A newt does not change its spots: using pattern mapping for the identification of individuals in large populations of newt species

The correct identification of individuals is a requirement of capture-mark-recapture (CMR) methods, and it is commonly achieved by applying artificial marks or by mutilation of study-animals. An alternative, non-invasive method to identify individuals is to utilize the patterns of their natural body markings. However, the use of pattern mapping is not yet widespread, mainly because it is considered time consuming, particularly in large populations and/or long-term CMR studies. Here we explore the use of pattern mapping for the identification of adult individuals in the alpine (Ichthyosaura alpestris) and smooth (Lissotriton vulgaris) newts (Amphibia, Salamandridae), using the freely available, open-source software Wild-ID. Our photographic datasets comprised nearly 4000 captured animals’ images, taken during a 3-year period. The spot patterns of individual newts of both species did not change through time, and were sufficiently varied to allow their individual identification, even in the larger datasets. The pattern-recognition algorithm of Wild-ID was highly successful in identifying individual newts in both species. Our findings indicate that pattern mapping can be successfully employed for the identification of individuals in large populations of a broad range of animals that exhibit natural markings. The significance of pattern-mapping is accentuated in CMR studies that aim in obtaining long-term information on the demography and population dynamics of species of conservation interest, such as many amphibians facing population declines.     

Validating the use of colouration patterns for individual recognition in the worm pipefish using a novel set of microsatellite markers

In studies of behaviour, ecology and evolution, identification of individual organisms can be an invaluable tool, capable of unravelling otherwise cryptic information regarding group structure, movement patterns, population size and mating strategies. The use of natural markings is arguably the least invasive method for identification. However, to be truly useful natural markings must be sufficiently variable to allow for unique identification, while being stable enough to permit long‐term studies. Non‐invasive marking techniques are especially important in fishes of the Family Syngnathidae (pipefishes, seahorses and seadragons), as many of these taxa are of conservation concern or used extensively in studies of sexual selection. Here, we assessed the reliability of natural markings as a character for individual identification in a wild population of Nerophis lumbriciformis by comparing results from natural markings to individual genetic assignments based on eight novel microsatellite loci. We also established a minimally invasive method based on epithelial cell swabbing to sample DNA. All pipefish used in the validation of natural markings, independently of sex or time between recaptures, were individually recognized through facial colouration patterns. Their identities were verified by the observation of the same multilocus genotype at every sampling event for each individual that was identified on the basis of natural markings. Successful recaptures of previously swabbed pipefish indicated that this process probably did not induce an elevated rate of mortality. Also, the recapture of newly pregnant males showed that swabbing did not affect reproductive behaviour.     

Computer-Assisted Photo Identification Outperforms Visible Implant Elastomers in an Endangered Salamander,Eurycea tonkawae

Despite recognition that nearly one-third of the 6300 amphibian species are threatened with extinction, our understanding of the general ecology and population status of many amphibians is relatively poor. A widely-used method for monitoring amphibians involves injecting captured individuals with unique combinations of colored visible implant elastomer (VIE). We compared VIE identification to a less-invasive method – computer-assisted photographic identification (photoID) – in endangered Jollyville Plateau salamanders (Eurycea tonkawae), a species with a known range limited to eight stream drainages in central Texas. We based photoID on the unique pigmentation patterns on the dorsal head region of 1215 individual salamanders using identification software Wild-ID. We compared the performance of photoID methods to VIEs using both ‘high-quality’ and ‘low-quality’ images, which were taken using two different camera types and technologies. For high-quality images, the photoID method had a false rejection rate of 0.76% compared to 1.90% for VIEs. Using a comparable dataset of lower-quality images, the false rejection rate was much higher (15.9%). Photo matching scores were negatively correlated with time between captures, suggesting that evolving natural marks could increase misidentification rates in longer term capture-recapture studies. Our study demonstrates the utility of large-scale capture-recapture using photo identification methods for Eurycea and other species with stable natural marks that can be reliably photographed.     

