Isolation and characterization of 12 microsatellite loci from Golden‐winged Warblers (Vermivora chrysoptera) with broad cross‐taxon utility in emberizine songbirds

We describe 12 microsatellite loci from the Golden‐winged Warbler (Vermivora chrysoptera), a bird species of conservation concern owing to competition and hybridization with the Blue‐winged Warbler (V. pinus). Directional mitochondral DNA introgression from V. pinus into V. chrysoptera has been reported within the hybrid zone. Frequent hybrid phenotypes suggest that nuclear introgression is also prevalent, although this has not been demonstrated conclusively with molecular markers. These microsatellites will foster explorations of the genetic interactions between these avian congeners, and amplification tests suggest that they have high cross‐species utility for closely allied bird genera and families.     

Synergistic effects of climate and land‐use change influence broad‐scale avian population declines

Climate and land‐use changes are expected to be the primary drivers of future global biodiversity loss. Although theory suggests that these factors impact species synergistically, past studies have either focused on only one in isolation or have substituted space for time, which often results in confounding between drivers. Tests of synergistic effects require congruent time series on animal populations, climate change and land‐use change replicated across landscapes that span the gradient of correlations between the drivers of change. Using a unique time series of high‐resolution climate (measured as temperature and precipitation) and land‐use change (measured as forest change) data, we show that these drivers of global change act synergistically to influence forest bird population declines over 29 years in the Pacific Northwest of the United States. Nearly half of the species examined had declined over this time. Populations declined most in response to loss of early seral and mature forest, with responses to loss of early seral forest amplified in landscapes that had warmed over time. In addition, birds declined more in response to loss of mature forest in areas that had dried over time. Climate change did not appear to impact populations in landscapes with limited habitat loss, except when those landscapes were initially warmer than the average landscape. Our results provide some of the first empirical evidence of synergistic effects of climate and land‐use change on animal population dynamics, suggesting accelerated loss of biodiversity in areas under pressure from multiple global change drivers. Furthermore, our findings suggest strong spatial variability in the impacts of climate change and highlight the need for future studies to evaluate multiple drivers simultaneously to avoid potential misattribution of effects.     

The effects of force‐fledging and premature fledging on the survival of nestling songbirds

Despite the broad consensus that force‐fledging of nestling songbirds lowers their probability of survival and therefore should be generally avoided by researchers, that presumption has not been tested. We used radiotelemetry to monitor the survival of fledglings of Ovenbirds Seiurus aurocapilla and Golden‐winged Warblers Vermivora chrysoptera that we unintentionally force‐fledged (i.e. nestlings left the nest in response to our research activities at typical fledging age), that fledged prematurely (i.e. nestlings left the nest earlier than typical fledging age), and that fledged independently of our activities. Force‐fledged Ovenbirds experienced significantly higher survival than those that fledged independent of our activities, and prematurely fledged Ovenbirds had a similarly high survival to those that force‐fledged at typical fledging age. We observed a similar, though not statistically significant, pattern in Golden‐winged Warbler fledgling survival. Our results suggest that investigator‐induced force‐fledging of nestlings, even when deemed premature, does not necessarily result in reduced fledgling survival in these species. Instead, our results suggest that a propensity or ability to fledge in response to disturbance may be a predictor of a higher probability of fledgling survival.     

A method for estimating population sex ratio for sage‐grouse using noninvasive genetic samples

Population sex ratio is an important metric for wildlife management and conservation, but estimates can be difficult to obtain, particularly for sexually monomorphic species or for species that differ in detection probability between the sexes. Noninvasive genetic sampling (NGS) using polymerase chain reaction (PCR) has become a common method for identifying sex from sources such as hair, feathers or faeces, and is a potential source for estimating sex ratio. If, however, PCR success is sex‐biased, naively using NGS could lead to a biased sex ratio estimator. We measured PCR success rates and error rates for amplifying the W and Z chromosomes from greater sage‐grouse (Centrocercus urophasianus) faecal samples, examined how success and error rates for sex identification changed in response to faecal sample exposure time, and used simulation models to evaluate precision and bias of three sex assignment criteria for estimating population sex ratio with variable sample sizes and levels of PCR replication. We found PCR success rates were higher for females than males and that choice of sex assignment criteria influenced the bias and precision of corresponding sex ratio estimates. Our simulations demonstrate the importance of considering the interplay between the sex bias of PCR success, number of genotyping replicates, sample size, true population sex ratio and accuracy of assignment rules for designing future studies. Our results suggest that using faecal DNA for estimating the sex ratio of sage‐grouse populations has great potential and, with minor adaptations and similar marker evaluations, should be applicable to numerous species.     

