Conservation Genetics

Detecting dugongs at low densities remains a major challenge for conservation monitoring, particularly in remote or turbid coastal habitats where conventional surveys are difficult. Environmental DNA (eDNA) offers a complementary approach, but robust species-specific tools are needed before it can be confidently applied to dugong surveillance. We developed and analytically validated a quantitative PCR TaqMan assay for detecting dugong (Dugong dugon) eDNA and evaluated its performance across controlled aquarium conditions in Australia and Abu Dhabi, and wild populations in northern Australia and the Arabian/Persian Gulf. The assay detected dugong eDNA in aquarium and field samples, with Sanger sequencing confirming amplification of dugong mitochondrial DNA from positive field detections. Our assay provides a robust and geographically transferable tool for targeted dugong eDNA detection, performing reliably across contrasting coastal environments and widely separated populations. This approach can strengthen local-scale surveillance of low-density or difficult-to-detect dugong populations.

Kashmir voles (Hyperacrius) are poorly studied fossorial arvicolines of the southern Himalayan slopes, with two species recognized. We sequenced Cytb from voles collected in northern Pakistan and combined these data with existing GenBank sequences, yielding 194 tips spanning nearly all arvicoline genera. We analyzed five data sets with maximum likelihood and compared external measurements among species using principal component and linear discriminant analyses. We recovered five well-supported clades of Hyperacrius separated by ~12-14% p-distance, which we treat as distinct species. We recognize clade 1 as H. wynnei, and populations referred to H. w. traubi are genetically indistinguishable from H. w. wynnei. We recognize H. zygomaticus, new rank (clade 2), and tentatively allocate clade 5 to H. brachelix. Clades 3 and 4 likely represent undescribed species. Morphometric analyses of external measurements clearly distinguish H. wynnei but not clades 3 and 4 (overall classification accuracy 70.3%). Phylogenetic analyses including nuclear genes placed Hyperacrius as sister to a clade containing Microtini, Arvicola, Lagurini, Dinaromys, and Ellobiusini, whereas mitochondrial analyses weakly allied it with Ellobiusini. Its traditional placement in Clethrionomyini is thus unsupported, although its exact position remains unresolved. Alticola is nonmonophyletic, with A. macrotis sequences nested within Clethrionomys, likely reflecting mitochondrial capture.

Arctic ecosystems are undergoing rapid climate warming, providing an opportunity to examine molecular evolution under environmental stress. Here, we used whole-genome resequencing to assess population structure, genetic diversity, and recent demographic history in the snowy owl (Bubo scandiacus), a highly mobile Arctic apex predator classified as Vulnerable by the IUCN. We analysed genome-wide data from individuals sampled across the species' circumpolar range using a chromosome-level reference genome. Population genomic analyses revealed an absence of detectable population structure, indicating extensive gene flow and near-panmixia across continents. Genome-wide nucleotide diversity was low ({pi}= 2.47 x 10^(-4)), while runs of homozygosity were dominated by short tracts, indicating limited standing genetic variation despite little evidence for recent close inbreeding. Our demographic inference revealed a sustained decline in effective population size over the past ~1,600 years, a pronounced reduction coinciding with the Medieval Warm Period followed by a partial recovery during the subsequent cooler interval. This direct, measurable effect of past climate warming on effective population size raises concerns for the long-term survival of the snowy owl. Our results demonstrate that high connectivity and low levels of recent inbreeding have not necessarily buffered this species against climate-driven demographic erosion of genetic diversity. This study highlights the value of using whole-genome population genomics to inform genomic monitoring and conservation decision-making in rapidly changing environments.

