Improved methods for estimating abundance and related demographic parameters from mark‐resight data

Over the past decade, there has been much methodological development for the estimation of abundance and related demographic parameters using mark‐resight data. Often viewed as a less‐invasive and less‐expensive alternative to conventional mark recapture, mark‐resight methods jointly model marked individual encounters and counts of unmarked individuals, and recent extensions accommodate common challenges associated with imperfect detection. When these challenges include both individual detection heterogeneity and an unknown marked sample size, we demonstrate several deficiencies associated with the most widely used mark‐resight models currently implemented in the popular capture‐recapture freeware Program MARK. We propose a composite likelihood solution based on a zero‐inflated Poisson log‐normal model and find the performance of this new estimator to be superior in terms of bias and confidence interval coverage. Under Pollock's robust design, we also extend the models to accommodate individual‐level random effects across sampling occasions as a potentially more realistic alternative to models that assume independence. As a motivating example, we revisit a previous analysis of mark‐resight data for the New Zealand Robin (Petroica australis) and compare inferences from the proposed estimators. For the all‐too‐common situation where encounter rates are low, individual detection heterogeneity is non‐negligible, and the number of marked individuals is unknown, we recommend practitioners use the zero‐inflated Poisson log‐normal mark‐resight estimator as now implemented in Program MARK.     

Combining capture–recapture data and known ages allows estimation of age‐dependent survival rates

In many animal populations, demographic parameters such as survival and recruitment vary markedly with age, as do parameters related to sampling, such as capture probability. Failing to account for such variation can result in biased estimates of population‐level rates. However, estimating age‐dependent survival rates can be challenging because ages of individuals are rarely known unless tagging is done at birth. For many species, it is possible to infer age based on size. In capture–recapture studies of such species, it is possible to use a growth model to infer the age at first capture of individuals. We show how to build estimates of age‐dependent survival into a capture–mark–recapture model based on data obtained in a capture–recapture study. We first show how estimates of age based on length increments closely match those based on definitive aging methods. In simulated analyses, we show that both individual ages and age‐dependent survival rates estimated from simulated data closely match true values. With our approach, we are able to estimate the age‐specific apparent survival rates of Murray and trout cod in the Murray River, Australia. Our model structure provides a flexible framework within which to investigate various aspects of how survival varies with age and will have extensions within a wide range of ecological studies of animals where age can be estimated based on size.     

Simultaneous cloning of multiple nuclear genes by pooling PCR products of variable size: a cost‐effective method of improving efficiency in large‐scale genetic analyses

I present a simple approach to overcome the high cost and low efficiency of cloning polymerase chain reaction (PCR) products for individuals in wide‐scale population genetic analyses. The methodology reduces the number of cloning reactions per individual by engineering a suite of genetic markers that differ in size and pooling these PCR products prior to cloning. Alleles from each gene are then recovered by screening transformed bacterial colonies and identifying the inserts corresponding to each gene based on size. I demonstrate the utility of this technique by presenting the results I obtained from cloning four nuclear genes in 118 individuals from three species of sea urchins (Strongylocentrotus purpuratus, S. droebachiensis and S. pallidus). Of the 472 different PCR products I cloned, I recovered at least one allele for 432 of them (91.5%) by screening between 16 and 32 bacterial colonies for each individual. There existed a bias with respect to recovery efficiency: the two largest fragments (1130–800 bp) were recovered 100% of the time, while the two smaller fragments (580–650 bp) were recovered in 85.6% and 81.4% of the experiments, respectively. I discuss the promise of this application for wide‐scale genetic analyses.     

multimark: an R package for analysis of capture–recapture data consisting of multiple “noninvasive” marks

