The structure of tropical bat-plant interaction networks during an extreme El Niño-Southern Oscillation event.

Interaction network structure reflects the ecological mechanisms acting within biological communities, which are affected by environmental conditions. In tropical forests, higher precipitation usually increases fruit production, which may lead frugivores to increase specialization, resulting in more modular and less nested animal-plant networks. In these ecosystems, El Niño is a major driver of precipitation, but we still lack knowledge of how species interactions change under this influence. To understand bat-plant network structure during an extreme El Niño-Southern Oscillation event, we determined the links between plantivorous bat species and the plants they consume by DNA barcoding seeds and pulp in bat faeces. These interactions were recorded in the dry forest and rainforest of Costa Rica, during the dry and the wet seasons of an extreme El Niño year. From these we constructed seasonal and whole-year bat-plant networks and analysed their structures and dissimilarities. In general, networks had low nestedness, had high modularity, and were dominated by one large compartment which included most species and interactions. Contrary to our expectations, networks were less nested and more modular in drier conditions, both in the comparison between forest types and between seasons. We suggest that increased competition, when resources are scarce during drier seasons and habitats, lead to higher resource partitioning among bats and thus higher modularity. Moreover, we have found similar network structures between dry and rainforests during El Niño and non-El Niño years. Finally, most interaction dissimilarity among networks occurred due to interaction rewiring among species, potentially driven by seasonal changes in resource availability.

Detection rates of aphid DNA in the guts of larval hoverflies and potential links to the provision of floral resources.

Aphidophagous hoverflies (Diptera, Syrphidae, Syrphinae) are common flower visitors and aphid predators in a range of flowering plants, including fruit crops. Here, we investigate whether aphid prey DNA can be detected in the gut contents of hoverfly larvae from a commercial strawberry field as a proof of concept that a molecular approach can be used to measure agricultural biocontrol. We used high-throughput sequencing (HTS) to target insect DNA and compared the resulting data to reference databases containing aphid and hoverfly DNA sequences. We explored what impact incorporating wildflowers within polythene-clad tunnels may have on aphid DNA detection rates in hoverfly larvae. In a randomized block experiment, coriander (Coriandrum sativum), field forget-me-not (Myosotis arvensis) and corn mint (Mentha arvensis) plants were inserted in rows of strawberries. Their effect on aphid DNA detection rates was assessed. Aphid DNA was found in 55 of 149 specimens (37%) validating the method in principle for measuring agricultural services provided by hoverflies. Interestingly, detection rates were higher near plots with forget-me-not than plots with coriander, though detection rates in control plots did not differ significantly from either wildflower species. These findings confirm that hoverflies predate aphids in UK strawberry fields, and that HTS is a viable method of identifying aphid DNA in predatory hoverflies. We comment on the need for further method development to narrow down identifications of both predator and prey. We furthermore provide some evidence that there is an effect of intercropping strawberry crops with wildflowers which may affect aphid consumption in hoverfly larvae.

Non-target effects of agri-environmental schemes on solitary bees and fungi in the United Kingdom.

Agri-environmental schemes (AES) are used to enhance pollinator diversity on agricultural farms within the UK. Though the impacts of these schemes on archetypal pollinator species such as the bumblebee (Bombus) and honeybee (Apis) are well-studied, the effects on non-target bee species like solitary bees, in the same environment, are generally lacking. One goal of AES is to alter floral provision and taxonomic composition of plant communities to provide better forage for pollinators, however, this may potentially impact other ecological communities such as fungal diversity associated with plant-bee communities. Fungi are integral in these bee communities as they can impact bee species both beneficially and detrimentally. We test the hypothesis that alteration of the environment through provision of novel plant communities has non-target effects on the fungi associated with solitary bee communities. We analyse fungal diversity and ecological networks formed between fungi and solitary bees present on 15 agricultural farms in the UK using samples from brood cells. The farms were allocated to two categories, low and high management, which differ in the number of agri-environmental measures implemented. Using internal transcribed spacer metabarcoding, we identified 456 fungal taxa that interact with solitary bees. Of these, 202 (approximately 44%) could be assigned to functional groups, the majority being pathotrophic and saprotrophic species. A large proportion was Ascosphaeraceae, a family of bee-specialist fungi. We considered the connectance, nestedness, modularity, nestedness overlap and decreasing fill, linkage density and fungal generality of the farms' bee-fungi ecological networks. We found no difference in the structure of bee-fungi ecological networks between low and high management farms, suggesting floral provision by AES has no significant impact on interactions between these two taxonomic groups. However, bee emergence was lower on the low management farms compared to high management, suggesting some limited non-target effects of AES. This study characterizes the fungal community associated with solitary bees and provides evidence that floral provision through AES does not impact fungal interactions.

