Wild Andean camelids promote rapid ecosystem development after glacier retreat
In mountainous regions, climate change drives range shifts of species and biomes, as organisms track suitable climates – , with some regions experiencing amplified warming and precipitation changes at higher elevations . Under warming temperatures, cold-adapted species including many high-elevation species can be at risk of population declines and range contractions which may lead to biodiversity loss , , . Warming is also associated with a global loss of ice and snow in mountain regions . As glaciers retreat, new ecosystems and species assemblages develop in glacier forefields , , but a major concern is that plant species shifts into glacier forefields could substantially lag behind climatic changes because glacier forefield soils are oligotrophic and develop slowly – . Understanding mechanisms that facilitate primary succession and post-glacial ecosystem emergence is urgent given the rapid pace of glacier loss in high-mountain environments worldwide – . In the tropical Andes, plant colonization is limited even 150 years after glacial retreat because newly exposed soils are severely nutrient limited – . The lag in plant primary succession creates a climatic debt that constrains plant distributions and hinders the response of tropical alpine communities to climate change , . The uncoupling between climate and plant colonization may make high-elevation ecosystems such as the vast carbon-sequestering, biodiverse Andean puna grasslands located just below glacial forelands far more vulnerable to range contraction than currently recognized (e.g ). by failing to account for the lag in upslope shifts. Glacial forefields have long been model systems for understanding changes in community composition and structure over time following disturbance – . Decades of research show that microbes are initial colonizers of recently exposed soils – and complex autotroph and decomposer communities occupy unvegetated soils before plant invasion , – . Microbial succession shapes soil formation, creates labile nutrient pools, facilitates colonization by vascular plants, and influences development of complex communities following glacier retreat , . Later colonization can be facilitated by positive plant–plant interactions (e.g., nurse plants) primarily by ameliorating extreme physical conditions , , . However, even nurse plants are unable to facilitate plant primary succession on timescales relevant to the rapid rate of glacial retreat in high-elevation environments . A key unknown is whether other natural mechanisms might facilitate broader community and ecosystem development on timescales relevant to the unprecedented recent glacier retreat in the tropical Andes. A growing body of literature suggests that environmental heterogeneity influences vegetation dynamics within glacial forefields , , but past studies have not recognized potentially important allogenic sources of natural heterogeneity in soil development that may provide natural mechanisms for community establishment in glacial forefields. Globally, mammals enrich soil nutrients and influence plant diversity and community composition – . In the Andes region, native ungulates (Artiodactyla: Camelidae) including domesticated alpaca ( Vicugna pacos ) and llama ( Lama glama glama ), and wild vicuña ( Vicugna vicugna ) and guanaco ( Lama glama guanicoe ), exhibit social behaviors that influence the landscape. Native camelids typically live in family groups and create discrete communal dung piles (“latrines”) which alter edaphic properties and plant communities in both established grasslands – and proglacial landscapes , , suggesting camelids may play an important role in the emergence of new high-Andean ecosystems following glacier loss. Interestingly, animal and microbial factors may interact, with pulses of animal nutrients also affecting microbial community composition and potentially dispersing mycorrhizal fungi , . Animal–plant and animal–microbe facilitation might be a more ubiquitous mechanism in the process of ecosystem development in proglacial areas worldwide than currently is recognized, and may represent an overlooked mechanism by which ecological communities can become established following the rapid loss of glacier cover. To better understand the role of large animals in the emergence of new ecosystems resulting from glacial retreat, we studied how nutrient transfer into the proglacial zone by native, wild camelids facilitates primary succession. Our study took place in the Tropical Andes, where new land is being rapidly exposed by glacier retreat , , . In this study we explore the impact of nutrient release by wild vicuñas ( Vicugna vicugna ) on edaphic properties, vegetation cover, and microbial community characteristics at latrines compared to paired glacial till controls. We hypothesized that soil modification by camelids would accelerate primary succession after glacial retreat and tested the following predictions: (1) soils in vicuña latrines would have less extreme temperature ranges, and greater moisture, organic material, and nutrient concentrations than controls; (2) animal-modified soils would be associated with higher plant cover, soil DNA concentrations, microbial richness, and a distinct microbial community; and (3) plant roots would have greater amounts of fungal endophyte colonization due to spore dispersal by vicuñas in latrines compared to surrounding soils.   