong investment in early reproduction, there was no evidence of an associated trade‐off with immune investment. Phenoloxidase activity increased most at cooler temperatures before plateauing, while hemocyte counts increased linearly with temperature. Resistance to bacterial challenge displayed a complex pattern, whereas survival after a viral challenge increased with rearing temperature. These results demonstrate that different immune system components and different pathogens can respond in distinct ways to changes in temperature. Overall, these data highlight the scope for significant changes in immunity, disease resistance, and host–parasite population dynamics to arise from small, biologically relevant changes to environmental temperature. In light of global warming, understanding these complex interactions is vital for predicting the potential impact of insect disease vectors and crop pests on public health and food security.     

The roles of head movements in the search and capture strategy of a tern (Aves, Laridae)

 Gull-billed terns (Gelochelidon nilotica) were video-filmed while searching for and capturing fiddler crabs. Search consists of a vertical head nystagmus, with fast upward flicks and downward slow phases made at the angular speed of the substrate in the approximate direction of the bill. The bill points down at about 60° during hunting, but is brought up to 15° from time to time, which brings the visual streak into line with the horizon; 45° roll movements of the head are consistent with alternation between the use of the temporal and central foveas to view the same object. When a crab has been detected the nystagmus is suspended, and the tern tracks the crab continuously as it manoeuvres into a catching position. This may involve tucking the head under the body so that the bill is 45° behind the vertical, and flying up and backwards for some metres, straightening up the head at the same time. Accepted: 7 November 1998     

In silico,in vitro,and in vivo machine learning in synthetic biology and metabolic engineering

Among the main learning methods reviewed in this study and used in synthetic biology and metabolic engineering are supervised learning, reinforcement and active learning, and in vitro or in vivo learning.In the context of biosynthesis, supervised machine learning is being exploited to predict biological sequence activities, predict structures and engineer sequences, and optimize culture conditions.Active and reinforcement learning methods use training sets acquired through an iterative process generally involving experimental measurements. They are applied to design, engineer, and optimize metabolic pathways and bioprocesses.The nascent but promising developments with in vitro and in vivo learning comprise molecular circuits performing simple tasks such as pattern recognition and classification.     

New optics sheds light on biology. Advances in microscopy, genetics and biochemistry are together increasingly enabling scientists to watch molecules in action