Altered structure of bat-prey interaction networks in logged tropical forests revealed by metabarcoding.

Habitat degradation is pervasive across the tropics and is particularly acute in Southeast Asia, with major implications for biodiversity. Much research has addressed the impact of degradation on species diversity; however, little is known about how ecological interactions are altered, including those that constitute important ecosystem functions such as consumption of herbivores. To examine how rainforest degradation alters trophic interaction networks, we applied DNA metabarcoding to construct interaction networks linking forest-dwelling insectivorous bat species and their prey, comparing old-growth forest and forest degraded by logging in Sabah, Borneo. Individual bats in logged rainforest consumed a lower richness of prey than those in old-growth forest. As a result, interaction networks in logged forests had a less nested structure. These network structures were associated with reduced network redundancy and thus increased vulnerability to perturbations in logged forests. Our results show how ecological interactions change between old-growth and logged forests, with potentially negative implications for ecosystem function and network stability.

Leech blood-meal invertebrate-derived DNA reveals differences in Bornean mammal diversity across habitats.

The application of metabarcoding to environmental and invertebrate-derived DNA (eDNA and iDNA) is a new and increasingly applied method for monitoring biodiversity across a diverse range of habitats. This approach is particularly promising for sampling in the biodiverse humid tropics, where rapid land-use change for agriculture means there is a growing need to understand the conservation value of the remaining mosaic and degraded landscapes. Here we use iDNA from blood-feeding leeches (Haemadipsa picta) to assess differences in mammalian diversity across a gradient of forest degradation in Sabah, Malaysian Borneo. We screened 557 individual leeches for mammal DNA by targeting fragments of the 16S rRNA gene and detected 14 mammalian genera. We recorded lower mammal diversity in the most heavily degraded forest compared to higher quality twice logged forest. Although the accumulation curves of diversity estimates were comparable across these habitat types, diversity was higher in twice logged forest, with more taxa of conservation concern. In addition, our analysis revealed differences between the community recorded in the heavily logged forest and that of the twice logged forest. By revealing differences in mammal diversity across a human-modified tropical landscape, our study demonstrates the value of iDNA as a noninvasive biomonitoring approach in conservation assessments.

Differential effects of fertilisers on pollination and parasitoid interaction networks.

