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Shifting up a gear with iDNA: from mammal detection events to standardized surveys
Abstract
Invertebrate-derived DNA (iDNA), in combination with high throughput sequencing, has been proposed as a cost-efficient and powerful tool to survey vertebrate species. Previous studies, however, have only provided evidence that vertebrates can be detected using iDNA, but have not taken the next step of placing these detection events within a statistical framework that allows for robust biodiversity assessments. Here, we compare concurrent iDNA and camera-trap surveys. Leeches were repeatedly collected in close vicinity to 64 camera-trap stations in Sabah, Malaysian Borneo. We analyze iDNA-derived mammalian detection events in a modern occupancy model that accounts for imperfect detection and compare the results with those from occupancy models parameterized with camera-trap-derived detection events. We also combine leech-iDNA and camera-trap data in a single occupancy model. We found consistent estimates of occupancy probabilities produced by our camera-trap and leech datasets. This indicates that the metabarcoding of leech-iDNA method provides reasonable estimates of occupancy and can be a suitable method for studying and monitoring mammal species in tropical rainforests. However, we also show that a more extensive collection of leeches would be needed to assess mammal biodiversity with a similar robustness as with camera traps. As certain taxa were only detected in leeches, we see great potential in complementing camera-trap studies with the iDNA approach, as long as the collection of leeches follows a similar robust and standardized sampling scheme. Synthesis and applications: The approach we describe here is not restricted to the processing of leech samples, but can be used for the analysis of other iDNA and environmental DNA (eDNA) data. Our study is the first step to shift the application of e/iDNA studies from opportunistic ad-hoc collections to systematic surveys required for long-term wildlife populations and biodiversity monitoring programs.
... Average raw reads per amplicon per sample 31 Pre-print Leech gut contents Targeted (vertebrates) Illumina MiSeq Unknown 32 Peer-reviewed Herbivore feces General biodiversity IonTorrent PGM 4,156 (post-filtering) 33 Peer-reviewed Soil Targeted (Phytophthora) Roche GS Junior Unknown 34 Peer-reviewed Freshwater ecosystem Targeted: planktonic protists Illumina MiSeq 150,000* 35 Peer-reviewed Turbid freshwater Targeted (vertebrates) Illumina MiSeq 7,181 (post-filtering) 36 Peer-reviewed Sea water General biodiversity Roche GS-FLX 21,192* (post-filtering) 37 Pre-print Brackish water General biodiversity Illumina MiSeq 200,185 (post-filtering) 38 Peer-reviewed High-salinity lake General biodiversity Illumina HiSeq 2500 124,779 for bacteria ~37,000 for archaea and eukaryota (post-fitlering) 39 Pre-print Wheat and oilseed rape residues General biodiversity Illumina MiSeq 80,000* (raw) 40 Peer-reviewed Scat Targeted www.nature.com/scientificreports www.nature.com/scientificreports/ ...
The characterization of biodiversity is a crucial element of ecological investigations as well as environmental assessment and monitoring activities. Increasingly, amplicon-based environmental DNA metabarcoding (alternatively, marker gene metagenomics) is used for such studies given its ability to provide biodiversity data from various groups of organisms simply from analysis of bulk environmental samples such as water, soil or sediments. The Illumina MiSeq is currently the most popular tool for carrying out this work, but we set out to determine whether typical studies were reading enough DNA to detect rare organisms (i.e., those that may be of greatest interest such as endangered or invasive species) present in the environment. We collected sea water samples along two transects in Conception Bay, Newfoundland and analyzed them on the MiSeq with a sequencing depth of 100,000 reads per sample (exceeding the 60,000 per sample that is typical of similar studies). We then analyzed these same samples on Illumina’s newest high-capacity platform, the NovaSeq, at a depth of 7 million reads per sample. Not surprisingly, the NovaSeq detected many more taxa than the MiSeq thanks to its much greater sequencing depth. However, contrary to our expectations this pattern was true even in depth-for-depth comparisons. In other words, the NovaSeq can detect more DNA sequence diversity within samples than the MiSeq, even at the exact same sequencing depth. Even when samples were reanalyzed on the MiSeq with a sequencing depth of 1 million reads each, the MiSeq’s ability to detect new sequences plateaued while the NovaSeq continued to detect new sequence variants. These results have important biological implications. The NovaSeq found 40% more metazoan families in this environment than the MiSeq, including some of interest such as marine mammals and bony fish so the real-world implications of these findings are significant. These results are most likely associated to the advances incorporated in the NovaSeq, especially a patterned flow cell, which prevents similar sequences that are neighbours on the flow cell (common in metabarcoding studies) from being erroneously merged into single spots by the sequencing instrument. This study sets the stage for incorporating eDNA metabarcoding in comprehensive analysis of oceanic samples in a wide range of ecological and environmental investigations.
The characterization of biodiversity is a crucial element of ecological investigations as well as environmental assessment and monitoring activities. Increasingly, amplicon-based environmental DNA metabarcoding (alternatively, marker gene metagenomics) is used for such studies given its ability to provide biodiversity data from various groups of organisms simply from analysis of bulk environmental samples such as water, soil or sediments. The Illumina MiSeq is currently the most popular tool for carrying out this work, but we set out to determine whether typical studies were reading enough DNA to detect rare organisms (i.e., those that may be of greatest interest such as endangered or invasive species) present in the environment. We collected sea water samples along two transects in Conception Bay, Newfoundland and analyzed them on the MiSeq with a sequencing depth of 100,000 reads per sample (exceeding the 60,000 per sample that is typical of similar studies). We then analyzed these same samples on Illumina's newest high-capacity platform, the NovaSeq, at a depth of 7 million reads per sample. Not surprisingly, the NovaSeq detected many more taxa than the MiSeq thanks to its much greater sequencing depth. However, contrary to our expectations this pattern was true even in depth-for-depth comparisons. In other words, the NovaSeq can detect more DNA sequence diversity within samples than the MiSeq, even at the exact same sequencing depth. Even when samples were reanalyzed on the MiSeq with a sequencing depth of 1 million reads each, the MiSeq's ability to detect new sequences plateaued while the NovaSeq continued to detect new sequence variants. These results have important biological implications. The NovaSeq found 40% more metazoan families in this environment than the MiSeq, including some of interest such as marine mammals and bony fish so the real-world implications of these findings are significant. These results are most likely associated to the advances incorporated in NovaSeq especially patterned flow cell, which prevents similar sequences that are neighbours on the flow cell (common in metabarcoding studies) from being erroneously merged into single spots by the sequencing instrument. This study sets the stage for incorporating eDNA metabarcoding in comprehensive analysis of oceanic samples in a wide range of ecological and environmental investigations.
Background
The use of environmental DNA for species detection via metabarcoding is growing rapidly. We present a co-designed lab workflow and bioinformatic pipeline to mitigate the 2 most important risks of environmental DNA use: sample contamination and taxonomic misassignment. These risks arise from the need for polymerase chain reaction (PCR) amplification to detect the trace amounts of DNA combined with the necessity of using short target regions due to DNA degradation.