Soil properties In 2018–2023 we identified vicuña latrines within glacial moraine soils that were exposed since the Little Ice Age maximum, including four latrines which we had first observed in a previous visit to the site in 2013 (Fig. ). Soils within vicuña latrines were significantly enriched in all edaphic properties that we measured, except for nitrate, compared to control glacial till soils adjacent to the latrines (Table ; Fig. ). However, mean nitrate levels showed considerable variation among the six sampled latriens compared to the uniformly very low nitrate values in moraine reference soils (Fig. ). Ammonium levels were also very low in glacial till control soils, and were significantly greater in latrine soils. Likewise, levels of soil organic matter were very high in latrine soils (mean = 62%), as were soil moisture levels (mean = 121%, Fig. ), compared to control soils. Daily maximum temperatures were consistently lower in latrine soils than in control soils in both the dry and wet seasons (Table , Fig. ). Latrine soil maximum temperatures were on average 5.4 °C colder than control soils in the dry season, and 5.0 °C colder in the wet season. Daily minimum temperatures were consistently warmer in latrine soils than in control soils in both the dry and wet seasons (Table , Fig. ). Latrine soils minimum temperatures were on average 2.1 °C warmer than control soils in the dry season and 1.9 °C in the wet season. Soil biota Soil biotic properties also showed significant differences between latrine soils and nearby control soils. For example, DNA concentrations in latrine soils (23.2 ± 8.8 µg DNA g soil −1 ) were significantly higher compared to control soils (0.62 ± 0.27 µg DNA g soil −1 ; Table ; Fig. f). While there were no differences in prokaryote ASV richness, eukaryote ASV richness was significantly greater in latrines than controls (Table ; Fig. ). Community composition at the ASV level was significantly different between latrines and controls for both bacteria (PERMANOVA, p = 0.004, F = 8.04, R 2 = 0.45, Fig. c) and eukaryotes (PERMANOVA, p = 0.002, F = 4.68, R 2 = 0.32, Fig. d). There were no differences in the amount of variation in communities within the latrine and control soil types (PERMDISP, p > 0.05). Differences in community composition were primarily driven by individual bacterial ASVs from (a) the cyanobacterial genera Nostoc , Wilmottia , and Tychonema that were higher in the controls and the actinobacterium genus Citricoccus (higher in latrines), and (b) by eukaryote ASVs from the rotifer genus Adineta the nematode genus Longidorus , the class Arachnida, and the genus Euglypha , which were more abundant in controls, and the cercozoan genus Trinema (higher in latrines). In addition to these specific ASVs, broad taxonomic differences in bacterial phyla included significantly lower relative abundances of Cyanobacteria and significantly greater relative abundances of Bacteroidetota, Firmicutes, Planctomycetota, Proteobacteria, and Verrucomicrobiota in latrine soils (Table ; Fig. ). For eukaryotes, there were lower relative abundances of the Metazoa kingdom in latrine soils but increases in the Cercozoa phylum and the Tubulinea class (Table ; Fig. ). Plant cover and endophytes Vegetation cover was significantly greater in latrines than controls in both the dry ( N = 6 pairs; Table ; Fig. e) and wet seasons (based on 114 quadrats from N = 24 locations including the original six; Χ 2 = 1353.2, df = 1, p-value < 0.001; mean percent cover ± SE on latrine plots = 42.3 ± 5.30, and moraine control plots = 8.6 ± 1.11). All three focal plants studied contained arbuscular mycorrhizal fungi, dark septate endophytes, and fine root endophytes. Percent root colonization by arbuscular mycorrhizae was significantly greater in Ephedra collected from latrines than controls (Table ; Fig. ). There were no significant differences in fungal colonization in the other two plant species studied (Table ; Fig. ).   In 2018–2023 we identified vicuña latrines within glacial moraine soils that were exposed since the Little Ice Age maximum, including four latrines which we had first observed in a previous visit to the site in 2013 (Fig. ). Soils within vicuña latrines were significantly enriched in all edaphic properties that we measured, except for nitrate, compared to control glacial till soils adjacent to the latrines (Table ; Fig. ). However, mean nitrate levels showed considerable variation among the six sampled latriens compared to the uniformly very low nitrate values in moraine reference soils (Fig. ). Ammonium levels were also very low in glacial till control soils, and were significantly greater in latrine soils. Likewise, levels of soil organic matter were very high in latrine soils (mean = 62%), as were soil moisture levels (mean = 121%, Fig. ), compared to control soils. Daily maximum temperatures were consistently lower in latrine soils than in control soils in both the dry and wet seasons (Table , Fig. ). Latrine soil maximum temperatures were on average 5.4 °C colder than control soils in the dry season, and 5.0 °C colder in the wet season. Daily minimum temperatures were consistently warmer in latrine soils than in control soils in both the dry and wet seasons (Table , Fig. ). Latrine soils minimum temperatures were on average 2.1 °C warmer than control soils in the dry season and 1.9 °C in the wet season.   