IFN‐γ+IL‐17+Th17 cells regulate fibrosis through secreting IL‐21 in systemic scleroderma

This study aimed to explore the function of IFN‐γ⁺IL‐17⁺Th17 cells on fibrosis in systemic scleroderma (SSc). Blood and skin samples were collected from 20 SSc cases and 10 healthy individuals. The percentage of IFN‐γ⁺IL‐17⁺Th17 cells was detected using flow cytometry. The in vitro induction of IFN‐γ⁺IL‐17⁺Th17 cells was performed adopting PHA and rIL‐12. Gene expression was detected via quantitative real‐time polymerase chain reaction (qRT‐PCR), whereas western blot analysis was adopted for protein analysis. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with SSc stages (P = .031), disease duration (P = .016), activity (P = .025) and skin scores (P < .001). In vitro, IFN‐γ⁺IL‐17⁺Th17 cells could promote the expressions of α‐SMA and COL1A1, revealing increased fibroblasts’ proliferation and enhanced collagen‐secreting capacity. In addition, IL‐21 expression was significantly increased in co‐culture medium of IFN‐γ⁺IL‐17⁺Th17 cells and fibroblasts (P < .001). IL‐21 neutralizer treatment resulted in the down‐regulation of α‐SMA and COL1A1. IL‐21 was confirmed as an effector of IFN‐γ⁺IL‐17⁺Th17 cells in fibrosis process. The distribution of IFN‐γ⁺IL‐17⁺Th17 cells was significantly increased in SSc cases and positively correlated with disease activity. IFN‐γ⁺IL‐17⁺Th17 cells could promote fibroblast proliferation and enhance collagen‐secreting ability via producing IL‐21, thus contributing to fibrosis in SSc.     

3,6'‐disinapoyl sucrose attenuates Aβ1‐42‐ induced neurotoxicity in Caenorhabditis elegans by enhancing antioxidation and regulating autophagy

The aggregation of β‐amyloid (Aβ) has the neurotoxicity, which is thought to play critical role in the pathogenesis of Alzheimer''s disease (AD). Inhibiting Aβ deposition and neurotoxicity has been considered as an important strategy for AD treatment. 3,6''‐Disinapoyl sucrose (DISS), one of the oligosaccharide esters derived from traditional Chinese medicine Polygalae Radix, possesses antioxidative activity, neuroprotective effect and anti‐depressive activity. This study was to explore whether DISS could attenuate the pathological changes of Aβ_1‐42 transgenic Caenorhabditis elegans (C. elegans). The results showed that DISS (5 and 50 μM) treatment significantly prolonged the life span, increased the number of egg‐laying, reduced paralysis rate, decreased the levels of lipofuscin and ROS and attenuated Aβ deposition in Aβ_1‐42 transgenic C. elegans. Gene analysis showed that DISS could up‐regulate the mRNA expression of sod‐3, gst‐4, daf‐16, bec‐1 and lgg‐1, while down‐regulate the mRNA expression of daf‐2 and daf‐15 in Aβ_1‐42 transgenic C. elegans. These results suggested that DISS has the protective effect against Aβ_1‐42‐induced pathological damages and prolongs the life span of C. elegans, which may be related to the reduction of Aβ deposition and neurotoxicity by regulating expression of genes related to antioxidation and autophagy.     

Low base‐substitution mutation rate and predominance of insertion‐deletion events in the acidophilic bacterium Acidobacterium capsulatum

Analyses of spontaneous mutation have shown that total genome‐wide mutation rates are quantitatively similar for most prokaryotic organisms. However, this view is mainly based on organisms that grow best around neutral pH values (6.0–8.0). In particular, the whole‐genome mutation rate has not been determined for an acidophilic organism. Here, we have determined the genome‐wide rate of spontaneous mutation in the acidophilic Acidobacterium capsulatum using a direct and unbiased method: a mutation‐accumulation experiment followed by whole‐genome sequencing. Evaluation of 69 mutation accumulation lines of A. capsulatum after an average of ~2900 cell divisions yielded a base‐substitution mutation rate of 1.22 × 10⁻¹⁰ per site per generation or 4 × 10⁻⁴ per genome per generation, which is significantly lower than the consensus value (2.5−4.6 × 10⁻³) of mesothermophilic (~15–40°C) and neutrophilic (pH 6–8) prokaryotic organisms. However, the insertion‐deletion rate (0.43 × 10⁻¹⁰ per site per generation) is high relative to the base‐substitution mutation rate. Organisms with a similar effective population size and a similar expected effect of genetic drift should have similar mutation rates. Because selection operates on the total mutation rate, it is suggested that the relatively high insertion‐deletion rate may be balanced by a low base‐substitution rate in A. capsulatum, with selection operating on the total mutation rate.     