Findings
Our high-throughput workflow minimizes these risks via a 4-step strategy: (i) technical replication with 2 PCR replicates and 2 extraction replicates; (ii) using multi-markers (12S,16S,CytB); (iii) a “twin-tagging,” 2-step PCR protocol; and (iv) use of the probabilistic taxonomic assignment method PROTAX, which can account for incomplete reference databases. Because annotation errors in the reference sequences can result in taxonomic misassignment, we supply a protocol for curating sequence datasets. For some taxonomic groups and some markers, curation resulted in >50% of sequences being deleted from public reference databases, owing to (i) limited overlap between our target amplicon and reference sequences, (ii) mislabelling of reference sequences, and (iii) redundancy. Finally, we provide a bioinformatic pipeline to process amplicons and conduct PROTAX assignment and tested it on an invertebrate-derived DNA dataset from 1,532 leeches from Sabah, Malaysia. Twin-tagging allowed us to detect and exclude sequences with non-matching tags. The smallest DNA fragment (16S) amplified most frequently for all samples but was less powerful for discriminating at species rank. Using a stringent and lax acceptance criterion we found 162 (stringent) and 190 (lax) vertebrate detections of 95 (stringent) and 109 (lax) leech samples.
Conclusions
Our metabarcoding workflow should help research groups increase the robustness of their results and therefore facilitate wider use of environmental and invertebrate-derived DNA, which is turning into a valuable source of ecological and conservation information on tetrapods.
• Population declines and local extirpation trends are widespread among freshwater species, but the responsible drivers of these trends are poorly understood. Identifying the potential drivers of population declines is essential to effective conservation planning. However, conventional detection methods used to monitor cryptic and elusive freshwater species are inefficient. Integrating new surveying and modelling techniques may allow for a more comprehensive assessment of population declines.
• We used environmental DNA (eDNA) sampling methods and detailed historical records to identify drivers of local extirpation in a declining, long‐lived giant salamander, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis) in West Virginia, U.S.A. We used a site occupancy and detection modelling framework (SODM) to test the influence of current land use, historical mining, hydrogeomorphic and water quality variables on model‐based predictions of hellbender extirpation and detection.
• We failed to detect hellbender eDNA at 51% (naïve 1 – Ψ) of historical sites, suggesting local extirpation at a broad spatial scale in West Virginia. Our best‐supported SODM model suggested catchment‐scale road density was the best predictor of hellbender extirpation, and that 38% (predicted 1 – Ψ) of historical sites may be locally extirpated. Estimates of hellbender occupancy probability were extremely low in highly developed catchments. Water turbidity and conductivity were the best predictors of eDNA detection, both negatively influencing detection probability.
• Roads can increase sedimentation rates and alter water chemistry of freshwater ecosystems, identifying landscape alteration/human development and water quality declines as possible drivers of hellbender extirpation trends in West Virginia. Our findings also suggest that water conductivity and turbidity may act as polymerase chain reaction inhibitors and decrease eDNA detection in lotic systems.
• This study emphasises the negative impacts of urban development on freshwater ecosystems and the sensitivity of long‐lived amphibian species to rapid environmental change. Our findings may aid in conservation planning by providing a sampling framework that integrates eDNA data within a SODM framework to rapidly and accurately assesses relational changes in aquatic species' occupancy at historical sites.
Cost-efficient repeatable methods to track biodiversity changes are important for forest and wildlife managers to improve management practices and target conservation efforts at the local scale.
The aim of this user’s guide is to provide practitioners step-by-step instructions for biodiversity assessment and monitoring of tropical forest mammals using camera-traps and e/iDNA. This includes guidance on the project design and standardized methodologies for data collection, data management, laboratory and data analysis, which
should enable users to produce standardized and more comparable biodiversity data collected in tropical rainforests.
In PART I METHODS AND DATA COLLECTION of this user’s guide we will first introduce the two main field methods, camera-trapping and iDNA, and highlight the advantages and disadvantages, of both techniques.
In PART II ANALYTICAL METHODS we introduce the most common methods used to analyse camera-trapping and e/iDNA datasets.
In PART III CASE STUDIES we provide key examples from the SCREENFORBIO project.
The final SUMMARY AND PERSPECTIVE section highlights the potential that these approaches have for the monitoring of terrestrial mammals in tropical rainforests, but also identifies areas in which further research is needed.
User's guide and supplementary material can be freely downloaded here:
http://www.izw-berlin.de/userguide.html
The use of environmental DNA (eDNA) has become an applicable non‐invasive tool with which to obtain information about biodiversity. A sub‐discipline of eDNA is iDNA (invertebrate‐derived DNA), where genetic material ingested by invertebrates is used to characterise the biodiversity of the species that served as hosts. While promising, these techniques are still in their infancy, as they have only been explored on limited numbers of samples from only a single or a few different locations. In this study, we investigate the suitability of iDNA extracted from more than 3,000 haematophagous terrestrial leeches as a tool for detecting a wide range of terrestrial vertebrates across five different geographical regions on three different continents. These regions cover almost the full geographical range of haematophagous terrestrial leeches, thus representing all parts of the world where this method might apply. We identify host taxa through metabarcoding coupled with high‐throughput sequencing on Illumina and IonTorrent sequencing platforms to decrease economic costs and workload and thereby make the approach attractive for practitioners in conservation management. We identified hosts in four different taxonomic vertebrate classes: mammals, birds, reptiles, and amphibians, belonging to at least 42 different taxonomic families. We find that vertebrate blood ingested by haematophagous terrestrial leeches throughout their distribution is a viable source of DNA with which to examine a wide range of vertebrates. Thus, this study provides encouraging support for the potential of haematophagous terrestrial leeches as a tool for detecting and monitoring terrestrial vertebrate biodiversity.
This article is protected by copyright. All rights reserved.
ABSTRACT: Environmental DNA (eDNA) detection is a technique used to non-invasively detect cryptic, low density, or logistically difficult-to-study species, such as imperiled manatees. For eDNA measurement, genetic material shed into the environment is concentrated from water samples and analyzed for the presence of target species. Cytochrome b quantitative PCR and droplet digital PCR eDNA assays were developed for the 3 Vulnerable manatee species: African, Amazonian,
and both subspecies of the West Indian (Florida and Antillean) manatee. Environmental DNA assays can help to delineate manatee habitat ranges, high use areas, and seasonal population changes. To validate the assay, water was analyzed from Florida’s east coast containing a high-density manatee population and produced 31 564 DNA molecules l−1 on average and high occurrence (ψ) and detection (p) estimates (ψ = 0.84 [0.40−0.99]; p = 0.99 [0.95−1.00]; limit of detection 3 copies μl−1). Similar occupancy estimates were produced in the Florida Panhandle (ψ = 0.79 [0.54−0.97]) and Cuba (ψ = 0.89 [0.54−1.00]), while occupancy estimates in Cameroon were lower (ψ = 0.49 [0.09−0.95]). The eDNA-derived detection estimates were higher than those generated using aerial survey data on the west coast of Florida and may be effective for population monitoring.
Subsequent eDNA studies could be particularly useful in locations where manatees are (1) difficult to identify visually (e.g. the Amazon River and Africa), (2) are present in patchy distributions or are on the verge of extinction (e.g. Jamaica, Haiti), and (3) where repatriation efforts are proposed (e.g. Brazil, Guadeloupe). Extension of these eDNA techniques could be applied to other imperiled marine mammal populations such as African and Asian dugongs.
