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Identification of Southeastern Bat Species Using Non-invasive Genetic Sampling of Individual Guano Pellets
Abstract
The impact of white-nose syndrome on North American bat populations may limit the effectiveness of traditional monitoring methods, including roost surveys, mist netting, and acoustic monitoring, and, in turn, determination of bat species occurrence. Genetic markers from deoxyribonucleic acid (DNA) extracted from feces (i.e., guano pellets) may provide an effective alternative method for assessing occurrence. We used an existing genetic marker from the 16S ribosomal subunit, mitochondrial DNA, to create a DNA sequence database for the 16 species of bats known to occur in Tennessee. We used our database to identify bat species from DNA extracted from 68 guano pellets collected from accumulations found in buildings of Great Smoky Mountains National Park from May to August 2015. No bats were directly observed at 19 roost buildings (55.9% of all identified roost buildings), where genetic analysis of guano was the only method available to determine species occurrence. Two of the species we detected roosting in buildings using DNA from guano, the little brown myotis Myotis lucifugus and northern long-eared myotis M. septentrionalis, are of special concern as a result of declines from white-nose syndrome. There are no records of the northern long-eared myotis roosting in Great Smoky Mountains National Park buildings, and no records of the little brown myotis roosting in buildings since white-nose syndrome became established in Great Smoky Mountains National Park. Our findings emphasize the utility of these genetic techniques for detecting bat species when visual or acoustic methods may be compromised by species rarity, elusive behavior, or similarities in species morphology and call characteristics.
... Rich soils, a hot climate with a good rainfall and copious underground water foster good crop growth in Jamaica. Agricultural activities occupy nearly 50% of the island, where the most common crops are sugar cane, coconut, bananas, citrus, coffee, maize, cocoa and rice plantation (Blume, 1974;Genoways et al., 2005). Changes in vegetation caused by agriculture influence pollen assemblages. ...
... Identification of bats from guano-derived DNA dates back to the early 2000 (Zinck et al., 2004). This approach has been successfully applied to fresh and modern -up to 20 years -samples (e.g., Brown et al., 2017;Walker et al., 2019;Guan et al., 2020). Overall, in all fecal DNA metabarcoding studies, the gold standard is to collect fresh feces for maximum DNA recovery. ...
The discovery of two undisturbed caves in Jamaica with 14 C and 210 Pb dating indicating that the oldest layers of guano were ca. 200 years old in the first cave (81 cm long core), and as much as 4300 years old in the second cave (129 cm long core) provides exciting possibilities to examine past ecological communities. We analyzed genetic and pollen profiles in these ancient bat guano deposits and revealed DNA sequences most similar to arthropods, mainly insects such as caddisflies (Trichoptera), butterflies (Lepidoptera) and mayflies (Ephemer-optera), suggesting a potential source for past dietary data. Palynological analysis failed to recover well-preserved pollen grains and spores older than ca. 200 years, however in layers preserving pollen, angiosperms were the most abundant plants observed, with a regular occurrence of the potato (Solanaceae) and pepper families (Piperaceae), which are frequently bat-dispersed. In general, changes in frequency of particular plant taxa appear to reflect changes of vegetation and land use in the cave vicinity; however, some changes could be linked to hurricanes, leading to forest canopy damage and promoting the growth of light-demanding species such as guarumo (Cecropia). Higher amounts of mangrove (Rhizophora) pollen have also been recorded in the periods of heavy hurricane activities. Our data highlight the value of bat guano deposits in caves as a resource for the analysis of past ecological systems and stress the conservation values of these deposits.