Grassland fertilisation drives non-random plant loss resulting in areas dominated by perennial grass species. How these changes cascade through linked trophic levels, however, is not well understood. We studied how grassland fertilisation propagates change through the plant assemblage into the plant-flower-visitor, plant-leaf miner and leaf miner-parasitoid networks using a year's data collection from a long-term grassland fertiliser application experiment. Our experiment had three fertiliser treatments each applied to replicate plots 15 m2 in size: mineral fertiliser, farmyard manure, and mineral fertiliser and farmyard manure combined, along with a control of no fertiliser. The combined treatment had the most significant impact, and both plant species richness and floral abundance decreased with the addition of fertiliser. While insect species richness was unaffected by fertiliser treatment, fertilised plots had a significantly higher abundance of leaf miners and parasitoids and a significantly lower abundance of bumblebees. The plant-flower-visitor and plant-herbivore networks showed higher values of vulnerability and lower modularity with fertiliser addition, while leaf miner-parasitoid networks showed a rise in generality. The different groups of insects were impacted by fertilisers to varying degrees: while the effect on abundance was the highest for leaf miners, the vulnerability and modularity of flower-visitor networks was the most affected. The impact on the abundance of leaf miners was positive and three times higher than the impact on parasitoids, and the impact on bumblebee abundance was negative and double the magnitude of impact on flower abundance. Overall, our results show that while insect species richness was unaffected by fertilisers, network structure changed significantly as the replacement of forbs by grasses resulted in changes in relative abundance across trophic levels, with the direction of change depending on the type of network. Synthesis. By studying multiple networks simultaneously, we were able to rank the relative impact of habitat change on the different groups of species within the community. This provided a more holistic picture of the impact of agricultural intensification and provides useful information when deciding on priorities for mitigation.

Exploratory analysis reveals arthropod consumption in 10 lemur species using DNA metabarcoding.

Arthropods (insects, spiders, etc.) can fulfill major nutritional requirements for primates, particularly in terms of proteins, fats, vitamins, and minerals. Yet, for many primate species we know very little about the frequency and importance of arthropod consumption. Traditional methods for arthropod prey identification, such as behavioral observations and fecal dissections, offer limited taxonomic resolution and, as a result, underestimate true diversity. Metabarcoding arthropod DNA from primate fecal samples provides a promising but underused alternative. Here, we inventoried arthropod prey diversity in wild lemurs by sequencing two regions of the CO1 gene. Samples were collected opportunistically from 10 species of lemurs inhabiting three national parks in southern Madagascar using a combination of focal animal follows and live trapping. In total, we detected arthropod DNA in 98 of the 170 fecal samples analyzed. Although all lemur species included in these analyses showed evidence of arthropod consumption, those within the family Cheirogaleidae appeared to consume the highest frequency and diversity of arthropods. To our knowledge, this study presents the first evidence of arthropod consumption in Phaner pallescens, Avahi peyrierasi, and Propithecus verreauxi, and identifies 32 families of arthropods as probable food items that have not been published as lemur dietary items to date. Our study emphasizes the importance of arthropods as a nutritional source and the role DNA metabarcoding can play in elucidating an animal's diet.

DNA metabarcoding reveals changes in the contents of carnivorous plants along an elevation gradient.

Resource variation along abiotic gradients influences subsequent trophic interactions and these effects can be transmitted through entire food webs. Interactions along abiotic gradients can provide clues as to how organisms will face changing environmental conditions, such as future range shifts. However, it is challenging to find replicated systems to study these effects. Phytotelmata, such as those found in carnivorous plants, are isolated aquatic communities and thus form a good model for the study of replicated food webs. Due to the degraded nature of the prey, molecular techniques provide a useful tool to study these communities. We studied the pitcher plant Sarracenia purpurea L. in allochthonous populations along an elevational gradient in the Alps and Jura. We predicted that invertebrate richness in the contents of the pitcher plants would decrease with increasing elevation, reflecting harsher environmental conditions. Using metabarcoding of the COI gene, we sequenced the invertebrate contents of these pitcher plants. We assigned Molecular Operational Taxonomic Units at ordinal level as well as recovering species-level data. We found small but significant changes in community composition with elevation. These recovered sequences could belong to invertebrate prey, rotifer inquilines, pollinators and other animals possibly living inside the pitchers. However, we found no directional trend or site-based differences in MOTU richness with elevational gradient. Use of molecular techniques for dietary or contents analysis is a powerful way to examine numerous degraded samples, although factors such as DNA persistence and the relationship with species presence still have to be completely determined.

Counting with DNA in metabarcoding studies: How should we convert sequence reads to dietary data?