In this article we describe eDNAoccupancy, an R package for fitting Bayesian, multi-scale occupancy models. These models are appropriate for occupancy surveys that include three, nested levels of sampling: primary sample units within a study area, secondary sample units collected from each primary unit, and replicates of each secondary sample unit. This design is commonly used in occupancy surveys of environmental DNA (eDNA). eDNAoccupancy allows users to specify and fit multi-scale occupancy models with or without covariates, to estimate posterior summaries of occurrence and detection probabilities, and to compare different models using Bayesian model-selection criteria. We illustrate these features by analyzing two published data sets: eDNA surveys of a fungal pathogen of amphibians and eDNA surveys of an endangered fish species.
Understandably, given the fast pace of biodiversity loss, there is much interest in using Earth observation technology to track biodiversity, ecosystem functions and ecosystem services. However, because most biodiversity is invisible to Earth observation, indicators based on Earth observation could be misleading and reduce the effectiveness of nature conservation and even unintentionally decrease conservation effort. We describe an approach that combines automated recording devices, highthroughput DNA sequencing and modern ecological modelling to extract much more of the information available in Earth observation data. This approach is achievable now, offering efficient and near-real-time monitoring of management impacts on biodiversity and its functions and services.
Metabarcoding of vertebrate DNA derived from carrion flies has been proposed as a promising tool for biodiversity monitoring. To evaluate its efficacy, we conducted metabarcoding surveys of carrion flies on Barro Colorado Island (BCI), Panama, which has a well-known mammal community, and compared our results against diurnal transect counts and camera-trapping. We collected 1084 flies in 29 sampling days, conducted metabarcoding with mammal-specific (16S) and vertebrate-specific (12S) primers, and sequenced amplicons on Illumina MiSeq. For taxonomic assignment, we compared BLAST with the new program PROTAX, and we found that PROTAX improved species identifications. We detected 20 mammal, four bird, and one lizard species from carrion fly metabarcoding, all but one of which are known from BCI. Fly metabarcoding detected more mammal species than concurrent transect counts (29 sampling days, 13 species) and concurrent camera-trapping (84 sampling days, 17 species), and detected 67% of the number of mammal species documented by eight years of transect counts and camera-trapping combined, although fly metabarcoding missed several abundant species. This study demonstrates that carrion fly metabarcoding is a powerful tool for mammal biodiversity surveys, and has the potential to detect a broader range of species than more commonly used methods.
A guide to data collection, modeling and inference strategies for biological survey data using Bayesian and classical statistical methods. This book describes a general and flexible framework for modeling and inference in ecological systems based on hierarchical models, with a strict focus on the use of probability models and parametric inference. Hierarchical models represent a paradigm shift in the application of statistics to ecological inference problems because they combine explicit models of ecological system structure or dynamics with models of how ecological systems are observed. The principles of hierarchical modeling are developed and applied to problems in population, metapopulation, community, and metacommunity systems. The book provides the first synthetic treatment of many recent methodological advances in ecological modeling and unifies disparate methods and procedures. The authors apply principles of hierarchical modeling to ecological problems, including * occurrence or occupancy models for estimating species distribution * abundance models based on many sampling protocols, including distance sampling * capture-recapture models with individual effects * spatial capture-recapture models based on camera trapping and related methods * population and metapopulation dynamic models * models of biodiversity, community structure and dynamics * Wide variety of examples involving many taxa (birds, amphibians, mammals, insects, plants) * Development of classical, likelihood-based procedures for inference, as well as Bayesian methods of analysis * Detailed explanations describing the implementation of hierarchical models using freely available software such as R and WinBUGS * Computing support in technical appendices in an online companion web site.
Camera trapping is a widely applied method to study mammalian biodiversity and is still gaining popularity. It can quickly generate large amounts of data which need to be managed in an efficient and transparent way that links data acquisition with analytical tools.
We describe the free and open‐source R package camtrapR , a new toolbox for flexible and efficient management of data generated in camera trap‐based wildlife studies. The package implements a complete workflow for processing camera trapping data. It assists in image organization, species and individual identification, data extraction from images, tabulation and visualization of results and export of data for subsequent analyses. There is no limitation to the number of images stored in this data management system; the system is portable and compatible across operating systems.
The functions provide extensive automation to minimize data entry mistakes and, apart from species and individual identification, require minimal manual user input. Species and individual identification are performed outside the R environment, either via tags assigned in dedicated image management software or by moving images into species directories.
Input for occupancy and (spatial) capture–recapture analyses for density and abundance estimation, for example in the R packages unmarked or secr , is computed in a flexible and reproducible manner. In addition, survey summary reports can be generated, spatial distributions of records can be plotted and exported to gis software, and single‐ and two‐species activity patterns can be visualized.
camtrapR allows for streamlined and flexible camera trap data management and should be most useful to researchers and practitioners who regularly handle large amounts of camera trapping data.
Environmental DNA (eDNA) monitoring approaches promise to greatly improve detection of rare, endangered, and invasive species in comparison to traditional field approaches. Herein, eDNA approaches and traditional seining methods were applied at 29 research locations to compare method-specific estimates of detection and occupancy probabilities for endangered tidewater goby (Eucyclogobius newberryi). At each location multiple paired seine hauls and water samples for eDNA analysis were taken, ranging from two to 23 samples per site, depending upon habitat size. Analysis using a multi-method occupancy modeling framework indicated that the probability of detection using eDNA was nearly double (0.74) the rate of detection for seining (0.39). The higher detection rates afforded by eDNA allowed determination of tidewater goby occupancy at two locations where they have not been previously detected and at one location considered to be locally extirpated. Additionally, eDNA concentration was positively related to tidewater goby catch per unit effort, suggesting eDNA could potentially be used as a proxy for local tidewater goby abundance. Compared to traditional field sampling, eDNA provided improved occupancy parameter estimates, and can be applied to increase management efficiency across a broad spatial range and within a diversity of habitats. This article is protected by copyright. All rights reserved.
Invertebrate-derived DNA (iDNA) from terrestrial haematophagous leeches has recently been proposed as a powerful non-invasive tool with which to detect vertebrate species and thus to survey their populations. However, to date little attention has been given to whether and how this, or indeed any other iDNA-derived data, can be combined with state-of-the-art analytical tools to estimate wildlife abundances, population dynamics and distributions. In this review, we discuss the challenges that face the application of existing analytical methods such as site-occupancy and spatial capture-recapture (SCR) models to terrestrial leech iDNA, in particular, possible violations of key assumptions arising from factors intrinsic to invertebrate parasite biology. Specifically, we review the advantages and disadvantages of terrestrial leeches as a source of iDNA and summarize the utility of leeches for presence, occupancy, and spatial capture-recapture models. The main source of uncertainty that attends species detections derived from leech gut contents is attributable to uncertainty about the spatio-temporal sampling frame, since leeches retain host-blood for months and can move after feeding. Subsequently, we briefly address how the analytical challenges associated with leeches may apply to other sources of iDNA. Our review highlights that despite the considerable potential of leech (and indeed any) iDNA as a new survey tool, further pilot studies are needed to assess how analytical methods can overcome or not the potential biases and assumption violations of the new field of iDNA. Specifically we argue that studies to compare iDNA sampling with standard survey methods such as camera trapping, and those to improve our knowledge on leech (and other invertebrate parasite) physiology, taxonomy, and ecology will be of immense future value.
