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Identification of windthrow eventsLandsat images from January 1st 2018 to December 31st 2019 were filtered on 20% or less of cloud coverage, and only the least cloudy image at each location was selected to make an image composite covering the entire Amazon region. In total, 395 least cloudy Landsat 8 images within the Amazon boundary during 2018–2019 were selected and displayed in false color (red: shortwave infrared band, green: near-infrared band, blue: red band) on Google Earth Engine for windthrow events identification (Supplementary Fig. 6). Hollow regions on Supplementary Fig. 6 (2.8% of the total area of the Amazon region) indicated that no clear images with 1 year before the identification were displayed in bright green colors (due to reflectance in near-infrared band from the pioneer species). “Old” windthrows account for ~80% of total identified windthrows, and they were verified using historical Landsat images that can go as far as 1984 (when Landsat 5 was launched). “Old” windthrows were validated once they were found with clear shape and more distinguish color on the historical Landsat images (Supplementary Fig. 7c). 10–15% of “old” windthrows without fan-shape were eliminated from this study because it was hard to identify if they were windthrows or other types of forest disturbance. The minimum size of windthrows identified in this study was 25,000 m2. This process generated the location and rough size of 1012 visible (both “old” and “new”) windthrow scars with fan-shaped patch, scattered small disturbance pixels tails, and an area of over 25,000 m2 (Supplementary Fig. 8). Based on a gap-size probability distribution function that simulates the entire disturbance gradient from all sizes of windthrows19, the proportion of total tree mortality represented by large windthrows ( >25,000 m2) identified in this study is 0.5–1.1%.Among 1012 visual identified windthrows, the occurrence year of 125 windthrows were identified using Landsat 5,7,8, MODIS, and TRMM dataset (Supplementary Table 2), and 38 windthrows from these 125 windthrows had clear remote sensing evidence to validate their occurring date (Supplementary Table 3). It is difficult to get the accurate year and date of occurrence of all identified windthrows. Previous studies showed that windthrows in the northwestern Amazon took ~20 years to recover to 90% of “pre-disturbance” biomass from all damage classes while forests in the central Amazon took ~40 years to recover40. The biomass recovery depends on the windthrow severity and time since disturbance33. Based on the recovery time (20–40 years) and the time of windthrow identification (2018–2019), we estimated that these 1012 windthrows most likely occurred within 30 years (between 1990 and 2019), and the estimated occurrence period was validated using the range of the occurrence year (1986–2019) of 125 windthrow cases.Windthrow density dataThe windthrow density shown in Fig. 1b was generated using 1012 windthrow points in QGIS45. We created a 2.5° by 2.5° grid map, and the windthrow density was calculated by counting the number of windthrows in each grid. These values were then converted to a density with units of counts of windthrows per 10,000 km2. We chose 2.5 degrees to aggregate the data to make sure that over 50% of grids have at least 1 windthrow event while still preserving the spatial distribution of mean afternoon CAPE over the Amazon. The contour lines displayed in Fig. 1c were generated using the “Contour” function on the windthrow density map in QGIS.Meteorological dataTo derive the correlation between windthrow density and meteorological variables, we used ERA 5 global reanalysis hourly CAPE on single levels from 1979 to present at 0.25° × 0.25° resolution provided by the European Center for Medium-Range Weather Forecasts. ERA 5 CAPE was computed by considering parcels of air departing at different pressure levels below the 35 kPa level, with maximum–unstable algorithm under a pseudo-adiabatic assumption46. Afternoon mean CAPE map was calculated as the average of hourly CAPE data from 17:00–23:00 UTC (13:00–19:00 local time in Amazon) over all the months between 1990 and 2019. We chose to average CAPE over 30 years because these windthrow events occurred in these 30 years and calculating the average can help capture the overall spatial pattern of CAPE and minimize the influence of interannual climate variability on windthrow events.To project future windthrow density in the Amazon for the end of the 21st century, we analyzed meteorological output from 10 ESMs that participated in CMIP6 (https://www.wcrp-climate.org/wgcm-cmip/wgcm-cmip6). The models used in this research were listed in Supplementary Table 1. We extracted daily surface temperature (tas), specific humidity (huss), surface pressure (ps), temperature (ta) from these models to calculate daily nondilute, near-surface-based, adiabatic CAPE. CMIP6 CAPE was calculated by considering the buoyancy of a near-surface parcel lifted adiabatically to a series of discrete pressure levels (100 kPa to 10 kPa in increments of 10 kPa). CMIP6 CAPE is calculated as follows:$${CAPE}=mathop{sum }limits_{i=1}^{10}{{{{{rm{d}}}}}}p{{{{{rm{H}}}}}}({b}_{i}){b}_{i}$$
