Evaluating species boundaries remains a significant challenge in rare, difficult to collect, and/or understudied groups because of a lack of available

The chaetotaxy of 15 species of eastern North American Entomobrya is redescribed in order to determine potential characters for the diagnosis of cryptic lineages and evaluate the diagnostic and phylogenetic utility of chaetotaxy. As a result, four new species (E. citrensis Katz & Soto-Adames, sp. n., E. jubata Katz & Soto-Adames, sp. n., E. neotenica Katz & Soto-Adames, sp. n. and E. unifasciata Katz & Soto-Adames, sp. n.) are described, and new diagnoses are provided for E. assuta Folsom, E. atrocincta Schött, E. decemfasciata (Packard), E. ligata Folsom, E. multifasciata (Tullberg), and E. quadrilineata (Bueker). Furthermore, previously undocumented levels of intraspecific variation in macrosetal pattern are reported, tempering the exclusive use of chaetotaxy for species delimitation. Phylogenetic relationships, estimated using both morphological and molecular data, indicate that Entomobrya is likely paraphyletic. The phylogenies also suggest that unreliable character homology, likely fostered by Entomobrya’s profusion of macrosetae, may limit the phylogenetic utility of chaetotaxy in groups characterized by an abundance of dorsal macrosetae.

Arthropods can strongly impact ecosystems through pollination, herbivory, predation, and parasitism. As such, characterizing arthropod biodiversity is vital to understanding ecosystem health, functions, and services. Emerging environmental DNA (eDNA) methods targeting trace arthropod eDNA left behind on flowers have the potential to track arthropod biodiversity and interactions. The goal of this study was to determine the extent to which eDNA metabarcoding can identify plant‐arthropod and arthropod‐arthropod interactions and assess eDNA metabarcoding compared to conventional sampling. We deployed camera traps to document arthropod activity on specific flowers, sampled eDNA from those same flowers, then performed a metabarcoding analysis that targets a partial fragment of the cytochrome c oxidase subunit I gene (COI) to determine all arthropod eDNA present. We found that our eDNA metabarcoding analysis detected small arthropod pollinators, plant pests, and parasites, and shed light on potential predator–prey interactions while detecting 55 species compared to just 21 species from conventional camera trapping. The camera trapping survey, however, detected larger, more conspicuous nectarivores more successfully. We also explored the ecology of residual arthropod eDNA, finding that rainfall had a significant negative effect on the ability to detect residual arthropod eDNA. Preliminary evidence also indicates flower species may impact the amount of arthropod eDNA that can be detected. We found that eDNA metabarcoding can provide clues to potential predator–prey interactions on flowers and highlights the potential insights that can be gained from future eDNA metabarcoding studies. We show that eDNA metabarcoding is a valuable tool for not only detecting pollinator communities but for revealing potential interactions among plants, pollinators, pests, parasites, and predators. Future research should focus on how to improve the detection of large pollinators/nectivores and studying the ecology of residual arthropod eDNA to further explore this method's utility.

Successful conservation of rare, threatened, or endangered (RTE) species is dependent upon rapid and accurate assessment of their distribution and abundance. However, assessments are challenging as RTE species typically exist as numerically small populations in often fragmented habitats and can possess complex natural histories. Environmental DNA (eDNA) analysis may provide a rapid, cost‐effective means of assessing RTE species presence/absence in viable habitat patches. We evaluated the efficacy of eDNA surveillance for the Louisiana Pinesnake (Pituophis ruthveni), an elusive, semi‐fossorial, nonvenomous colubroid snake endemic to Louisiana and Texas, USA, that has dramatically declined in both distribution and abundance. We developed two quantitative polymerase chain reaction (qPCR) assays that target the mitochondrial cytochromecoxidase subunit I (COI) and mitochondrially encoded ATP synthase membrane subunit 6 (ATP6) genes. We validated each assayin silico,in vitro, andin situ, and investigated the influence of eDNA extraction method and genetic marker on assay performance. Both assays were highly sensitive and successfully detected the Louisiana Pinesnake under artificial and field conditions, including bedding samples collected from captive snake enclosures (100%), soil samples from Louisiana Pinesnake release sites (100%), and soil samples from sites where Louisiana Pinesnakes were documented via radio telemetry (45%). Although differences between genetic markers were negligible, assay performance was strongly influenced by eDNA extraction method. Informed by our results, we discuss methodological and environmental factors influencing Louisiana Pinesnake eDNA detection and quantification, broader implications for management and conservation of the Louisiana Pinesnake and other terrestrial reptiles and provide recommendations for future research. We suggest that eDNA surveys can more effectively assess Louisiana Pinesnake occupancy than conventional sampling, highlighting the need for comprehensive eDNA monitoring initiatives to better identify suitable habitat that will promote persistence of this imperiled species going forward.