Genotyping validates photo‐identification by the head scale pattern in a large population of the European adder (Vipera berus)

Capture‐mark‐recapture procedures are a basic tool in population studies and require that individual animals are correctly identified throughout their lifetime. A method that has become more and more popular uses photographic records of natural markings, such as pigmentation pattern and scalation configuration. As with any other marking tool, the validity of the photographic identification technique should be evaluated thoroughly. Here, we report on a large‐scale double‐marking study in which European adders (Vipera berus) were identified by both microsatellite genetic markers and by the pattern of head scalation. Samples that were successfully genotyped for all nine loci yielded 624 unique genotypes, which matched on a one‐to‐one basis with the individual assignments based on the head scalation pattern. Thus, adders considered as different individuals by their genotypes were also identified as different individuals by their head scalation pattern, and vice versa. Overall, variation in the numbers, shape, and arrangement of the head scales enabled us to distinguish among 3200+ photographed individual snakes. Adders that were repeatedly sequenced genetically over intervals of 2–3 years showed no indication whatsoever for a change in the head scale pattern. Photographic records of 900+ adders that were recaptured over periods of up to 12 years showed a very detailed and precise match of the head scale characteristics. These natural marks are thus robust over time and do not change during an individual's lifetime. With very low frequency (0.3%), we detected small changes in scalation that were readily discernible and could be attributed to physical injury or infection. Our study provides a conclusive validation for the use of photo‐identification by head scale patterns in the European adder.     

Use of unique brook trout spot patterns over a short duration for a mark-recapture study

Environmental Biology of Fishes - Identifying individuals by their natural body markings is an excellent surrogate for artificial tags. Photo identification has been primarily tested in oceanic...     

Nicks and notches of the dorsal ridge: Promising mark types for the photo-identification of narwhals

The narwhal is a hunted species for which we have many knowledge gaps. Photo-identification, which uses photographs of natural markings to identify individuals, is widely used in cetacean studies and can address a broad range of biological questions. However, it has not been developed for narwhals. The marks used for other cetaceans are inappropriate for this species either because narwhals lack the body part on which these marks are found or because the marks are known to change with time. We investigated the marks apparent in photographs of narwhals. Nicks and notches on the dorsal ridge are the mark types most promising for photo-identification. They are found on 91%–98% of the individuals, thus allowing the identification of a large part of the population. They can be used to differentiate between individuals, in part because they are variable in their location, numbers, shape, and size. Although our results suggest that nicks and notches are relatively stable over time, rates of change should be formally measured to assess the probability of photographic matches over multiple years. However, we are confident that these marks can be used in studies spanning at least a field season.     

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.     

Generalized spatial mark–resight models with incomplete identification: An application to red fox density estimates

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Identification of individual adult female Javan lutungs (Trachypithecus auratus sondaicus) by using patterns of dark pigmentation in the pubic area

In a series of field surveys of wild Javan lutungs (Trachypithecus auratus sondaicus) conducted at Pangandaran Nature Reserve in West Java, Indonesia, from 2011 to 2012, we tried to use a method of individual identification by using individual-specific patterns of dark pigmentation in the pubic area. During the 2011 dry season, we used a digital SLR camera with a 400-mm telephoto lens to photograph the pubic area of each individual of a habituated group. These photographs were the basis for identifying 14 different adult females. During the rainy season of 2011 and the dry season of 2012, we checked the presence/absence of each of the identified individuals and found that these patterns were stable, at least during our study period. We found that two adult females and one adult female disappeared from the subject group between the first and second and between the second and third surveys, respectively, and that one adult female gave birth between the first and second surveys, but the infant had disappeared from the group between the second and third surveys. We could not confirm the validity of the method for juvenile females because of the dense white hair in their pubic areas and the fact that few individuals had clear patterns. Furthermore, we could not use this method for males because of the lack of pigmentation in the pubic area. As patterns of pigmentation in the pubic area are known to be present in other Trachypithecus species, our method can be useful for identification of individual adult females of these species, on which few individual-based behavioral studies have been conducted. Collecting individual-based behavioral data would enable us to track the presence of individuals in groups or movements between groups; determine the effects of social rank and age on within-group competition and copulation; and examine population data.     