Soil biotic properties also showed significant differences between latrine soils and nearby control soils. For example, DNA concentrations in latrine soils (23.2 ± 8.8 µg DNA g soil −1 ) were significantly higher compared to control soils (0.62 ± 0.27 µg DNA g soil −1 ; Table ; Fig. f). While there were no differences in prokaryote ASV richness, eukaryote ASV richness was significantly greater in latrines than controls (Table ; Fig. ). Community composition at the ASV level was significantly different between latrines and controls for both bacteria (PERMANOVA, p = 0.004, F = 8.04, R 2 = 0.45, Fig. c) and eukaryotes (PERMANOVA, p = 0.002, F = 4.68, R 2 = 0.32, Fig. d). There were no differences in the amount of variation in communities within the latrine and control soil types (PERMDISP, p > 0.05). Differences in community composition were primarily driven by individual bacterial ASVs from (a) the cyanobacterial genera Nostoc , Wilmottia , and Tychonema that were higher in the controls and the actinobacterium genus Citricoccus (higher in latrines), and (b) by eukaryote ASVs from the rotifer genus Adineta the nematode genus Longidorus , the class Arachnida, and the genus Euglypha , which were more abundant in controls, and the cercozoan genus Trinema (higher in latrines). In addition to these specific ASVs, broad taxonomic differences in bacterial phyla included significantly lower relative abundances of Cyanobacteria and significantly greater relative abundances of Bacteroidetota, Firmicutes, Planctomycetota, Proteobacteria, and Verrucomicrobiota in latrine soils (Table ; Fig. ). For eukaryotes, there were lower relative abundances of the Metazoa kingdom in latrine soils but increases in the Cercozoa phylum and the Tubulinea class (Table ; Fig. ).   Vegetation cover was significantly greater in latrines than controls in both the dry ( N = 6 pairs; Table ; Fig. e) and wet seasons (based on 114 quadrats from N = 24 locations including the original six; Χ 2 = 1353.2, df = 1, p-value < 0.001; mean percent cover ± SE on latrine plots = 42.3 ± 5.30, and moraine control plots = 8.6 ± 1.11). All three focal plants studied contained arbuscular mycorrhizal fungi, dark septate endophytes, and fine root endophytes. Percent root colonization by arbuscular mycorrhizae was significantly greater in Ephedra collected from latrines than controls (Table ; Fig. ). There were no significant differences in fungal colonization in the other two plant species studied (Table ; Fig. ).   Our study shows that animals can have profound effects on both the abiotic and biotic characteristics of recently deglaciated high-mountain soils. Vicuña latrines buffer the wide temperature fluctuations that occur daily in near-surface soils at these elevations, are enriched in organic matter and soil nutrients, and are characterized by higher plant cover than glacial moraine reference soils. In addition, latrine soils have significantly higher DNA concentrations, higher eukaryote richness, and distinct communities of bacteria and eukaryotes compared to unmodified moraine soils. Our results suggest that native, wild camelids that transfer limiting nutrients into newly exposed glacier forefield ecosystems create biogeochemical and biodiversity hotspots. Soil modification by animals thus ameliorates some of the harsh conditions in glacier forefields, providing a mechanism for accelerated soil development and primary succession, promoting the establishment of ecological communities long before the broader landscape is heavily colonized by plants. Soil modifications made by vicuñas on proglacial habitat and resource availability will likely persist on the landscape and, given the observed longevity of their dung piles, represent important legacy effects (sensu ). We observed some latrines that have been present for at least 11 years based on repeated visits to the site. Soil chemistry data indicate higher resource levels (e.g., ammonia, organic matter, and water) in vicuña latrines than in the surrounding soils. Soil organic matter was also very high on latrines (mean of 62%) similar to “organic soils” or Histosols . For comparison, productive agricultural soils have between 3 and 6% organic matter , and deglaciated moraine soils that have been exposed for 85 years at this locale typically contain ~ 1.5% TOC . A high level of organic material development on latrines contrasts sharply with the surrounding soils where organic matter was close to 0%. Vicuña dung likely contributes directly to the input of organic matter as their diets are high in relatively indigestible plant fiber and lignin , and additional organic matter development likely occurs from accumulation of dead plant material on latrine patches. In contrast, organic matter inputs in other developing high-elevation soils that lack large animals are limited primarily to aeolian inputs and cyanobacteria , , . Soil moisture levels were also surprisingly high in the latrine soils despite having been sampled in the dry season, suggesting that latrines have higher water availability even at a time when water may be a limiting resource in the surrounding glacial soils. Moisture likely is influenced by the higher organic matter content and soil texture properties as well as shading from the high plant cover. We suspect the vicuñas are primarily transporting nutrients from established ecosystems below the proglacial zone and depositing them in concentrated hotspots inside the recently deglaciated areas (e.g . described nutrient transport by vicuñas among different established grassland communities). However, we did not determine the origin of organic matter on latrines and thus cannot rule out the interesting possibility that vicuñas may contribute both to allochthonous nutrient transfer into the proglacial zone and redistribution and cycling within the proglacial zone because they are feeding on plants in their own dung piles within the glacier forefields and because gut passage time may be as little as ~ 24 h . We suspect the bulk of nutrients and organic matter in vicuña dung is likely derived from sources outside the glacier forefield because overall plant cover is so low in the newly exposed proglacial areas. Vicuña-created hotspots likely provide suitable habitat patches that attract other animals into the recently deglaciated zone, and the nutrient transfer could act as an important subsidy for a wide range of other organisms such as insects, lizards, birds, and other mammals. Thus, vicuña (and other animals that create and modify habitats in this way in other regions) likely also structure species abundances and biodiversity in a facilitation cascade (sensu ), making the proglacial zone into