Ecological Indicators

Animal COI (mitochondrial cytochrome oxidase I) metabarcoding of environmental DNA (eDNA) is increasingly used to assess biodiversity in complex substrates such as soil. However, due to read-length constraints of second-generation sequencing platforms, mini-barcodes have been used instead of the full barcode region. Long-read sequencing technologies now enable the recovery of full-length barcode sequences, and are more commonly applied for studying microbes, but their use for metabarcoding the full-length standard COI barcoding region in animals remains limited. In this study, we compared three COI amplicon sets -- 313 bp, 660 bp, and 1,256 bp -- amplified from soil eDNA samples and sequenced using Illumina and PacBio platforms to evaluate their overall concurrence, the effectiveness of identifying nuclear mitochondrial DNA segments (NUMTs) and chimeras, as well as their respective taxonomic resolution. The long-read datasets exhibited a higher identification rate of NUMTs and true chimeras, suggesting that longer sequences improve the detection of noise in COI metabarcoding data, thereby reducing the occurrence of spurious taxa. Taxonomy assignment confidence was similar between the 313 bp and 660 bp datasets, whereas extending the amplicon beyond the standard COI barcode region (1,256 bp) reduced confidence, likely because longer reads extend into regions poorly represented in barcode reference databases. Despite substantially lower sequencing depth in the 660 bp dataset, per-sample OTU richness did not differ significantly from that recovered with the Illumina 313 bp amplicon set. Similarly, the relationships between samples were strongly correlated across the detected OTU communities, indicating consistent ecological interpretations between short and long amplicons. We conclude that the standard ~658 bp COI barcode is an optimal marker for soil animal metabarcoding from eDNA, balancing target recovery, artifact detection, taxonomic assignment and ecological interpretability. As COI eDNA metabarcoding becomes increasingly used in biodiversity assessment and is increasingly adopted in large-scale monitoring initiatives, this study provides methodological guidance for improving the robustness of soil animal community biomonitoring.

Freshwater monitoring programmes like the European Unions Water Framework Directive (WFD) provide a wealth of data on European lake status, including water quality and macrophytes (aquatic plants) as critical habitat features that support health of humans and wildlife. Easier WFD data access can enable external management and research to better safeguard human and natural freshwater use. We demonstrate a replicable workflow to easily download and process multi-year (2007-2024) observations of lake macrophytes (425 sites) and complementary water quality variables (202 sites) from Swedish WFD data. Then, we illustrate the value of improved data access to address ecological questions that drive conservation, investigating how spatial scales influence macrophyte richness and associated water quality relationships using a spatial random intercept model. Decomposing the spatial intercept links small scales (<10 km) to site-level gradients and large scales (>100 km) to biogeographical drivers. Stochastic and environmentally-structured processes coexisted at intermediate scales (10-100 km). Adding water quality rarely improved overall predictive performance of macrophyte diversity models but consistently influences the role of different spatial scales. Water quality variables showed consistent spatially structured variation at intermediate scales and unique spatial patterns in tandem, overlapping with large-scale biogeographical influences. Altogether, we show context-dependencies for spatial model interpretation and provide guidance in accounting for spatial confounding to improve inferential and predictive performance. Our workflow and results show a clear way forward for accessing high-quality macrophyte and water quality data sets and their utility for addressing ecological questions that guide macrophyte protection under the WFD. HighlightsO_LIyears Swedish of macrophyte and water quality monitoring data were extracted. C_LIO_LIrichness showed scale-specific patterns linked to geographic gradients. C_LIO_LIbest predictive models for richness had no water quality at all. C_LIO_LIoverlap in their spatial scales and must be carefully separated. C_LIO_LIpen access data and multiscale analysis can apply to many ecological questions. C_LI

1. Detecting environmental DNA (eDNA) from rare or low-abundance aquatic species remains a major challenge, particularly when it is highly degraded, present at low concentrations, and dominated by DNA from non-target taxa. These challenges are further amplified in sedimentary ancient DNA (sedaDNA) studies, where thousands of years can degrade eDNA further, making the detection and quantitative interpretation of weak biological signals difficult. 2. Metabarcoding is commonly used to produce high-throughput community-level data from eDNA but is inherently compositional and influenced by amplification biases. Nonetheless, metabarcoding read abundance or PCR replicate detection frequency are increasingly used as proxies for relative DNA concentration, but their quantitative interpretation has rarely been evaluated against independent measures of absolute DNA abundance. 3. We used droplet digital PCR (ddPCR) to quantify mitochondrial DNA from Atlantic cod (Gadus morhua) and Atlantic herring (Clupea harengus) in 136 ancient eDNA extracts from Icelandic marine sediment cores spanning the last three millennia. We compared ddPCR copy number estimates with metabarcoding (18S) derived relative abundance and detection frequency, and evaluated whether temporal DNA trends corresponded with proxy reconstructed sea surface temperature (SST) variability. 4. We found that ddPCR-measured fish sedaDNA abundance was positively correlated with the proportion of metabarcoding PCR replicates for both Atlantic cod and Atlantic herring. Moreover, temporal trends in Atlantic herring DNA abundance were consistent with proxy reconstructed SST variability, supporting the ecological relevance of the molecular signal. 5. Overall, our results show that ddPCR-derived DNA concentrations and metabarcoding PCR replicate detection frequency capture consistent patterns in low-abundance fish sedaDNA from marine sediments. The observed agreement between approaches supports the use of PCR replicate detection frequency as a semi-quantitative proxy for low-abundance sedaDNA.