(1)
Where ({{{{{rm{d}}}}}}p) = 10 kPa, H is the Heaviside unit step function, and ({b}_{i}=frac{1}{{rho }_{i}}-frac{1}{{rho }_{e,i}}), with ({rho }_{i}) being the parcel density at pressure level i and ({rho }_{e,i}) being the environmental density at pressure level i.The future projections in our analysis were based on SSP585, a high-emission scenario with high radiative forcing by the end of the century. We calculated mean daily CAPE over 1990–2015 as current CMIP6 CAPE and mean daily CAPE over 2070–2099 as future CMIP6 CAPE. Since different approaches were used to calculate ERA 5 CAPE and CMIP6 CAPE47, the absolute CAPE values of the two datasets are not comparable. Therefore, for each ESM model, we scaled future CMIP6 CAPE by multiplying, grid-wise, the delta CAPE generated from an individual model in CMIP6 with the ERA 5 current mean afternoon CAPE (Fig. 1c) as follows:$${delta},{CAPE}=(CAPE_{CMIP6_{,future}},-CAPE_{CMIP6_current})/CAPE_{CMIP6_current}$$
(2)
$$CAP{E}_{scaled_CMIP6_,future}=(1+delta,CAPE)times CAP{E}_{ERA5}{_}_{current}$$
(3)
The delta CAPE indicated the projected increase in CAPE from 1990–2015 to the end of the 21st century. In this way, a scaled CMIP6 future CAPE map was generated for each model, and an ensemble-mean scaled CMIP6 CAPE map over 10 ESM models can be found in Supplementary Fig. 5b. The scaled CMIP6 future CAPE values were within plausible range compared to the ERA 5 current mean afternoon CAPE values, and both current and future CAPE maps were used to produce the increase in area with high CAPE values ( >1023 J kg−1) in Table 1. However, it is worth noting that the scaling with relative changes in delta CAPE (%) is more sensitive to CMIP historical baseline conditions than absolute changes of CAPE (J kg−1), which will likely introduce a larger scaled spread (min/max CAPE changes).The increase in area with storm-favorable environments was calculated as follows:$$Increase=(are{a}_{future}-area_{current})/are{a}_{current}$$
(4)
Where areacurrent is the area of CAPE  > 1023 J kg−1 for current ERA 5 CAPE, and areafuture is the area of CAPE  > 1023 J kg−1 for the scaled CMIP6 future CAPE.A model of windthrow densityWe developed a model based on the relationship between satellite-derived windthrow density and mean afternoon CAPE from the ERA 5 reanalysis over 1990–2019. The non-parametric model provides a look-up table of windthrow density as a function of CAPE within the range of observations. Counts of observed windthrow events and Amazon’s area were separately binned by CAPE using the same bins, producing two histograms of CAPE. The ratio of the former to the latter gives the density of windthrow events (windthrow events per area) as a function of CAPE. To avoid noise at the tails of the histograms, the six CAPE bins were chosen such that each bin would have about the same number of windthrow events (either 168 or 169). The total number of windthrow events is given by the sum over bins of the product of windthrow density and area. The minimum and maximum of current ERA 5 mean afternoon CAPE was 42 and 1549. The minimum CAPE value of the first bin was extended to 0 and the maximum CAPE value of the last bin was extended to infinity under the assumption that the windthrow density is similar for neighboring values. Based on the windthrow density and CAPE relationship used in the model, it is the increase in the area with high CAPE that then leads to an increase in the number of windthrow events.It is worth noting that the future windthrow density produced by models may be underestimated because the windthrow observations within regions with high CAPE were incomplete due to high cloud coverage. Moreover, the non-parametric model makes the conservative assumption that the windthrow density does not increase at higher, as-yet unobserved values of mean afternoon CAPE.Future projections of windthrow densityWe combined the non-parametric relationship (Fig. 2a) with the future CAPE map generated from the ten CMIP6 ESMs (adjusted by ERA 5 mean CAPE values) to estimate the changes in windthrow density at the end of the 21st century. We estimated uncertainties for windthrow density projections by combining information about model-to-model differences. The analysis yielded a set of 10 estimates. The overall windthrow density increase and uncertainty were estimated using the mean increase and one standard deviation from the ensemble of the 10 models. More