Megadiverse countries concentrate most of the World's biodiversity. These countries encompass different levels of research infrastructure, and nearly all of them have been historically committed to the Convention of Biological Diversity, often setting precedents and leading new approaches. Here, we ask whether the assessment of genetic diversity indicators provide novel insights for biodiversity management and conservation in one of those countries, Mexico. We evaluated the first national application of the Kunming-Montreal Global Biodiversity Framework (GBF), genetic diversity indicators in Mexico. The data covered 97 taxa across diverse ecosystem types and taxonomic groups, including crop wild relatives, to assess the capacity of genetic diversity indicators to provide novel insights, as well as their relevance for strengthening national conservation strategies. We calculated two indicators: the proportion of populations with an effective population size greater than 500 (Ne 500) and the proportion of populations maintained (PM). Data were sufficient to estimate the Ne 500 indicator for 47 taxa and the PM for 28 taxa, with mean indicator values of 0.217 and 0.936, respectively. Species' geographic range was positively associated with Ne 500, whereas national endemicity had no significant effect. Comparisons with the IUCN Red List and Mexico's national extinction-risk legislation revealed that genetic vulnerability occurs even among taxa not currently recognized as threatened. Our findings highlight a gap in legal recognition of genetic diversity protection that could be strengthened by incorporating population-level assessments focused on maintaining adaptive potential in the long-term. Additionally, the indicators allowed to highlight conservation concerns within crop wild relatives, though fully realizing their potential requires integrating the entire wild-to-domesticated complex. The Mexican experience highlights that the main challenge for scaling the monitoring of genetic diversity is not only generating new data, but integrating existing information into systems that strengthen collaboration and communication among researchers, local communities, and government institutions.

Aim: Given that much of Earth's biodiversity is contained within species, inferring intraspecific genetic diversity and identifying its ecological correlates is essential for understanding evolutionary processes and informing conservation. Here, we investigate broad-scale variation in species-level mitochondrial genetic diversity and evaluate the relative importance of alternative ecological hypotheses proposed to explain it. Location: Brazilian Atlantic Forest, Iberian Peninsula, northwestern Africa, Arabian Peninsula, Madagascar and Australian Monsoonal Tropics. Taxon: Amphibians and reptiles Methods: We assembled a comprehensive dataset by integrating and manually curating mitochondrial sequences, occurrence records, environmental data and species traits from multiple open-access sources. We used multiple mitochondrial genes to estimate mean nucleotide diversity for each study species and quantified regional variation in genetic diversity. We then used random forests models to evaluate the relative importance of selected variables representing hypotheses related to water-energy availability, topography, historical climatic stability, land-cover characteristics, species geographic characteristics, life-history traits, evolutionary history and conservation status. Results: The final dataset comprised 247 amphibians and 295 reptiles, for which 43 and 52 explanatory variables were evaluated, respectively. Overall, the strongest support was found for hypotheses related to life-history traits, topographic heterogeneity and water-energy availability. In amphibians, higher genetic diversity was associated with smaller body size and greater environmental heterogeneity, particularly broader elevational and precipitation gradients. In reptiles, genetic diversity was primarily associated with life-history traits, with species of smaller body size and lower reproductive output exhibiting higher diversity. Long-term climatic stability showed comparatively little explanatory power for both taxa. Main conclusions: Our comprehensive study demonstrates that integrating extensive manual data curation with comparative macrogenetic analyses provides a robust framework for identifying broad-scale ecological correlates of mitochondrial genetic diversity. These findings improve our understanding of the factors associated with contemporary patterns of genetic diversity and provide a foundation for future conservation assessments in data-limited systems.