Integrating broad‐scale data to assess demographic and climatic contributions to population change in a declining songbird

Climate variation and trends affect species distribution and abundance across large spatial extents. However, most studies that predict species response to climate are implemented at small spatial scales or are based on occurrence‐environment relationships that lack mechanistic detail. Here, we develop an integrated population model (IPM) for multi‐site count and capture‐recapture data for a declining migratory songbird, Wilson's warbler (Cardellina pusilla), in three genetically distinct breeding populations in western North America. We include climate covariates of vital rates, including spring temperatures on the breeding grounds, drought on the wintering range in northwest Mexico, and wind conditions during spring migration. Spring temperatures were positively related to productivity in Sierra Nevada and Pacific Northwest genetic groups, and annual changes in productivity were important predictors of changes in growth rate in these populations. Drought condition on the wintering grounds was a strong predictor of adult survival for coastal California and Sierra Nevada populations; however, adult survival played a relatively minor role in explaining annual variation in population change. A latent parameter representing a mixture of first‐year survival and immigration was the largest contributor to variation in population change; however, this parameter was estimated imprecisely, and its importance likely reflects, in part, differences in spatio‐temporal distribution of samples between count and capture‐recapture data sets. Our modeling approach represents a novel and flexible framework for linking broad‐scale multi‐site monitoring data sets. Our results highlight both the potential of the approach for extension to additional species and systems, as well as needs for additional data and/or model development.     

Considerations for monitoring population trends of colonial waterbirds using the effective number of breeders and census estimates

Detecting trends in population size fluctuations is a major focus in ecology, evolution, and conservation biology. Populations of colonial waterbirds have been monitored using demographic approaches to determine annual census size (N_a). We propose the addition of genetic estimates of the effective number of breeders (N_b) as indirect measures of the risk of loss of genetic diversity to improve the evaluation of demographics and increase the accuracy of trend estimates in breeding colonies. Here, we investigated which methods of the estimation of N_b are more precise under conditions of moderate genetic diversity, limited sample sizes and few microsatellite loci, as often occurs with natural populations. We used the wood stork as a model species and we offered a workflow that researchers can follow for monitoring bird breeding colonies. Our approach started with simulations using five estimators of N_b and the theoretical results were validated with empirical data collected from breeding colonies settled in the Brazilian Pantanal wetland. In parallel, we estimated census size using a corrected method based on counting active nests. Both in simulations and in natural populations, the approximate Bayesian computation (ABC) and sibship assignment (SA) methods yielded more precise estimates than the linkage disequilibrium, heterozygosity excess, and molecular coancestry methods. In particular, the ABC method performed best with few loci and small sample sizes, while the other estimators required larger sample sizes and at least 13 loci to not underestimate N_b. Moreover, according to our N_b/N_a estimates (values were often ≤0.1), the wood stork colonies evaluated could be facing the loss of genetic diversity. We demonstrate that the combination of genetic and census estimates is a useful approach for monitoring natural breeding bird populations. This methodology has been recommended for populations of rare species or with a known history of population decline to support conservation efforts.     