Advances in DNA sequencing technology have revolutionized the field of molecular analysis of trophic interactions, and it is now possible to recover counts of food DNA sequences from a wide range of dietary samples. But what do these counts mean? To obtain an accurate estimate of a consumer's diet should we work strictly with data sets summarizing frequency of occurrence of different food taxa, or is it possible to use relative number of sequences? Both approaches are applied to obtain semi-quantitative diet summaries, but occurrence data are often promoted as a more conservative and reliable option due to taxa-specific biases in recovery of sequences. We explore representative dietary metabarcoding data sets and point out that diet summaries based on occurrence data often overestimate the importance of food consumed in small quantities (potentially including low-level contaminants) and are sensitive to the count threshold used to define an occurrence. Our simulations indicate that using relative read abundance (RRA) information often provides a more accurate view of population-level diet even with moderate recovery biases incorporated; however, RRA summaries are sensitive to recovery biases impacting common diet taxa. Both approaches are more accurate when the mean number of food taxa in samples is small. The ideas presented here highlight the need to consider all sources of bias and to justify the methods used to interpret count data in dietary metabarcoding studies. We encourage researchers to continue addressing methodological challenges and acknowledge unanswered questions to help spur future investigations in this rapidly developing area of research.

Approaches to integrating genetic data into ecological networks.

As molecular tools for assessing trophic interactions become common, research is increasingly focused on the construction of interaction networks. Here, we demonstrate three key methods for incorporating DNA data into network ecology and discuss analytical considerations using a model consisting of plants, insects, bats and their parasites from the Costa Rica dry forest. The simplest method involves the use of Sanger sequencing to acquire long sequences to validate or refine field identifications, for example of bats and their parasites, where one specimen yields one sequence and one identification. This method can be fully quantified and resolved and these data resemble traditional ecological networks. For more complex taxonomic identifications, we target multiple DNA loci, for example from a seed or fruit pulp sample in faeces. These networks are also well resolved but gene targets vary in resolution and quantification is difficult. Finally, for mixed templates such as faecal contents of insectivorous bats, we use DNA metabarcoding targeting two sequence lengths (157 and 407 bp) of one gene region and a MOTU, BLAST and BIN association approach to resolve nodes. This network type is complex to generate and analyse, and we discuss the implications of this type of resolution on network analysis. Using these data, we construct the first molecular-based network of networks containing 3,304 interactions between 762 nodes of eight trophic functions and involving parasitic, mutualistic and predatory interactions. We provide a comparison of the relative strengths and weaknesses of these data types in network ecology.

Assessing niche partitioning of co-occurring sibling bat species by DNA metabarcoding.

Niche partitioning through foraging is a mechanism likely involved in facilitating the coexistence of ecologically similar and co-occurring animal species by separating their use of resources. Yet, this mechanism is not well understood in flying insectivorous animals. This is particularly true of bats, where many ecologically similar or cryptic species coexist. The detailed analysis of the foraging niche in sympatric, cryptic sibling species provides an excellent framework to disentangle the role of specific niche factors likely involved in facilitating coexistence. We used DNA metabarcoding to determine the prey species consumed by a population of sympatric sibling Rhinolophus euryale and Rhinolophus mehelyi whose use of habitat in both sympatric and allopatric ranges has been well established through radio tracking. Although some subtle dietary differences exist in prey species composition, the diet of both bats greatly overlapped (Ojk  = 0.83) due to the consumption of the same common and widespread moths. Those dietary differences we did detect might be related to divergences in prey availabilities among foraging habitats, which prior radio tracking on the same population showed are differentially used and selected when both species co-occur. This minor dietary segregation in sympatry may be the result of foraging on the same prey-types and could contribute to reduce potential competitive interactions (e.g., for prey, acoustic space). Our results highlight the need to evaluate the spatial niche dimension in mediating the co-occurrence of similar insectivorous bat species, a niche factor likely involved in processes of bat species coexistence.

Barcoding the food chain: from Sanger to high-throughput sequencing.