... We extracted fecal DNA from individual pellets using PowerSoil DNA Isolation Kits (Mo Bio Laboratories, Carlsbad, CA, USA), which we then amplified by polymerase chain reaction (PCR). We sequenced positive PCR results via Sanger sequencing on an Applied Biosystems 3730 DNA analyzer (Applied Biosystems, Foster City, CA, USA), and then compared results to a reference database for bat species of the southeastern United States in Sequencher version 5.0.1 (GeneCodes, Ann Arbor, MI, USA; Brown et al. 2017). Capture, handling, and sample collection protocols were authorized under scientific collection permits from the USFWS (TE35313B-3) and NPS (GRSM-2015-SCI-1228), approved by the University of Tennessee Institutional Animal Care and Use Committee (IACUC 2026-0515) and by the American Society of Mammalogists (Sikes et al. 2016). ...
... Discerning between day, night, and alternate roosts, however, is not possible at roosts indicated only by guano. Genetic analysis of DNA extracted from guano, in buildings or natural roost sites, is useful for detecting bat species that are cryptic or exhibit frequent roost switching, and is an important tool for researchers and managers in light of declines in populations of North America bats as a result of WNS (Turner et al. 2011, Frick et al. 2015, Brown et al. 2017). ...
Bats roosting in buildings are a challenge for wildlife managers because of their conservation needs, capacity to transmit disease, and potential to damage structures. An understanding of roost selection by bats in buildings is important for effective management but was lacking in the southeastern United States. During 2015 and 2016, we surveyed 140 buildings in Great Smoky Mountains National Park in eastern Tennessee and western North Carolina, USA for bats, identifying 48 roost sites and detecting 5 species. We compared the microclimate conditions, building features, and habitat patch characteristics of buildings used and unused by bats using an information-theoretic approach. Averaged parameter estimates from logistic regression models developed with survey data indicated bat presence was more likely in old buildings with dark conditions surrounded by low road density. Of all roost buildings surveyed, 65.9% were accessed regularly by tourists and 68.2% were managed as historical structures. We present alternative management strategies that may be implemented to ensure human health and safety and preserve historical sites while protecting bats during the reproductive period. © 2017 The Wildlife Society.
... The authors successfully extracted DNA from the big brown bat (Eptesicus fuscus) feces and demonstrated that the genotypes derived from fecal material completely matched those obtained from tissue-derived DNA of the same individuals. Recently, Brown et al. (2017) extracted DNA from guano pellets accumulated in buildings at the Great Smoky Mountains National Park (Tennessee, USA), and sequenced 175-185 bp of 16S rRNA. They were able to identify the bat species roosting at the study site, many not previously observed there, including some with Neotropical distribution. ...
Although all living beings modify their environment, human beings have acquired the ability to do so on a superlative space-time scale. As a result of industrialization and the use of new technologies, the anthropogenic impact has been increasing in the last centuries, causing reductions in the sizes or the extinction of numerous wild populations. In this sense, from the field of conservation genetics, various efforts have been made in recent decades to provide new knowledge that contributes to the conservation of populations, species, and habitats. In this book, we summarize the concrete contributions of researchers to the conservation of the Neotropical mammals using Molecular Ecology techniques.
The book is divided into three major sections. The first section provides an up-to-date review of the conservation status of Neotropical mammals, the applications of the molecular markers in its conservation, and the use of non-invasive and forensic genetic techniques. The second and third sections present, respectively, a series of case studies in various species or taxonomic groups of Neotropical mammals.
... The authors successfully extracted DNA from the big brown bat (Eptesicus fuscus) feces and demonstrated that the genotypes derived from fecal material completely matched those obtained from tissue-derived DNA of the same individuals. Recently, Brown et al. (2017) extracted DNA from guano pellets accumulated in buildings at the Great Smoky Mountains National Park (Tennessee, USA), and sequenced 175-185 bp of 16S rRNA. They were able to identify the bat species roosting at the study site, many not previously observed there, including some with Neotropical distribution. ...