BEEtag: A Low-Cost,Image-Based Tracking System for the Study of Animal Behavior and Locomotion

A fundamental challenge common to studies of animal movement, behavior, and ecology is the collection of high-quality datasets on spatial positions of animals as they change through space and time. Recent innovations in tracking technology have allowed researchers to collect large and highly accurate datasets on animal spatiotemporal position while vastly decreasing the time and cost of collecting such data. One technique that is of particular relevance to the study of behavioral ecology involves tracking visual tags that can be uniquely identified in separate images or movie frames. These tags can be located within images that are visually complex, making them particularly well suited for longitudinal studies of animal behavior and movement in naturalistic environments. While several software packages have been developed that use computer vision to identify visual tags, these software packages are either (a) not optimized for identification of single tags, which is generally of the most interest for biologists, or (b) suffer from licensing issues, and therefore their use in the study of animal behavior has been limited. Here, we present BEEtag, an open-source, image-based tracking system in Matlab that allows for unique identification of individual animals or anatomical markers. The primary advantages of this system are that it (a) independently identifies animals or marked points in each frame of a video, limiting error propagation, (b) performs well in images with complex backgrounds, and (c) is low-cost. To validate the use of this tracking system in animal behavior, we mark and track individual bumblebees (Bombus impatiens) and recover individual patterns of space use and activity within the nest. Finally, we discuss the advantages and limitations of this software package and its application to the study of animal movement, behavior, and ecology.     

Phylogenetic characterization and in situ detection of bacterial communities associated with seahorses (Hippocampus guttulatus) in captivity

Although there are several studies describing bacteria associated with marine fish, the bacterial composition associated with seahorses has not been extensively investigated since these studies have been restricted to the identification of bacterial pathogens. In this study, the phylogenetic affiliation of seahorse-associated bacteria was assessed by 16S rRNA gene sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rRNA analysis. Both methods revealed that Vibrionaceae was the dominant population in Artemia sp. (live prey) and intestinal content of the seahorses, while Rhodobacteraceae was dominant in water samples from the aquaculture system and cutaneous mucus of the seahorses. To our knowledge, this is the first time that bacterial communities associated with healthy seahorses in captivity have been described.     

A quantitative measure of migratory connectivity

The study of the extent of the connection between areas where populations of birds breed and areas where they winter has flourished in recent years mainly thanks to the development of new techniques, but also due to traditional ringing and recovery schemes, which allow tracking of individuals or populations linking wintering and breeding distributions. Currently, studies on migratory connectivity focus on retention of breeding population spatial structure on the non-breeding grounds and vice versa.Here we propose a method to quantify migratory connectivity based on Mantel correlation coefficients and to statistically test for deviations of the observed connectivity from a random mix of individuals. In addition, we propose a procedure, based on clustering algorithms, to identify whether observed connectivity depends on aggregation of individuals or on rigid transference of distribution patterns between areas.We applied this method to a large dataset of ringing recoveries of barn swallows (Hirundo rustica L) migrating from their Western Palearctic breeding areas to sub-Saharan winter quarters. We show that migration of barn swallow populations connects specific breeding and wintering areas, and that the “sub-populations” quantitatively identified by our method are consistent with qualitative patterns of migratory connectivity identified by studies of individual geographical populations based on other methods. Finally, we tested the performance of the method by running simulations under different scenarios. Such simulations showed that the method is robust and able to correctly detect migratory connectivity even with smaller datasets and when a strong geographical pattern is not present in the population. Our method provides a quantitative measure of migratory connectivity and allows for the identification of populations showing high connectivity between the breeding and wintering areas. This method is suitable for a generalized application to diverse animal taxa as well as to large scale analyses of connectivity for conservation purposes.     