I describe an open‐source R package, multimark , for estimation of survival and abundance from capture–mark–recapture data consisting of multiple “noninvasive” marks. Noninvasive marks include natural pelt or skin patterns, scars, and genetic markers that enable individual identification in lieu of physical capture. multimark provides a means for combining and jointly analyzing encounter histories from multiple noninvasive sources that otherwise cannot be reliably matched (e.g., left‐ and right‐sided photographs of bilaterally asymmetrical individuals). The package is currently capable of fitting open population Cormack–Jolly–Seber (CJS) and closed population abundance models with up to two mark types using Bayesian Markov chain Monte Carlo (MCMC) methods. multimark can also be used for Bayesian analyses of conventional capture–recapture data consisting of a single‐mark type. Some package features include (1) general model specification using formulas already familiar to most R users, (2) ability to include temporal, behavioral, age, cohort, and individual heterogeneity effects in detection and survival probabilities, (3) improved MCMC algorithm that is computationally faster and more efficient than previously proposed methods, (4) Bayesian multimodel inference using reversible jump MCMC, and (5) data simulation capabilities for power analyses and assessing model performance. I demonstrate use of multimark using left‐ and right‐sided encounter histories for bobcats (Lynx rufus) collected from remote single‐camera stations in southern California. In this example, there is evidence of a behavioral effect (i.e., trap “happy” response) that is otherwise indiscernible using conventional single‐sided analyses. The package will be most useful to ecologists seeking stronger inferences by combining different sources of mark–recapture data that are difficult (or impossible) to reliably reconcile, particularly with the sparse datasets typical of rare or elusive species for which noninvasive sampling techniques are most commonly employed. Addressing deficiencies in currently available software, multimark also provides a user‐friendly interface for performing Bayesian multimodel inference using capture–recapture data consisting of a single conventional mark or multiple noninvasive marks.     

Genetic fingerprinting proves cross‐correlated automatic photo‐identification of individuals as highly efficient in large capture–mark–recapture studies

Capture–mark–recapture (CMR) approaches are the backbone of many studies in population ecology to gain insight on the life cycle, migration, habitat use, and demography of target species. The reliable and repeatable recognition of an individual throughout its lifetime is the basic requirement of a CMR study. Although invasive techniques are available to mark individuals permanently, noninvasive methods for individual recognition mainly rest on photographic identification of external body markings, which are unique at the individual level. The re‐identification of an individual based on comparing shape patterns of photographs by eye is commonly used. Automated processes for photographic re‐identification have been recently established, but their performance in large datasets (i.e., > 1000 individuals) has rarely been tested thoroughly. Here, we evaluated the performance of the program AMPHIDENT, an automatic algorithm to identify individuals on the basis of ventral spot patterns in the great crested newt (Triturus cristatus) versus the genotypic fingerprint of individuals based on highly polymorphic microsatellite loci using GENECAP. Between 2008 and 2010, we captured, sampled and photographed adult newts and calculated for 1648 samples/photographs recapture rates for both approaches. Recapture rates differed slightly with 8.34% for GENECAP and 9.83% for AMPHIDENT. With an estimated rate of 2% false rejections (FRR) and 0.00% false acceptances (FAR), AMPHIDENT proved to be a highly reliable algorithm for CMR studies of large datasets. We conclude that the application of automatic recognition software of individual photographs can be a rather powerful and reliable tool in noninvasive CMR studies for a large number of individuals. Because the cross‐correlation of standardized shape patterns is generally applicable to any pattern that provides enough information, this algorithm is capable of becoming a single application with broad use in CMR studies for many species.     

Lek fidelity and movement among leks by male Greater Sage‐grouse Centrocercus urophasianus: a capture‐mark‐recapture approach

Males in lek mating systems tend to exhibit high fidelity to breeding leks despite substantial evidence of skewed mating success among males. Although movements between leks are often reported to be rare, such movements provide a mechanism for an individual to improve lifetime fitness in response to heterogeneity in reproductive conditions. Additionally, estimates of apparent movements among leks are potentially biased due to unaccounted variation in detection probability across time and space. We monitored breeding male Greater Sage‐grouse Centrocercus urophasianus on 13 leks in eastern Nevada over a 10‐year period, and estimated movement rates among leks using capture‐mark‐recapture methods. We expected that male movement rates among leks would be low, despite predictions of low breeding success for most males, and that detection rates would be highly variable among leks and years. We used a robust design multistate analysis in Program mark to estimate probability of movements among leks, while accounting for imperfect detection of males. Male Sage‐grouse were extremely faithful to their leks; the annual probability of a male moving away from its original lek of capture was approximately 3% (se = 0.01). Detection probabilities varied substantially among leks (range = 0.21–0.95), and among years (range = 0.30–0.76), but remained relatively constant within years at each lek. These results suggest that male Sage‐grouse dispersal is either rare, or consists primarily of dispersal of sub‐adults from their natal areas prior to the breeding season. The study highlights the benefits of robust design multistate models over standard ‘live‐encounter’ analyses, as they not only permit estimation of additional parameters, such as movement rates, but also allow for more precise parameter estimates that are less sensitive to heterogeneity in detection rates. Additionally, as these data were collected using capture‐mark‐recapture methods, our approach to estimating movement rates would be beneficial in systems where radiotagging is detrimental to the study organism.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Effect of Tooth Removal on Recaptures of Raccoons