Reliable assessment of animal populations is a long‐standing challenge in wildlife ecology. Technological advances have led to widespread adoption of camera traps ( CT s) to survey wildlife distribution, abundance and behaviour. As for any wildlife survey method, camera trapping must contend with sources of sampling error such as imperfect detection. Early applications focused on density estimation of naturally marked species, but there is growing interest in broad‐scale CT surveys of unmarked populations and communities. Nevertheless, inferences based on detection indices are controversial, and the suitability of alternatives such as occupancy estimation is debatable.
We reviewed 266 CT studies published between 2008 and 2013. We recorded study objectives and methodologies, evaluating the consistency of CT protocols and sampling designs, the extent to which CT surveys considered sampling error, and the linkages between analytical assumptions and species ecology.
Nearly two‐thirds of studies surveyed more than one species, and a majority used response variables that ignored imperfect detection (e.g. presence–absence, relative abundance). Many studies used opportunistic sampling and did not explicitly report details of sampling design and camera deployment that could affect conclusions.
Most studies estimating density used capture–recapture methods on marked species, with spatially explicit methods becoming more prominent. Few studies estimated density for unmarked species, focusing instead on occupancy modelling or measures of relative abundance. While occupancy studies estimated detectability, most did not explicitly define key components of the modelling framework (e.g. a site) or discuss potential violations of model assumptions (e.g. site closure). Studies using relative abundance relied on assumptions of equal detectability, and most did not explicitly define expected relationships between measured responses and underlying ecological processes (e.g. animal abundance and movement).
Synthesis and applications . The rapid adoption of camera traps represents an exciting transition in wildlife survey methodology. We remain optimistic about the technology's promise, but call for more explicit consideration of underlying processes of animal abundance, movement and detection by cameras, including more thorough reporting of methodological details and assumptions. Such transparency will facilitate efforts to evaluate and improve the reliability of camera trap surveys, ultimately leading to stronger inferences and helping to meet modern needs for effective ecological inquiry and biodiversity monitoring.
Rigorous and cost-effective methods are essential to efficiently assess wildlife populations and obtain accurate data to inform conservation and management decisions. In the UK, available data on terrestrial mammal species are distinctly lacking, many populations are in decline and survey methods are technically demanding and labour-intensive. There is, therefore, much need to investigate alternative methodologies to ensure that resource use is efficient and data are reliable. Camera-trapping presents a relatively new approach for surveying mammals, though in the UK, the extent to which camera traps have been used has not been quantified and their performance has not yet been compared relative to existing methods. This study uses biological parameters and economic and logistic costs to assess the efficiency and reliability of camera-trapping and transect-sampling during winter field trials. Tracks and sign surveys and sightings surveys were conducted simultaneously and where appropriate, investigated independently. In addition, a nationally-distributed questionnaire was used to investigate surveyor competence and identify temporal trends in method use in the UK. Field trials concluded that camera-trapping was the most labour-efficient method for producing a species inventory, and frequently recorded more species per sampling site than did transect-sampling. However, when the total sampling period was limited, species were encountered at a faster rate by the detection of tracks and signs than by the alternative methods investigated. The single density estimate derived from camera trap data was higher than that from transect-sampling, and no differences were observed within the three alpha diversity index estimates derived by each survey method. The questionnaire suggests that the reliability of species presence/absence data derived from tracks and signs surveys is probably compromised by surveyor confidence of species identification. A multi-evidence approach is, therefore, recommended for less-competent surveyors. Despite greater initial economic costs, it is advocated that camera-trapping may be an efficient, rigorous and cost-effective method for large-scale long-term monitoring programmes. Furthermore, data suggest that camera trap use will become increasingly frequent in the UK. More research is required to investigate the relationships between method efficiency and season, species density and habitat, and to assess the accuracy of species density estimates.
The continuous decline in Earth’s biodiversity represents a major crisis and challenge for the 21st century, and there is international political agreement to slow down or halt this decline. The challenge is in large part impeded by the lack of knowledge on the state and distribution of biodiversity – especially since the majority of species on Earth are un-described by science. All conservation efforts to save biodiversity essentially depend on the monitoring of species and populations to obtain reliable distribution patterns and population size estimates. Such monitoring has traditionally relied on physical identification of species by visual surveys and counting of individuals. However, traditional monitoring techniques remain problematic due to difficulties associated with correct identification of cryptic species or juvenile life stages, a continuous decline in taxonomic expertise, non-standardized sampling, and the invasive nature of some survey techniques. Hence, there is urgent need for alternative and efficient techniques for large-scale biodiversity monitoring. Environmental DNA (eDNA) – defined here as: genetic material obtained directly from environmental samples (soil, sediment, water, etc.) without any obvious signs of biological source material – is an efficient, non-invasive and easy-to-standardize sampling approach. Coupled with sensitive, cost-efficient and ever-advancing DNA sequencing technology, it may be an appropriate candidate for the challenge of biodiversity monitoring. Environmental DNA has been obtained from ancient as well as modern samples and encompasses single species detection to analyses of ecosystems. The research on eDNA initiated in microbiology, recognizing that culture-based methods grossly misrepresent the microbial diversity in nature. Subsequently, as a method to assess the diversity of macro-organismal communities, eDNA was first analyzed in sediments, revealing DNA from extinct and extant animals and plants, but has since been obtained from various terrestrial and aquatic environmental samples. Results from eDNA approaches have provided valuable insights to the study of ancient environments and proven useful for monitoring contemporary biodiversity in terrestrial and aquatic ecosystems. In the future, we expect the eDNA-based approaches to move from single-marker analyses of species or communities to meta-genomic surveys of entire ecosystems to predict spatial and temporal biodiversity patterns. Such advances have applications for a range of biological, geological and environmental sciences. Here we review the achievements gained through analyses of eDNA from macro-organisms in a conservation context, and discuss its potential advantages and limitations for biodiversity monitoring.
Environmental DNA (eDNA) metabarcoding is increasingly used to study present and past biodiversity. eDNA analyses often rely on amplification of very small quantities or degraded DNA. To avoid missing detection of taxa that are actually present (false negatives), multiple extractions and amplifications of the same samples are often performed. However, the level of replication needed for reliable estimates of presence / absence patterns remains an unaddressed topic. Furthermore, degraded DNA and PCR/sequencing errors might produce false positives. We used simulations and empirical data to evaluate the level of replication required for accurate detection of targeted taxa in different contexts, and to assess the performance of methods used to reduce the risk of false detections. Furthermore, we evaluated whether statistical approaches developed to estimate occupancy in presence of observational errors can successfully estimate true prevalence, detection probability, and false positive rates. Replications reduced the rate of false negatives; the optimal level of replication was strongly dependent on the detection probability of taxa. Occupancy models successfully estimated true prevalence, detection probability, and false positive rates, but their performance increased with the number of replicates. At least eight PCR replicates should be performed if detection probability is not high, such as in ancient DNA studies. Multiple DNA extractions from the same sample yielded consistent results; in some cases collecting multiple samples from the same locality allowed detecting more species. The optimal level of replication for accurate species detection strongly varies among studies, and could be explicitly estimated to improve the reliability of results.This article is protected by copyright. All rights reserved.
As human threats continue to impact natural habitats, there is an increasing need to regularly monitor the trends in large vertebrate populations. Conservation efforts must be directed appropriately, but field work necessary for data collection is often limited by time and availability of people. Camera traps are used as an efficient method to insure continuous sampling and to work in difficult to access areas. In the present study, we illustrate how this instrument is serving a diverse field of studies, such as animal behavior, population monitoring and fauna-flora interaction. By looking at the material and technical aspects of various models of camera trap for implementation in different field studies in animal ecology, we highlight the need to choose appropriate camera trap models for the target species and to set up solid sampling protocols to successfully achieve study objectives.