Advances in science and technology enable scientists to peek at cellular and molecular events in vivo. The same technologies also provide new tools for cancer diagnostics and monitoring treatmentAdvances in light microscopy are about to transform molecular biology by enabling biologists to observe molecular processes in vivo. Although biochemical analysis or genomic sequencing, along with imaging techniques such as X-ray diffraction and electron microscopy, can provide detailed information about cellular structures and the products of particular processes, it is light microscopy that allows scientists to capture dynamic events in the cell. Basic research is not the only enterprise that benefits from improvements in microscopy; new optical techniques also have an increasing role in diagnosis and surgery—in particular for some cancers—to analyse tissue in real time.…it is light microscopy that allows scientists to capture dynamic events in the cellFor decades, light microscopy was regarded as the poor relative of electron microscopy and X-ray crystallography, both of which enable much greater resolutions, down to atomic scales around 0.1 nm. Yet both techniques come at the expense of the object because they can only analyse fixed samples—crystals in the case of X-ray crystallography and thin sections for electron microscopy. This has always been the advantage of light microscopy: to capture events in living systems. Now, various developments over the past two decades have come together to introduce real-time imaging. “Revolutionary improvements in laser light sources, detectors, or cameras, and the advent of computerized digital light and fluorescence microscopy have been essential for enabling real-time imaging and micro-endoscopy in living systems,” said Frank Chuang, associate director of research and education at the Centre for Biophotonics and Research at the University of California, Davis in Sacramento, USA.The first major step for real-time light microscopy was the development of fluorescence methods for labelling proteins in vivo. These methods exploit the fact that fluorescent compounds absorb electromagnetic radiation at one wavelength and emit light at a different wavelength. A sample can be illuminated by electromagnetic radiation at the prescribed wavelength, and only the fluorescent components of the sample, as well as areas very close to them, are observed. Using fluorophores as markers for specific proteins, scientists can obtain functional information as this allows them to track their movement.The opening move was the discovery of the green fluorescence protein (GFP) in the jellyfish Aequorea victoria in the late 1970s (Tsien, 1998). GFP is composed of 238 amino-acid residues and gives off bright green fluorescence on exposure to blue light. Since its discovery, GFP has been further improved by protein design and has been applied to studies of proteins that are marked with GFP in bacteria, yeast and fish, as well as plant and mammalian cells. For these discoveries, Martin Chalfie, Osamu Shimomura and Roger Tsien received the Nobel Prize in Chemistry in 2008.The first major step for real-time light microscopy was the development of fluorescence methods for labelling proteins in vivoGFP can be spliced into the genome next to the gene of the protein of interest, which is then expressed as a fluorescent fusion protein. GFP can also be introduced into target cells to analyse, for example, brain circuitry or the entry of viruses into cells during infection. “That was definitely a turning point […] and people can now capture not only stills but also dynamic scenarios of these proteins, to observe how they are moving, and where they are localized in living samples ranging from bacteria to entire living animals,” commented Michael Knop, professor of molecular biology at Heidelberg University, Germany, who applies live-cell microscopic imaging to studying protein function in meiosis.One problem though was that in traditional so-called wide-field light microscopy—in which the light source illuminates the whole sample—background light interferes with the area of interest. This was addressed by the confocal microscope, which eliminates the background by shining light through a pinhole so that it can be focused on a small target zone. This improves optical resolution immensely, but at the expense of signal intensity, because most of the light is blocked at the pinhole. Images have to be exposed for longer periods or at increased intensity, which causes greater photo damage to the object.This problem was eventually addressed by another important advance for light microscopy, the development of fast, ultra-sensitive charged-coupled-device (CCD) chips for photodetection. “This really started 10–12 years ago when a new generation of CCDs allowed the construction of cameras that could be used for very low light detection,” said Knop. CCD cameras help to reduce photo damage to samples and capture images more quickly, which increases the scope for making time-lapsed movies.These improvements led the way for other developments, notably the light-sheet microscope in 2003. It was pioneered at the European Molecular Biology Laboratory in Heidelberg, Germany, by a group headed by Ernst Stelzer, now Professor in Advanced Light Microscopy at the University of Frankfurt, Germany. The microscope works on the basis of illuminating the specimen from the side rather than from above; a sheet of light exposes only the narrow slice on which the microscope is focused at the time. Light-sheet microscopy involves two independently operated lenses for illumination and detection. By focusing the light only on the narrow optical section within the focal plane of observation, it further reduces photo damage or photo-bleaching.The other goal of light-microscopy development is improving resolution, which is needed to observe individual proteins and detail at the molecular levelLight-sheet microscopy has since been combined with other techniques for improving contrast and manipulating specimens to create even better three-dimensional images. It enabled the dramatic in vivo recording of embryonic development in a zebrafish, from the early 32-cell stage to the neurulation period, when cells start to differentiate to form organs. Individual cells were visualized with 60–90 seconds between samples, and four-megapixel image frames were recorded at a rate of five per second. This recording speed was important because cell movement could then be tracked with sufficient accuracy to study migration. The work showed that light-sheet microscopy exposed the embryo to 200 times less energy than a conventional microscope and 5,000–6,000 times less than a confocal fluorescence microscope (Keller et al, 2008).The other major goal of light-microscopy development is improving resolution, which is needed to observe individual proteins and detail at the molecular level. It requires overcoming the diffraction-limit rule, which states that the resolution cannot be better than half the wavelength of light, around 200 nm, which is insufficient even to observe organelles within cells. This limit occurs because the wavelength defines the minimum spot size that can be resolved from a single ray of light. Any smaller details will be blurred by interference from light emitted from neighbouring points in the sample.Even when a sample is being observed in vivo, the preparation—involving excitation with light—is not normalHowever, ways have been found to extract information from samples at higher resolution. One such technique is called STED (stimulated emission depletion) fluorescence microscopy, described in 1994 by Stefan Hell at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany (Hell & Wichmann, 1994). STED is based on the idea of blocking light that is emitted from around the point of interest, to prevent interference. It combines the normal pulse of light that stimulates fluorescence with a second pulse tuned to the frequency of the excited radiation, which cancels it out. This second pulse is directed close to the focal point from a doughnut-shaped emitter. By transmitting the second cancelling pulse from the rim of the doughnut only, the light is focused onto a ring around the focal point so that only a small area in the centre receives the pulse that generates fluorescence. This creates a coherent spot of emitted light corresponding to the molecules in the centre of the ring only, which yields a sharp image at a resolution beyond the diffraction limit. Furthermore, by increasing the intensity of the cancelling light that is emitted from the rim of the doughnut, the diameter of the coherent spot in the middle can be reduced to further increase resolution, in principle, down to the size of a single atom. In practice, one of the highest resolutions obtained so far is 2.6 nm, which is in the range of the size of individual proteins (Rittweger et al, 2009).Although STED and light-sheet microscopy—combined with other techniques and tools—promise to revolutionize cell biology by providing image sequences at higher resolutions and shorter timescales, they also bring new challenges or accentuate existing ones. The two main issues concern disturbing the system being observed and introducing visual artefacts that cannot be immediately identified, because the images are constructed, at least partly, by computers. This boils down to one fundamental problem: how to tell whether the image reflects the system being observed under normal environmental conditions. Even when a sample is being observed in vivo, the preparation—involving excitation with light—is not normal. “This is a major issue, because when you basically set up conditions to detect a protein of interest, you also interfere with the system,” commented Knop. “One of the typical problems in researching mammalian cells for example is that many proteins are over expressed when people label them.”In the case of the zebrafish embryo, it might seem as though normal development has occurred, but even when a healthy animal is produced it is impossible to be sure that some details of the mechanisms involved have not been changed by the observational process. This can be addressed by validating findings obtained by high-resolution optical microscopy in other ways. “Depending on the organism there are different options,” said Knop. “In yeast we can use genetics.”…the increasing power and resolution of light microscopy also increases the sensitivity of the observations to small fluctuations in the environmentFurthermore, the increasing power and resolution of light microscopy also increases the sensitivity of the observations to small fluctuations in the environment. This was an issue for Peter Sudbery at the University of Sheffield in the UK, who uses a microscope called Deltavision to study the growth of the fungus Candida. “Candida only forms hyphae at 37 °C, and even a small drop in temperature changes its behaviour,” he said. “Also small fluctuations in temperature of less than 0.1 °C cause the image to lose focus. So while the Deltavision can be programmed to make time-lapse movies with the exposures taken automatically at preset intervals, for videos longer than a few minutes the microscope has to be manually monitored constantly to check the focus and temperature.”Although artefacts and environmental control are important issues at the cutting edge of research near the limits of resolution, they are fortunately less critical for medical applications. Here, resolution at the cell level is usually not necessary; the main issue lies in developing sufficiently small systems that can accurately and conveniently observe the region of interest during diagnosis and surgery.The most prevalent application in medical imaging is for tumour detection—both during diagnosis and surgery—either to replace traditional biopsy, which is invasive and time-consuming, or to replace direct visual inspection, which can be inaccurate. One method is near-infra-red (NIR) microscopy, in which the patient ingests compounds that cause tumour cells to emit radiation in the NIR spectrum in response to excitation. This can either be used to identify specific tumours or general cancers, according to Stijn Keereweer, research fellow at the Erasmus Medical Centre in Rotterdam, the Netherlands. “Agents can target specific epitopes which differ between various tumours, but can also be targeted against general characteristics, such as MMP [matrix metalloproteinase] activity or increased glucose uptake,” said Keereweer. He added that by mounting cameras on laparoscopic probes, the technique could be extended to internal abdominal-cancer surgery, as well as for neck and head cancers when the observation can be performed by external systems.The advantage of these optical systems is that the surgical margin required to be sure that all malignant tissue has been removed can be reduced, thereby minimizing risk and injury to the patient. One of the hurdles for this technique is regulatory approval of the tumour-specific agents that are needed to elicit the fluorescence.However, there are emerging techniques that do not require such agents, by using conventional confocal laser microscopy without fluorescence. A study at the Garvan Institute of Medical Research in Sydney, Australia, showed that in vivo analysis of surface and near-surface tissue could be performed successfully in real time by using a contrast agent administered intravenously to show structural features (Nguyen et al, 2009).Techniques that avoid the need for patients to ingest an agent—for contrast or to generate fluorescence—would be ideal as they would reduce preparation time. This can be achieved by exploiting auto-fluorescence instead of adding fluorescent compounds, and has been shown to successfully identify oesophageal cancer, according to Bevin Lin at the Department of Biomedical Engineering at the University of California Davis. “Regarding oesophageal carcinoma, current screening methods can take 20–30 minutes,” he said. “There is a need for a rapid method of imaging that is less complicated and faster than topical or intravenous contrast agents. Therefore, significant advantages of auto-fluorescence microscopy include the elimination of contrast agents, preparation time, and complex instrumentation, which could directly translate to reduced cost and improved patient care.” As Lin pointed out, regulatory approval from the US Food and Drug Administration is still required for the endoscopy system, but he believes that there is great potential for optical systems. “Hopefully, optical methods will become as clinically mainstream as traditional methods,” he said.The most prevalent application in medical imaging is for tumour detection…There is similar expectation that real-time optical imaging will become as widely used in research once teething troubles associated with recognition of artefacts and disturbances to the systems under observation are overcome. “Effective, affordable and usable super-resolution imaging that could be used on living, non-fixed cells with GFP and variants would transform the whole of cell biology,” said Sudbery.     