Abstract: Researchers have extensively used mark—recapture techniques to obtain information on demographic parameters of wildlife populations. However, researchers have recognized that a number of factors can influence capture probabilities of wildlife species, which in turn can bias mark—recapture estimates of demographic parameters. Tooth extraction, which is a commonly used technique in studies of mesopredator species to obtain precise age estimates and to monitor the use of vaccine baits, is an aspect of animal handling that clearly might affect the recapture probability of individuals. However, the effect that tooth removal has on the individual recapture probabilities of wildlife species is unknown. During 2005, we trapped and marked 91 raccoons (Procyon lotor) in northern Indiana, USA, as part of a mark—recapture study designed specifically to determine if tooth extractions have an effect on recapture probabilities of individuals. We performed tooth extractions on 50% of the raccoons at the time of capture, and we attempted to balance tooth extractions with respect to sex and age of raccoons. We used logistic regression to model the effects of sex, age, and tooth removal on recapture probabilities, and we used Mann—Whitney U-tests to examine the effect of tooth removal on the number of times we recaptured individuals. The probability of recapture differed between sexes but did not differ as a function of tooth removal or among age classes. In addition, we failed to detect any difference in the mean number of times that we recaptured raccoons between the tooth removed and non—tooth-removed groups. Our results suggest that managers can use tooth extractions as an effective management tool without biasing population estimates or compromising other management objectives.     

Computation vs. cloning: evaluation of two methods for haplotype determination

Nuclear sequence data, often from multiple loci, are increasingly being employed in analyses of population structure and history, yet there has been relatively little evaluation of methods for accurately and efficiently separating the alleles or haplotypes in heterozygous individuals. We compared the performance of a computational method of haplotype reconstruction and standard cloning methods using a highly variable intron (ornithine decarboxylase, intron 6) in three closely related species of dabbling ducks (genus Anas). Cloned sequences from 32 individuals were compared to results obtained from phase 2.1.1 . phase correctly identified haplotypes in 28 of 30 heterozygous individuals when the underlying model assumed no recombination. Haplotypes of the remaining two individuals were also inferred correctly except for unique polymorphisms, the phase of which was appropriately indicated as uncertain (phase probability = 0.5). For a larger set of 232 individuals, results were essentially identical regardless of the recombination model used and haplotypes for all 30 of the tested heterozygotes were correctly inferred, with the exception of uncertain phase for unique polymorphisms in one individual. In contrast, initial sequences of one clone per sample yielded accurate haplotype determination in only 26 of 30 individuals; polymerase chain reaction (PCR)/cloning errors resulting from misincorporation of individual nucleotides could be recognized and avoided by comparison to direct sequences, but errors due to PCR recombination resulted in incorrect haplotype reconstruction in four individuals. The accuracy of haplotypes reconstructed by phase , even when dealing with a relatively small number of samples and numerous variable sites, suggests broad utility of computational approaches for reducing the cost and improving the efficiency of data collection from nuclear sequence loci.     

Comparison of photo‐matching algorithms commonly used for photographic capture–recapture studies

Photographic capture–recapture is a valuable tool for obtaining demographic information on wildlife populations due to its noninvasive nature and cost‐effectiveness. Recently, several computer‐aided photo‐matching algorithms have been developed to more efficiently match images of unique individuals in databases with thousands of images. However, the identification accuracy of these algorithms can severely bias estimates of vital rates and population size. Therefore, it is important to understand the performance and limitations of state‐of‐the‐art photo‐matching algorithms prior to implementation in capture–recapture studies involving possibly thousands of images. Here, we compared the performance of four photo‐matching algorithms; Wild‐ID, I3S Pattern+, APHIS, and AmphIdent using multiple amphibian databases of varying image quality. We measured the performance of each algorithm and evaluated the performance in relation to database size and the number of matching images in the database. We found that algorithm performance differed greatly by algorithm and image database, with recognition rates ranging from 100% to 22.6% when limiting the review to the 10 highest ranking images. We found that recognition rate degraded marginally with increased database size and could be improved considerably with a higher number of matching images in the database. In our study, the pixel‐based algorithm of AmphIdent exhibited superior recognition rates compared to the other approaches. We recommend carefully evaluating algorithm performance prior to using it to match a complete database. By choosing a suitable matching algorithm, databases of sizes that are unfeasible to match “by eye” can be easily translated to accurate individual capture histories necessary for robust demographic estimates.     