Chromosome‐level genome assembly for the horned‐gall aphid provides insights into interactions between gall‐making insect and its host plant

The aphid Schlechtendalia chinensis is an economically important insect that can induce horned galls, which are valuable for the medicinal and chemical industries. Up to now, more than twenty aphid genomes have been reported. Most of the sequenced genomes are derived from free‐living aphids. Here, we generated a high‐quality genome assembly from a galling aphid. The final genome assembly is 271.52 Mb, representing one of the smallest sequenced genomes of aphids. The genome assembly is based on contig and scaffold N50 values of the genome sequence are 3.77 Mb and 20.41 Mb, respectively. Nine‐seven percent of the assembled sequences was anchored onto 13 chromosomes. Based on BUSCO analysis, the assembly involved 96.9% of conserved arthropod and 98.5% of the conserved Hemiptera single‐copy orthologous genes. A total of 14,089 protein‐coding genes were predicted. Phylogenetic analysis revealed that S. chinensis diverged from the common ancestor of Eriosoma lanigerum approximately 57 million years ago (MYA). In addition, 35 genes encoding salivary gland proteins showed differentially when S. chinensis forms a gall, suggesting they have potential roles in gall formation and plant defense suppression. Taken together, this high‐quality S. chinensis genome assembly and annotation provide a solid genetic foundation for future research to reveal the mechanism of gall formation and to explore the interaction between aphids and their host plants.     

Using fractal self‐similarity to increase precision of shrub biomass estimates

We show that aerial tips are self‐similar fractals of whole shrubs and present a field method that applies this fact to improves accuracy and precision of biomass estimates of tall‐shrubs, defined here as those with diameter at root collar (DRC) ≥ 2.5 cm. Power function allometry of biomass to stem diameter generates a disproportionate prediction error that increases rapidly with diameter. Thus, biomass should be modeled as a single measure of stem diameter only if stem diameter is less than a threshold D_max. When stem diameter exceeds D_max, then the stem internode should be treated as a conic frustrum requiring two additional measures: a second, node‐adjacent diameter and a length. If the second diameter is less than D_max, then the power function allometry can be applied to the aerial tip; otherwise an additional internode is measured. This “two‐component” allometry—internodes as frustra and aerial tips as shrubs—can reduce estimated biomass error propagated to the plot‐level by as much as 50% or more where very large shrubs are present D_max is any diameter such that the ratio of single‐component to two‐component uncertainty exceeds the ratio of two‐component to single‐component measurement time. Guidelines for estimating D_max based on pilot field data are provided. Tall shrubs are increasing in abundance and distribution across Arctic, alpine, boreal, and dryland ecosystems. Estimating their biomass is important for both ecological studies and carbon accounting. Reducing field‐sample prediction error increases precision in multi‐stage modeling because additional measures efficiently improve plot‐level biomass precision, reducing uncertainty for shrub biomass estimates.     

Rapid adaptive responses of rosette‐type macrophyte Vallisneria natans juveniles to varying water depths: The role of leaf trait plasticity