Society faces the complex challenge of supporting biodiversity and ecosystem functioning, while ensuring food security by providing safe traceable food through an ever-more-complex global food chain. The increase in human mobility brings the added threat of pests, parasites, and invaders that further complicate our agro-industrial efforts. DNA barcoding technologies allow researchers to identify both individual species, and, when combined with universal primers and high-throughput sequencing techniques, the diversity within mixed samples (metabarcoding). These tools are already being employed to detect market substitutions, trace pests through the forensic evaluation of trace "environmental DNA", and to track parasitic infections in livestock. The potential of DNA barcoding to contribute to increased security of the food chain is clear, but challenges remain in regulation and the need for validation of experimental analysis. Here, we present an overview of the current uses and challenges of applied DNA barcoding in agriculture, from agro-ecosystems within farmland to the kitchen table.

The effects of parameter choice on defining molecular operational taxonomic units and resulting ecological analyses of metabarcoding data.

The combination of DNA barcoding and high-throughput (next-generation) sequencing (metabarcoding) provides many promises but also serious challenges. Generating a reliable comparable estimate of biodiversity remains a central challenge to the application of the technology. Many approaches have been used to turn millions of sequences into distinct taxonomic units. However, the extent to which these methods impact the outcome of simple ecological analyses is not well understood. Here we performed a simple analysis of dietary overlap by skinks and shrews on Ile Aux Aigrettes, Mauritius. We used a combination of filtering thresholds and clustering algorithms on a COI metabarcoding dataset and demonstrate that all bioinformatics parameters will have interacting effects on molecular operational taxonomic unit (MOTU) recovery rates. These effects generated estimates covering two orders of magnitude. However, the effect on a simple ecological analysis was not large and, despite the wide variation in estimates of niche overlap, the same ecological conclusion was drawn in most cases. We advise that a conservative clustering programme coupled with larger sequence divergences to define a cluster, the removal of singletons, rigorous length filtering, and stringent match criteria for Molecular Identifier tags are preferable to avoid MOTU inflation and that the same parameters be used in all comparative analyses.

Dietary overlap and seasonality in three species of mormoopid bats from a tropical dry forest.

Competing hypotheses explaining species' use of resources have been advanced. Resource limitations in habitat and/or food are factors that affect assemblages of species. These limitations could drive the evolution of morphological and/or behavioural specialization, permitting the coexistence of closely related species through resource partitioning and niche differentiation. Alternatively, when resources are unlimited, fluctuations in resources availability will cause concomitant shifts in resource use regardless of species identity. Here, we used next-generation sequencing to test these hypotheses and characterize the diversity, overlap and seasonal variation in the diet of three species of insectivorous bats of the genus Pteronotus. We identified 465 prey (MOTUs) in the guano of 192 individuals. Lepidoptera and Diptera represented the most consumed insect orders. Diet of bats exhibited a moderate level of overlap, with the highest value between Pteronotus parnellii and Pteronotus personatus in the wet season. We found higher dietary overlap between species during the same seasons than within any single species across seasons. This suggests that diets of the three species are driven more by prey availability than by any particular predator-specific characteristic. P. davyi and P. personatus increased their dietary breadth during the dry season, whereas P. parnellii diet was broader and had the highest effective number of prey species in all seasons. This supports the existence of dietary flexibility in generalist bats and dietary niche overlapping among groups of closely related species in highly seasonal ecosystems. Moreover, the abundance and availability of insect prey may drive the diet of insectivores.

Island bat diets: does it matter more who you are or where you live?

