An empirical comparison of character‐based and coalescent‐based approaches to species delimitation in a young avian complex

The process of discovering species is a fundamental responsibility of systematics. Recently, there has been a growing interest in coalescent‐based methods of species delimitation aimed at objectively identifying species early in the divergence process. However, few empirical studies have compared these new methods with character‐based approaches for discovering species. In this study, we applied both a character‐based and a coalescent‐based approaches to delimit species in a closely related avian complex, the light‐vented/Taiwan bulbul (Pycnonotus sinensis/Pycnonotus taivanus). Population aggregation analyses of plumage, mitochondrial and 13 nuclear intron character data sets produced conflicting species hypotheses with plumage data suggesting three species, mitochondrial data suggesting two species, and nuclear intron data suggesting one species. Such conflict is expected among recently diverged species, and by integrating all sources of data, we delimited three species verified with independently congruent character evidence as well as a more weakly supported fourth species identified by a single character. Attempts to validate species hypothesis using Bayesian Phylogenetics and Phylogeography (BPP), a coalescent‐based method of species delimitation, revealed several issues that can seemingly affect statistical support for species recognition. We found that θ priors had a dramatic impact on speciation probabilities, with lower values consistently favouring splitting and higher values consistently favouring lumping. More resolved guide trees also resulted in overall higher speciation probabilities. Finally, we found suggestive evidence that BPP is sensitive to the divergent effects of nonrandom mating caused by intraspecific processes such as isolation‐with‐distance, and therefore, BPP may not be a conservative method for delimiting independently evolving population lineages. Based on these concerns, we questioned the reliability of BPP results and based our conclusions about species limits exclusively on character data.     

“How” and “what” matters: Sampling method affects biodiversity estimates of reef fishes

Understanding changes in biodiversity requires the implementation of monitoring programs encompassing different dimensions of biodiversity through varying sampling techniques. In this work, fish assemblages associated with the “outer” and “inner” sides of four marinas, two at the Canary Islands and two at southern Portugal, were investigated using three complementary sampling techniques: underwater visual censuses (UVCs), baited cameras (BCs), and fish traps (FTs). We firstly investigated the complementarity of these sampling methods to describe species composition. Then, we investigated differences in taxonomic (TD), phylogenetic (PD) and functional diversity (FD) between sides of the marinas according to each sampling method. Finally, we explored the applicability/reproducibility of each sampling technique to characterize fish assemblages according to these metrics of diversity. UVCs and BCs provided complementary information, in terms of the number and abundances of species, while FTs sampled a particular assemblage. Patterns of TD, PD, and FD between sides of the marinas varied depending on the sampling method. UVC was the most cost‐efficient technique, in terms of personnel hours, and it is recommended for local studies. However, for large‐scale studies, BCs are recommended, as it covers greater spatio‐temporal scales by a lower cost. Our study highlights the need to implement complementary sampling techniques to monitor ecological change, at various dimensions of biodiversity. The results presented here will be useful for optimizing future monitoring programs.     

The Role of Serum High Mobility Group Box 1 and Interleukin‐6 Levels in Acute Pancreatitis: A Meta‐Analysis

The purpose of this meta‐analysis was to comprehensively investigate the correlation between high mobility group box 1 (HMGB1) and interleukin‐6 (IL‐6) in relation to acute pancreatitis. A highly regulated exploration of various electronic databases, supplemented by manual searching methods, was performed in an attempt to identify pertinent articles of a useful nature. Subsequently, high‐quality cohort studies that were deemed to comply with the arduous inclusion and exclusion criteria were selected for our meta‐analysis. The extensive data analyses reported in our meta‐analysis were conducted in connection with the Comprehensive Meta‐analysis 2.0 (CMA 2.0). A total of 395 studies (135 Chinese studies and 260 English studies) were initially retrieved. 27 of those studies were selected for our meta‐analysis, comprising of 896 cases of mild acute pancreatitis (MAP), 700 cases of severe acute pancreatitis (SAP) as well as 312 healthy controls. Pooled data suggested that serum HMGB1 and IL‐6 levels of SAP and MAP patients were higher than in healthy controls. Moreover, serum HMGB1 and IL‐6 levels of SAP patients exhibited significantly higher levels than in that of MAP patients. Based on the rigorous investigation of our meta‐analysis, it was concluded that serum HMGB1 and IL‐6 levels might be used as effective indicators for pancreatic lesions as well as the degree of inflammatory response, owing ultimately to the observations and data analyses, suggesting that serum HMGB1 and IL‐6 levels share a close correlation with the severity of pancreatitis. J. Cell. Biochem. 119: 616–624, 2018. © 2017 Wiley Periodicals, Inc.     