The vast mammal diversity of the Neotropics is the result of a long evolutionary history. Among mammals, rodents comprise more than half of all Neotropical mammal species, and South America is home to about a quarter of the entire world’s rodent species. However, the economic growth of the region still depends strongly on natural resource exploitation and has high rates of environmental degradation and biodiversity loss. In this context, conservation genetics offers means to help reducing current extinction rates and to preserve biodiversity. In this chapter, we discuss conservation genetics using examples from studies developed in the Neotropical region with rodents from the Sigmodontinae sub-family and rodents from the genus Ctenomys (Caviomorpha). Although some species are well studied and present a large amount of data based on different approaches, long-term genetic studies are needed to understand better the combined impacts of all ‘non-genetic’ and genetic threats faced by populations. Such studies are important because understanding these issues is still far from being accomplished for caviomorphs and sigmodontines in the Neotropical region. Moreover, conservation genetics is essential because it also addresses the conservation of processes like speciation and hybridization.
... The authors successfully extracted DNA from the big brown bat (Eptesicus fuscus) feces and demonstrated that the genotypes derived from fecal material completely matched those obtained from tissue-derived DNA of the same individuals. Recently, Brown et al. (2017) extracted DNA from guano pellets accumulated in buildings at the Great Smoky Mountains National Park (Tennessee, USA), and sequenced 175-185 bp of 16S rRNA. They were able to identify the bat species roosting at the study site, many not previously observed there, including some with Neotropical distribution. ...
Bats constitute the second most diverse group of mammals, accounting for ca. 20% of mammalian diversity, with 1400 extant species recognized worldwide. Of these, about 400 taxa occur in the Neotropical realm. Bats provide a number of environmental services, playing a crucial role in maintaining integrity of ecosystems. Like many other organisms, bats are struggling to persist in face of the dramatic environmental and climatic changes caused by human activities. The use of DNA-based methods has greatly contributed to study Neotropical Chiroptera in many aspects such as systematics, ecology, and basic biology, including dispersal, mating systems and foraging patterns, population genetic structure, and the identification of Management and Evolutionarily Significant Units deserving special attention with regard to conservation. Here, we illustrate some of the research questions that have been addressed in Neotropical bats using conservation genetics tools. We discuss current limitations and challenges, and identify knowledge gaps and questions for future research, which could be answered using innovative methods and technological advances in this field.
... The authors successfully extracted DNA from the big brown bat (Eptesicus fuscus) feces and demonstrated that the genotypes derived from fecal material completely matched those obtained from tissue-derived DNA of the same individuals. Recently, Brown et al. (2017) extracted DNA from guano pellets accumulated in buildings at the Great Smoky Mountains National Park (Tennessee, USA), and sequenced 175-185 bp of 16S rRNA. They were able to identify the bat species roosting at the study site, many not previously observed there, including some with Neotropical distribution. ...
Molecular genetic data are increasingly used to assist in species identification and delimitation, and provide a powerful tool to detect conservation units (population/s of an organism considered to be different for conservation purposes). In combination with distributional and ecological information, molecular genetic data can contribute to establishing the conservation status of species and populations. Here, we review these applications to rodent species in Argentina, a diverse assemblage of some 200 species. We capitalize on recent efforts to establish the conservation status of mammals in Argentina and combine this information with available molecular genetic data for rodents in this country. This portion of the southern South America offers exceptional cases of population differentiation and species diversification related to a wide and diverse geography, which includes varied landscapes such as the Patagonian steppe, Andean highlands, tropical and subtropical forests, and the Monte and Pampa regions. Many species are data deficient (including no or limited genetic data) and only known from their type localities. Among the remaining species, most have at least some available mitochondrial DNA sequence data and these, coupled with morphological, karyotypic and (less frequently) nuclear DNA sequence or microsatellite data have been instrumental in identifying and delimiting species. In general, multilocus data with the desirable geographical density are lacking for all but a few species that have been studied in greater detail. Studies coupling genetic and ecological data (e.g. landscape or ecological genetics) are limited to a handful of more intensively studied cases. Given that habitat loss and fragmentation are among the major threats for survival of natural populations, assessing the extent of these threatening processes is crucial in conservation management and a priority in studies of conservation biology.