Rosette‐type submerged macrophytes are widely distributed across a range of water depths in shallow lakes and play a key role in maintaining ecosystem structures and functions. However, little is known about the rapid adaptive responses of such macrophytes to variations in water depth, especially at the juvenile stage. Here, we conducted a short‐term in situ mesocosm experiment, in which the juveniles of Vallisneria natans were exposed to a water depth gradient ranging from 20 to 360 cm. Twenty‐two leaf‐related traits were examined after 4 weeks of growth in a shallow lake. Most (18) traits of V. natans generally showed high plasticity in relation to water depth. Specifically, juveniles allocated more biomass to leaves and had higher specific leaf area, leaf length‐to‐width ratio, chlorophyll content, and carotenoids content in deep waters, displaying trait syndrome associated with high resource acquisition. In contrast, V. natans juveniles in shallow waters had higher leaf dry matter content, leaf soluble carbohydrate content, carotenoids per unit chlorophyll, and peroxidase activity, pertaining to resource conservation. Notably, underwater light intensity was found to be the key factor explaining the trait plasticity along the water depth gradient, and 1.30 mol photons m⁻² d⁻¹ (at 270 cm) could be the optimal irradiance level based on the total biomass of V. natans juveniles. The present study highlights the significance of leaf trait plasticity for rosette‐type macrophytes in response to variations in water depth and sheds new light on the differences between trade‐offs in deep‐ and shallow‐water areas.     

Declines in occurrence of plants characteristic for a nutrient‐poor meadow habitat are partly explained by their responses to nutrient addition and competition

Species losses and local extinctions are alarmingly common, frequently as a consequence of habitat destruction. Nevertheless, many intact habitats also face species losses, most likely due to environmental changes. However, the exact drivers, and why they affect some species more than others in apparently intact habitats, are still poorly understood. Addressing these questions requires data on changes in occurrence frequency of many species, and comparisons of the responses of those species to experimental manipulations of the environment. Here, we use historic (1911) and contemporary (2017) data on the presence–absence of 42 plant species in 14 seemingly intact Molinia meadows around Lower Lake Constance to quantify changes in occurrence frequency. Then, we performed a common‐garden experiment to test whether occurrence frequencies in 1911 and changes therein by 2017 could be explained by responses of the 42 species to nutrient addition and competition with the acquisitive generalist grass Poa pratensis. Within the 14 still intact Molinia meadows, 36 of the 42 species had declined since 1911. As expected, nutrient addition generally led to increased biomass production of the 42 target species, and competition with P. pratensis had a negative effect. The latter was stronger at high nutrient availability. The more frequent species were in 1911 and the more they declined in frequency between 1911 and 2017, the less above‐ground biomass they produced in our experiment. Competition with P. pratensis magnified this effect. Our work highlights that environmental change can contribute to local extinction of species in otherwise intact habitat remnants. Specifically, we showed that increased nutrient availability negatively affected formerly widespread Molinia‐meadow species in competition with P. pratensis. Our study thus identified a likely mechanism for the decline in occurrence frequency of species in the remaining Molinia meadows.     

Fibronectin type III domain‐containing 5 improves aging‐related cardiac dysfunction in mice

Aging is an important risk factor for cardiovascular diseases, and aging‐related cardiac dysfunction serves as a major determinant of morbidity and mortality in elderly populations. Our previous study has identified fibronectin type III domain‐containing 5 (FNDC5) and its cleaved form, irisin, as the cardioprotectant against doxorubicin‐induced cardiomyopathy. Herein, aging or matched young mice were overexpressed with FNDC5 by adeno‐associated virus serotype 9 (AAV9) vectors, or subcutaneously infused with irisin to uncover the role of FNDC5 in aging‐related cardiac dysfunction. To verify the involvement of nucleotide‐binding oligomerization domain‐like receptor with a pyrin domain 3 (NLRP3) and AMP‐activated protein kinase α (AMPKα), Nlrp3 or Ampkα2 global knockout mice were used. Besides, young mice were injected with AAV9‐FNDC5 and maintained for 12 months to determine the preventive effect of FNDC5. Moreover, neonatal rat cardiomyocytes were stimulated with tumor necrosis factor‐α (TNF‐α) to examine the role of FNDC5 in vitro. We found that FNDC5 was downregulated in aging hearts. Cardiac‐specific overexpression of FNDC5 or irisin infusion significantly suppressed NLRP3 inflammasome and cardiac inflammation, thereby attenuating aging‐related cardiac remodeling and dysfunction. In addition, irisin treatment also inhibited cellular senescence in TNF‐α‐stimulated cardiomyocytes in vitro. Mechanistically, FNDC5 activated AMPKα through blocking the lysosomal degradation of glucagon‐like peptide‐1 receptor. More importantly, FNDC5 gene transfer in early life could delay the onset of cardiac dysfunction during aging process. We prove that FNDC5 improves aging‐related cardiac dysfunction by activating AMPKα, and it might be a promising therapeutic target to support cardiovascular health in elderly populations.     