The probability that a site has at least one individual of a species (‘occupancy’) has come to be
widely used as a state variable for animal population monitoring. The available statistical theory for
estimation when detection is imperfect applies particularly to habitat patches or islands, although it is also
used for arbitrary plots in continuous habitat. The probability that such a plot is occupied depends on plot
size and home-range characteristics (size, shape and dispersion) as well as population density. Plot size is
critical to the definition of occupancy as a state variable, but clear advice on plot size is missing from the
literature on the design of occupancy studies. We describe models for the effects of varying plot size and
home-range size on expected occupancy. Temporal, spatial, and species variation in average home-range
size is to be expected, but information on home ranges is difficult to retrieve from species presence/absence
data collected in occupancy studies. The effect of variable home-range size is negligible when plots are very
large (>100 x area of home range), but large plots pose practical problems. At the other extreme, sampling
of ‘point’ plots with cameras or other passive detectors allows the true ‘proportion of area occupied’ to be
estimated. However, this measure equally reflects home-range size and density, and is of doubtful value for
population monitoring or cross-species comparisons. Plot size is ill-defined and variable in occupancy
studies that detect animals at unknown distances, the commonest example being unlimited-radius point
counts of song birds. We also find that plot size is ill-defined in recent treatments of ‘‘multi-scale’’
occupancy; the respective scales are better interpreted as temporal (instantaneous and asymptotic) rather
than spatial. Occupancy is an inadequate metric for population monitoring when it is confounded with
home-range size or detection distance.
Society responds to changes in climate and land use via mitigation measures, including rainwater retention and storage in rewetted and newly constructed wetlands. Humans living close to these wetlands express concerns about future mosquito nuisance situations, and request the necessary distance between human occupation and wetlands to avoid such problems. Wetland managers need to know the distance required, as well as the type of management needed for such buffer or barrier zones. Here we performed an extensive literature survey to collect quantitative information on mosquito flight distance and the relevant environmental conditions. Mosquitoes have an average maximum flight distance of between 50 m to 50 km, depending on the species. Long-distance or migratory flights are strongly related to species ecological preferences and physiology, are survived by few specimens, and do not relate to nuisance situations. Nuisance-related or non-oriented flights are also species-specific and cover much shorter distances-between 25 m and 6 km for the 23 species analyzed. Based on these results, we made regression-based estimations of the percentages of the population that cross certain distances. A 90% reduction in breeding site population density would require minimal distances of 56 m for Anopheles saperoi and 8.6 km for Anopheles sinensis, and much greater distances for Aedes vexans, Culex quinquefasciatus, and Culiseta morsitans. Little useful information was available regarding the environmental conditions under which non-oriented flights took place. Qualitatively, the review showed that flight capacity was influenced by landscape structure, meteorological conditions (temperature, humidity, and illumination), and species physiology (energy available for flight). Overall, our findings suggest that predictions regarding the construction of barrier zones around breeding sites can be made based on mosquito and host density and human nuisance perception, and that barrier zone usefulness strongly depends on the mosquito species involved. Additional quantitative research is needed to better document the non-oriented dispersal patterns of the mosquitoes that populate rewetted and newly constructed wetlands, and the effects of vegetation types in barrier zones on mosquito densities.
The proliferation of camera-trapping studies has led to a spate of extensions in the known distributions of many wild cat species, not least in Borneo. However, we still do not have a clear picture of the spatial patterns of felid abundance in Southeast Asia, particularly with respect to the large areas of highly-disturbed habitat. An important obstacle to increasing the usefulness of camera trap data is the widespread practice of setting cameras at non-random locations. Non-random deployment interacts with non-random space-use by animals, causing biases in our inferences about relative abundance from detection frequencies alone. This may be a particular problem if surveys do not adequately sample the full range of habitat features present in a study region. Using camera-trapping records and incidental sightings from the Kalabakan Forest Reserve, Sabah, Malaysian Borneo, we aimed to assess the relative abundance of felid species in highly-disturbed forest, as well as investigate felid space-use and the potential for biases resulting from non-random sampling. Although the area has been intensively logged over three decades, it was found to still retain the full complement of Bornean felids, including the bay cat Pardofelis badia, a poorly known Bornean endemic. Camera-trapping using strictly random locations detected four of the five Bornean felid species and revealed inter- and intra-specific differences in space-use. We compare our results with an extensive dataset of >1,200 felid records from previous camera-trapping studies and show that the relative abundance of the bay cat, in particular, may have previously been underestimated due to the use of non-random survey locations. Further surveys for this species using random locations will be crucial in determining its conservation status. We advocate the more wide-spread use of random survey locations in future camera-trapping surveys in order to increase the robustness and generality of inferences that can be made.
Environmental DNA (eDNA) methods for detecting aquatic species are advancing rapidly, but with little evaluation of field protocols or precision of resulting estimates. We compared sampling results from traditional field methods with eDNA methods for two amphibians in 13 streams in central Idaho, USA. We also evaluated three water collection protocols and the influence of sampling location, time of day, and distance from animals on eDNA concentration in the water. We found no difference in detection or amount of eDNA among water collection protocols. eDNA methods had slightly higher detection rates than traditional field methods, particularly when species occurred at low densities. eDNA concentration was positively related to field-measured density, biomass, and proportion of transects occupied. Precision of eDNA-based abundance estimates increased with the amount of eDNA in the water and the number of replicate subsamples collected. eDNA concentration did not vary significantly with sample location in the stream, time of day, or distance downstream from animals. Our results further advance the implementation of eDNA methods for monitoring aquatic vertebrates in stream habitats.
Recent studies suggest that vertebrate genetic material ingested by invertebrates (iDNA) can be used to investigate vertebrate ecology. Given the ubiquity of invertebrates that feed on vertebrates across the globe, iDNA might qualify as a very powerful tool for 21st century population and conservation biologists. Here, we identify some invertebrate characteristics that will likely influence iDNA retrieval and elaborate on the potential uses of invertebrate-derived information. We hypothesize that beyond inventorying local faunal diversity, iDNA should allow for more profound insights into wildlife population density, size, mortality, and infectious agents. Based on the similarities of iDNA with other low-quality sources of DNA, a general technical framework for iDNA analyses is proposed. As it is likely that no such thing as a single ideal iDNA sampler exists, forthcoming research efforts should aim at cataloguing invertebrate properties relevant to iDNA retrieval so as to guide future usage of the invertebrate tool box.
Large-scale monitoring schemes are essential in assessing global mammalian biodiversity, and in this framework, leeches have recently been promoted as an indirect source of DNA from terrestrial mammal species. Carrion feeding flies are ubiquitous and can be expected to feed on many vertebrate carcasses. Hence, we tested whether fly-derived DNA analysis may also serve as a novel tool for mammalian diversity surveys. We screened DNA extracted from 201 carrion flies collected in tropical habitats of Côte d'Ivoire and Madagascar for mammal DNA using multiple PCR systems and retrieved DNA sequences from a diverse set of species (22 in Côte d'Ivoire, four in Madagascar) exploiting distinct forest strata and displaying a broad range of body sizes. Deep sequencing of amplicons generated from pools of flies performed equally well as individual sequencing approaches. We conclude that the analysis of fly-derived DNA can be implemented in a very rapid and cost-effective manner and will give a relatively unbiased picture of local mammal diversity. Carrion flies therefore represent an extraordinary and thus far unexploited resource of mammal DNA, which will probably prove useful for future inventories of wild mammal communities.