... Using COI sequences mined from Genbank and new sequences from collected samples, Korstian et al. (2016) tested the use of COI locus for DNA barcoding of all 47 bat species found in the United States; 80% of species examined had distinct barcodes, including most species currently listed as threatened or endangered. Using a different molecular marker, Brown et al. (2017) screened sequences from the mitochondrial 16S ribosomal subunit to create a DNA sequence database for 16 species of bats known to occur in Tennessee, USA and demonstrated its usefulness in identifying southeastern bat species using guano. ...
Species identification is often difficult, especially for early life-history stages, poorly known species within diverse taxa, and microbes. Molecular genetics has contributed the technique of DNA barcoding, offering a low-tech, potentially high-impact tool for identification of species. After briefly describing a range of applications, this review focus on its use for identification of larval fishes. Molecular identification of larval fishes would increase knowledge of larval fish ecology, providing insights into reproductive ecology and population dynamics, and contribute to identification and protection of critical habitat. Other applications of environmental interest include identification of species from fecal starting material and forensic investigation. Limiting application of DNA barcoding is the environmental community's unfamiliarity withthe technique and limited development of DNA sequence archives for some taxa.
... of confirmed Gray Bats (Brown et al. 2017). On 22 July 2016, we resurveyed the same location on Cosby Creek. ...
We captured 3 male federally endangered Myotis grisescens (Gray Bat) in the Cosby Creek area of Great Smoky Mountains National Park (GRSM) in Cocke County, TN, during summer mist-netting surveys (15 and 22 July 2016). Although GRSM is known for its diversity of bat species (n = 11), these specimens represent the first Gray Bats, and twelfth bat species, captured within the GRSM boundary. The documentation of this species is important, as it is the third federally listed bat species (along with Myotis sodalis [Indiana Bat] and Myotis septentrionalis [Northern Long-eared Bat]) known to occur within GRSM. We recommend further research be conducted on Gray Bats in GRSM to understand the species’ habitat use and potential implications for park management.
... Large maternity colonies of this species were observed roosting in buildings before WNS became established in GRSM (Harvey, 1987), and recent documentation of sparse little brown bat roosts in buildings appear consistent with observed population declines throughout eastern North America (Brown et al., 2017;Fagan et al., 2017). We found that attitudes toward bats, perceived severity of threats to bats, and perceived importance of ecosystem services were important factors influencing support for bat management. ...
In the human dimensions of wildlife management, evaluating stakeholder perceptions of target species helps inform effective conservation efforts. Stakeholder perceptions are invaluable when managing taxa like bats, which may have historically negative cultural preconceptions. However, insectivorous bats provide critical ecosystem services in North America through agricultural insect pest control, and many of these species are threatened by white-nose syndrome (WNS), a disease caused by an invasive fungal pathogen. In Great Smoky Mountains National Park (GRSM), the most visited National Park in the United States (USA), bats are regularly observed roosting in historical buildings by visitors and park employees during summer. As a result, natural and cultural resource managers seek to ensure public safety and protect historical structures while minimizing impacts on bats, especially in light of declines in bat populations as a result of WNS. However, managers lacked information on visitor perceptions of bats and support for potential management action regarding the taxon. From June to August 2016, we surveyed 420 park visitors at three sites in the Cades Cove area of GRSM on their attitudes toward bats, perception of threats to and ecosystem services provided by bats, and support for management of bats. Most respondents supported management action to protect bats in buildings in Cades Cove during summer (76%). Standardized parameter estimates from a multiple linear regression developed with survey data indicated that attitudes toward bats and perception of threats to bats had the greatest effects on support for bat management. Wildlife management and conservation agencies seeking to further cultivate support for management of bats roosting in public spaces may apply these results in the design of tailored programming and outreach materials.
In the original article,Box 1 was not printed in its entirety, and two references were badly quoted in page 3 of the pdf and were missing in the reference list.