A single‐nucleotide polymorphism‐based approach for rapid and cost‐effective genetic wolf monitoring in Europe based on noninvasively collected samples

Noninvasive genetics based on microsatellite markers has become an indispensable tool for wildlife monitoring and conservation research over the past decades. However, microsatellites have several drawbacks, such as the lack of standardisation between laboratories and high error rates. Here, we propose an alternative single‐nucleotide polymorphism (SNP)‐based marker system for noninvasively collected samples, which promises to solve these problems. Using nanofluidic SNP genotyping technology (Fluidigm), we genotyped 158 wolf samples (tissue, scats, hairs, urine) for 192 SNP loci selected from the Affymetrix v2 Canine SNP Array. We carefully selected an optimised final set of 96 SNPs (and discarded the worse half), based on assay performance and reliability. We found rates of missing data in this SNP set of <10% and genotyping error of ~1%, which improves genotyping accuracy by nearly an order of magnitude when compared to published data for other marker types. Our approach provides a tool for rapid and cost‐effective genotyping of noninvasively collected wildlife samples. The ability to standardise genotype scoring combined with low error rates promises to constitute a major technological advancement and could establish SNPs as a standard marker for future wildlife monitoring.     

A combined inverse method and multivariate approach for exploring population trends of Florida manatees

Given the expense and time required to monitor marine mammal populations effectively, approaches that fully exploit the resulting data certainly are warranted. We employed a two‐step modeling approach to estimate key demographic parameters, including immigration, from aerial surveys of manatees (Trichechus manatus latirostris) in the Northwest management unit of Florida. Abundances of adults and calves were predicted by multivariate adaptive regression spline (MARS) models, after accounting for heterogeneous detection rates caused by variable environmental conditions. The resulting predictions were incorporated into a stage‐structured, deterministic model that used an inverse method to estimate parameters with and without immigration. The model without immigration estimated mean survival probabilities of 0.966, 0.923, and 0.794 for adults, subadults and calves, respectively, with a per capita reproductive rate of 0.135. These parameter estimates yielded an overall mean population growth rate of approximately 1.037, which is comparable to rates from mark‐recapture studies. When we added an immigration term that accounted for the greater slope in adult counts since 1999, as identified by the MARS model, the estimated per capita reproductive rate was 0.122, with survival probabilities for adults, subadults and calves of 0.926, 0.920, and 0.833, respectively. These rates were coupled with an estimated mean winter immigration rate corresponding to roughly 5.2% of the adult and subadult population. In this latter scenario, the number of manatees in the core population of the Northwest management unit was predicted to remain constant, with a population growth rate near one, and additional manatees counted during aerial surveys were deemed to be immigrants. While further studies could certainly expound on the potential effects of migrants on population indices, we present this first published immigration estimate for wintering manatees in northwest Florida.     

Simulations inform design of regional occupancy‐based monitoring for a sparsely distributed,territorial species

Sparsely distributed species attract conservation concern, but insufficient information on population trends challenges conservation and funding prioritization. Occupancy‐based monitoring is attractive for these species, but appropriate sampling design and inference depend on particulars of the study system. We employed spatially explicit simulations to identify minimum levels of sampling effort for a regional occupancy monitoring study design, using white‐headed woodpeckers (Picoides albolvartus), a sparsely distributed, territorial species threatened by habitat decline and degradation, as a case study. We compared the original design with commonly proposed alternatives with varying targets of inference (i.e., species range, space use, or abundance) and spatial extent of sampling. Sampling effort needed to achieve adequate power to observe a long‐term population trend (≥80% chance to observe a 2% yearly decline over 20 years) with the previously used study design consisted of annually monitoring ≥120 transects using a single‐survey approach or ≥90 transects surveyed twice per year using a repeat‐survey approach. Designs that shifted inference toward finer‐resolution trends in abundance and extended the spatial extent of sampling by shortening transects, employing a single‐survey approach to monitoring, and incorporating a panel design (33% of units surveyed per year) improved power and reduced error in estimating abundance trends. In contrast, efforts to monitor coarse‐scale trends in species range or space use with repeat surveys provided extremely limited statistical power. Synthesis and applications. Sampling resolutions that approximate home range size, spatially extensive sampling, and designs that target inference of abundance trends rather than range dynamics are probably best suited and most feasible for broad‐scale occupancy‐based monitoring of sparsely distributed territorial animal species.     