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Systematic paleontologySauropterygia Owen, 186038.Eosauropterygia Rieppel, 199439.Pachypleurosauroidea Huene, 195640.Pachypleurosauridae Nopcsa, 192841.Luopingosaurus imparilis gen. et sp. nov.EtymologyThe genus name refers to the Luoping County, at which the fossil site is located. Species epithet imparilis (Latin) means peculiar and unusual.HolotypeA ventrally exposed skeleton with a posterior part of the caudal missing, IVPP V19049.Locality and horizonLuoping, Yunnan, China; Second (Upper) Member of Guanling Formation, Pelsonian (~ 244 Ma), Anisian, Middle Triassic37.DiagnosisA pachypleurosaurid distinguishable from other members of this family by the following combination of features (those unique among pachypleurosaurids identified with an asterisk): snout (preorbital portion) long and anteriorly pointed, 55.0% of skull length (*); orbital length about one quarter of skull length; internal naris retracted, without contribution from premaxilla; nasal ending at level of anterior margin of prefrontal; dentary length 71.7% of mandibular length; hyoid length 9.7% of mandibular length; presence of entepicondylar foramen in humerus; 21 cervical and 27 dorsal vertebrae (*); distinct expansions of distal heads of posterior two sacral ribs; six pairs of caudal ribs; phalangeal formula 2–3-5–5-3 for manus and 2–3-4–6-4 for pes (*); Metatarsal I short and stout with expanded proximal end, 56.4% of Metatarsal V in length (*); and Metatarsal IV being longest phalange in pes.Comparative descriptionThe holotype and only currently known specimen of Luopingosaurus has a preserved length of 46.2 cm from the rostral tip to the 30th caudal vertebra (for measurements, see Table 1). The estimated total length of the body may have reached 64 cm, assuming similar tail proportions of pachypleurosaurids. As such, Luopingosaurus is longer than most of other pachypleurosauroids that are small-sized with a maximum total length rarely exceeding 50 cm4,9,10,11,12,14,15,16,18,23,25, although some pachypleurosaurids are notably larger (e.g., 88 cm in Diandongosaurus cf. acutidentatus22, ~ 120 cm in Neusticosaurus edwardsii8, and ~ 130 cm in Wumengosaurus delicatomandibularis13).Table 1 Measurements (in mm) of the holotype (IVPP V19049) of Luopingosaurus imparilis gen. et sp. nov. R, right.Full size tableThe pre-orbital portion, distinctly longer than the postorbital region, measures 55% of the total skull length (the premaxillary symphysis to the occipital condyle) and 51% of the mandibular length. The paired premaxillae form most of the snout anterior to the naris with a pointed anterior tip, similar to the conditions in Wumengosaurus13,30 and Honghesaurus23. By contrast, other pachypleurosauroids uniformly have a blunt rostrum4,6,7,8,9,10,11,12,14,15,16,18,22,25. The premaxilla bears a long posteromedial process inserting between the anterior parts of the elongate nasals (Fig. 3). The premaxillary teeth are homodont with a tall peduncle and a short, conical crown, but the tooth number is hard to estimate because of occlusion of jaws. The posterior parts of nasals contact each other medially, and posteriorly, they contact the frontals in an interdigitating suture at the level of the anterior margin of the prefrontal. In Honghesaurus23, Wumengosaurus30, Neusticosaurus8 and Serpianosaurus9, the even longer nasal extends posteriorly beyond this level and ends at the anterior portion of the orbit.Figure 3Skull and mandible of Luopingosaurus imparilis gen. et sp. nov., IVPP V19049. Head before (a) and after (b) dusted with ammonium chloride. (c), Line- drawing. (d, e), two computed laminography scanning slices. (f), reconstruction in ventral view. ac, acetabulum; an, angular; ar, articular; ax, axis; c, cervical vertebra; den, dentary; eo, exoccipital; f, frontal; hy, hyoid; in, internal naris; j, jugal; m, maxilla; n, nasal; p, parietal; par, prearticular; pof, postfrontal; prf, prefrontal; pt, pterygoid; q, quadrate; qj, quadratojugal; sa, surangular; sp, splenial; sq, squamosal; stf, supratemporal fossa; v, vomer.Full size imageThe orbit is oval and large, measuring 24.8% of the skull length (Fig. 3). The lateral margin of the frontal contacts the prefrontal anteriorly and the postfrontal posteriorly, and defines most of the medial border of the orbit. The L-shaped jugal, together with the posterolateral process of the maxilla, forms the