usable habitat for other species decades before the broader landscape is colonized by grassland plants. Future research will determine the degree to which vicuña latrines consist of organic matter transported from lower elevations and subsidize the trophic structure of emerging glacier forefield animal communities. In such high-elevation, low-latitude environments, one of the most extreme abiotic characteristics of the environment is a wide daily temperature range. At our study site, high insolation can result in a 39 °C diurnal temperature range near the soil surface . Our data show that latrines buffer extreme low and high temperatures during both the dry and wet seasons. Thermal buffering on latrines may reduce cryoturbation and thermal stress, two factors that could limit the establishment of a wide range of plants and microbes from lower elevations. Buffering of high temperatures on latrines in the wet season may occur because of higher soil moisture leading to greater evaporative cooling potential, and latrines may also retain an insulating layer of snow cover longer than controls. Soil DNA concentrations were almost two orders of magnitude higher in latrine soils compared to controls. For comparison, DNA concentrations in latrine soils are greater than concentrations in low-elevation grassland soils in Colorado, USA (9.9 ± 2.0 µg DNA g soil −1 ) that were measured using the same protocols and instrumentation . Thus, using DNA concentration as a proxy for biomass , it appears that latrines have sped the rate of development such that belowground biomass of microbes and plant roots has reached or exceeded ecosystems with developed grassland plant communities. Future studies using more direct quantification methods for microbes and plant roots will help determine the relative contributions of each group to the belowground buildup of biomass. Latrine soils contained distinct bacterial/archaeal communities compared to nearby unvegetated glacial till soils. These patterns resulted from taxa turnover and different relative abundances rather than increases or decreases in richness and are not surprising given the differences in abiotic properties (e.g., water, ammonium, temperature; Figs. and ) observed on latrines compared to controls. Control soils are characterized by greater relative abundances of oligotrophic and/or autotrophic taxa such as cyanobacteria while latrine soils have greater relative abundances of eutrophic or fast-growing taxa that have likely outcompeted others in the high-resource conditions. Such differences in community structure have also been shown between unvegetated and vegetated soils in other studies of high elevation soils , . Latrine soils are enriched in Bacteroidota, which are common soil bacteria and have been shown to increase in relative abundance in high-elevation tundra soils that receive chronic N inputs and during primary succession at the nearby Puca Glacier chronosequence , in line with our findings of increased nitrogen concentrations in the latrine soils. While Bacteroidota is also a phylum with members abundant in the mammalian gut, the Bacteroidota families found in the latrine soils (e.g., Cytophagaceae, Chitinophagaceae, Flavobacteriaceae, Sphingobacteriaceae) are not the typical gut-associated families such as Prevotellaceae and Rikenellaceae. In fact, these two families and the other typical bacteria and parasites of the mammalian gut such as Ruminococcaceae, Lachnospiraceae, Erysipelotrichaceae, Bifidobacteriaceae, nematodes, and trichomonads were either absent or in very low abundance in latrine soils. Their absence suggests that differences with control soils are not driven by inputs of gut taxa, but by selection of soil microbes that thrive in the environmental conditions of the latrine soils. Further evidence that the bacteria found in the latrine soils do not originate from the gut flora of vicuñas comes from examining the dominant Bacteroidota ASVs found in the present study. Both the Spirosoma (Cytophagaceae) and Ferruginibacter (Chitinophagaceae) ASVs found in the present study have been previously described in soils from the same area. The Ferruginibacter ASV is 100% identical (across 253 base pairs) with a sequence (acc. no. GQ306056) found in 2001 near our highest latrine pile at the “Spit” site (5410 m.a.s.l ). Likewise, the Spiromonas ASV is 99.6% similar to sequences (DQ514240, DQ514129) previously found at a site about 300 m from our lowest sampled latrine pile during a survey of the forefield of the Puca Glacier in 2003 . Very similar ASVs have been found in other cold early successional soils (KP913388 ) and polar deserts (FJ490274 ), further supporting the hypothesis that these bacteria are indigenous to cold, dry climates and not animal guts. Latrine soils contained greater richness of eukaryote ASVs than controls, which is likely due to the less stressful rhizosphere environment in the latrines . Latrine soils also contained unique eukaryotic communities relative to controls, which were driven by both protist and animal taxa such as insects and mites. Fungi had generally low relative abundance in both moraine and latrine soils, with some patches of high abundance in several moraine soils. Unfortunately, there were many unassigned taxa in the eukaryote dataset, or taxonomy that was only resolved at broad levels. Key eukaryote ASVs that were significantly more relatively abundant in control soils compared to latrine soils were diverse, featuring a nematode, a mite, a cercozoan protist, and a rotifer (Fig. ). One explanation for the increase in the dorylaim nematode (genus Longidorus ) in controls compared to latrines is that they feed on cyanobacteria, as has been noted in biological soil crusts in other systems . On the other hand, increases in the testate amoeba ASV (genus Trinema ) in latrines compared to