Nondetection of a species at a site does not imply that the species is absent unless the probability of detection is 1. We propose a model and likelihood-based method for estimating site occupancy rates when detection probabilities are 1. The model provides a flexible framework enabling covariate information to be included and allowing for missing observations. Via computer simulation, we found that the model provides good estimates of the occupancy rates, generally unbiased for moderate detection probabilities (0.3). We estimated site occupancy rates for two anuran species at 32 wetland sites in Maryland, USA, from data collected during 2000 as part of an amphibian monitoring program, Frog-watch USA. Site occupancy rates were estimated as 0.49 for American toads (Bufo amer-icanus), a 44% increase over the proportion of sites at which they were actually observed, and as 0.85 for spring peepers (Pseudacris crucifer), slightly above the observed proportion of 0.83.
Three species of land leeches, including a new combination Haemadipsa rjukjuanacomb. n., a new record for Haemadipsa picta Moore, as well as an updated description for Tritetrabdella taiwana (Oka), are reported in this study. Morphological characters and DNA barcode analysis were used to identify these species. In addition, since Haemadipsa rjukjuana had been regarded as a variety of the Japanese land leech Haemadipsa japonica for a century, morphological differences between these two species were also compared.
Southern Asia is a biodiversity hotspot both for terrestrial mammals and for leeches. Many small-mammal groups are under-studied in this region, while other mammals are of known conservation concern. In addition to standard methods for surveying mammals, it has recently been demonstrated that residual bloodmeals within leeches can be sequenced to find mammals in a given area. While these invertebrate-parasite-derived DNA (iDNA) methods are promising, most of the leech species utilized for this type of survey remain unevaluated, notwithstanding that their diversity varies substantially. Here we examine approximately 750 individual leech specimens in the genus Haemadipsa across a large range in southern Asia (Bangladesh, Cambodia, and China), specifically reviewing the diversity of mammals they feed on and their own genetic structuring. Leeches were found to feed on a considerable variety of mammals, corroborating prior studies. Additionally, leeches were found to have fed both on bats and on birds, neither of which has previously been recorded with this method. The genetic structuring of the leeches themselves revealed 15 distinct clades of which only two precisely corresponded to previously characterized species, indicating that much work is needed to finalize classifications in this genus. Most importantly, with regards to mammal conservation, leeches in these clades appear to feed on a broad range of mammals. © The Trustees of the Natural History Museum, London 2018. All Rights Reserved.
Measuring mammal biodiversity in tropical rainforests is challenging, and methods that reduce effort while maximizing success are crucial for long‐term monitoring programmes. Commonly used methods to assess mammal biodiversity may require substantial sampling effort to be effective. Genetic methods are a new and important sampling tool on the horizon, but obtaining sufficient DNA samples can be a challenge.
We evaluated the efficacy of using parasitic leeches Haemadipsa spp., as compared to camera trapping, to sample biodiversity. We collected 200 leeches from four forest patches in northeast Bangladesh, and identified recent vertebrate hosts using Sanger sequencing of the 16S rRNA gene extracted from each individual leech's blood meals. We then compared these data to species data from camera trapping conducted in the same forest patches.
Overall, 41.9% of sequenced leeches contained amplifiable non‐human mammal DNA. Four days of collecting leeches led to the identification of 12 species, compared to 26 species identified in 1,334 camera trap nights.
Synthesis and applications . After assessing the cost, effort and power of each technique, there are pros and cons to both camera trapping and leech blood meal analysis. Camera trapping and leech collection appear to be complementary approaches. When used together, they may provide a more complete monitoring tool for mammal biodiversity in tropical rainforests. Managers should consider adding leech collection to their biodiversity monitoring toolkit, as improved information will allow managers to create more effective conservation programmes.
Fungi are a megadiverse group of organisms, they play major roles in ecosystem functioning, and are important for human health, food production, and nature conservation. Our knowledge on fungal diversity and fungal ecology is however still very limited, in part because surveying and identifying fungi is time demanding and requires expert knowledge. We present a method that allows anyone to generate a list of fungal species likely to occur in a region of interest, with minimal effort and without requiring taxonomical expertise. The method consists of using a cyclone sampler to acquire fungal spores directly from the air to an Eppendorf tube, and applying DNA barcoding with probabilistic species identification to generate a list of species from the sample. We tested the feasibility of the method by acquiring replicate air samples from different geographical regions within Finland. Our results show that air sampling is adequate for regional-level surveys, with samples collected >100 km apart varying but samples collected <10 km apart not varying in their species composition. The data show marked phenology, and thus that obtaining a representative species list requires aerial sampling that covers the entire fruiting season. In sum, aerial sampling combined with probabilistic molecular species identification offers a highly effective method for generating a species list of air dispersing fungi. The method presented here has the potential to revolutionize fungal surveys, as it provides a highly cost-efficient way to include fungi as a part of large-scale biodiversity assessments and monitoring programs.
A crucial step in the use of DNA markers for biodiversity surveys is the assignment of Linnaean taxonomies (species, genus, etc.) to sequence reads. This allows the use of all the information known based on the taxonomic names. Taxonomic placement of DNA barcoding sequences is inherently probabilistic because DNA sequences contain errors, because there is natural variation among sequences within a species, and because reference data bases are incomplete and can have false annotations. However, most existing bioinformatics methods for taxonomic placement either exclude uncertainty, or quantify it using metrics other than probability.
In this paper we evaluate the performance of the recently proposed probabilistic taxonomic placement method PROTAX by applying it to both annotated reference sequence data as well as to unknown environmental data. Our four case studies include contrasting taxonomic groups (fungi, bacteria, mammals and insects), variation in the length and quality of the barcoding sequences (from individually Sanger‐sequenced sequences to short Illumina reads), variation in the structures and sizes of the taxonomies (800–130 000 species) and variation in the completeness of the reference data bases (representing 15–100% of known species).
Our results demonstrate that PROTAX yields essentially unbiased probabilities of taxonomic placement, which means its quantification of species identification uncertainty is reliable. As expected, the accuracy of taxonomic placement increases with increasing coverage of taxonomic and reference sequence data bases, and with increasing ratio of genetic variation among taxonomic levels over within taxonomic levels.
We conclude that reliable species‐level identification from environmental samples is still challenging and that neglecting identification uncertainty can lead to spurious inference. A key aim for future research is the completion of taxonomic and reference sequence data bases and making these two types of data compatible.
Motivation:
When targeted to a barcoding region, high-throughput sequencing can be used to identify species or operational taxonomical units from environmental samples, and thus to study the diversity and structure of species communities. Although there are many methods which provide confidence scores for assigning taxonomic affiliations, it is not straightforward to translate these values to unbiased probabilities. We present a probabilistic method for taxonomical classification (PROTAX) of DNA sequences. Given a pre-defined taxonomical tree structure that is partially populated by reference sequences, PROTAX decomposes the probability of one to the set of all possible outcomes. PROTAX accounts for species that are present in the taxonomy but that do not have reference sequences, the possibility of unknown taxonomical units, as well as mislabeled reference sequences. PROTAX is based on a statistical multinomial regression model, and it can utilize any kind of sequence similarity measures or the outputs of other classifiers as predictors.