Sequence capture across large phylogenetic scales by using pooled PCR‐generated baits: A case study of Lepidoptera

Sequence capture across large phylogenetic scales is not easy because hybridization capture is only effective when the genetic distance between the bait and target is small. Here, we propose a simple but effective strategy to tackle this issue: pooling DNA from a number of selected representative species of different clades to prepare PCR‐generated baits to minimize the genetic distance between the bait and target. To demonstrate the utility of this strategy, we newly developed a set of universal nuclear markers (including 94 nuclear protein‐coding genes) for Lepidoptera, a superdiverse insect group. We used a DNA pool from six lepidopteran species (representing six superfamilies) to prepare PCR baits for the 94 markers. These homemade PCR baits were used to capture sequence data from 43 species of 17 lepidopteran families, and 94% of the target loci were recovered. We constructed two data sets from the obtained data (one containing ~90 kb target coding sequences and the other containing ~120 kb target + flanking coding sequences). Both data sets yielded highly similar and well‐resolved trees with 90% of nodes having >95% bootstrap support. Our capture experiment indicated that using DNA mixtures pooled from different clade‐representative species of Lepidoptera to prepare PCR baits can reliably capture a large number of targeted nuclear markers across different Lepidoptera lineages. We hope that this newly developed nuclear marker set will serve as a new phylogenetic tool for Lepidoptera phylogenetics, and the PCR bait preparation strategy can facilitate the application of sequence capture techniques by researchers to accelerate data collection.     

Trap‐shyness subsidence is a threshold function of mark–recapture interval in brown mudfish Neochanna apoda populations

The influence of capture interval on trap shyness, and temperature, rainfall and drought on capture probability (p) in 827 brown mudfish Neochanna apoda was quantified using mark–recapture models. In particular, it was hypothesized that the loss of trapping memory in marked N. apoda would lead to a capture‐interval threshold required to minimize trap shyness. Neochanna apoda trap shyness approximated a threshold response to capture interval, declining rapidly with increasing capture intervals up to 16·5 days, after which p remained constant. Tests for detecting trap‐dependent capture probability in Cormack–Jolly–Seber models failed to detect trap shyness in N. apoda capture histories with capture intervals averaging 16 days. This confirmed the applicability of the 16 day capture‐interval threshold for mark–recapture studies. Instead, N. apoda p was positively influenced by water temperature and rainfall during capture. These results imply that a threshold capture interval is required to minimize the trade‐off between the competing assumptions of population closure and p homogeneity between capture occasions in closed mark–recapture models. Moreover, environmental factors that influence behaviour could potentially confound abundance indices, and consequently abundance trends should be interpreted with caution in the face of long‐term climate change, such as with global warming.     

Synchrony in capture dates suggests cryptic social organization in sea snakes (Emydocephalus annulatus,Hydrophiidae)

Abstract Complex sociality is widespread in lizards, but the difficulties of directly observing social interactions in free‐ranging snakes have precluded such studies for most snake species. However, a type of data already available from mark‐recapture studies (dates of capture and recapture of individually marked animals) can reveal social substructure within snake populations. If individuals associate with each other in social groups, we expect synchrony in the dates of capture and recapture of those animals. A field study of turtle‐headed sea snakes (Emydocephalus annulatus) in New Caledonia reveals exactly this phenomenon. For example, animals that were captured on the same day in one year often were recaptured on the same day the following year. Analysis rejects non‐social interpretations of these data (such as spatial‐temporal confounding in sampling, intrapopulation heterogeneity in cues for activity), suggesting instead that many individual sea snakes belong to ‘social’ groups that consistently move about together. The phenomenon of capture synchrony during mark‐recapture studies can provide new insights into the occurrence and correlates of cryptic social aggregations.     