Galangin as a direct inhibitor of vWbp protects mice from Staphylococcus aureus‐induced pneumonia

The surge in multidrug resistance in Staphylococcus aureus (S. aureus) and the lag in antibiotic discovery necessitate the development of new anti‐infective strategies to reduce S. aureus infections. In S. aureus, von Willebrand factor‐binding protein (vWbp) is not only the main coagulase that triggers host prothrombin activation and formation of fibrin cables but also bridges the bacterial cell wall and von Willebrand factor, thereby allowing S. aureus to bind to platelets and endothelial cells, playing a vital role in pathogenesis of S. aureus infections. Here, we have identified that galangin, a bioactive compound found in honey and Alpinia officinarum Hance, is a potent and direct inhibitor of vWbp by coagulation activity inhibition assay, thermal shift assay and biolayer interferometry assay. Molecular dynamic simulations and verification experiments revealed that the Trp‐64 and Leu‐69 residues are necessary for the binding of galangin to vWbp. Significantly, galangin attenuated S. aureus virulence in a mouse S. aureus‐induced pneumonia model. In addition, we also identified that galangin can enhance the therapeutic effect of latamoxef on S. aureus‐induced pneumonia. Taken together, the results suggest that galangin may be used for the development of therapeutic drugs or utilized as adjuvants to combine with antibiotics to combat S. aureus‐related infections.     

Trade‐off between flight capability and reproduction in Acridoidea (Insecta: Orthoptera)

In many insect taxa, there is a well‐established trade‐off between flight capability and reproduction. The wing types of Acridoidea exhibit extremely variability from full length to complete loss in many groups, thus, provide a good model for studying the trade‐off between flight and reproduction. In this study, we completed the sampling of 63 Acridoidea species, measured the body length, wing length, body weight, flight muscle weight, testis and ovary weight, and the relative wing length (RWL), relative flight muscle weight (RFW), and gonadosomatic index (GSI) of different species were statistically analyzed. The results showed that there were significant differences in RWL, RFW, and GSI among Acridoidea species with different wing types. RFW of long‐winged species was significantly higher than that of short‐winged and wingless species (p < .01), while GSI of wingless species was higher than that of long‐winged and short‐winged species. The RWL and RFW had a strong positive correlation in species with different wing types (correlation coefficient r = .8344 for male and .7269 for female, and p < .05), while RFW was strong negatively correlated with GSI (r = −.2649 for male and −.5024 for female, and p < .05). For Acridoidea species with wing dimorphism, males with relatively long wings had higher RFW than that of females with relatively short wings, while females had higher GSI. Phylogenetic comparative analysis showed that RWL, RFW, and GSI all had phylogenetic signals and phylogenetic dependence. These results revealed that long‐winged individuals are flight capable at the expense of reproduction, while short‐winged and wingless individuals cannot fly, but has greater reproductive output. The results support the trade‐off between flight and reproduction in Acridoidea.     

Genetic diversity and sex‐biased dispersal in the brown spotted pitviper (Protobothrops mucrosquamatus): Evidence from microsatellite markers

Dispersal plays a vital role in the geographical distribution, population genetic structure, quantity dynamics, and evolution of a species. Sex‐biased dispersal is common among vertebrates and many studies have documented a tendency toward male‐biased dispersal in mammals and female‐biased dispersal in birds. However, dispersal patterns in reptiles remain poorly understood. In this study, we explored the genetic diversity and dispersal patterns of the widely distributed Asian pitviper Protobothrops mucrosquamatus. In total, 16 polymorphic microsatellite loci were screened in 150 snakes (48 males, 44 females, 58 samples without sex information) covering most of their distribution. Microsatellite analysis revealed high genetic diversity in P. mucrosquamatus. Bayesian clustering of population assignment identified two major clusters for all populations, somewhat inconsistent with the mitochondrial DNA phylogeny of P. mucrosquamatus reported in previous research. Analyses based on 92 sex‐determined and 37 samples of P. mucrosquamatus from three small sites in Sichuan, China (Mingshan, Yibin, and Zizhong) consistently suggested female‐biased dispersal in P. mucrosquamatus, which is the first example of this pattern in snakes. The female‐biased dispersal patterns in P. mucrosquamatus may be explained by local resource competition.     