Information on Western bongo (Tragelaphus eurycerus eurycerus) across its range remains scarce and fragmented, leaving the status of populations poorly understood, limiting effective conservation planning. We conducted three camera-trap surveys across Semuliki National Park, Uganda, between October 2019 and August 2020 (90 camera locations; 11,640 camera days), during which we detected the only currently known population of Western bongo in East Africa. We assessed the status of this population using data from the final survey (March-August 2020), when bongos were detected, applying an individual identification system adapted from Eastern bongo (Tragelaphus eurycerus isaaci) and spatially explicit capture-recapture (SECR) modelling to estimate density and abundance. To reflect known aspects of bongo ecology, we incorporated sex and habitat covariates derived from remotely sensed data as predictors of density. Bongos were detected on 17 occasions, and we identified 18 individuals (11 females and 7 males), of which seven immature individuals (5 females and 2 males). The top-ranking SECR model included the tasseled cap greenness index and sex as predictors of density. The model estimated a population size of 56.49 individuals (SE = 7.69; 95% CI = 41.43-71.56), with an estimated density of 0.58 individuals km-2 (SE = 0.171; 95% CI = 0.32-1.09) within Semuliki National Park. These results provide the first quantitative baseline for this population and suggest the presence of an established breeding population, highlighting the need for structured monitoring to support its long-term conservation.

Habitat fragmentation and destruction in old-growth forests represents a primary threat to specialist species, frequently resulting in the erosion of genetic diversity. The Hermit Beetle, Osmoderma barnabita, is a protected relic of primeval forests and an obligate saproxylic specialist whose larval development depends exclusively on the wood-mould of hollow trees. This study provides the first high-resolution, genome-wide assessment of O. barnabita across seven Polish forest complexes using double-digestion Restriction-site Associated DNA sequencing data from 49 individuals. Following rigorous quality control and reference bias assessment, we evaluate contemporary population structure. To distinguish between evolutionary processes, we isolated 2,920 putatively adaptive outliers from the neutral background, evaluating both neutral and adaptive genomic signals. The analyses revealed four distinct genetic clusters, confirming that the Polish metapopulation is severely subdivided. Crucially, the westernmost population in the Lower Silesian Forest emerged as the most divergent evolutionary unit (FST up to 0.360), suggesting potential hybrid origin or long-term isolation. We observed a striking linear correlation (R2=0.986) between neutral diversity and adaptive potential, indicating that genetic drift in small and isolated forest fragments is actively depleting the variation required for long-term persistence of this species. Our findings demonstrate significant gene flow restriction across all sites, suggesting that current conservation frameworks fail to encompass these highly differentiated lineages. To mitigate this genetic vulnerability, we recommend reclassifying these clusters as independent Management Units and Evolutionary Significant Units. Maintaining forest continuity and protecting local micro-habitats are essential to prevent further isolation and preserve the genomic integrity of this saproxylic flagship species.

During biogeographical ancestry (BGA) analysis, an unknown individual for whom ancestry informative markers (AIMs) were analyzed is often assigned to one of a few reference populations. For some individuals, such an assignment is not meaningful: the two parents of the individual may come from different populations, or the individual may come from a population not included in the reference database. In this study, we examined classification methods in combination with 104 AIMs from the VISAGE Enhanced Tool to provide one of the following three possible outcomes: (i) in-reference (IR), with an assignment to one of the reference populations; (ii) admixed (AD), with parents from two in-reference populations; and (iii) out-of-reference (OR), where the individual's ancestry is not sufficiently represented by the reference populations. We therefore compared seven classifiers in combination with class-specific acceptance thresholds for the OR decision. The best-performing method according to our selection criterion was a naive Bayes classifier over pairs of populations, which we call the recent naive Bayes (RNB) classifier. Populations held out of the reference panel were correctly rejected in $84.9\%$ of cases. We further tested the method on $800$ individuals from seven additional groups not included in the reference panel. Only $29.4\%$ of individuals from four non-admixed populations were rejected, resembling joint ancestry with reference populations. In addition, we investigated the prediction accuracy in a newly collected dataset of 28 individuals with self-reported ancestry by analyzing AIMs using massively parallel sequencing.