lateral border of the orbit. No distinct lacrimal is discernable; the bone is probably absent as in other sauropterygians. The postfrontal contacts the dorsal process of the triradiate postorbital ventrally, and both bones define the posterior border of the orbit. Additionally, the posterior process of the postorbital contacts the anterior process of the squamosal, forming the bar between the supratemporal fossa and the ventrally open infratemporal fenestra. The jugal extends beyond the ventral margin of the postorbital and also contacts the anterior process of the squamosal, resembling the conditions in Wumengosaurus30, Honghesaurus23 and Diandongosaurus15. This contact is absent in other pachypleurosauroids4,6,7,8,9,10,11,12.A pair of vomers and pterygoids and a right palatine are discernable in the palate (Fig. 3a–c). The vomer is elongate and slender, extending anteriorly well beyond the nasal. The internal naris, partly covered by the detached splenial, is longitudinally retracted, corresponding to a retracted external naris (Fig. 3d–f). The medial margin of the naris is defined by the nasal, without contribution from the premaxilla. A retracted naris is otherwise present in Wumengosaurus13,30, Qianxisaurus16 and Honghesaurus23. Similar to the condition in Honghesaurus23, the retracted naris of Luopingosaurus is relatively short, having a longitudinal diameter distinctly less than half of the longitudinal diameter of the orbit. By contrast, other pachypleurosauroids4,6,7,8,9,10,11,12,25 generally have an oval-shaped naris. The elongate palatine has a slightly convex medial margin suturing with the pterygoid. Because of the coverage of the detached splenial, the lateral portion of the palatine is unexposed, and it is hard to know whether an ectopterygoid is present or not. The pterygoid is the largest and longest element of the palate, measuring 55.2% of the mandibular length. It has an anterior projection that contacts the vomer anteromedially, and does not participate in the margin of the internal naris. At the level of the posterior orbital margin, the pterygoid has a triangular lateral extension, which was termed as the ectopterygoid process of the pterygoid in Neusticosaurus8. The pterygoid extends back to the occipital condyle, and covers the basicranium and parietals in ventral view. Additionally, the bone has a broad posterolateral process that is set off from the palatal surface by a distinct ridge, resembling the conditions in Serpianosaurus9 and Neusticosaurus8. Posteriorly, the basioccipital is exposed in ventral view, showing the area for attachment to the right exoccipital.The left quadrate is exposed in lateral view with its dorsal process extending underneath the base of the descending process of the squamosal. The posterior margin of the quadrate is excavated, as in many other pachypleurosaurids (e.g., Serpianosaurus9 and Honghesaurus23). The quadratojugal is narrow and splint-like, flanking the anterior margin of the quadrate. A pair of hyoids are ossified. They are rod-like, slightly expanded at both ends. The dentary is wedge-shaped, being 71.7% of the mandibular length. Laterally, it bears a longitudinal series of pores and grooves parallel to the oral margin of the bone (Fig. 3a). The elongate angular tapers at both ends, contacting the dentary anterodorsally and the surangular dorsally in ventral view. The surangular, slightly shorter than the angular, contacts the articular posterodorsally, with a pointed anterior tip wedging into the notched posterior margin of the dentary. The retroarticular process of the articular is very short with a rounded posterior margin. Medially, the splenial and prearticular form most of the inner wall of the mandible. The splenial tapers at both ends and enters the mandibular symphysis anteriorly, having a length similar to the dentary. The relatively slender prearticular contacts the splenial anterodorsally, extends posteriorly and abuts the articular dorsally, measuring 41.1% of the mandibular length.The whole series of 21 cervical vertebrae (including the atlas-axis complex) is well exposed ventrally. The atlas centrum is oval, much smaller than the axis centrum (Fig. 3c). From the axis, the cervical vertebrae increase gradually in size toward the trunk vertebrae posteriorly. The bicephalous cervical ribs have typical free anterior and posterior processes as in other pachypleurosauroids8,9. The trunk is relatively long, including 27 dorsal vertebrae. The posterior dorsal ribs show certain pachyostosis (Fig. S1). Each gastralium consists of five elements (a short and more massive median element and two slender rods in line towards each side; Figs. 3, 4a, b, S1), similar to the conditions