controls were likely driven by the vegetation composition and increased cover, as vegetation type has been shown to be a key factor structuring the composition of montane amoebae . Given the magnitude of vicuña latrine effects on edaphic properties and microbial communities, it is not surprising that plant cover on latrines was much higher than on glacial till controls. Clearly these latrines are hotspots of biological activity that can facilitate plant community establishment at elevations that are otherwise mostly unvegetated. The plant community composition and origin of plant seeds on vicuña-modified proglacial soils is the subject of future work, which will investigate whether the plants present in latrines came from seeds that were already present in the soil and then were able to germinate, blew in and settled after latrine development, or were transported via endo- and exozoochory. Zimmer et al. demonstrated that domestic llamas can deposit viable seeds into proglacial environments. Given foraging differences among camelids and differences between native species and non-native sheep, goats, and cattle , there may be differences in plant transport based on the presence of different species in a particular region. With the exception of arbuscular mycorrhizae in Ephedra , our hypothesis of increased endophyte colonization in plant roots in latrines was generally not supported by the data. Increases in plant cover are most likely due to increased nutrient concentrations rather than by facilitation by arbuscular mycorrhizal fungi, dark septate endophytes, or fine root endophytes. Even if vicuñas are potentially transporting fungal spores, mycorrhizal colonization may remain low in latrine soils because nutrients are abundant and it is not worth the carbon investment by the plant to support more mycorrhizae to facilitate nutrient acquisition . These data combined with low fungal relative abundances in 18 S sequencing data suggest that fungi are not playing an important role with regards to nutrient cycling and plant facilitation in vicuña latrines. The lack of soil phosphorus data is a limitation of this study. However, we suspect that phosphorus levels increase on latrines because phosphorus levels are positively correlated with organic matter and nitrogen levels in other high elevation, early successional ecosystems , and available phosphorus concentrations are uniformly very low (< 1 ppm) and represent the limiting nutrient in the unvegetated soils in this basin , . Our research provides a better understanding of the ecosystem ecology of one of the most common wild ungulates in the Andes. A social behavior of these animals influences nutrient transfer into an emerging ecosystem that is highly nutrient limited , providing a mechanism by which soil and community development may decrease the lag time between deglaciation and primary succession on a timescale relevant to the rapid pace of climate change. Although the spatial scale of latrines is currently limited to isolated patches, latrine hotspots may: (1) have outsized influence on trophic structure in ecological communities in the recently deglaciated zone, (2) their area of influence may increase over time as precipitation causes runoff that redistributes nutrients from the piles into surrounding areas (especially since these high-elevation tropical sites are projected to experience rapid increases in mixed precipitation instead of only frozen precipitation; e.g ). , and (3) hotspots may provide crucial seed banks, microhabitats, and source populations for puna grassland species to colonize proglacial areas as soils develop on a broader scale. Camelids in the Andes are not the sole example of animals facilitating ecosystem development in proglacial systems. Nesting seabirds , , and potentially other mammals such as mule deer ( Odocoileus hemionus ), mountain goats ( Oreamnos americanus ), and rodents could represent important sources of nutrient subsidy into emerging glacier forefield ecosystems. Animal nutrient inputs likely have long-term effects in primary successional landscapes, where animals create long-term modification to basal habitat conditions and provide limiting resources that facilitate secondary and tertiary organisms. At our study site, while part of the landscape is emerging following classic microbe-plant and plant-plant facilitation , we have demonstrated that a parallel and accelerated natural process occurs simultaneously in patches modified by animals. Failure to account for how animals facilitate ecosystem development and jumpstart microbial–plant and plant–plant interactions limits our understanding of how animal-modified soils may interact with the surrounding developing soils over time, and more broadly limits a realistic understanding of how ecosystem emergence happens following a large disturbance such as glacial retreat. Finally, our study suggests that areas with recovered and reintroduced vicuña populations, thanks to decades of successful conservation following their near-extinction in the 1960s , may show different ecosystem responses to climate change than areas where vicuña populations are relatively low or absent. Vicuña latrines provide insights into a possible nature-based solution to both ecological and socio-ecological impacts of climate change sensu Seddon et al. which threaten the livelihoods of indigenous high-Andean pastoralists . Rewilding with or directing native camelids into proglacial areas might be useful for altering ecosystem formation following glacier retreat , in a way that supports local communities. Thus, conservation policies and management practices for animals that venture into proglacial areas may influence ecosystem responses to climate change.   