A new method for studying population genetics of cyst nematodes based on Pool‐Seq and genomewide allele frequency analysis

Cyst nematodes are important agricultural pests responsible for billions of dollars of losses each year. Plant resistance is the most effective management tool, but it requires a close monitoring of population genetics. Current technologies for pathotyping and genotyping cyst nematodes are time‐consuming, expensive and imprecise. In this study, we capitalized on the reproduction mode of cyst nematodes to develop a simple population genetic analysis pipeline based on genotyping‐by‐sequencing and Pool‐Seq. This method yielded thousands of SNPs and allowed us to study the relationships between populations of different origins or pathotypes. Validation of the method on well‐characterized populations also demonstrated that it was a powerful and accurate tool for population genetics. The genomewide allele frequencies of 23 populations of golden nematode, from nine countries and representing the five known pathotypes, were compared. A clear separation of the pathotypes and fine genetic relationships between and among global populations were obtained using this method. In addition to being powerful, this tool has proven to be very time‐ and cost‐efficient and could be applied to other cyst nematode species.     

Hybridization selects for prime‐numbered life cycles in Magicicada: An individual‐based simulation model of a structured periodical cicada population

We investigate competition between separate periodical cicada populations each possessing different life‐cycle lengths. We build an individual‐based model to simulate the cicada life cycle and allow random migrations to occur between patches inhabited by the different populations. We show that if hybridization between different cycle lengths produces offspring that have an intermediate life‐cycle length, then predation acts disproportionately to select against the hybrid offspring. This happens because they emerge in low densities without the safety‐in‐numbers provided by either parent population. Thus, prime‐numbered life cycles that can better avoid hybridization are favored. However, we find that this advantage of prime‐numbered cycles occurs only if there is some mechanism that can occasionally synchronize emergence between local populations in sufficiently many patches.     

Phytotyping4D: a light‐field imaging system for non‐invasive and accurate monitoring of spatio‐temporal plant growth

Integrative studies of plant growth require spatially and temporally resolved information from high‐throughput imaging systems. However, analysis and interpretation of conventional two‐dimensional images is complicated by the three‐dimensional nature of shoot architecture and by changes in leaf position over time, termed hyponasty. To solve this problem, Phytotyping^4D uses a light‐field camera that simultaneously provides a focus image and a depth image, which contains distance information about the object surface. Our automated pipeline segments the focus images, integrates depth information to reconstruct the three‐dimensional architecture, and analyses time series to provide information about the relative expansion rate, the timing of leaf appearance, hyponastic movement, and shape for individual leaves and the whole rosette. Phytotyping^4D was calibrated and validated using discs of known sizes, and plants tilted at various orientations. Information from this analysis was integrated into the pipeline to allow error assessment during routine operation. To illustrate the utility of Phytotyping^4D, we compare diurnal changes in Arabidopsis thaliana wild‐type Col‐0 and the starchless pgm mutant. Compared to Col‐0, pgm showed very low relative expansion rate in the second half of the night, a transiently increased relative expansion rate at the onset of light period, and smaller hyponastic movement including delayed movement after dusk, both at the level of the rosette and individual leaves. Our study introduces light‐field camera systems as a tool to accurately measure morphological and growth‐related features in plants.     

Survival of Greater Sage‐Grouse broods: survey method affects disturbance and age‐specific detection probability

Investigators rely on brood surveys to estimate annual fecundity of game birds. However, investigators often do not account for factors that influence brood detection probability nor rarely document how much females and their broods are disturbed (flush rates) during surveys, which could lead to biased survival estimates. We used 45 radio‐tagged female Greater Sage‐Grouse (Centrocercus urophasianus) with broods to compare detection probabilities and document disturbance among four survey methods to allow future investigators to select the method that best meets their objectives. These methods included daytime flush, daytime visual, nocturnal spotlight, and fecal surveys at nocturnal roost sites, with the latter being a novel method. We used Cormack–Jolly–Seber (CJS) models to compare detection probability and daily survival estimates for visual and fecal surveys of broods 0–47 d post‐hatch and a double‐survey approach to compare detection probabilities among flush, fecal, and spotlight surveys ~42 d post‐hatch when investigators often determine brood fate. From CJS models, detection probability for visual surveys increased with brood age (0.618–0.881), whereas detection probability for fecal surveys did not (0.748). Daily survival probability estimates increased with brood age and differed annually based on fecal surveys (2016: 0.978–1.000 and 2017: 0.839–0.998). We detected age‐specific daily survival probability with visual surveys (0.956–0.997), but not annual differences. Based on the double‐survey approach, detection probability was high (0.857–1.000) for all methods. We flushed ~310–750% fewer females and broods during fecal and spotlight surveys than during both types of daytime surveys. Our results highlight the need to account for detection probabilities among methods and document disturbance to hens and broods that can help investigators design surveys to minimize impacts to birds. Furthermore, our result suggest that actions to improve brood survival during the first week post‐hatch may improve local recruitment.     