Results:
We demonstrate the performance of PROTAX by using as predictors the output from BLAST, the phylogenetic classification software TIPP, and the RDP classifier. We show that PROTAX improves the predictions of the baseline implementations of TIPP and RDP classifiers, and that it is able to combine complementary information provided by BLAST and TIPP, resulting in accurate and unbiased classifications even with very challenging cases such as 50% mislabeling of reference sequences.
Availability:
Perl/R implementation of PROTAX is available at http://www.helsinki.fi/science/metapop/Software.htm CONTACT: panu.somervuo@helsinki.fi SUPPLEMENTARY INFORMATION: Supplement is available at Bioinformatics online.
Most tropical mammal species are threatened or data-deficient. Data collection is impeded by the traditional monitoring approaches which can be laborious, expensive and struggle to detect cryptic diversity. Monitoring approaches using mammal DNA derived from invertebrates are emerging as cost- and time-effective alternatives. As a step towards development of blowfly-derived DNA as an effective method for mammal monitoring in the biodiversity hotspot of Peninsular Malaysia, our objectives were (i) to determine the persistence period of amplifiable mammal mtDNA in blowfly guts through a laboratory feeding experiment (ii) to design and test primers that can selectively amplify mammal COI DNA mini-barcodes in the presence of high concentrations of blowfly DNA. The persistence period of amplifiable mammal mtDNA in blowfly guts was 24 h to 96 h post-feeding indicating the need for collecting flies within 24 h of capture to detect mammal mtDNA of sufficient quantity and quality. We designed a new primer combination for a COI DNA mini-barcode that did not amplify blowfly DNA and showed 89% amplification success for a dataset of mammals from Peninsular Malaysia. The short (205 bp) DNA mini-barcode could distinguish most mammal species (including separating dark taxa) and is of suitable length for high-throughput sequencing. Our new DNA mini-barcode target and a standardized trapping protocol with retrieval of blowflies every 24 h could point the way forward in the development of blow-fly-derived DNA as an effective method for mammal monitoring.
DNA analysis from carrion flies (iDNA analysis) has recently been promoted as a powerful tool for cost and time efficient monitoring of wildlife. While originally applied to identify any mammalian species present in an area, it should also allow for targeted detection of species and individuals. Using carrion flies captured in the Taï National Park, Côte d'Ivoire, we assessed this possibility by i) screening carrion fly DNA extracts with non-specific and species-specific PCR systems respectively targeting mitochondrial DNA (mtDNA) fragments of any mammal or of Jentink's duiker (Cephalophus jentinki), three colobine monkeys (subfamily Colobinae) and sooty mangabey (Cercocebus atys) and ii) genotyping carrion fly extracts containing sooty mangabey mtDNA. In comparison with the non-specific PCR assay, the use of specific PCRs increased the frequency of detection of target species up to three-fold. Detection rates partially reflected relative abundances of target species in the area. Amplification of seven microsatellite loci from carrion flies positive for sooty mangabey mtDNA yielded an average PCR success of 46%, showing that the identification of individuals is, to some extent, possible. Regression analysis of microsatellite PCR success and mtDNA concentration revealed that, among all carrion flies analysed for this study, 1% contained amounts of mammal mtDNA sufficient to attempt genotyping with potentially high success. We conclude that carrion fly-derived DNA analysis represents a promising tool for targeted monitoring of mammals in their natural habitat.This article is protected by copyright. All rights reserved.
Extraction and identification of DNA from an environmental sample has proven noteworthy recently in detecting and monitoring not only common species, but also those that are endangered, invasive, or elusive. Particular attributes of so-called environmental DNA (eDNA) analysis render it a potent tool for elucidating mechanistic insights in ecological and evolutionary processes. Foremost among these is an improved ability to explore ecosystem-level processes, the generation of quantitative indices for analyses of species, community diversity, and dynamics, and novel opportunities through the use of time-serial samples and unprecedented sensitivity for detecting rare or difficult-to-sample taxa. Although technical challenges remain, here we examine the current frontiers of eDNA, outline key aspects requiring improvement, and suggest future developments and innovations for research.
Estimates of species richness and diversity are central to community and macroecology and are frequently used in conservation planning. Commonly used diversity metrics account for undetected species primarily by controlling for sampling effort. Yet the probability of detecting an individual can vary among species, observers, survey methods, and sites. We review emerging methods to estimate alpha, beta, gamma, and metacommunity diversity through hierarchical multispecies occupancy models (MSOMs) and multispecies abundance models (MSAMs) that explicitly incorporate observation error in the detection process for species or individuals. We examine advantages, limitations, and assumptions of these detection-based hierarchical models for estimating species diversity. Accounting for imperfect detection using these approaches has influenced conclusions of comparative community studies and creates new opportunities for testing theory.
1. The use of environmental DNA (eDNA) to detect species in aquatic environments such as ponds and streams is a powerful new technique with many benefits. However, species detection in eDNA-based surveys is likely to be imperfect, which can lead to underestimation of the distribution of a species.
Site occupancy models account for imperfect detection and can be used to estimate the proportion of sites where a species occurs from presence/absence survey data, making them ideal for the analysis of eDNA-based surveys. Imperfect detection can result from failure to detect the species during field work (e.g. by water samples) or during laboratory analysis (e.g. by PCR).
To demonstrate the utility of site occupancy models for eDNA surveys, we reanalysed a data set estimating the occurrence of the amphibian chytrid fungus Batrachochytrium dendrobatidis using eDNA. Our reanalysis showed that the previous estimation of species occurrence was low by 5–10%. Detection probability was best explained by an index of the number of hosts (frogs) in ponds.
Per-visit availability probability in water samples was estimated at 0·45 (95% CRI 0·32, 0·58) and per-PCR detection probability at 0·85 (95% CRI 0·74, 0·94), and six water samples from a pond were necessary for a cumulative detection probability >95%. A simulation study showed that when using site occupancy analysis, researchers need many fewer samples to reliably estimate presence and absence of species than without use of site occupancy modelling.
Our analyses demonstrate the benefits of site occupancy models as a simple and powerful tool to estimate detection and site occupancy (species prevalence) probabilities despite imperfect detection. As species detection from eDNA becomes more common, adoption of appropriate statistical methods, such as site occupancy models, will become crucial to ensure that reliable inferences are made from eDNA-based surveys.
1. The proportion of sites occupied by a species is a state variable commonly used in ecology. Replicate surveys at sampled sites can be used to address the problem of imperfect detection to obtain an asymptotically unbiased estimator of species occupancy. Replication can be achieved in various ways, including visiting a series of spatial subunits within each surveyed site. Depending on how these spatial subunits are sampled, bias can be induced in the estimator of site occupancy.
2. This paper explores and discusses the consequences of sampling with or without replacement when using spatial replication in occupancy studies and the species is absent from some of the replicates within occupied sites. Simulations are used to illustrate the findings, and results are compared with previously published recommendations.
3. The study shows that sampling without replacement does not induce bias in the estimator of site occupancy when each spatial subunit has a constant probability of occupancy, regardless of the occupancy status of other subunits within the same sampling site.
4. The study further demonstrates that sampling with replacement may in this case induce bias in the site occupancy estimator. It is thus argued that choosing to sample with or without replacement should be based on considerations of the system in question.