Tetrahedral DNA nanostructure improves transport efficiency and anti‐fungal effect of histatin 5 against Candida albicans

ObjectivesAnti‐microbial peptides (AMPs) have been comprehensively investigated as a novel alternative to traditional antibiotics against microorganisms. Meanwhile, Tetrahedral DNA nanostructures (TDNs) have gained attention in the field of biomedicine for their premium biological effects and transportation efficiency as delivery vehicles. Hence, in this study, TDN/Histatin 5 (His‐5) was synthesized and the transport efficiency and anti‐fungal effect were measured to evaluate the promotion of His‐5 modified by TDNs.Materials and MethodsTetrahedral DNA nanostructures/His‐5 complex was prepared via electrostatic attraction and characterized by transmission electron microscopy (TEM), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The anti‐fungal effect of the TDN/His‐5 complex was evaluated by determining the growth curve and colony‐forming units of C. albicans. The morphological transformation of C. albicans was observed by light microscope and scanning electron microscope (SEM). Immunofluorescence was performed, and potassium efflux was detected to mechanistically demonstrate the efficacy of TDN/His‐5.ResultsThe results showed that Histatin 5 modified by TDNs had preferable stability in serum and was effectively transported into C. albicans, leading to the increased formation of intracellular reactive oxygen species, higher potassium efflux and enhanced anti‐fungal effect against C. albicans.ConclusionsOur study showed that TDN/His‐5 was synthesized successfully. And by the modification of TDNs, His‐5 showed increased transport efficiency and improved anti‐fungal effect.     

Fijian sea krait behavior relates to fine‐scale environmental heterogeneity in old‐growth coastal forest: The importance of integrated land–sea management for protecting amphibious animals

The importance of terrestrial coastal ecosystems for maintaining healthy coral reef ecosystems remains understudied. Sea kraits are amphibious snakes that require healthy coral reefs for foraging, but little is known about their requirements of terrestrial habitats, where they slough their skin, digest prey, and breed. Using concurrent microclimate measurements and behavior surveys, we show that a small, topographically flat atoll in Fiji with coastal forest provides many microhabitats that relate to the behaviors of Yellow Lipped Sea Kraits, Laticauda colubrina. Microclimates were significantly related to canopy cover, leaf litter depth, and distance from the high‐water mark (HWM). Sea kraits were almost exclusively observed in coastal forest within 30 m of the HWM. Sloughing of skins only occurred within crevices of mature or dying trees. Resting L. colubrina were significantly more likely to occur at locations with higher mean diurnal temperatures, lower leaf litter depths, and shorter distances from the HWM. On Leleuvia, behavior of L. colubrina therefore relates to environmental heterogeneity created by old‐growth coastal forests, particularly canopy cover and crevices in mature and dead tree trunks. The importance of healthy coastal habitats, both terrestrial and marine, for L. colubrina suggests it could be a good flagship species for advocating integrated land‐sea management. Furthermore, our study highlights the importance of coastal forests and topographically flat atolls for biodiversity conservation. Effective conservation management of amphibious species that utilize land‐ and seascapes is therefore likely to require a holistic approach that incorporates connectivity among ecosystems and environmental heterogeneity at all relevant scales.     

Oral intake of Lactobacillus plantarum L‐14 extract alleviates TLR2‐ and AMPK‐mediated obesity‐associated disorders in high‐fat‐diet‐induced obese C57BL/6J mice