Visualizing the “sandwich” structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3‐photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700‐nm window enables the highest optical transmittance through the skull. Using label‐free third‐harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140‐μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the “sandwich” structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.      

Investigating the bifunctionality of cyclizing and “classical” 5‐aminolevulinate synthases

The precursor to all tetrapyrroles is 5‐aminolevulinic acid, which is made either via the condensation of glycine and succinyl‐CoA catalyzed by an ALA synthase (the C4 or Shemin pathway) or by a pathway that uses glutamyl‐tRNA as a precursor and involves other enzymes (the C5 pathway). Certain ALA synthases also catalyze the cyclization of ALA‐CoA to form 2‐amino‐3‐hydroxycyclopent‐2‐en‐1‐one. Organisms with synthases that possess this second activity nevertheless rely upon the C5 pathway to supply ALA for tetrapyrrole biosynthesis. The C₅N units are components of a variety of secondary metabolites. Here, we show that an ALA synthase used exclusively for tetrapyrrole biosynthesis is also capable of catalyzing the cyclization reaction, albeit at much lower efficiency than the dedicated cyclases. Two absolutely conserved serines present in all known ALA‐CoA cyclases are threonines in all known ALA synthases, suggesting they could be important in distinguishing the functions of these enzymes. We found that purified mutant proteins having single and double substitutions of the conserved residues are not improved in their respective alternate activities; rather, they are worse. Protein structural modeling and amino acid sequence alignments were explored within the context of what is known about the reaction mechanisms of these two different types of enzymes to consider what other features are important for the two activities.     

Phylum Tardigrada: an “individual” approach

Phylum Tardigrada consists of ～1000 tiny, hardy metazoan species distributed throughout terrestrial, limno‐terrestrial and oceanic habitats. Their phylogenetic status has been debated, with current evidence placing them in the Ecdysozoa. Although there have been efforts to explore tardigrade phylogeny using both morphological and molecular data, limitations such as their few morphological characters and low genomic DNA concentrations have resulted in restricted taxonomic coverage. Using a protocol that allows us to identify and extract DNA from individuals, we have sequenced 18S rDNA from 343 tardigrades from across the globe. Using maximum parsimony and Bayesian analyses we have found support for dividing Order Parachela into three super‐families and further evidence that indicates the traditional taxonomic perspective of families in the class Eutardigrada are nonmonophyletic and require re‐working. It appears that conserved morphology within Tardigrada has resulted in conservative taxonomy as we have found cases of several discrete lineages grouped into single genera. Although this work substantially adds to the understanding of the evolution and taxonomy of the phylum, we highlight that inferences gained from this work are likely to be refined with the inclusion of further taxa—specifically representatives of the nine families yet to be sampled. © The Willi Hennig Society 2008.     

Using expert knowledge to incorporate uncertainty in cause‐of‐death assignments for modeling of cause‐specific mortality

Implicit and explicit use of expert knowledge to inform ecological analyses is becoming increasingly common because it often represents the sole source of information in many circumstances. Thus, there is a need to develop statistical methods that explicitly incorporate expert knowledge, and can successfully leverage this information while properly accounting for associated uncertainty during analysis. Studies of cause‐specific mortality provide an example of implicit use of expert knowledge when causes‐of‐death are uncertain and assigned based on the observer's knowledge of the most likely cause. To explicitly incorporate this use of expert knowledge and the associated uncertainty, we developed a statistical model for estimating cause‐specific mortality using a data augmentation approach within a Bayesian hierarchical framework. Specifically, for each mortality event, we elicited the observer's belief of cause‐of‐death by having them specify the probability that the death was due to each potential cause. These probabilities were then used as prior predictive values within our framework. This hierarchical framework permitted a simple and rigorous estimation method that was easily modified to include covariate effects and regularizing terms. Although applied to survival analysis, this method can be extended to any event‐time analysis with multiple event types, for which there is uncertainty regarding the true outcome. We conducted simulations to determine how our framework compared to traditional approaches that use expert knowledge implicitly and assume that cause‐of‐death is specified accurately. Simulation results supported the inclusion of observer uncertainty in cause‐of‐death assignment in modeling of cause‐specific mortality to improve model performance and inference. Finally, we applied the statistical model we developed and a traditional method to cause‐specific survival data for white‐tailed deer, and compared results. We demonstrate that model selection results changed between the two approaches, and incorporating observer knowledge in cause‐of‐death increased the variability associated with parameter estimates when compared to the traditional approach. These differences between the two approaches can impact reported results, and therefore, it is critical to explicitly incorporate expert knowledge in statistical methods to ensure rigorous inference.     