Marine species with broad distributions often exhibit complex patterns of genetic connectivity, shaped by historical and contemporary processes. Understanding these patterns is critical for conserving taxa threatened by ongoing and future environmental change. Here, we use reduced-representation and whole-genome sequencing to investigate population genetic patterns in three co-occurring sea snake species (Hydrophis stokesii, Hydrophis major, and Aipysurus laevis) across northern Australia and New Caledonia. Broadscale analyses revealed major genetic breaks across all species between: (1) Australia and New Caledonia, (2) northwestern and northeastern Australia, and (3) the offshore Timor Sea reefs and adjacent northwestern continental shelf of Australia. More subtle genetic differentiation was observed between Shark Bay in Western Australia and northern localities. All species showed relatively large effective population sizes and broadscale connectivity across ~1,300 km of Australia's northwestern continental shelf. This likely reflects substantial historical range expansion over late Quaternary timescales, which may have contributed to the observed genetic connectivity across the region. However, subtle patterns of isolation by distance emphasised the contribution of species-specific traits in shaping connectivity. The isolated offshore reef populations are genetically distinct and potentially threatened by environmental change. Disjunct populations in Shark Bay and the Timor Sea maintain similar levels of genetic diversity to the extensive continental shelf populations, while New Caledonian populations exhibit reduced diversity. Our results support the delineation of spatial units for sea snake conservation based on shared phylogeographic structure, while also finding important population- and species-specific differences in connectivity at finer spatial and temporal scales.

Bottom trawling is the most widespread human disturbance on the seafloor, known to affect seabed structure and benthic biota. Yet, its impact on benthic communities remains documented mainly for taxa identifiable through morphology, leaving microscopic benthic communities largely overlooked. The Gulf of Lion continental shelf has been trawled for decades by French and Spanish fleets, to the point that encountering untrawled reference areas became elusive. Here, we examined prokaryote, protist and Animalia benthic communities at eight stations, using eDNA metabarcoding of core sediment samples, with five primer-pairs. Benthic communities were cross-referenced with contrasted trawling impacts, integrated across three periods: over one month, three years or ten years preceding the sampling. The community composition was primarily structured by ecological habitats, yet, trawling pressure showed association with prokaryotes and Animalia diversity. Diversity was higher at sites trawled more intensively in the preceding month, whereas long-term trawling was associated with a significant erosion of Animalia alpha diversity. Unexpectedly, communities did not display consistent vertical structure and the relative contributions of trawling and hydrodynamic reworking remain unresolved due to the absence of untrawled sites. Overall, our results show that trawling effects extend to prokaryotic compartments rarely considered in impact assessments, providing a proof of concept for eDNA-based assessment, and underline a pervasive long-term effect on Animalia communities. This study also highlights the importance of the timescale used to quantify fishing pressure and underscores the complexity of impact assessment when monitoring begins too late to establish a clear baseline, particularly when no reference site remains.

Management of marine protected areas (MPAs) require long-term monitoring and evaluation that is often limited by the cost and effort of conventional visual based surveys. We evaluated a community science-based eDNA approach to MPA monitoring over two years in the Point Dume State Marine Reserve, the Point Dume State Marine Conservation Area, and two paired unprotected reference sites. Across the sampling period, we detected 119 species of fish and marine mammals, including 19 of the 22 regional management-priority species for Southern CA MPAs. Protected and unprotected comparison sites were broadly similar in overall assemblage with 93 species shared across all sites. Multivariate analyses revealed site-level differences, although season and year explained more variation than protection status. Species presence also shifted during a La-Nina transition, highlighting the importance of long-term monitoring under different climatological contexts. Community scientists greatly expanded the temporal and spatial scope of eDNA sampling and generated high-quality data. Subsampling analysis showed that quarterly sampling, with three sample replicates and sequencing depths between 700k-990k, recovered 95% of observed biodiversity of the full time series. These results demonstrate that a community science-based eDNA program could provide a standardized, scalable, low-cost, and management-relevant approach for long-term monitoring across California's MPA network.