Study setting and field sampling Our study took place in the rapidly deglaciating Sibinacocha watershed of the Cordillera Vilcanota (hereafter “CV”; 13.7° S, 71.0° W), Cusco Department, southeastern Peru (Fig. ). The CV is the second most heavily glacierized range in Peru and is located at the western margin of the Amazon Basin and near the northern extent of the Peruvian–Bolivian altiplano. The climate in the CV is characterized by pronounced wet and dry seasons. Most precipitation falls from October–April, with only 1–2% of annual precipitation occurring during peak dry season (June–August ). There have been average annual temperature increases of 0.1–0.4 °C/decade (1965–2009; Salzmann et al. 2013 ), similar to the positive temperature trends observed across the Central Andes region . The CV has been the focus of a decades-long research effort to understand the climatological, ecological, and social impacts of rapid climate change (e.g , , – ). Massive loss of ice (30% of area and about 45% of volume between 1985 and 2006 ) at a rapid and increasing rate in the CV, including the projected disappearance by the 2050s of Quelccaya, the largest tropical ice cap in the world , has resulted in a large amount of new land emerging from retreating ice. Organisms ranging from microbes to vertebrates (e.g., anurans, lizards, ungulates, and puma) are moving upslope to colonize the recently deglaciated terrain at elevations above 5000 masl , , . In this environment, the first bacterial colonizers are betaproteobacteria and cyanobacteria, and the first vascular plant colonizers are predominantly grasses (e.g., genera Cinnagrostis ( Calamagrostis) and Festuca ) and forbs in the genus Ephedra . Microbial and plant colonization here are strongly nutrient limited . Global high-elevation records have been documented across the CV for potato cultivation, orchids, amphibians, pelecypods, lizards, and tarantulas , , . We mapped and studied vicuña latrines that were found in glacial moraines that we determined to have been exposed since the glacial maximum extent during the Little Ice Age (hereafter “LIA”; estimated at 1850–1900 in this area ). We used a comparative experimental design wherein we sampled six focal latrines and six nearby paired glacial till controls located 5–15 m away from each latrine. Four study sites were located on soils situated between the LIA and 1931 isolines, and two study sites were located on soils situated between the 1980 and 2005 isolines , providing an estimate of the maximum age since exposure for each site. The soils underlying each pair of latrine and reference plots are located within the same glacial retreat isolines and are expected to be of nearly the same age because of their close proximity to one other. All latrines sampled had vicuña dung present. Soil properties The underlying geology consists of fluvioglacial and recent moraine deposits. Unconsolidated glacial moraines are comprised of blocks and gravel of mixed granite, metamorphic (primarily slate, quartzite), and sedimentary (shales, sandstones) parent materials in a silt-sand or clay-sand matrix . Soils were sampled to a depth of 4 cm with a sterilized trowel (after removing dung) in the early dry season, April and May, 2019 ( N = 6 pairs). Each sample consisted of soil collected from four randomly selected locations within the study plot that was thoroughly mixed inside the sample bag. Soils were stored in sealed bags on ice in the field and then kept frozen until analysis. We measured soil organic matter, gravimetric soil moisture, and soil inorganic nitrogen (NH 4 + and NO 3 − ). Organic matter was calculated using the loss-on-ignition method after the soils had been treated with acid to remove carbonates . Soil moisture was calculated gravimetrically by oven drying 5 g of soil for 12 h at 60 °C (sufficient for dry sandy soils). Inorganic nitrogen was extracted on freshly collected samples by mixing with 2 M KCl for 1 h, filtering out the solute, and analyzing on an OI Analytical Flow Solution 3000 Flow Injection Analyzer at the Colorado State University Soil Water and Plant Testing Laboratory (Fort Collins, CO). Soil temperature was measured in the field with HOBO pendant temperature/light data loggers (Onset Computer Corp., MA, USA) deployed in the dry season (8/18/2018–9/18/2018) and the wet season (12/18/2018–1/18/2019) at a depth of 4 cm at two paired latrines and controls. DNA extraction and sequencing We extracted DNA from 0.25 g of soil ( N = 6 pairs) sampled as described for Soil Properties in the dry season with the Qiagen DNeasy PowerSoil extraction kit (Hilden, Germany) following the manufacturer’s protocols. DNA concentrations were measured with a Qubit 3.0 fluorometer (Thermo Fisher Scientific, MA, USA). Extracted DNA was amplified via polymerase chain reaction using the 515 F/806R and 1391f/EukBr primer pairs for the 16 S (for bacteria and archaea) and 18 S (for eukaryotes) regions of the genome, respectively, following the methods of the Earth Microbiome Project – . Amplified DNA was normalized with the SequalPrep Normalization Kit (Invitrogen Inc., Carlsbad, CA) and sequenced on an Illumina MiSeq (paired-end 2 × 150 bp) at the BioFrontiers Next Generation Sequencing Facility (Boulder, CO). Sequencing data were demultiplexed with the “idemp” tool, adapters were cut from sequences with the “cutadapt” tool ( https://cutadapt.readthedocs.io/en/stable/ ), and sequences were then analyzed with the DADA2 pipeline to trim and quality-filter reads, infer amplicon sequence variants (ASVs), merge forward and reverse reads, remove chimeras, and assign taxonomy using the SILVA version 138.1 database 16 S and the PR version 5.0.1 database for 18 S . 