Nondetection sampling bias in marked presence‐only data

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Nonindependence and sensitivity analyses in ecological and evolutionary meta‐analyses

Meta‐analysis is an important tool for synthesizing research on a variety of topics in ecology and evolution, including molecular ecology, but can be susceptible to nonindependence. Nonindependence can affect two major interrelated components of a meta‐analysis: (i) the calculation of effect size statistics and (ii) the estimation of overall meta‐analytic estimates and their uncertainty. While some solutions to nonindependence exist at the statistical analysis stages, there is little advice on what to do when complex analyses are not possible, or when studies with nonindependent experimental designs exist in the data. Here we argue that exploring the effects of procedural decisions in a meta‐analysis (e.g. inclusion of different quality data, choice of effect size) and statistical assumptions (e.g. assuming no phylogenetic covariance) using sensitivity analyses are extremely important in assessing the impact of nonindependence. Sensitivity analyses can provide greater confidence in results and highlight important limitations of empirical work (e.g. impact of study design on overall effects). Despite their importance, sensitivity analyses are seldom applied to problems of nonindependence. To encourage better practice for dealing with nonindependence in meta‐analytic studies, we present accessible examples demonstrating the impact that ignoring nonindependence can have on meta‐analytic estimates. We also provide pragmatic solutions for dealing with nonindependent study designs, and for analysing dependent effect sizes. Additionally, we offer reporting guidelines that will facilitate disclosure of the sources of nonindependence in meta‐analyses, leading to greater transparency and more robust conclusions.     

Factors associated with leucism in the common blackbird Turdus merula

Leucism is the total or partial lack of melanins in the skin and associate structures (i.e. hair or feathers). Little is known about the factors influencing this chromatic aberration although some local studies suggest that there is an effect of habitat, age and sex. To test these hypotheses and expand our knowledge on leucism, we carried out a large‐scale study using common blackbirds Turdus merula as our model species. Given the poor information available on this topic and the variability of methodological approaches, we used three different methods to assess the effect of these variables in the presence of leucism: transects, bird captures and citizen science information (pictures from internet). We found an effect of habitat indicating that there are more leucistic blackbirds in cities than in non‐urban areas. In addition, we found a positive association between presence of white feathers and age providing the first large‐scale support for the progressive graying hypothesis in birds. This chromatic aberration was also influenced by sex, with males showing higher probability of leucism than females, although only for the capture data, indicating that the method used to study this phenomenon can partly influence our conclusions, and therefore suggesting caution when planning future studies in this topic.     

Overcoming the “Light‐Soaking” Issue in Inverted Organic Solar Cells by the Use of Al:ZnO Electron Extraction Layers

A common phenomenon of organic solar cells (OSCs) incorporating metal‐oxide electron extraction layers is the requirement to expose the devices to UV light in order to improve device characteristics – known as the so‐called “light‐soaking” issue. This behaviour appears to be of general validity for various metal‐oxide layers, various organic donor/acceptor systems, and regardless if single junction devices or multi stacked cells are considered. The requirement of UV exposure of OSCs may impose severe problems if substrates with limited UV transmission, UV blocking filters or UV to VIS down‐conversion concepts are applied. In this paper, we will demonstrate that this issue can be overcome by the use of Al doped ZnO (AZO) as electron extraction interlayer. In contrast to devices based on TiO_x and ZnO, the AZO devices show well‐behaved solar cell characteristics with a high fill factor (FF) and power conversion efficiency (PCE) even without the UV spectral components of the AM1.5 solar spectrum. As opposed to previous claims, our results indicate that the origin of s‐shaped characteristics of the OSCs is the metal‐oxide/organic interface. The electronic structures of the TiO_x/fullerene and AZO/fullerene interfaces are studied by photoelectron spectroscopy, revealing an electron extraction barrier for the TiO_x/fullerene case and facilitated electron extraction for AZO/fullerene. These results are of general relevance for organic solar cells based on various donor acceptor active systems.     