Acoustic Identification of Individuals within Large Avian Populations: A Case Study of the Brownish-Flanked Bush Warbler, South-Central China

Acoustic identification is increasingly being used as a non-invasive method for identifying individuals within avian populations. However, most previous studies have utilized small samples of individuals (<30). The feasibility of using acoustic identification of individuals in larger avian populations has never been seriously tested. In this paper, we assess the feasibility of using distinct acoustic signals to identify individuals in a large avian population (139 colour-banded individuals) of Brownish-flanked Bush Warbler (Cettia fortipes) in the Dongzhai National Nature Reserve, south-central China. Most spectro-temporal variables we measured show greater variation among individuals than within individual. Although there was slight decline in the correct rate of individual identification with increasing sample sizes, the total mean correct rate yielded by discriminant function analysis was satisfactory, with more than 98% of songs correctly recognized to the corresponding individuals. We also found that using a part of randomly selected measured variables was sufficient to obtain a high correct rate of individual identification. We believe that our work will increase confidence in the use of using acoustic recognition techniques for avian population monitoring programs.     

Where have all the tadpoles gone? Individual genetic tracking of amphibian larvae until adulthood

Reliably marking larvae and reidentifying them after metamorphosis is a challenge that has hampered studies on recruitment, dispersal, migration and survivorship of amphibians for a long time, as conventional tags are not reliably retained through metamorphosis. Molecular methods allow unique genetic fingerprints to be established for individuals. Although microsatellite markers have successfully been applied in mark–recapture studies on several animal species, they have never been previously used in amphibians to follow individuals across different life cycle stages. Here, we evaluate microsatellites for genetic across‐stages mark–recapture studies in amphibians and test the suitability of available software packages for genotype matching. We sampled tadpoles of the dendrobatid frog Allobates femoralis, which we introduced on a river island in the Nature Reserve ‘Les Nouragues’ in French Guiana. In two subsequent recapture sessions, we searched for surviving juveniles and adults, respectively. All individuals were genotyped at 14 highly variable microsatellite loci, which yielded unique genetic fingerprints for all individuals. We found large differences in the identification success of the programs tested. The pairwise‐relatedness‐based approach, conducted with the programs kingroup or ML‐Relate, performed best with our data set. Matching ventral patterns of juveniles and adult individuals acted as a control for the reliability of the genetic identification. Our results demonstrate that microsatellite markers are a highly powerful tool for studying amphibian populations on an individual basis. The ability to individually track amphibian tadpoles throughout metamorphosis until adulthood will be of substantial value for future studies on amphibian population ecology and evolution.     

An automated program to find animals and crop photographs for individual recognition

Detailed data on individual animals are critical to ecological and evolutionary studies, but attaching identifying marks can alter individual fates and behavior leading to biases in parameter estimates and ethical issues. Individual-recognition software has been developed to assist in identifying many species from non-invasive photographic data. These programs utilize algorithms to find unique individual characteristics and compare images to a catalogue of known individuals. Currently, all applications for individual identification require manual processing to crop images so only the area of interest remains, or the area of interest must be manually delineated in each image. Thus, one of the main bottlenecks in processing data from photographic capture-recapture surveys is in cropping to an area of interest so that matching algorithms can identify the individual. Here, we describe the development and testing of an automated cropping program. The methods and techniques we describe are broadly applicable to any system where raw photos must be cropped down to a specific area of interest before pattern recognition software can be used for individual identification. We developed and tested the program for use with identification photos of wild giraffes.     

Genotyping validates the efficacy of photographic identification in a capture‐mark‐recapture study based on the head scale patterns of the prairie lizard (Sceloporus consobrinus)