Reliable estimates of population size are fundamental to understanding wildlife population status and informing conservation. Obtaining such estimates remains challenging for unmarked species, particularly in tropical countries where logistical and financial constraints limit intensive monitoring. The endangered dhole (Cuon alpinus), a highly social canid without natural markings, is one such species for which robust population estimates remain scarce. We estimated dhole density in the Valparai plateau, a human-dominated agroforest-forest mosaic in India's Western Ghats. We compared Spatially Explicit Capture-Recapture (SECR) models based on individual identification from fecal DNA and Space-to-Event (STE) models based on camera-trap detections. We additionally applied both approaches to individually identifiable leopards (Panthera pardus) to cross-validate STE versus SECR estimates. SECR estimated dhole density at 22 individuals per 100 sq.km, with STE estimates varying substantially depending on how the camera viewshed area was defined. STE estimates based on post hoc viewshed measurements, particularly after reducing the viewshed by 10%, were comparable to SECR estimates. In contrast, STE underestimated leopard density relative to SECR (7 versus 12 per 100 sq.km), although uncertainty intervals overlapped. Ecologically, our results indicate that human-use agroforest mosaics can support dhole densities comparable to, or exceeding, those reported from several Protected Areas, highlighting their potential importance for dhole conservation. More broadly, we argue that STE models may offer a practical and relatively inexpensive approach for baseline monitoring of dholes from camera-trap surveys, but their reliability depends strongly on accurate characterization of the effective viewshed and may vary across species and landscapes.

Urban protected areas (PAs) have become important habitats for wildlife in rapidly expanding human-dominated landscapes. These PAs may provide refuge and continuously abundant resources, which may enable some adaptable carnivores to persist and attain high abundances. However, little is known about the population ecology and interactions between carnivores sharing such spatially restricted and disturbed urban habitats. We evaluated the densities and spatio-temporal interactions of leopards (Panthera pardus) and striped hyenas (Hyaena hyaena) in Jhalana-Amagarh Conservation Reserve, a protected area (PA) embedded within the metropolis of Jaipur, India. During May-July 2025, we deployed 30 paired camera-traps and used spatially explicit capture-recapture models to estimate density and individual activity centres. We further quantified their diel activity and temporal overlap using kernel density estimation. Leopard density was estimated at 7.03 {+/-} 1.31 individuals/10 km sq and striped hyena density at 4.06 {+/-} 0.96 individuals/10 km sq, with activity centres of both species occurring in close proximity and frequently towards the reserve periphery. Both species were predominantly nocturnal and showed high temporal overlap ({Delta}4 = 0.801, CI=0.73-0.86). Our results report exceptionally high leopard and striped hyena densities in an urban PA and exemplify the importance of small PAs within rapidly expanding cities, stressing the need for finer-scale studies more broadly to understand the mechanisms shaping interactions between the two species. However, such systems require careful management of food subsidies, habitat quality and connectivity along with long-term monitoring of population viability, disease dynamics and human-wildlife conflict.

At large scales, our ability to compare genetic diversity across species is often challenged by the difficulty of extracting range-wide sequence data from online databases. Using mitochondrial DNA from amphibians and reptiles spanning across six regions: Brazilian Atlantic Forest, Iberian Peninsula, northwestern Africa, Arabian Peninsula, Madagascar and Australian Monsoonal Tropics, we develop and implement a framework to overcome common limitations in macrogenetics. We compiled and manually curated a spatially explicit genetic dataset from public repositories, quantified the spatial representativeness of genetic sampling across species' ranges and developed a novel interpolation framework to estimate and map species-specific patterns of intraspecific genetic diversity (ISD) while accounting for heterogeneous spatial sampling. We estimated mean nucleotide diversity across species to produce regional maps for each genetic marker. Our final dataset comprised 182 amphibian and 239 reptile species, encompassing 184 genera from 54 families. Range representativeness of genetic sampling was generally high, despite some variation across study regions. Spatial patterns of ISD revealed geographic heterogeneity, and were consistent across the best-sampled mitochondrial markers. By combining automated and manual georeferencing, quantifying range representativeness, and accounting for bias in spatial interpolations, our pipeline is marker-agnostic and directly applicable to nuclear and genomic data as they become available.