Use of time‐lapse photography and digital image analysis to estimate breeding success of a cliff‐nesting seabird

Development of new methods for obtaining basic demographic data from difficult‐to‐access breeding colonies and easily disturbed species is an important challenge in studies of seabirds. We describe a method that can generate data concerning annual breeding success of cliff‐nesting seabirds or other colonial birds with open nests. Our method requires only a single visit to a colony every second or third year, and is based on the use of automated time‐lapse photography. To test our method, we used time‐lapse photos to examine the breeding success of Thick‐billed Murres (Uria lomvia) in two breeding colonies in Greenland during the years 2011, 2012, and 2014. Based on the analysis of time‐lapse photos, we found that hatching success during the 3 yr of our study ranged from 60% to 81%, fledging success from 89% to 95%, and breeding success from 53% to 74% (Table 1). Use of digital image analysis made it possible to differentiate between breeding and non‐breeding birds and determine if and when breeding attempts failed or succeeded. The key to making our method a realistic long‐term monitoring technique is the use of an automated, formal image analysis to process the thousands of photos from the time‐lapse cameras and, more specifically, to reduce a large number of photos to a manageable number. Using our method, we needed 12–22 h per study plot, depending on the number of breeding sites per plot (range = 47–127) and whether it was the first or the second time the plot was analyzed, to obtain our estimates of hatching, fledging, and breeding success. This included time for data preparation, image analyses, visual inspections, and summarizing data in a spreadsheet. We found that our estimates of breeding success were comparable to those obtained by direct observation in the field. An important aspect of using time‐lapse technology is to foresee potential reasons why time‐lapse cameras might stop taking pictures, for example, equipment failure (camera, timer, or battery) or interference by visitors (e.g., vandalism or theft). As such, thorough testing of time‐lapse systems and selecting camera locations less likely to be disturbed are most important. We believe that use of time‐lapse photography in combination with digital image analysis to estimate breeding success can be useful for determining the breeding success of other cliff‐nesting seabirds and, more generally, other birds that breed in colonies, especially those located in remote areas and where direct observation may disturb birds.     

Harnessing the MinION: An example of how to establish long‐read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora

Long‐read sequencing technologies are transforming our ability to assemble highly complex genomes. Realizing their full potential is critically reliant on extracting high‐quality, high‐molecular‐weight (HMW) DNA from the organisms of interest. This is especially the case for the portable MinION sequencer which enables all laboratories to undertake their own genome sequencing projects, due to its low entry cost and minimal spatial footprint. One challenge of the MinION is that each group has to independently establish effective protocols for using the instrument, which can be time‐consuming and costly. Here, we present a workflow and protocols that enabled us to establish MinION sequencing in our own laboratories, based on optimizing DNA extraction from a challenging plant tissue as a case study. Following the workflow illustrated, we were able to reliably and repeatedly obtain >6.5 Gb of long‐read sequencing data with a mean read length of 13 kb and an N50 of 26 kb. Our protocols are open source and can be performed in any laboratory without special equipment. We also illustrate some more elaborate workflows which can increase mean and average read lengths if this is desired. We envision that our workflow for establishing MinION sequencing, including the illustration of potential pitfalls and suggestions of how to adapt it to other tissue types, will be useful to others who plan to establish long‐read sequencing in their own laboratories.     

High intraspecific diversity of Restorer‐of‐fertility‐like genes in barley

Nuclear restorer of fertility (Rf) genes suppress the effects of mitochondrial genes causing cytoplasmic male sterility (CMS), a condition in which plants fail to produce viable pollen. Rf genes, many of which encode RNA‐binding pentatricopeptide repeat (PPR) proteins, are applied in hybrid breeding to overcome CMS used to block self‐pollination of the seed parent. Here, we characterise the repertoire of restorer‐of‐fertility‐like (RFL) PPR genes in barley (Hordeum vulgare). We found 26 RFL genes in the reference genome (‘Morex’) and an additional 51 putative orthogroups (POGs) in a re‐sequencing data set from 262 barley genotypes and landraces. Whereas the sequences of some POGs are highly conserved across hundreds of barley accessions, the sequences of others are much more variable. High sequence variation strongly correlates with genomic location – the most variable genes are found in a cluster on chromosome 1H. A much higher likelihood of diversifying selection was found for genes within this cluster than for genes present as singlets. This work includes a comprehensive analysis of the patterns of intraspecific variation of RFL genes. The RFL sequences characterised in this study will be useful for the development of new markers for fertility restoration loci.     