An improved extraction method enables the comprehensive analysis of lipids,proteins, metabolites and phytohormones from a single sample of leaf tissue under water‐deficit stress

Phytohormones play essential roles in the regulation of growth and development in plants. Plant hormone profiling is therefore essential to understand developmental processes and the adaptation of plants to biotic and/or abiotic stresses. Interestingly, commonly used hormone extraction and profiling methods do not adequately resolve other molecular entities, such as polar metabolites, lipids, starch and proteins, which would be required to comprehensively describe the continuing biological processes at a systematic level. In this article we introduce an updated version of a previously published liquid:liquid metabolite extraction protocol, which not only allows for the profiling of primary and secondary metabolites, lipids, starch and proteins, but also enables the quantitative analysis of the major plant hormone classes, including abscisic acid, auxins, cytokinins, jasmonates and salicylates, from a single sample aliquot. The optimization of the method, which uses the introduction of acidified water, enabling the complete purification of major plant hormones into the organic (methyl‐tert‐butyl‐ether) phase, eliminated the need for solid‐phase extraction for sample clean‐up, and therefore reduces both sampling time and cost. As a proof‐of‐concept analysis, Arabidopsis thaliana plants were subjected to water‐deficit stress, which were then profiled for hormonal, metabolic, lipidomic and proteomic changes. Surprisingly, we determined not only previously described molecular changes but also significant changes regarding the breakdown of specific galactolipids, followed by the substantial accumulation of unsaturated fatty‐acid derivatives and diverse jasmonates in the course of adaptation to water‐deficit stress.     

salmonnb: a program for computing cohort‐specific effective population sizes (Nb) in Pacific salmon and other semelparous species using the temporal method

We describe a new method and a computer program salmonn b to calculate the effective number of breeders (N_b) per year in semelparous species with variable age at maturity. The existing temporal method for the ‘salmon’ life history produces an estimate of the harmonic mean N_b in the two sampled years. salmonn b reads genotypic data in standard formats and computes yearly N_b values by combining information from pairwise comparisons of samples taken in 3 or more years. Simulations show that the new method produces unbiased estimates of yearly N_b, and precision is inversely proportional to true effective size.     

Consequences of multiple mating‐system shifts for population and range‐wide genetic structure in a coastal dune plant

Evolutionary transitions from outcrossing to selfing can strongly affect the genetic diversity and structure of species at multiple spatial scales. We investigated the genetic consequences of mating‐system shifts in the North American, Pacific coast dune endemic plant Camissoniopsis cheiranthifolia (Onagraceae) by assaying variation at 13 nuclear (n) and six chloroplast (cp) microsatellite (SSR) loci for 38 populations across the species range. As predicted from the expected reduction in effective population size (N_e) caused by selfing, small‐flowered, predominantly selfing (SF) populations had much lower nSSR diversity (but not cpSSR) than large‐flowered, predominantly outcrossing (LF) populations. The reduction in nSSR diversity was greater than expected from the effects of selfing on N_e alone, but could not be accounted for by indirect effects of selfing on population density. Although selfing should reduce gene flow, SF populations were not more genetically differentiated than LF populations. We detected five clusters of nSSR genotypes and three groups of cpSSR haplotypes across the species range consisting of parapatric groups of populations that usually (but not always) differed in mating system, suggesting that selfing may often initiate ecogeographic isolation. However, lineage‐wide genetic variation was not lower for selfing clusters, failing to support the hypothesis that selection for reproductive assurance spurred the evolution of selfing in this species. Within three populations where LF and SF plants coexist, we detected genetic differentiation among diverged floral phenotypes suggesting that reproductive isolation (probably postzygotic) may help maintain the striking mating‐system differentiation observed across the range of this species.