16 S reads were not trimmed while 18 S reads were trimmed to 130 bp. Sequences were aligned with MUSCLE and phylogenetic trees constructed using the FastTree algorithm in the QIIME program . Chloroplast and mitochondria sequences were removed from the 16 S dataset while plants, bacteria, and archaea were removed from the 18 S dataset. Any ASVs that were unassigned at the domain level were also removed from both datasets. Then, data were rarefied to the lowest number of sequences per sample (5111 for 16 S, 3733 for 18 S) with the mctoolsr R package before downstream analyses. Sequencing data were deposited to NCBI under BioProject accession number PRJNA1145100. Plant cover, root collection, and microscopy We estimated percent ground cover using the point intercept method on two to four 1 m 2 quadrats per site, with 100 observations per quadrat. Cover was classified into the following categories: bare ground (i.e., sand, rocks, dung) and plant cover (i.e., grass, forb, shrub, dead plant). The proportion of plant cover was compared for paired quadrats at six latrine and moraine reference sites from the dry season (2018), and plant cover count was compared for 24 latrine sites with moraine reference plots in the wet season 2023. Roots were collected to study endophyte colonization from three common plant species, the forb Ephedra rupestris , and the grasses Cinnagrostis nitidula and Festuca floribunda on the six paired latrine and moraine reference plots. Roots were immediately rinsed in the field and stored in 70% ethanol until staining in the lab. We only used roots that could be traced back to the individual plant to confirm identity. Roots were cleared with 10% KOH for 1 h at 90 °C, reacidified in 1% HCl for 20 min, and stained overnight in acidic glycerol trypan blue . Stained roots were transferred to acidic glycerol to destain and were stored at 4 °C until microscopy (maximum 5-month storage time). We placed roots horizontally on slides and made passes up and down the slide at random intervals such that 100 intersections between the root and ocular crosshair were observed. The presence or absence of arbuscular mycorrhizal fungi, fine root endophytes, and dark septate endophytes was recorded at each intersection . Statistical analyses Soil properties, vegetation cover (2019 dry season data), fungal root colonization, and microbial ASV richness were analyzed with paired t-tests to compare differences between latrines and paired unvegetated reference (control) plots adjacent to the latrines. We season vegetation cover (2023 data) was compared on latrine and moraine soils with a generalized linear mixed effects model that specified Poisson errors and a random effect of location to account for replicate quadrat samples from each site (lmer package ), followed by ANOVA (car package; Fox and Weisberg 2019 . Differences in microbial community composition were tested with permutational multivariate analysis of variance (PERMANOVA, Anderson 2001) implemented in the vegan R package on a weighted UniFrac distance matrix to account for phylogenetic relatedness, calculated with the phyloseq R package . All analyses were performed in the software R (version 4.2.1 ).   Our study took place in the rapidly deglaciating Sibinacocha watershed of the Cordillera Vilcanota (hereafter “CV”; 13.7° S, 71.0° W), Cusco Department, southeastern Peru (Fig. ). The CV is the second most heavily glacierized range in Peru and is located at the western margin of the Amazon Basin and near the northern extent of the Peruvian–Bolivian altiplano. The climate in the CV is characterized by pronounced wet and dry seasons. Most precipitation falls from October–April, with only 1–2% of annual precipitation occurring during peak dry season (June–August ). There have been average annual temperature increases of 0.1–0.4 °C/decade (1965–2009; Salzmann et al. 2013 ), similar to the positive temperature trends observed across the Central Andes region . The CV has been the focus of a decades-long research effort to understand the climatological, ecological, and social impacts of rapid climate change (e.g , , – ). Massive loss of ice (30% of area and about 45% of volume between 1985 and 2006 ) at a rapid and increasing rate in the CV, including the projected disappearance by the 2050s of Quelccaya, the largest tropical ice cap in the world , has resulted in a large amount of new land emerging from retreating ice. Organisms ranging from microbes to vertebrates (e.g., anurans, lizards, ungulates, and puma) are moving upslope to colonize the recently deglaciated terrain at elevations above 5000 masl , , . In this environment, the first bacterial colonizers are betaproteobacteria and cyanobacteria, and the first vascular plant colonizers are predominantly grasses (e.g., genera Cinnagrostis ( Calamagrostis) and Festuca ) and forbs in the genus Ephedra . Microbial and plant colonization here are strongly nutrient limited . Global high-elevation records have been documented across the CV for potato cultivation, orchids, amphibians, pelecypods, lizards, and tarantulas , , . We mapped and studied vicuña latrines that were found in glacial moraines that we determined to have been exposed since the glacial maximum extent during the Little Ice Age (hereafter “LIA”; estimated at 1850–1900 in this area ). We used a comparative experimental design wherein we sampled six focal latrines and six nearby paired glacial till controls located 5–15 m away from each latrine. Four study sites were located on soils situated between the LIA and 1931 isolines, and two study sites were located on soils situated between the 1980 and 2005 isolines , providing an estimate of the maximum age since exposure for each site. The soils underlying each pair of latrine and reference plots are located within the same glacial retreat isolines and are expected to be of nearly the same age because of their close proximity to one other. All latrines sampled had vicuña dung present.   