The terminal 5′ phosphate and proximate phosphorothioate promote ligation‐independent cloning

Function studies of many proteins are waited to develop after genome sequencing. High‐throughout technology of gene cloning will strongly promote proteins' function studies. Here we describe a ligation‐independent cloning (LIC) method, which is based on the amplification of target gene and linear vector by PCR using phosphorothioate‐modified primers and the digestion of PCR products by λ exonuclease. The phosphorothioate inhibits the digestion and results in the generation of 3′ overhangs, which are designed to form complementary double‐stranded DNA between target gene and linear vector. We compared our phosphorothioate primer cloning methods with several LIC methods, including dU primer cloning, hybridization cloning, T4 DNA polymerase cloning, and in vivo recombination cloning. The cloning efficiency of these LIC methods are as follows: phosphorothioate primer cloning > dU primer cloning > hybridization cloning > T4 DNA polymerase cloning >> in vivo recombination cloning. Our result shows that the 3′ overhangs is a better cohesive end for LIC than 5′ overhang and the existence of 5′phosphate promotes DNA repair in Escherichia coli, resulting in the improvement of cloning efficiency of LIC. We succeeded in constructing 156 expression plasmids of Aeropyrum pernix genes within a week using our method.     

Laying date,body mass and tick infestation of nestling tropical Roseate Terns Sterna dougallii predict fledging success,first‐year survival and age at first return to the natal colony

Both intrinsic and extrinsic factors recorded at individual nests can predict offspring fitness and survival but few studies have examined these effects in the tropics. We recorded nestling survival, post‐fledging survival and age at first return of Roseate Terns breeding at Aride Island, Seychelles, over a 12‐year period (1998–2009). Nest data recorded at the egg, nestling and fledging stages were collected during six breeding seasons (1998, 2001–2005) and a capture‐mark‐recapture dataset of six cohorts of fledglings was obtained from 2001–2009. Logistic regression models were used to assess the predictive effect of reproductive variables on fledging success, while multistate capture‐mark‐recapture models were used to estimate post‐fledging survival and return–recruitment probabilities to the natal site. Nestling survival probability increased with earliness of laying and was negatively affected by tick infestation during the growth period (0–23 days). Fledging probability was also positively related to chick body condition, whereas other pre‐fledging reproductive parameters such as clutch size and egg size were not influential. A multistate modelling of age‐specific survival and return–recruitment (transition) rates found that first‐year survival differed between cohorts and was also negatively affected by tick infestation. Annual survival stabilized from age 2 onwards at 0.83 ± 0.02. Transition rates were positively related to body condition at fledging, with heavier individuals returning for the first time to the natal colony at a younger age compared with lighter individuals. These results highlight the importance of local conditions encountered by tropical seabirds during the breeding season in shaping demographic parameters.     

Continuous Covariates in Mark‐Recapture‐Recovery Analysis: A Comparison of Methods

Summary Time varying, individual covariates are problematic in experiments with marked animals because the covariate can typically only be observed when each animal is captured. We examine three methods to incorporate time varying, individual covariates of the survival probabilities into the analysis of data from mark‐recapture‐recovery experiments: deterministic imputation, a Bayesian imputation approach based on modeling the joint distribution of the covariate and the capture history, and a conditional approach considering only the events for which the associated covariate data are completely observed (the trinomial model). After describing the three methods, we compare results from their application to the analysis of the effect of body mass on the survival of Soay sheep (Ovis aries) on the Isle of Hirta, Scotland. Simulations based on these results are then used to make further comparisons. We conclude that both the trinomial model and Bayesian imputation method perform best in different situations. If the capture and recovery probabilities are all high, then the trinomial model produces precise, unbiased estimators that do not depend on any assumptions regarding the distribution of the covariate. In contrast, the Bayesian imputation method performs substantially better when capture and recovery probabilities are low, provided that the specified model of the covariate is a good approximation to the true data‐generating mechanism.     

Estimation of capture probabilities using generalized estimating equations and mixed effects approaches

Modeling individual heterogeneity in capture probabilities has been one of the most challenging tasks in capture–recapture studies. Heterogeneity in capture probabilities can be modeled as a function of individual covariates, but correlation structure among capture occasions should be taking into account. A proposed generalized estimating equations (GEE) and generalized linear mixed modeling (GLMM) approaches can be used to estimate capture probabilities and population size for capture–recapture closed population models. An example is used for an illustrative application and for comparison with currently used methodology. A simulation study is also conducted to show the performance of the estimation procedures. Our simulation results show that the proposed quasi‐likelihood based on GEE approach provides lower SE than partial likelihood based on either generalized linear models (GLM) or GLMM approaches for estimating population size in a closed capture–recapture experiment. Estimator performance is good if a large proportion of individuals are captured. For cases where only a small proportion of individuals are captured, the estimates become unstable, but the GEE approach outperforms the other methods.     