Remote and vast ecosystems present a challenge for biodiversity monitoring. The Central Kalahari Game Reserve in Botswana is one such ecosystem spanning 52,800 km2, a mosaic of fossilized rivers, clay pans, semi-arid sand dunes, scrub, and woodland. It is one of the last remaining untouched wildernesses in the world and hosts unique biodiversity, including populations of threatened mammals. Due to its extreme arid environment, size, and inaccessibility, surveying biodiversity across the reserve is both logistically and technically difficult. Here we demonstrate that a low-effort approach of sampling soil across the reserve is an efficient way to rapidly assess the terrestrial mammal biodiversity using eDNA metabarcoding. We found that eDNA from soil near waterholes performed better at capturing local biodiversity than soil from roads. eDNA metabarcoding detected 38 mammal species, 15 bird species and two amphibians and a simultaneously conducted camera trapping study detected 29 mammal species. eDNA was particularly powerful for detecting small-bodied taxa, including murids, mongooses and bats, which were largely missed by camera traps, while camera trapping was aimed at and performed well for detecting felids. Mammal species that had a higher activity nearby eDNA sampling points (i.e., a higher number of camera trapping recordings nearby) had a higher probability of being detected via eDNA. We propose that simple soil collection for eDNA metabarcoding, particularly close to animal congregation points such as savanna waterholes, offers a practical, scalable and cost-effective strategy for DNA-based biomonitoring in large, remote areas such those in Southern Africa.

Eucalyptus recurva (Mongarlowe Mallee) is Critically Endangered, with only six known adult individuals persisting across two sites in the Southern Tablelands of New South Wales, Australia. Its extreme rarity, uniquely long lifespan, and limited reproductive output make it a priority for conservation genomics. Here we report the first genome assembly of E. recurva: a haplotype-resolved, gapless, telomere-to-telomere (T2T) assembly produced from Oxford Nanopore Technologies (ONT) long-read sequencing. Both haplotypes span 11 chromosomes (consistent with the conserved Eucalyptus karyotype of 2n = 22), with assembly sizes of 521.1 Mb (Hap 1) and 501.5 Mb (Hap 2), BUSCO completeness >99.6%, and quality values of >QV 62. Comparative analyses place E. recurva within section Maidenaria as sister to Eucalyptus viminalis and reveal high synteny between the two species. Inter-haplotype comparison identified 3.6 million SNPs and modest structural variation, suggesting that, despite extreme demographic bottlenecking, E. recurva retains meaningful genomic heterozygosity. We also characterise the chloroplast genome, which exhibits heteroplasmy, and report an apparently bipartite mitochondrial genome. This reference genome provides an essential resource for conservation management, population genetics, and the study of eucalypt genome evolution.

Self-fertilization results in reduction of heterozygosity over time, accelerated by small population sizes, restricted ranges, and fragmented habitats. Yet some species maintain heterozygosity despite these factors. One is Corallorhiza bentleyi, a threatened, mycoheterotrophic, self-pollinating orchid restricted to Virginia and West Virginia, USA. Reduced representation sequencing was employed across five of the ~12 known localities of C. bentleyi, including representatives of sister species C. involuta from Mexico, to quantify patterns of population structure and genomic diversity. Surveys of C. bentleyi revealed a predominance of the cleistogamous (closed-flowered) growth form. Differentiation was observed between the two species but with weak population structure in C. bentleyi. Surprisingly, excess heterozygosity and negative inbreeding were recovered in both species, counter to expectations. Analyses provided evidence against bioinformatic artifacts (collapsed paralogs), small-sample bias (Robertson effect), and accumulation of mutations along chromosomes due to low recombination associated with selfing (Meselson effect) as explanations for excess heterozygosity. Demographic analyses favored a model of post-glacial divergence among C. bentleyi and C. involuta lacking bottlenecks. Finally, simulations under alternative selection scenarios favored models of overdominance vs. those of negative frequency dependent selection. It was concluded that overdominance, namely selection against selfed homozygotes, is the most likely explanation for elevated heterozygosity in C. bentleyi. Conservation concerns are discussed in light of these findings and the threats of extinction for one of the most vulnerable orchid species in North America.