The underlying geology consists of fluvioglacial and recent moraine deposits. Unconsolidated glacial moraines are comprised of blocks and gravel of mixed granite, metamorphic (primarily slate, quartzite), and sedimentary (shales, sandstones) parent materials in a silt-sand or clay-sand matrix . Soils were sampled to a depth of 4 cm with a sterilized trowel (after removing dung) in the early dry season, April and May, 2019 ( N = 6 pairs). Each sample consisted of soil collected from four randomly selected locations within the study plot that was thoroughly mixed inside the sample bag. Soils were stored in sealed bags on ice in the field and then kept frozen until analysis. We measured soil organic matter, gravimetric soil moisture, and soil inorganic nitrogen (NH 4 + and NO 3 − ). Organic matter was calculated using the loss-on-ignition method after the soils had been treated with acid to remove carbonates . Soil moisture was calculated gravimetrically by oven drying 5 g of soil for 12 h at 60 °C (sufficient for dry sandy soils). Inorganic nitrogen was extracted on freshly collected samples by mixing with 2 M KCl for 1 h, filtering out the solute, and analyzing on an OI Analytical Flow Solution 3000 Flow Injection Analyzer at the Colorado State University Soil Water and Plant Testing Laboratory (Fort Collins, CO). Soil temperature was measured in the field with HOBO pendant temperature/light data loggers (Onset Computer Corp., MA, USA) deployed in the dry season (8/18/2018–9/18/2018) and the wet season (12/18/2018–1/18/2019) at a depth of 4 cm at two paired latrines and controls.   We extracted DNA from 0.25 g of soil ( N = 6 pairs) sampled as described for Soil Properties in the dry season with the Qiagen DNeasy PowerSoil extraction kit (Hilden, Germany) following the manufacturer’s protocols. DNA concentrations were measured with a Qubit 3.0 fluorometer (Thermo Fisher Scientific, MA, USA). Extracted DNA was amplified via polymerase chain reaction using the 515 F/806R and 1391f/EukBr primer pairs for the 16 S (for bacteria and archaea) and 18 S (for eukaryotes) regions of the genome, respectively, following the methods of the Earth Microbiome Project – . Amplified DNA was normalized with the SequalPrep Normalization Kit (Invitrogen Inc., Carlsbad, CA) and sequenced on an Illumina MiSeq (paired-end 2 × 150 bp) at the BioFrontiers Next Generation Sequencing Facility (Boulder, CO). Sequencing data were demultiplexed with the “idemp” tool, adapters were cut from sequences with the “cutadapt” tool ( https://cutadapt.readthedocs.io/en/stable/ ), and sequences were then analyzed with the DADA2 pipeline to trim and quality-filter reads, infer amplicon sequence variants (ASVs), merge forward and reverse reads, remove chimeras, and assign taxonomy using the SILVA version 138.1 database 16 S and the PR version 5.0.1 database for 18 S . 16 S reads were not trimmed while 18 S reads were trimmed to 130 bp. Sequences were aligned with MUSCLE and phylogenetic trees constructed using the FastTree algorithm in the QIIME program . Chloroplast and mitochondria sequences were removed from the 16 S dataset while plants, bacteria, and archaea were removed from the 18 S dataset. Any ASVs that were unassigned at the domain level were also removed from both datasets. Then, data were rarefied to the lowest number of sequences per sample (5111 for 16 S, 3733 for 18 S) with the mctoolsr R package before downstream analyses. Sequencing data were deposited to NCBI under BioProject accession number PRJNA1145100.   We estimated percent ground cover using the point intercept method on two to four 1 m 2 quadrats per site, with 100 observations per quadrat. Cover was classified into the following categories: bare ground (i.e., sand, rocks, dung) and plant cover (i.e., grass, forb, shrub, dead plant). The proportion of plant cover was compared for paired quadrats at six latrine and moraine reference sites from the dry season (2018), and plant cover count was compared for 24 latrine sites with moraine reference plots in the wet season 2023. Roots were collected to study endophyte colonization from three common plant species, the forb Ephedra rupestris , and the grasses Cinnagrostis nitidula and Festuca floribunda on the six paired latrine and moraine reference plots. Roots were immediately rinsed in the field and stored in 70% ethanol until staining in the lab. We only used roots that could be traced back to the individual plant to confirm identity. Roots were cleared with 10% KOH for 1 h at 90 °C, reacidified in 1% HCl for 20 min, and stained overnight in acidic glycerol trypan blue . Stained roots were transferred to acidic glycerol to destain and were stored at 4 °C until microscopy (maximum 5-month storage time). We placed roots horizontally on slides and made passes up and down the slide at random intervals such that 100 intersections between the root and ocular crosshair were observed. The presence or absence of arbuscular mycorrhizal fungi, fine root endophytes, and dark septate endophytes was recorded at each intersection .   Soil properties, vegetation cover (2019 dry season data), fungal root colonization, and microbial ASV richness were analyzed with paired t-tests to compare differences between latrines and paired unvegetated reference (control) plots adjacent to the latrines. We season vegetation cover (2023 data) was compared on latrine and moraine soils with a generalized linear mixed effects model that specified Poisson errors and a random effect of location to account for replicate quadrat samples from each site (lmer package ), followed by ANOVA (car package; Fox and Weisberg 2019 . Differences in microbial community composition were tested with permutational multivariate analysis of variance (PERMANOVA, Anderson 2001) implemented in the vegan R package on a weighted UniFrac distance matrix to account for phylogenetic relatedness, calculated with the phyloseq R package . All analyses were performed in the software R (version 4.2.1 ).   