Strong “bottom‐up” influences on small mammal populations: State‐space model analyses from long‐term studies

“Bottom‐up” influences, that is, masting, plus population density and climate, commonly influence woodland rodent demography. However, “top‐down” influences (predation) also intervene. Here, we assess the impacts of masting, climate, and density on rodent populations placed in the context of what is known about “top‐down” influences. To explain between‐year variations in bank vole Myodes glareolus and wood mouse Apodemus sylvaticus population demography, we applied a state‐space model to 33 years of catch‐mark‐release live‐trapping, winter temperature, and precise mast‐collection data. Experimental mast additions aided interpretation. Rodent numbers in European ash Fraxinus excelsior woodland were estimated (May/June, November/December). December–March mean minimum daily temperature represented winter severity. Total marked adult mice/voles (and juveniles in May/June) provided density indices validated against a model‐generated population estimate; this allowed estimation of the structure of a time‐series model and the demographic impacts of the climatic/biological variables. During two winters of insignificant fruit‐fall, 6.79 g/m² sterilized ash seed (as fruit) was distributed over an equivalent woodland similarly live‐trapped. September–March fruit‐fall strongly increased bank vole spring reproductive rate and winter and summer population growth rates; colder winters weakly reduced winter population growth. September–March fruit‐fall and warmer winters marginally increased wood mouse spring reproductive rate and September–December fruit‐fall weakly elevated summer population growth. Density dependence significantly reduced both species' population growth. Fruit‐fall impacts on demography still appeared after a year. Experimental ash fruit addition confirmed its positive influence on bank vole winter population growth with probable moderation by colder temperatures. The models show the strong impact of masting as a “bottom‐up” influence on rodent demography, emphasizing independent masting and weather influences; delayed effects of masting; and the importance of density dependence and its interaction with masting. We conclude that these rodents show strong “bottom‐up” and density‐dependent influences on demography moderated by winter temperature. “Top‐down” influences appear weak and need further investigation.     

Agroforestry coffee soils increase the insect‐suppressive potential offered by entomopathogenic fungi over full‐sun soils: A case proposing a “bait survival technique”

Entomopathogenic fungi are important natural enemies of insects. However, there is little information on the insect‐suppressive potential of these fungi and possible effects of farming management on this. Meanwhile, changes in natural landscapes due to agricultural intensification have caused considerable biodiversity loss and consequent decay of ecosystem services. However, the adoption of practices such as agroforestry in agroecosystems can foster abiotic and biotic conditions that conserve biodiversity, consequently restoring the provision of ecosystems services. Here, we assessed the effect of management systems (agroforestry or full‐sun) on the pest‐suppressive potential of entomopathogenic fungi in Brazilian coffee plantations. We used the insect bait method coupled with survival analyses to assess the speed of kill by entomopathogenic fungi and their presence in soil samples from both farming systems. We found that insects exposed to agroforestry soils died more quickly than insects exposed to full‐sun soils. Of the fungi isolated from the bait insects, Metarhizium was found most frequently, followed by Beauveria. Meanwhile, Fusarium was frequently isolated as primary or secondary infections. We propose that the differential survival of insects is indicative of a greater suppressive potential by entomopathogenic fungi in agroforestry, and that this could be promoted by the diversified landscape, microclimatic stability, and reduced soil disturbance in agroforestry systems. Furthermore, our results provide a useful demonstration of the potential use of the insect bait method to investigate pest‐suppressive potential through bait insect mortality, and we term this the “bait survival technique.”     

“Uncovering” Industrial Symbiosis

Since 1989, efforts to understand the nature of interfirm resource sharing in the form of industrial symbiosis and to replicate in a deliberate way what was largely self‐organizing in Kalundborg, Denmark have followed many paths, some with much success and some with very little. This article provides a historical view of the motivations and means for pursuing industrial symbiosis—defined to include physical exchanges of materials, energy, water, and by‐products among diversified clusters of firms. It finds that “uncovering” existing symbioses has led to more sustainable industrial development than attempts to design and build eco‐industrial parks incorporating physical exchanges. By examining 15 proposed projects brought to national and international attention by the U.S. President's Council on Sustainable Development beginning in the early 1990s, and contrasting these with another 12 projects observed to share more elements of self‐organization, recommendations are offered to stimulate the identification and uncovering of already existing “kernels” of symbiosis. In addition, policies and practices are suggested to identify early‐stage precursors of potentially larger symbioses that can be nurtured and developed further. The article concludes that environmentally and economically desirable symbiotic exchanges are all around us and now we must shift our gaze to find and foster them.