Haploid,diploid, and pooled exome capture recapitulate features of biology and paralogy in two non-model tree species

Despite their suitability for studying evolution, many conifer species have large and repetitive giga-genomes (16–31 Gbp) that create hurdles to producing high coverage SNP data sets that capture diversity from across the entirety of the genome. Due in part to multiple ancient whole genome duplication events, gene family expansion and subsequent evolution within Pinaceae, false diversity from the misalignment of paralog copies creates further challenges in accurately and reproducibly inferring evolutionary history from sequence data. Here, we leverage the cost-saving benefits of pool-seq and exome-capture to discover SNPs in two conifer species, Douglas-fir (Pseudotsuga menziesii var. menziesii (Mirb.) Franco, Pinaceae) and jack pine (Pinus banksiana Lamb., Pinaceae). We show, using minimal baseline filtering, that allele frequencies estimated from pooled individuals show a strong, positive correlation with those estimated by sequencing the same population as individuals (r > .948), on par with such comparisons made in model organisms. Further, we highlight the utility of haploid megagametophyte tissue for identifying sites that are probably due to misaligned paralogs. Together with additional minor filtering, we show that it is possible to remove many of the loci with large frequency estimate discrepancies between individual and pooled sequencing approaches, improving the correlation further (r > .973). Our work addresses bioinformatic challenges in non-model organisms with large and complex genomes, highlights the use of megagametophyte tissue for the identification of paralogous artefacts, and suggests the combination of pool-seq and exome capture to be robust for further evolutionary hypothesis testing in these systems.     