in most of other pachypleurosauroids9,11,18,25 (except Neusticosaurus8). Three sacral ribs are clearly revealed by X-ray computed microtomography (Fig. 4c–f). They are relatively short and stout, with the posterior twos bearing a distinct expansion on their distal heads. The distal expansion of the sacral rib is also present in Keichousaurus11, Prosantosaurus25, Qianxisaurus16 and Wumengosaurus13, but it is not pronounced in other pachypleurosauroids4,6,7,8,9,10. The caudal ribs are relatively few, six pairs in number. Additionally, several chevron bones are visible in the proximal caudal region, and they are gradually reduced in length posteriorly (Fig. 4d).Figure 4Girdles, limbs and vertebrae of Luopingosaurus imparilis gen. et sp. nov., IVPP V19049. Photo (a) and line-drawing (b) of pectoral girdle, forelimbs and anterior dorsal vertebrae. Photo (c), line-drawing (d) and two computed laminography scanning slices (e, f) of pelvic girdle, hind limbs and posterior vertebrae. as, astragalus; ca, caudal vertebra; cal, calcaneum; car, caudal rib; co, coracoid; d, dorsal vertebra; dltp, deltopectoral crest; enf, entepicondylar foramen; fe, femur; fi, fibula; h, humerus; il, ilium; int, intermedium; is, ischium; mc, metacarpal; mt, metatarsal; pu, pubis; s, sacral vertebra; sc, scapula; sr, sacral rib; ti, tibia; ul, ulna; uln, ulnare.Full size imageThe paired clavicles and the median interclavicle form a transverse bar at the 20th cervical vertebrae (Fig. 4a, b). The blade-like clavicle tapers posterolaterally with its distal projection overlapped by the scapula in ventral view. The left clavicle contacts the right one anterodorsally to the interclavicle. The interclavicle tapers laterally to a point at each end. The anterior margin of the interclavicle is convex and its posterior margin is slightly concave without a midline projection (contra the condition in Anarosaurus42). The scapula consists of a broad ventral portion and a relatively narrow and elongate dorsal wing that varies little through its length. The coracoid is hourglass-shaped with a slightly concave posterolateral margin and a conspicuously concave anteromedial margin. The medial margin is straight, along which the coracoids would articulate each other in the midline. The humerus is constricted at the middle portion with a nearly straight preaxial margin and a concave postaxial margin. A slit in the expanded distal portion of this bone indicates the possible presence of an entepicondylar foramen (Fig. 4a, b). The radius, slightly longer than the ulna, is more expanded proximally than distally. The ulna is straight with a slightly constricted shaft. In each forelimb, there is two nearly rounded carpals, ulnare and intermedium; the former is half the width of the latter. Five metacarpals are rod-like, slightly expanded at both ends. Among them, Metacarpal I is the shortest, 48% of the length of Metacarpal II. Metacarpal III is slightly shorter than Metacarpal IV, which is the longest. Metacarpal V is 71% of the length of Metacarpal IV. The phalangeal formula is 2–3–5–5–3 for the manus, indicating presence of hyperphalangy in Luopingosaurus (see Discussion below).In the pelvic girdle, the ilia, pubes and ischia are well exposed (Fig. 4c–f). The ilium is nearly triangular with a relatively long and tapering posterior process. The plate-like pubis is well constricted at its middle portion, with the medial portion nearly equal to the lateral portion. The obturator foramen is slit-like, located at the posterolateral corner of this bone (Fig. 4e). The ischium is also plate-like, having a relatively narrow lateral portion and an expanded medial portion that is notably longer than the medial portion of the pubis. The posterolateral ischial margin is highly concave. The posterior pubic margin and anterior ischial margin are moderately concave, and both together would enclose the thyroid fenestra. The femur is slightly longer than the humerus, with a constricted shaft and equally expanded ends (Fig. 4d). No internal trochanter is developed. The tibia is nearly equal to the fibula in length; the former is straight and thicker than the slightly curved latter. Two ossified tarsals, calcaneum and astragalus, are nearly rounded; the latter is significantly larger than the former. As in Honghesaurus23, the astragalus lacks a proximal concavity. The right metatarsals are well-preserved. Metatarsal I is the shortest and stoutest phalange with an expanded proximal end, and Metatarsal IV is the longest. Metatarsal II is nearly twice the length of Metatarsal I. Metatarsal III is slightly shorter than Metatarsal IV, and Metatarsal V is 76% of the length of Metatarsal IV. The phalangeal count is 2–3–4–6–4, which is complete judging from the appearance of the distal phalanges in the right pes (Fig. 4c). More