The processes of vicariance and dispersal are central to our understanding of diversification, yet determining the factors that influence these processes remains a significant challenge in evolutionary biology. Caves offer ideal systems for examining the mechanisms underlying isolation, divergence, and speciation. Intrinsic ecological differences among cavernicolous organisms, such as the degree of cave dependence, are thought to be major factors influencing patterns of genetic isolation in caves. Using a comparative phylogeographic approach, we employed mitochondrial and nuclear markers to assess the evolutionary history of two ecologically distinct groups of terrestrial cave‐dwelling springtails (Collembola) in the generaPygmarrhopalites(Arrhopalitidae) andPogonognathellus(Tomoceridae) that are codistributed in caves throughout the Salem Plateau—a once continuous karst region, now bisected by the Mississippi River Valley in Illinois and Missouri. Contrasting phylogeographic patterns recovered for troglobioticPygmarrhopalitessp. and eutroglophilicPogonognathellussp. suggests that obligate associations with cave habitats can restrict dispersal across major geographic barriers such as rivers and valleys, but may also facilitate subterranean dispersal between neighboring cave systems.Pygmarrhopalitessp. populations spanning the Mississippi River Valley were estimated to have diverged 2.9–4.8 Ma, which we attribute to vicariance resulting from climatic and geological processes involved in Mississippi River Valley formation beginning during the late Pliocene/early Pleistocene. Lastly, we conclude that the detection of many deeply divergent, morphologically cryptic, and microendemic lineages highlights our poor understanding of microarthropod diversity in caves and exposes potential conservation concerns.

Species detections often vary depending on the survey methods employed. Some species may go undetected when using only one approach in community‐level inventory and monitoring programs, which has management and conservation implications. We conducted a comparative study of terrestrial mammal and bird detections in the spring and summer of 2021 by placing camera traps at 30 locations across a large military installation in northern Michigan, USA and testing replicate soil samples from these sites for environmental DNA (eDNA) using an established vertebrate metabarcoding assay. We detected a total of 48 taxa from both survey methods: 26 mammalian taxa (excluding humans, 24 to species and two to genus) and 22 avian taxa (21 to species and one to genus). We detected a relatively even distribution of mammalian taxa on cameras (17) and via eDNA analysis (15), with seven taxa detected from both methods. Most medium‐to‐large carnivores were detected only on cameras, whereas semi‐fossorial small mammals were detected only via eDNA analysis. We detected higher bird diversity with camera traps (18 taxa) compared to eDNA analysis (eight taxa; four taxa were detected with both methods), but cameras alone were most effective at detecting smaller birds that frequently occupy arboreal environments. We also used Bayesian spatial occupancy models for two widely distributed game species (white‐tailed deer, Odocoileus virginianus, and ruffed grouse, Bonasa umbellus) that were moderately detected with both survey methods and found species‐specific site use (occupancy) estimates were similar between cameras and eDNA analysis. Concordant with similar studies, our findings suggest that a combination of camera trap and eDNA surveys could be most useful for assessing the composition of terrestrial mammal communities. Camera traps may be most efficient for assessing bird diversity but can be complemented with eDNA analysis, particularly for species that spend considerable time on the ground.