Differences in body size, echolocation call frequency and location may result in diet partitioning among bat species. Comparisons between island populations are one way to evaluate these competing hypotheses. We conducted a species-level diet analysis of three Rhinolophus and one Hipposideros species on the Philippine islands of Cebu, Bohol and Siquijor. We identified 655 prey (MOTUs) in the guano from 77 individual bats. There was a high degree of overlap among species' diets despite differences in body size and call frequency. For example, the diet of the 3 g-Hipposideros pygmaeus (mean CF = 102 kHz) exhibited a diet overlap higher than expected by chance with all three Rhinolophus species, even the 13 g-Rhinolophus inops (mean CF = 54 kHz). We observed more convergence in diet between Rhinolophus species and H. pygmaeus than between Rhinolophus species themselves, which may be explained by the broad diet of H. pygmaeus. There was less dietary overlap between Rhinolophus virgo from two islands than between R. virgo and congeners from Cebu. These data suggest that location causes convergence in diet, but specific species characteristics may drive niche specialization. The complex interplay between location and the perceptual ability of each species leads to a situation where simple explanations, for example body size, do not translate into predictable prey partitioning. In particular, our observations raise interesting questions about the foraging strategy and adaptability of the tiny H. pygmaeus.

An inordinate fondness for beetles? Variation in seasonal dietary preferences of night-roosting big brown bats (Eptesicus fuscus).

Generalist species with numerous food web interactions are thought to provide stability to ecosystem dynamics; however, it is not always clear whether habitat generality translates into dietary diversity. Big brown bats are common across North America and employ a flexible foraging strategy over water, dense forests, forest edges and rural and urban settings. Despite this generalist use of habitat, they are paradoxically characterized as beetle specialists. However, hard carapaces may preferentially survive digestion leading to over-representation during morphological analysis of diet. This specialization has not been evaluated independently using molecular analysis and species-level identification of prey. We used next-generation sequencing to assess the diet of big brown bats. Beetles were consumed in the highest frequency but Lepidoptera species richness was highest among identified prey. The consumption of species showed strong seasonal and annual variation. While Coleoptera consumption varied, Lepidoptera and Ephemeroptera were relatively constant dietary components. Dietary diversity increased in late summer when insect diversity decreases. Our results indicate that big brown bats are dietary generalists and, while beetles are an important component of the diet, Lepidoptera are equally important, and Lepidoptera and Ephemeroptera are the only stable prey resource exploited. As resources become limited, big brown bats may respond by increasing the species richness of prey and thus their connectedness in the ecosystem. This characterization of diet corresponds well with a generalist approach to foraging, making them an important species in encouraging and maintaining ecosystem stability.

Diet of the insectivorous bat Pipistrellus nathusii during autumn migration and summer residence.

Migration is widespread among vertebrates, yet bat migration has received little attention and only in the recent decades has a better understanding of it been gained. Migration can cause significant changes in behaviour and physiology, due to increasing energy demands and aerodynamic constraints. Dietary shifts, for example, have been shown to occur in birds before onset of migration. For bats, it is not known if a change in diet occurs during migration, although breeding season-related dietary preference has been documented. It is known that a diet rich in fats and the accumulation of fat deposits do increase the flight range of migratory bats. Some bat species can be regarded as long-distance migrants, covering up to 2000 km between summer and winter roosting areas. Pipistrellus nathusii (Vespertilionidae), a European long-distant migrant, travels each year along the Baltic Sea from north-eastern Europe to hibernate in central and southern Europe. This study presents data on the dietary habits of migrating Pipistrellus nathusii compared with those during the breeding season. We analysed faecal samples from bats on fall migration caught at the Ornithological Field Station in Pape, Latvia and from samples collected in North-Latvian summer roosts. We applied both morphological identification and molecular methods, as morphological methods also recognize life stages of prey and can contribute frequency data. The diets of bats on migration and breeding bats were similar, with Diptera and Lepidoptera comprising the major prey categories. However, certain prey groups could be explained by the different hunting habitats exploited during migration vs. summer residence.

Resource partitioning by insectivorous bats in Jamaica.