The use of DNA data is ubiquitous across animal sciences. DNA may be obtained from an organism for a myriad of reasons including identification and distinction between cryptic species, sex identification, comparisons of different morphocryptic genotypes or assessments of relatedness between organisms prior to a behavioural study. DNA should be obtained while minimizing the impact on the fitness, behaviour or welfare of the subject being tested, as this can bias experimental results and cause long-lasting effects on wild animals. Furthermore, minimizing impact on experimental animals is a key Refinement principle within the ‘3Rs’ framework which aims to ensure that animal welfare during experimentation is optimised. The term ‘non-invasive DNA sampling’ has been defined to indicate collection methods that do not require capture or cause disturbance to the animal, including any effects on behaviour or fitness. In practice this is not always the case, as the term ‘non-invasive’ is commonly used in the literature to describe studies where animals are restrained or subjected to aversive procedures. We reviewed the non-invasive DNA sampling literature for the past six years (380 papers published in 2013-2018) and uncovered the existence of a significant gap between the current use of this terminology (i.e. ‘non-invasive DNA sampling’) and its original definition. We show that 58% of the reviewed papers did not comply with the original definition. We discuss the main experimental and ethical issues surrounding the potential confusion or misuse of the phrase ‘non-invasive DNA sampling’ in the current literature and provide potential solutions. In addition, we introduce the terms ‘non-disruptive’ and ‘minimally disruptive’ DNA sampling, to indicate methods that eliminate or minimise impacts not on the physical integrity/structure of the animal, but on its behaviour, fitness and welfare, which in the literature reviewed corresponds to the situation for which an accurate term is clearly missing. Furthermore, we outline when these methods are appropriate to use.
Bat guano is a noninvasive, data-rich genetic resource. However, the constituent bat DNA is relatively scant, degraded, and complexed with polymerase chain reaction inhibitors. It also is comingled with a rich pool of nontarget DNA from microbes, parasites, and dietary items. We designed and tested new DNA assays for bat species identification (COX1-Bat) and sex identification (XGXYC) for use with guano and other challenging samples. We reviewed previously published assays that can be used with guano samples to obtain the same species and sex data, and attempted to validate these assays for species in which they had not previously been tested. Our results demonstrate that guano-derived DNA can be used successfully to 1) identify nearly all US and Canadian bats at the species level, or to one of three Myotis species clusters, and 2) identify the sex of at least 23 US and Canadian bat species. Our newly developed assays, and validation of previously published assays, for guano-based identification of species and sex in bats, significantly enhance the power of noninvasive sampling and genetic analysis for bat studies, management, and conservation.
In North America, bat research efforts largely have focused on summer maternity colonies and winter hibernacula, leaving the immediate pre- and post-hibernation ecology for many species unstudied. Understanding these patterns and processes is critical for addressing potential additive impacts to White-nose Syndrome (WNS)-affected bats, as autumn is a time of vital weight gain and fat resources are largely depleted in early spring in surviving individuals. Our study sought to examine autumn and spring bat activity patterns in the central Appalachian Mountains around three hibernacula to better understand spatio-temporal patterns during staging for hibernation and post-hibernation migration in the post-WNS environment. From early September through November 2015 and 2016, and from early March through April 2016 and 2017, we assessed the effects of distance to hibernacula and ambient conditions on nightly bat activity for Myotis spp. and big brown bats (Eptesicus fuscus) using zero-crossing frequency division bat detectors near cave entrances and 1 km, 2 km, and 3 km distant from caves. Following identification of echolocation calls, we used generalized linear mixed effects models to examine patterns of activity across the landscape over time and relative to weather. Overall bat activity was low at all sample sites during autumn and spring periods except at sites closest to hibernacula. Best-supported models describing bat activity varied, but date and ambient temperatures generally appeared to be major drivers of activity in both seasons. Total activity for all species had largely ceased by mid-November. Spring bat activity was variable across the sampling season, however, some activity was observed as early as mid-March, almost a month earlier than the historically accepted emergence time regionally. Current timing of restrictions on forest management activities that potentially remove day-roosts near hibernacula when bats are active on the landscape may be mismatched with actual spring post-hibernation emergence. Adjustments to the timing of these restrictions during the spring may help to avoid potentially additive negative impacts on WNS-impacted bat species.