Fully terrestrial eDNA sampling approaches offer a potentially powerful addition to biodiversity monitoring efforts23,24. However, protocols for using eDNA-based methods to characterize terrestrial biodiversity, and vertebrate communities in particular, are still nascent27,28,30. In this study, we show for the first time that an eDNA metabarcoding approach can be used to broadly characterize tree-dwelling mammal communities by sampling tree trunks and surrounding soil. Our findings add to recent work (e.g., for reptiles22,24) showing that surface eDNA collection methods, which are relatively untested compared with soil-based eDNA methods, can also be effective at detecting terrestrial vertebrates. Further, we demonstrate that supplementing metabarcoding detection with qPCR-based methods can greatly improve sensitivity, a potentially important consideration for monitoring schemes focused on rare taxa (e.g., Refs.11,12). Together, our results have significant implications for global biodiversity conservation as the broader guild of arboreal vertebrates includes highly threatened5,48,49, as well as invasive alien species50, that are often cryptic, inhabit inaccessible locations, and are therefore challenging to monitor.Our methods captured over 60% of the mammalian diversity expected at the sites, and a similar fraction of the subset of arboreal species, despite sampling only 21 trees. Species accumulation curves suggest that more species would likely have been added with increased sampling effort. These results broadly agree with those of Leempoel et al.23 who found that soil eDNA metabarcoding well characterized mammal communities in California chaparral. However, in both our study and that of Leempoel et al.23, some conspicuous absences were evident. Bats comprised all of the arboreal species that we expected but failed to detect at our sites using metabarcoding (Fig. 1A). Leempoel et al.23 also noted a lack of bat detections (2 of 14 possible taxa detected), which they suggested could be due to low efficiency of either the 12S primer set or of their soil sampling methods for that order. While both reasons could also apply to the lack of bats detected in our study (discussed further below), the performance of the 12S primer set very likely contributed to our lack of American black bear detections as MiMammal-U primers are known to be ineffective at amplifying bear DNA38. These challenges highlight the reality that false negatives and varying detectability among species are common issues to all survey approaches, including eDNA metabarcoding. Our study represents a rare example among metabarcoding studies in that it uses repeated sampling and community occupancy models to quantify false negative rates. This quantitative approach, coupled with continued experimentation with different molecular techniques and survey methods (e.g., Refs.23,27), will be vital to helping researchers decide how eDNA metabarcoding methods will fit into existing biodiversity monitoring efforts moving forward.Although our results suggest that sampling for tree-roosting bats using eDNA metabarcoding still requires further research and optimization, our approach likely has application to characterizing communities in a much broader range of arboreal species globally. Geographic regions with multiple elusive arboreal mammals of management interest—for example, gliders and tree kangaroos in Australasia, or primates in the global tropics—may be particularly suited for a metabarcoding approach for community-level assessments4,8,9,49. It may be especially useful for rapid biodiversity assessments (RBAs51) in remote forested environments, where the ability to collect multiple samples relatively rapidly without regard to time of day would be a key advantage27. Existing survey methods to monitor arboreal mammals tend to be optimized for particular groups of species, often segregated by body size and behavior, with no suitable single method available to characterize all members of the guild4,8,9,16,49. Diurnal and nocturnal species, for example, often require separate survey methods or timing8. While camera traps capture both diurnal and nocturnal species, they typically miss smaller species16,23. The need for multiple methods to survey for nocturnal and diurnal, or large and small, species separately raises the cost of sampling and can result in datasets that are difficult to compare across sites because of inherent sampling biases8. Excluding bats, we found encouraging results for both diurnal and nocturnal arboreal species of a broad range of body sizes, detecting all