In this investigation, we use variation in wing morphology, echolocation behaviour, patterns of habitat use and molecular diet analysis to demonstrate that six species of sympatric insectivorous bats in Jamaica show significant differences that could explain resource partitioning among the species. High-intensity echolocating species that used shorter, broadband signals and had shorter, broader wings (Pteronotus macleayii, Pteronotus quadridens, Mormoops blainvillii) foraged most in edge habitats, but differed in timing of peak activity. P. macleayii and M. blainvillii differed in diet, but low sample size precluded diet analysis for P. quadridens. High-intensity echolocating species that used longer, more narrowband signals and had longer, narrower wings (Molossus molossus, Tadarida brasiliensis) foraged most in open areas and differed in diet from the other species. Two disparate species were most active in clutter (dense vegetation). Pteronotus parnellii used high-duty-cycle echolocation apparently specialized for detecting fluttering targets in clutter. Macrotus waterhousii used low-intensity, broadband echolocation calls and presumably uses prey-generated sounds when foraging. These two species also differed in diet. Our data show that differences in morphology and echolocation behaviour coincide with differences in habitat use and diet, resulting in minimal overlap in resource use among species.

The diet of Myotis lucifugus across Canada: assessing foraging quality and diet variability.

Variation in prey resources influences the diet and behaviour of predators. When prey become limiting, predators may travel farther to find preferred food or adjust to existing local resources. When predators are habitat limited, local resource abundance impacts foraging success. We analysed the diet of Myotis lucifugus (little brown bats) from Nova Scotia (eastern Canada) to the Northwest Territories (north-western Canada). This distribution includes extremes of season length and temperature and encompasses colonies on rural monoculture farms, and in urban and unmodified areas. We recognized nearly 600 distinct species of prey, of which ≈30% could be identified using reference sequence libraries. We found a higher than expected use of lepidopterans, which comprised a range of dietary richness from ≈35% early in the summer to ≈55% by late summer. Diptera were the second largest prey group consumed, representing ≈45% of dietary diversity early in the summer. We observed extreme local dietary variability and variation among seasons and years. Based on the species of insects that were consumed, we observed that two locations support prey species with extremely low pollution and acidification tolerances, suggesting that these are areas without environmental contamination. We conclude that there is significant local population variability in little brown bat diet that is likely driven by seasonal and geographical changes in insect diversity, and that this prey may be a good indicator of environment quality.

Diversification and reproductive isolation: cryptic species in the only New World high-duty cycle bat, Pteronotus parnellii.

BACKGROUND: Molecular techniques are increasingly employed to recognize the presence of cryptic species, even among commonly observed taxa. Previous studies have demonstrated that bats using high-duty cycle echolocation may be more likely to speciate quickly. Pteronotus parnellii is a widespread Neotropical bat and the only New World species to use high-duty cycle echolocation, a trait otherwise restricted to Old World taxa. Here we analyze morphological and acoustic variation and genetic divergence at the mitochondrial COI gene, the 7th intron region of the y-linked Dby gene and the nuclear recombination-activating gene 2, and provide extensive evidence that P. parnellii is actually a cryptic species complex. RESULTS: Central American populations form a single species while three additional species exist in northern South America: one in Venezuela, Trinidad and western Guyana and two occupying sympatric ranges in Guyana and Suriname. Reproductive isolation appears nearly complete (only one potential hybrid individual found). The complex likely arose within the last ~6 million years with all taxa diverging quickly within the last ~1-2 million years, following a pattern consistent with the geological history of Central and northern South America. Significant variation in cranial measures and forearm length exists between three of the four groups, although no individual morphological character can discriminate these in the field. Acoustic analysis reveals small differences (5-10 kHz) in echolocation calls between allopatric cryptic taxa that are unlikely to provide access to different prey resources but are consistent with divergence by drift in allopatric species or through selection for social recognition. CONCLUSIONS: This unique approach, considering morphological, acoustic and multi-locus genetic information inherited maternally, paternally and bi-parentally, provides strong support to conclusions about the cessation of gene flow and degree of reproductive isolation of these cryptic species.