Mark‐recapture abundance estimate of tucuxi dolphins (Sotalia fluviatilis) in a lake system of the Central Amazon

The tucuxi (Sotalia fluviatilis) is a small dolphin endemic to the Amazon River basin. Because the abundance and trends are currently unknown for the species, this study aimed to estimate its abundance in a lake system of the Central Amazon. A total of 10 two‐day sampling periods were carried out from March to June of 2013 throughout a 13.5 km² area in the Mamirauá Reserve. In the 104 encounters with the species, a minimum number of 389 dolphins were sighted and photographed, which allowed the positive identification of 49 individuals. Mark‐recapture models were used to estimate an abundance of 119 individuals (95% CI = 105–150) (corrected for the proportion of identifiable individuals). This is the first estimation of S. fluviatilis abundance using mark‐recapture analyses and, together with the photo‐id catalog made available, provides a useful reference for future studies regarding tucuxi dolphins.     

Habitat‐driven life history variation in an amphibian metapopulation

Life‐history theory states that, during the lifetime of an individual, resources are allocated to either somatic maintenance or reproduction. Resource allocation tradeoffs determine the evolution and ecology of life‐history strategies and determine an organisms’ position along the fast–slow continuum. Theory predicts that environmental stochasticity is an important driver of resource allocation and therefore life‐history evolution. Highly stochastic environments are expected to increase uncertainty in reproductive success and select for iteroparity and a slowing down of the life history. To date, most empirical studies have used comparisons among species to examine these theoretical predictions. By contrast, few have investigated how environmental stochasticity affects life‐history strategies at the intraspecific level. In this study, we examined how variation in breeding site stochasticity (among‐year variability in pond volume and hydroperiod) promotes the co‐occurrence of different life‐history strategies in a spatially structured population, and determines life‐history position along the fast–slow continuum in the yellow‐bellied toad Bombina variegata. We collected mark–recapture data from a metapopulation and used multievent capture–recapture models to estimate survival, recruitment and breeding probabilities. We found higher survival and longer lifespans in populations inhabiting variable sites compared to those breeding in stable ones. In addition, probabilities of recruitment and skipping a breeding event were higher in variable sites. The temporal variance of survival and recruitment probabilities, as well as the probability to skip breeding, was higher in variable sites. Taken together, these findings indicate that populations breeding in variable sites experienced a slowing down of the life‐history. Our study thus revealed similarities in the macroevolutionary and microevolutionary processes shaping life‐history evolution.     

Uracil DNA glycosylase-mediated cloning of polymerase chain reaction-amplified DNA: application to genomic and cDNA cloning.

A simple and rapid method for cloning of amplification products directly from the polymerase chain reaction (PCR) has been developed. The method is based on the addition of a 12-base dUMP-containing sequence (CUACUACUACUA) to the 5' end of PCR primers. Incorporation of these primers during PCR results in the selective placement of dUMP residues into the 5' end of amplification products. Selective degradation of the dUMP residues in the PCR products with uracil DNA glycosylase (UDG) disrupts base pairing at the termini and generates 3' overhangs. Annealing of 3' protruding termini to vector DNA containing complementary 3' ends results in chimeric molecules which can be transformed, with high efficiency, without in vitro ligation. Directional cloning of PCR products has also been accomplished by incorporating different dU-containing sequences at the end of each PCR primer. Substitution of all dT residues in PCR primers with dU eliminates cloning of aberrant "primer dimer" products and enriches cloning of genuine PCR products. The method has been applied to cloning of inter-Alu DNA sequences from human placental DNA. Using a single primer, DNA sequences between appropriately oriented Alu sequences were amplified and cloned. Cloning of cDNA for the glyceraldehyde-3'-phosphate dehydrogenase gene from rat brain RNA was also demonstrated. The 3' end region of this gene was amplified by the 3' RACE method and the amplified DNA was cloned after UDG digestion. Characterization of cloned DNAs by sequence analysis showed accurate repair of the cloning junctions. The ligase-free cloning method with UDG should prove to be a widely applicable procedure for rapid cloning of PCR-amplified DNA.