seven expected species (Fig. 1A). While more work is needed to assemble robust genetic reference libraries before global arboreal mammal monitoring with eDNA metabarcoding will be broadly feasible, a clear advantage of the technique remains the power to detect a broad swath of species, with widely varying morphologies and behaviors, with a single method23,27,28,51.The promise of eDNA metabarcoding approaches for at least some arboreal guilds is well illustrated by our results for southern flying squirrel, Glaucomys volans. Like other flying squirrels (Tribe: Pteromyini), this species is strictly nocturnal, highly arboreal, and tends to get injured in live traps, making it difficult to directly observe and monitor48,52. Yet G. volans eDNA was readily detectable using metabarcoding in our study, occurring in 19–26% of soil samples and 47–52% of roller samples across both sites. Our similarly encouraging results for detecting other squirrels (Sciuridae) also bode well for management applications. For example, the methods would enable fine-scale mapping of habitat use in places such as the United Kingdom where native red squirrels (Sciurus vulgaris) are outcompeted by eastern gray squirrels, or the Delmarva peninsula (USA) to support the conservation efforts for the Delmarva fox squirrel (Sciurus niger cinereus)53. Further research is needed to determine the extent to which our results for squirrels generalize to other taxa with similar active tree-climbing lifestyles (e.g., gliders4, primates49).Our finding that soil samples revealed fewer species, had lower detection probability, and had lower read counts than roller samples, even for some non-arboreal species like white-tailed deer, likely reflects multiple factors. First, soil and tree bark represent markedly different biological and chemical environments that likely differ in eDNA quantity by species, eDNA persistence rates54,55, and microorganism abundance. The latter may be especially pertinent to our study as we observed a relatively large drop in the number of reads after removal of microorganism reads, especially for soil samples. This suggests that performing additional purification steps prior to sequencing could boost the ability of both methods, and especially soil eDNA, to detect target species by increasing mammalian sequencing depth. Other in-lab factors, such as method of extraction23 or choice of primers, similarly have the potential to influence the recovery and amplification of target species’ DNA and should be the focus of future research.Next, our focal trees were not chosen to occur near any special attractants or areas of multi-species use, such as saltlicks or water sources, which has proven successful in other vertebrate eDNA studies18,25,31,32,56. It is possible that adding a broader range of soil sampling sites, including some targeted towards other guilds (e.g., burrow users32,56), would have yielded a more complete inventory. Nevertheless, both soil and surface methods have advantages over the much more commonly-used metabarcoding approaches that rely on natural water bodies for assessing mammal communities16,17,27,28,29,30,38 as they are not limited to where these features occur. Our study is the first to suggest that surface eDNA metabarcoding methods can be a powerful supplement to established soil-based methods of characterizing mammal communities, especially for arboreal species.As noted, bats were especially lacking from our eDNA metabarcoding results, with only two of six likely species detected. Notably, our metabarcoding species list lacked two of the bat species that our sampling scheme was designed around (eastern red bat and northern long-eared bat) and for which we had confirmed recent presence at the sites (Table 1). The lack of northern long-eared bat detections may directly relate to recent precipitous population declines (~ 99%) caused by white-nose syndrome57. However, the lack of eastern red bat detections was especially surprising as roosting of this species was suspected based on telemetry in 17 of our 21 target trees. Reasons for this omission may relate to the fact that eastern red bats roost singly on small twigs and in leaf clusters, and therefore may not leave much DNA on tree trunks. Another possibility is low efficiency of the 12S primer set for bats, although we were unable to find information about this in the literature. It is notable that Leempoel et al.23 had a similarly poor representation of bats with comparable soil-based methods. However, our metabarcoding results did indicate that we are capable of detecting even uncommon, or at least unexpected, bat species with our methods. Eastern small-footed bat, which is typically viewed as