5. Synthesis and applications: In some occupancy studies, it may be convenient to obtain the replication required to account for imperfect detection by surveying various spatial subunits within each sampling site. Sampling with replacement has previously been recommended for this scenario. However, this sampling strategy can induce bias in the estimator of occupancy under realistic ecological conditions. The discussion provided here is of special interest for those involved in the design of occupancy studies.
The fraction of sampling units in a landscape where a target species is present (occupancy) is an extensively used concept in ecology. Yet in many applications the species will not always be detected in a sampling unit even when present, resulting in biased estimates of occupancy. Given that sampling units are surveyed repeatedly within a relatively short timeframe, a number of similar methods have now been developed to provide unbiased occupancy estimates. However, practical guidance on the efficient design of occupancy studies has been lacking.
In this paper we comment on a number of general issues related to designing occupancy studies, including the need for clear objectives that are explicitly linked to science or management, selection of sampling units, timing of repeat surveys and allocation of survey effort. Advice on the number of repeat surveys per sampling unit is considered in terms of the variance of the occupancy estimator, for three possible study designs.
We recommend that sampling units should be surveyed a minimum of three times when detection probability is high (> 0·5 survey ⁻¹ ), unless a removal design is used.
We found that an optimal removal design will generally be the most efficient, but we suggest it may be less robust to assumption violations than a standard design.
Our results suggest that for a rare species it is more efficient to survey more sampling units less intensively, while for a common species fewer sampling units should be surveyed more intensively.
Synthesis and applications . Reliable inferences can only result from quality data. To make the best use of logistical resources, study objectives must be clearly defined; sampling units must be selected, and repeated surveys timed appropriately; and a sufficient number of repeated surveys must be conducted. Failure to do so may compromise the integrity of the study. The guidance given here on study design issues is particularly applicable to studies of species occurrence and distribution, habitat selection and modelling, metapopulation studies and monitoring programmes.
Aim Spatial autocorrelation (SAC) in data, i.e. the higher similarity of closer samples, is a common phenomenon in ecology. SAC is starting to be considered in the analysis of species distribution data, and over the last 10 years several studies have incorporated SAC into statistical models (here termed ‘spatial models’). Here, I address the question of whether incorporating SAC affects estimates of model coefficients and inference from statistical models.
Methods I review ecological studies that compare spatial and non‐spatial models.
Results In all cases coefficient estimates for environmental correlates of species distributions were affected by SAC, leading to a mis‐estimation of on average c . 25%. Model fit was also improved by incorporating SAC.
Main conclusions These biased estimates and incorrect model specifications have implications for predicting species occurrences under changing environmental conditions. Spatial models are therefore required to estimate correctly the effects of environmental drivers on species present distributions, for a statistically unbiased identification of the drivers of distribution, and hence for more accurate forecasts of future distributions.
Summary • Occupancy estimation and modelling based on detection–nondetection data provide an effective way of exploring change in a species’ distribution across time and space in cases where the species is not always detected with certainty. Today, many monitoring programmes target multiple species, or life stages within a species, requiring the use of multiple detection methods. When multiple methods or devices are used at the same sample sites, animals can be detected by more than one method. • We develop occupancy models for multiple detection methods that permit simultaneous use of data from all methods for inference about method-specific detection probabilities. Moreover, the approach permits estimation of occupancy at two spatial scales: the larger scale corresponds to species’ use of a sample unit, whereas the smaller scale corresponds to presence of the species at the local sample station or site. • We apply the models to data collected on two different vertebrate species: striped skunks Mephitis mephitis and red salamanders Pseudotriton ruber. For striped skunks, large-scale occupancy estimates were consistent between two sampling seasons. Small-scale occupancy probabilities were slightly lower in the late winter/spring when skunks tend to conserve energy, and movements are limited to males in search of females for breeding. There was strong evidence of method-specific detection probabilities for skunks. As anticipated, large- and small-scale occupancy areas completely overlapped for red salamanders. The analyses provided weak evidence of method-specific detection probabilities for this species. • Synthesis and applications. Increasingly, many studies are utilizing multiple detection methods at sampling locations. The modelling approach presented here makes efficient use of detections from multiple methods to estimate occupancy probabilities at two spatial scales and to compare detection probabilities associated with different detection methods. The models can be viewed as another variation of Pollock's robust design and may be applicable to a wide variety of scenarios where species occur in an area but are not always near the sampled locations. The estimation approach is likely to be especially useful in multispecies conservation programmes by providing efficient estimates using multiple detection devices and by providing device-specific detection probability estimates for use in survey design.
With nearly one quarter of mammalian species threatened, an accurate description of their distribution and conservation status is needed [1]. For rare, shy or cryptic species, existing monitoring methods are often prohibitively expensive or unreliable. The problem is particularly acute in tropical forests, where a disproportionate number of species are listed by IUCN as 'data deficient'[2], due to the difficulty of monitoring with conventional approaches. This presents serious obstacles to conservation management. We, here, describe a new screening tool, the analysis of mammalian DNA extracted from haematophagous leeches. By demonstrating that PCR amplifiable mammalian blood DNA survives for at least four months post feeding in haematophagous Hirudo spp. leeches, we hypothesise that most wild caught adult leeches will contain DNA traces of their last blood meal. We subsequently demonstrate the efficacy of the method, by testing it in situ using terrestrial Haemadipsa spp. leeches caught in a tropical Vietnamese rainforest setting, and identify cryptic, rare and newly discovered mammalian species. We propose that DNA from leeches represents a quick, cost-effective and standardised way to obtain basic data on mammalian biodiversity and species occupancy, facilitating efficient use of limited conservation resources.
Ticks are among the most important vectors of disease in the Northern Hemisphere, and a better understanding of their feeding behaviour and life cycle is critical to the management and control of tick-borne zoonoses. DNA-based tools for the identification of residual bloodmeals in hematophagous arthropods have proven useful in the investigation of patterns of host use in nature. Using a blind test approach, we challenged the utility of the DNA barcode library for the identification of vertebrate bloodmeals in engorged, field-collected Ixodes scapularis. Universal vertebrate primers for the COI barcode region successfully amplified DNA from the host bloodmeal and only rarely amplified tick DNA. Of the 61 field-collected ticks, conclusive genus- and species-level identification was possible for 72% of the specimens. In all but two cases, barcode-based identification of the bloodmeal was consistent with the morphological identification of the vertebrate host the ticks were collected from. Possible explanations for mismatches or ambiguities are presented. This study validates the utility of the DNA barcode library as a valuable and reliable resource for the identification of unknown bloodmeals in arthropod vectors of disease. Future directions aimed at the refinement of these techniques to gain additional information and to improve the amplification success of digested vertebrate DNA in tick bloodmeals are discussed.
Autocorrelation is a very general statistical property of ecological variables observed across geographic space; its most common forms are patches and gradients. Spatial autocorrelation, which comes either from the physical forcing of environmental variables or from community processes, presents a problem for statistical testing because autocorrelated data violate the assumption of independence of most standard statistical procedures. The paper discusses first how autocorrelation in ecological variables can be described and measured, with emphasis on mapping techniques. Then, proper statistical testing in the presence of autocorrelation is briefly discussed. Finally, ways are presented of explicitly introducing spatial structures into ecological models. Two approaches are proposed; in the raw-data approach, the spatial structure takes the form of a polynomial of the x and y geographic coordinates of the sampling stations; in the matrix approach, the spatial structure is introduced in the form of a geog