Population studies often incorporate capture‐mark‐recapture (CMR) techniques to gather information on long‐term biological and demographic characteristics. A fundamental requirement for CMR studies is that an individual must be uniquely and permanently marked to ensure reliable reidentification throughout its lifespan. Photographic identification involving automated photographic identification software has become a popular and efficient noninvasive method for identifying individuals based on natural markings. However, few studies have (a) robustly assessed the performance of automated programs by using a double‐marking system or (b) determined their efficacy for long‐term studies by incorporating multi‐year data. Here, we evaluated the performance of the program Interactive Individual Identification System (I³S) by cross‐validating photographic identifications based on the head scale pattern of the prairie lizard (Sceloporus consobrinus) with individual microsatellite genotyping (N = 863). Further, we assessed the efficacy of the program to identify individuals over time by comparing error rates between within‐year and between‐year recaptures. Recaptured lizards were correctly identified by I³S in 94.1% of cases. We estimated a false rejection rate (FRR) of 5.9% and a false acceptance rate (FAR) of 0%. By using I³S, we correctly identified 97.8% of within‐year recaptures (FRR = 2.2%; FAR = 0%) and 91.1% of between‐year recaptures (FRR = 8.9%; FAR = 0%). Misidentifications were primarily due to poor photograph quality (N = 4). However, two misidentifications were caused by indistinct scale configuration due to scale damage (N = 1) and ontogenetic changes in head scalation between capture events (N = 1). We conclude that automated photographic identification based on head scale patterns is a reliable and accurate method for identifying individuals over time. Because many lizard or reptilian species possess variable head squamation, this method has potential for successful application in many species.     

Nosewitness Identification: Effects of Negative Emotion

Every individual has a unique body odor (BO), similar to a fingerprint. In forensic research, identification of culprit BOs has been performed by trained dogs, but not by humans. We introduce the concept of nosewitness identification and present the first experimental results on BO memory in witness situations involving violent crimes. Two experiments indicated that BO associated with male characters in authentic videos could later be identified in BO lineup tests well above chance. Moreover, culprit BO in emotional crime videos could be identified considerably better than the BO of a male person in neutral videos. This indicates that nosewitness identification benefits from emotional encoding. Altogether, the study testifies to the virtue of body odor as a cue to identify individuals observed under negative emotion.     

Accurate mass tag retention time database for urine proteome analysis by chromatography-mass spectrometry

Information about peptides and proteins in urine can be used to search for biomarkers of early stages of various diseases. The main technology currently used for identification of peptides and proteins is tandem mass spectrometry, in which peptides are identified by mass spectra of their fragmentation products. However, the presence of the fragmentation stage decreases sensitivity of analysis and increases its duration. We have developed a method for identification of human urinary proteins and peptides. This method based on the accurate mass and time tag (AMT) method does not use tandem mass spectrometry. The database of AMT tags containing more than 1381 AMT tags of peptides has been constructed. The software for database filling with AMT tags, normalizing the chromatograms, database application for identification of proteins and peptides, and their quantitative estimation has been developed. The new procedures for peptide identification by tandem mass spectra and the AMT tag database are proposed. The paper also lists novel proteins that have been identified in human urine for the first time.     

Chrysophaentins are competitive inhibitors of FtsZ and inhibit Z-ring formation in live bacteria

The bacterial cell division protein FtsZ polymerizes in a GTP-dependent manner to form a Z-ring that marks the plane of division. As a validated antimicrobial target, considerable efforts have been devoted to identify small molecule FtsZ inhibitors. We recently discovered the chrysophaentins, a novel suite of marine natural products that inhibit FtsZ activity in vitro. These natural products along with a synthetic hemi-chrysophaentin exhibit strong antimicrobial activity toward a broad spectrum of Gram-positive pathogens. To define their mechanisms of FtsZ inhibition and determine their in vivo effects in live bacteria, we used GTPase assays and fluorescence anisotropy to show that hemi-chrysophaentin competitively inhibits FtsZ activity. Furthermore, we developed a model system using a permeable Escherichia coli strain, envA1, together with an inducible FtsZ-yellow fluorescent protein construct to show by fluorescence microscopy that both chrysophaentin A and hemi-chrysophaentin disrupt Z-rings in live bacteria. We tested the E. coli system further by reproducing phenotypes observed for zantrins Z1 and Z3, and demonstrate that the alkaloid berberine, a reported FtsZ inhibitor, exhibits auto-fluorescence, making it incompatible with systems that employ GFP or YFP tagged FtsZ. These studies describe unique examples of nonnucleotide, competitive FtsZ inhibitors that disrupt FtsZ in vivo, together with a model system that should be useful for in vivo testing of FtsZ inhibitor leads that have been identified through in vitro screens but are unable to penetrate the Gram-negative outer membrane.