a rock-roosting species and is considered endangered by the International Union for Conservation of Nature (IUCN)58, was detected in both soil and surface eDNA samples from Morristown National Historic Park. This species was not otherwise confirmed as present at the site until a year later, in spring 2022, when it was caught in a mist net (BM, unpublished data). Our results with respect to bat detections, along with those of others23, underscore the need for further research to adapt eDNA metabarcoding methods to this vulnerable group, which could contribute much needed demographic and distribution information. This is especially urgent as 18% of bat species are listed as “data deficient” by the IUCN, while 57% lack basic population trend information5,58.Our comparison of qPCR to metabarcoding detection methods for big brown bat represents a hopeful result for the use of eDNA to monitor rare vertebrates that are of particular conservation interest. It is well-known that qPCR-based eDNA surveys targeted towards individual species return higher detection probabilities and have greater power at low abundance, than metabarcoding approaches59. Our results agree, showing for the first time that adding a qPCR step in the analysis of surface and soil eDNA samples can be effective for detecting bats in forested environments. The addition of a qPCR step opens the door for developing species-specific assays to increase detection power for endangered or elusive bat species, or other cryptic arboreal mammals49. Emerging molecular detection approaches such as droplet digital PCR have the potential to increase this sensitivity even further59. Like other eDNA-based tools and survey tools in general, careful consideration of sampling effort, the natural history of target species, and the configuration of different field and molecular methods will be key to optimizing our approach to characterize mammal communities, or to target a particular species, in different regions.Although eDNA surveys are not inexpensive given the need for both fieldwork and molecular analyses, they can be cheaper than conventional approaches, especially if such approaches require many hours of fieldwork or expensive equipment60. Thus, the relative cost-effectiveness of surface or soil eDNA surveys will depend heavily on the mammal communities of interest, the mix of methods that must be employed to effectively sample them, and the purpose of the sampling efforts. However, even if costs are increased, eDNA surveys can reduce field time to the extent that they can improve detection rates, either by replacing or supplementing conventional sampling methods (e.g., as a supplement to visual observations). With higher detection rates, fewer visits are required to achieve the same results. This operational efficiency would be especially advantageous when field conditions present safety risks, are intrusive to sensitive habitats, or are challenging to access. For example, adding surface eDNA sampling to existing visual surveys of eastern wood rats (Neotoma floridana), a cryptic mammal that inhabits steep, rocky slopes in the eastern US, could likely increase detection power, thereby reducing the need for additional risky and costly sampling visits. More studies involving direct comparisons among methods (e.g., Refs.23,24,30,60), in a variety of ecoregions, are needed to determine the extent to which incorporating our methods into existing vertebrate monitoring workflows would increase efficiency.Finally, we detected other vertebrates, including seven birds and one salamander, in soil and surface eDNA samples, despite our use of a mammal-specific primer set. This is similar to results from Leempoel et al.23 in California using the same primer set, in which six bird species were detected. We found that surface eDNA detected more bird species than soil, perhaps for the same reasons as for mammals (above). Our results provide evidence that surface eDNA surveys, with taxon-specific primers, could be used to survey bird communities, or used to target particularly rare species in forested ecosystems (e.g., Ref.61). Our detection of a salamander, coupled with recent promising research into reptile detection using surface eDNA methods22,24 suggests a broader potential for applications with other vertebrates as well. Finally, both surface and soil eDNA metabarcoding can be expanded beyond forests, providing insight into their effectiveness in other habitats (e.g., caves17 or talus slopes). Our study and others highlight that the potential of coupling surface and soil eDNA methods for detecting and monitoring mammalian biodiversity, and terrestrial organisms generally, has yet to be fully realized. More