Identifying species from non-invasively collected, mixed species samples for biodiversity monitoring can be difficult and expensive using typical molecular methods because samples are often degraded. This is the case when identifying the species of bats present in roosts where guano may be the only means of assessment without disturbing the bats themselves. To aid in such studies, we developed species-specific DNA probes and a microarray capable of identifying most bat species in the United States and Canada using our existing database of 16S mitochondrial DNA sequences. The microarray was sensitive enough to detect DNA diluted 1:500 for several species combinations and was able to detect the presence of more species in mixed guano samples collected from roosts than did direct sequencing from individual fecal pellets. We suggest that these DNA probes and the microarray could be a valuable tool with which to non-invasively monitor bat populations and roost use.
Resumen
Los lineamientos para el uso de especies de mamíferos de vida silvestre en la investigación con base en Sikes et al. (2011) se actualizaron. Dichos lineamientos cubren técnicas y regulaciones profesionales actuales que involucran el uso de mamíferos en la investigación y enseñanza; también incorporan recursos nuevos, resúmenes de procedimientos y requisitos para reportes. Se incluyen detalles acerca de captura, marcaje, manutención en cautiverio y eutanasia de mamíferos de vida silvestre. Se recomienda que los comités institucionales de uso y cuidado animal (cifras en inglés: IACUCs), las agencias reguladoras y los investigadores se adhieran a dichos lineamientos como fuente base de protocolos que involucren mamíferos de vida silvestre, ya sea investigaciones de campo o en cautiverio. Dichos lineamientos fueron preparados y aprobados por la ASM, en consulta con profesionales veterinarios experimentados en investigaciones de vida silvestre y IACUCS, de quienes cuya experiencia colectiva provee un entendimiento amplio y exhaustivo de la biología de mamíferos no-domesticados. La presente versión de los lineamientos y modificaciones posteriores están disponibles en línea en la página web de la ASM, bajo Cuidado Animal y Comité de Uso: (http://mammalogy.org/uploads/committee_files/CurrentGuidelines.pdf). Recursos adicionales relacionados con el uso de animales de vida silvestre para la investigación se encuentran disponibles en (http://www.mammalsociety.org/committees/animal-care-and-use#tab3).
Indiana bats (Myotis sodalis) are capable of exploiting ephemeral resources in structurally complex, interior forests. Consequently, surveying for this species is difficult, and even in areas harboring known populations, documenting presence remains a challenge. We aimed to determine what factors affected probability of detection (p) and site occupancy (ψ) for Indiana bats when sampling with acoustic bat detectors near an active maternity colony. From May to August 2012 and 2013, we passively sampled 71 random points near Indianapolis, Indiana, with Wildlife Acoustic SM2BAT+ detectors. On average, sampling occurred ~2.2 km from maternity colony roosts, which contained ≥ 150 bats by mid-July. Habitat and landscape covariates were measured in the field or via ArcGIS. We reduced model covariates with Pearson's correlation and principal component analysis. Indiana bat calls were identified using Bat Call ID software. We used Presence 6.1 software and Akaike's Information Criteria to assess models for detectability and occupancy. Detectability of Indiana bats increased as " forest closure " and mean nightly temperature increased, likely due to reduced clutter and increased bat activity, respectively. The null model best explained ψ, but models were likely underpowered from few Indiana bat detections. Higher rates for ψ were observed near agricultural lands, indicating this cover type may fulfill certain habitat requirements for Indiana bats within the study site. Overall, detection rates were low even when sampling near a concentrated population on its maternity grounds, likely due to limitations inherent to bat detector technologies. Managers should be aware of the limitations of using passive acoustic sampling as a means by which to document presence– absence for this endangered species.