Understanding floral visitor dynamics along elevational gradients is essential for assessing potential implications for pollen limitation and population genetic processes in fragmented populations of cliff-dwelling alpine endemics. Physoplexis comosa (Campanulaceae) is a chasmophytic paleoendemic restricted to vertical rock faces on dolomitic and limestone bedrock from 200 to 2,800 m a.s.l. The species exhibits a specific floral morphology with fused corolla apices, basal openings for nectar access, and secondary pollen presentation, yet its functional floral visitor assemblage has remained poorly characterized across the species' elevational range. In this pilot study, we tested a two-tier camera-monitoring setup combining action cameras for diurnal monitoring with Raspberry Pi-based systems equipped with IR illumination for combined diurnal and nocturnal recording. We conducted monitoring across five elevational sites (1,480 to 2,400 m a.s.l.) in the Eastern Alps. Recorded video material was downsampled and annotated using the Addax platform alongside manual cross-validation to record physical contact events. Preliminary results confirm the technical feasibility of continuous monitoring in steep terrain and suggest possible elevational variation in diurnal visitors, alongside nocturnal activity by Geometridae moths. Camera-based observations provided higher functional resolution and revealed a different visitor assemblage than previous eDNA metabarcoding trials. This proof-of-concept establishes a methodological foundation for forthcoming multi-day monitoring campaigns and complements ongoing studies of the mating system and population-genetic analyses.

Exotic species can reshape native communities, in the extreme, to local extirpation. Before that, natives may adjust where and when they use the landscape, in ways that allow coexistence and may be the earliest sign of trouble. Distinguishing coexistence built on such adjustment from one built on pre-existing niche differences requires comparing a native where the invader is absent with the same native where it is present, across sites at different invasion stages. Using eight years (2018-2026) of camera-trap data from 103 stations across Uruguay (154,590 camera-nights), we asked how the invasive axis deer (Axis axis) affects the space and time use of two native cervids: the forest-dwelling brown brocket (Subulo gouazoubira) and the grassland-restricted pampas deer (Ozotoceros bezoarticus). Activity-overlap, directional-avoidance, hierarchical activity, and two-species occupancy analyses showed sharply divergent dyads. A. axis and S. gouazoubira shared forest yet segregated in space and along the understory stratum the brocket requires and the invader avoids, and shifted their diel activity by context; understory cover fell with A. axis abundance, consistent with habitat modification. For A. axis and O. bezoarticus, segregation was intrinsic, opposite diel niches and habitats, with no support for a detectable shift in pampas deer behavior. The only behavioral signal was asymmetric: the invader, rather than the native, appeared to adjust, though few shared sites warrant caution. The same invader thus drives different mechanisms, but coexistence is provisional: A. axis is still expanding, far from equilibrium, so these shifts read best as early warning signals.

1. Assessing the conservation value of secondary forests and reforestation is important for conservation planning. Butterflies are widely used as indicators, and fruit-feeding butterflies are often used in biodiversity assessments in tropical landscapes. 2. We examined fruit-feeding butterfly diversity, abundance, and community composition across a primary forest, a secondary forest, and a plantation forest in northern Vietnam. We sampled butterflies nearly monthly during one year using baited traps. 3. Primary and secondary forest supported higher species richness than plantation forest. Moreover, while secondary forest had mainly a subset of species found in primary forest, the plantation forest had a distinct butterfly fauna. 4. Butterfly abundance and community composition varied across months, without showing a clear seasonal pattern. 5. Our results suggest that secondary and plantation forests do not fully replace primary forests, but they contribute meaningfully to butterfly conservation in human-modified tropical landscapes.