ObjectivesWhether periodic oral intake of postbiotics positively affects weight regulation and prevents obesity‐associated diseases in vivo is unclear. This study evaluated the action mechanism of Lactobacillus plantarum L‐14 (KTCT13497BP) extract and the effects of its periodic oral intake in a high‐fat‐diet (HFD) mouse model.Materials and methodsMouse pre‐adipocyte 3T3‐L1 cells and human bone marrow mesenchymal stem cells (hBM‐MSC) were treated with L‐14 extract every 2 days during adipogenic differentiation, and the mechanism underlying anti‐adipogenic effects was analysed at cellular and molecular levels. L‐14 extract was orally administrated to HFD‐feeding C57BL/6J mice every 2 days for 7 weeks. White adipose tissue was collected and weighed, and liver and blood serum were analysed. The anti‐adipogenic mechanism of exopolysaccharide (EPS) isolated from L‐14 extract was also analysed using Toll‐like receptor 2 (TLR2) inhibitor C29.ResultsL‐14 extract inhibited 3T3‐L1 and hBM‐MSC differentiation into mature adipocytes by upregulating AMPK signalling pathway in the early stage of adipogenic differentiation. The weight of the HFD + L‐14 group (31.51 ± 1.96 g) was significantly different from that of the HFD group (35.14 ± 3.18 g). L‐14 extract also significantly decreased the serum triacylglycerol/high‐density lipoprotein cholesterol ratio (an insulin resistance marker) and steatohepatitis. In addition, EPS activated the AMPK signalling pathway by interacting with TLR2, consequently inhibiting adipogenesis.ConclusionsEPS from L‐14 extract inhibits adipogenesis via TLR2 and AMPK signalling pathways, and oral intake of L‐14 extract improves obesity and obesity‐associated diseases in vivo. Therefore, EPS can be used to prevent and treat obesity and metabolic disorders.     

Long‐term changes in occurrence,relative abundance,and reproductive fitness of bat species in relation to arrival of White‐nose Syndrome in West Virginia,USA

White‐nose syndrome (WNS) is a disease caused by the fungus Pseudogymnoascus destructans which has resulted in the deaths of millions of bats across eastern North America. To date, hibernacula counts have been the predominant means of tracking the spread and impact of this disease on bat populations. However, an understanding of the impacts of WNS on demographic parameters outside the winter season is critical to conservation and recovery of bat populations impacted by this disease. We used long‐term monitoring data to examine WNS‐related impacts to summer populations in West Virginia, where WNS has been documented since 2009. Using capture data from 290 mist‐net sites surveyed from 2003 to 2019 on the Monongahela National Forest, we estimated temporal patterns in presence and relative abundance for each bat species. For species that exhibited a population‐level response to WNS, we investigated post‐WNS changes in adult female reproductive state and body mass. Myotis lucifugus (little brown bat), M. septentrionalis (northern long‐eared bat), and Perimyotis subflavus (tri‐colored bat) all showed significant decreases in presence and relative abundance during and following the introduction of WNS, while Eptesicus fuscus (big brown bat) and Lasiurus borealis (eastern red bat) responded positively during the WNS invasion. Probability of being reproductively active was not significantly different for any species, though a shift to earlier reproduction was estimated for E. fuscus and M. septentrionalis. For some species, body mass appeared to be influenced by the WNS invasion, but the response differed by species and reproductive state. Results suggest that continued long‐term monitoring studies, additional research into impacts of this disease on the fitness of WNS survivors, and a focus on providing optimal nonwintering habitat may be valuable strategies for assessing and promoting recovery of WNS‐affected bat populations.     

Assessment of medium and large‐sized mammals and their behavioral response toward anthropogenic activities in Jorgo‐Wato Protected Forest,Western Ethiopia

Medium and large‐sized mammals of Jorgo‐Wato Protected Forest have not yet been documented though the forest established before four decades. Hence, this study aims to document medium and large mammals and the behavioral responses of selected mammals toward anthropogenic activities in the study area. The study was conducted from February 2015 to June 2016, encompassing the wet and dry seasons. Data were collected mainly through camera traps, indirect and direct evidence. The study revealed about 23 medium and large‐sized mammals that belong to seven orders namely Bovidae, Carnivora, Primates, Rodentia, Tubulidentata, Lagomorpha, and Hyracoidea. Papio anubis, C. guereza, and C. aethiops were the most abundant large mammals in JWPF. Because of high anthropogenic activities, African buffalo shifted its activity period from diurnal into crepuscular and nocturnal. African buffalo traveled longer distances during the wet season (mean = 14.33 km, SD = 1.25 km) than during the dry season (mean = 9.00 km, SD = 2.16 km). This could be due to the fact that the local people were less likely to go to the forest for resource exploitation during the wet season as they are fully engaged in agricultural activities. However, low agricultural activities during the dry season allow the local people to extract resources and involve in bushmeat hunting which could limit the movement of mammals to their refugia. African buffalo preferred to rest on and adjacent to a gravel road (22.1%) in the forest, followed by on open rocky hilltops (14.7%) at night time, but rest in the bottomland thicket vegetation during the dry daytime. Regardless of high human pressure in the area, this study has revealed a good number of medium and large‐sized mammals that could be used as baseline information to design a sound conservation and management action plan of large mammals and their habitat in Jorgo‐Wato Protected Forest.     