Integrating stable isotopes,parasite, and ring‐reencounter data to quantify migratory connectivity—A case study with Barn Swallows breeding in Switzerland,Germany, Sweden,and Finland

Ecosystems around the world are connected by seasonal migration. The migrant animals themselves are influenced by migratory connectivity through effects on the individual and the population level. Measuring migratory connectivity is notoriously difficult due to the simple requirement of data conveying information about the nonbreeding distribution of many individuals from several breeding populations. Explicit integration of data derived from different methods increases the precision and the reliability of parameter estimates. We combine ring‐reencounter, stable isotope, and blood parasite data of Barn Swallows Hirundo rustica in a single integrated model to estimate migratory connectivity for three large scale breeding populations across a latitudinal gradient from Central Europe to Scandinavia. To this end, we integrated a non‐Markovian multistate mark‐recovery model for the ring‐reencounter data with normal and binomial mixture models for the stable isotope and parasite data. The integration of different data sources within a mark‐recapture modeling framework enables the most precise quantification of migratory connectivity on the given broad spatial scale. The results show that northern‐breeding populations and Southern Africa as well as southern‐breeding populations and Western–Central Africa are more strongly connected through Barn Swallow migration than central European breeding populations with any of the African wintering areas. The nonbreeding distribution of Barn Swallows from central European breeding populations seems to be a mixture of those populations breeding further north and south, indicating a migratory divide.     

Cerulean Warblers in the Ozark region: habitat selection,breeding biology,survival, and space use

Cerulean Warblers (Setophaga cerulea) are a species with declining populations that exhibit regional variation in habitat selection and demographic rates. The Ozark region of the south‐central United States likely provides important habitat for Cerulean Warblers, but little is known about their breeding biology in that region. We studied Cerulean Warblers in riparian forests of the Ozarks of Arkansas from 2018 to 2020. We assessed multi‐scale habitat selection for vegetative and topographic features, documented their breeding biology, estimated within‐season and annual apparent survival, and estimated territory sizes. We found that Cerulean Warblers selected riparian habitat characterized by large‐diameter trees across all spatial scales. Contrary to the results of previous studies, males appeared to avoid white oaks (Quercus spp., Section Quercus) at the territory scale, but this avoidance may reflect an underlying preference for riparian habitat. Our logistic‐exposure estimate of nest survival (0.32; 85% confidence interval: 0.21–0.46) was similar to the median of estimates reported in previous studies. Our results indicate that maintaining riparian forests with large trees is important to provide suitable habitat for Cerulean Warblers in the Ozark region. Because of similarities in habitat selection among regions, some management practices from other populations, including retaining large trees and promoting a heterogeneous canopy structure, may be useful for managing for Cerulean Warblers in riparian areas of the Ozarks. However, selection for topography and tree species by Cerulean Warblers in our study also suggests that region‐specific management strategies will be beneficial. Finally, our demographic rate estimates for this population should prove valuable in future full‐annual‐cycle population modeling efforts.     

Sharks modulate their escape behavior in response to predator size,speed and approach orientation

Escape responses are often critical for surviving predator–prey interactions. Nevertheless, little is known about how predator size, speed and approach orientation impact escape performance, especially in larger prey that are primarily viewed as predators. We used realistic shark models to examine how altering predatory behavior and morphology (size, speed and approach orientation) influences escape behavior and performance in Squalus acanthias, a shark that is preyed upon by apex marine predators. Predator models induced C-start escape responses, and increasing the size and speed of the models triggered a more intense response (increased escape turning rate and acceleration). In addition, increased predator size resulted in greater responsiveness from the sharks. Among the responses, predator approach orientation had the most significant impact on escapes, such that the head-on approach, as compared to the tail-on approach, induced greater reaction distances and increased escape turning rate, speed and acceleration. Thus, the anterior binocular vision in sharks renders them less effective at detecting predators approaching from behind. However, it appears that sharks compensate by performing high-intensity escapes, likely induced by the lateral line system, or by a sudden visual flash of the predator entering their field of view. Our study reveals key aspects of escape behavior in sharks, highlighting the modulation of performance in response to predator approach.