Phylogeography of sugar kelp: Northern ice‐age refugia in the Gulf of Alaska

Many Northeast (NE) Pacific fishes and invertebrates survived Pleistocene glaciations in northern refugia, but the extent that kelps survived in northern areas is uncertain. Here, we test the hypothesis that populations of sugar kelp (Saccharina latissima) persisted in the Gulf of Alaska during ice‐age maxima when the western margin of the Cordilleran ice sheet covered coastal areas around the NE Pacific Ocean. We estimated genetic diversities within and phylogeographical relationships among 14 populations along 2,800 km in the NE Pacific and Bering Sea with partial sequences of mitochondrial DNA 5′‐cytochrome oxidase subunit I (COI, bp = 624, n = 543), chloroplast DNA ribulose‐1,5‐bisphosphate carboxylase large subunit‐3′ (rbcL, bp = 735, n = 514), and 11 microsatellite loci. Concatenated sequences of rbcL and COI showed moderate levels of within‐population genetic diversity (mean h = 0.200) but substantial differences among populations (Φ_ST = 0.834, p < .0001). Microsatellites showed moderate levels of heterozygosity within populations (mean H _E = 0.391). Kelps in the same organellar lineage tended to cluster together, regardless of geographic origins, as indicated in a principal coordinate analysis (PCoA) of microsatellite genotypes. The PCoA also showed evidence of nuclear hybridizations between co‐occurring organellar lineages. Individual admixture plots with population clusters of K = 2, 6, and 9 showed increasing complexity with considerable historical admixture between some clusters. A time‐calibrated phylogeny placed divergences between rbcL‐COI lineages at 1.4 million years at most. The time frames of mutation in the rbcL‐COI lineages and microsatellite population clusters differed among locations. The existence of ancient lineages in the Gulf of Alaska, moderate levels of genetic diversity, and the absence of departures from neutrality are consistent with northern refugia during multiple Croll‐Milankovitch climate cycles in the Pleistocene Epoch.     

DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera‐recorded diets

Ecological research is often hampered by the inability to quantify animal diets. Diet composition can be tracked through DNA metabarcoding of fecal samples, but whether (complex) diets can be quantitatively determined with metabarcoding is still debated and needs validation using free‐living animals. This study validates that DNA metabarcoding of feces can retrieve actual ingested taxa, and most importantly, that read numbers retrieved from sequencing can also be used to quantify the relative biomass of dietary taxa. Validation was done with the hole‐nesting insectivorous Pied Flycatcher whose diet was quantified using camera footage. Size‐adjusted counts of food items delivered to nestlings were used as a proxy for provided biomass of prey orders and families, and subsequently, nestling feces were assessed through DNA metabarcoding. To explore potential effects of digestion, gizzard and lower intestine samples of freshly collected birds were subjected to DNA metabarcoding. For metabarcoding with Cytochrome Oxidase subunit I (COI), we modified published invertebrate COI primers LCO1490 and HCO1777, which reduced host reads to 0.03%, and amplified Arachnida DNA without significant changing the recovery of other arthropod taxa. DNA metabarcoding retrieved all commonly camera‐recorded taxa. Overall, and in each replicate year (N = 3), the relative scaled biomass of prey taxa and COI read numbers correlated at R = .85 (95CI:0.68–0.94) at order level and at R = .75 (CI:0.67–0.82) at family level. Similarity in arthropod community composition between gizzard and intestines suggested limited digestive bias. This DNA metabarcoding validation demonstrates that quantitative analyses of arthropod diet is possible. We discuss the ecological applications for insectivorous birds.