Freshwater salinization is of increasing concern for integrity and functioning of freshwater habitats worldwide. Experiments so far often have studied drastic salt additions, while gradient designs have been performed less commonly. We tested the effect of freshwater salinization in a mesocosm exposing the plankton community of the oligotrophic Lake Lunz, Austria, to a four-fold salinization gradient (control, 0.2, 1, a 5 ppt salt). Salinity was manipulated in a factorial design with enrichment, with 10 g L-1 and 30 g L-1 phosphorus, resulting in 8 treatments with 3 replicates each. We followed the effects of salinization on diversity, community composition and resource use over 36 days. Community composition was assessed by amplicon sequencing, Diversity loss and community turnover followed upon salt addition. All levels of salinization caused pronounced changes in community composition, with 5 ppt causing the most drastic changes. Salinization caused trophic downgrading by kicking out especially protistan consumers and rotifers, while some green algae and chrysophytes were especially tolerant, resulting in reduced phylogenetic and functional diversity with increasing salinization. In line with reduced top down control, salinization affected temporal variability in chlorophyll-a (chl-a) and resource use (RUE), with higher salinity causing more extreme fluctuations in chl-a and RUE. Enrichment overall aggravated salinization, enhancing temporal turnover and temporal fluctuations in resource use.

Italy, one of Europe's most biodiverse countries, is expected to undergo landscape-wide nature recovery thanks to the EU Nature Restoration Regulation. National red lists represent critical instruments for informing conservation strategies at finer geographic resolutions and prioritizing taxa of national importance; yet the taxonomic completeness of Italian red lists remains unquantified in relation to national biodiversity. Equally, no systematic evaluation has been conducted on the representation of threatened and endemic Italian taxa within European and international conservation directives and treaties. We present the first comprehensive review of threat status and policy inclusion of Italian biodiversity, encompassing animals, plants, fungi, lichens, and algae. We cross-referenced national species checklists with Italian, European, Mediterranean, and global IUCN red lists, alongside policy annexes from the Birds and Habitats Directives, the Bern and Barcelona Conventions, and CITES. Our dataset comprised 76,845 taxa, of which 8,389 are endemic; yet red list assessments exist for only 10% of this total (7,349 taxa, including 1,700 endemics). Conservation policy coverage is even more restrictive: only 1,346 taxa are listed under at least one legislative instrument, with only half of these classified as threatened. This constitutes a compounding double bottleneck with most of the Italian biodiversity remaining both unassessed and unprotected and systematically biasing conservation policy toward an unrepresentative fraction of national biodiversity. We recommend accelerated national assessments and urgent establishment of a national biodiversity priority list founded on transparent prioritization protocols. This would complement ecosystem-based interventions mandated by the Nature Restoration Regulation while correcting for vertebrate-centred biases.

Ectomycorrhizal (EM) fungi are vital to forest ecosystems, supporting tree growth and survival. However, their inclusion in conservation policy and action remains limited and little is known about the status of their genetic diversity, which is essential for their long-term survival and adaptation. The Global Biodiversity Framework adopted a genetic indicator based on the effective population size, Ne, to monitor genetic diversity in all species. To date, it is still uncertain how Ne, a key parameter, can be reliably assessed in species with complex life history traits. Ectomycorrhizal fungi are a highly diverse group of taxa displaying haplodiplontic life cycles with partially clonal reproduction. Here, we review the literature to understand how these life history traits might affect Ne and its estimation in six species of EM fungi. We estimated Ne in 19 populations using eight genetic and genomic datasets from selected studies. We compared Ne estimates using Linkage Disequilibrium (LD) and Sibship Frequency (SF) methods. We tested how Ne estimates change due to partial clonality and genetic structure gradients and whether the number of genetic markers influence the precision of the estimates. We show a systematic bias in Ne estimations when large clones are present and when populations are not correctly delimited. We found both methods are not robust to these factors, which makes them unreliable for conservation assessment purposes in EM fungi. This study provides new perspectives for further research into the links between life history traits and the effective population size of ectomycorrhizal fungi.

Habitat degradation and biodiversity loss in the Black and Azov Seas necessitate improved tools for spatially explicit conservation planning. We employed stacked species distribution modelling (SSDM) to assess habitat quality for the three resident cetacean species, the common dolphin (Delphinus delphis ponticus), the bottlenose dolphin (Tursiops truncatus ponticus), and the harbour porpoise (Phocoena phocoena relicta), which serve as apex predators and indicators of ecosystem health. Occurrence data were compiled from the Global Biodiversity Information Facility (GBIF), and ensemble species distribution models (ESDMs) were constructed using nine algorithms within the SSDM framework, with eight environmental predictors extracted from Bio-ORACLE v3.0. Individual ESDMs demonstrated excellent predictive performance (AUC: from 0.82 to 0.83; TSS: from 0.65 to 0.67; prop.correct: from 0.82 to 0.83). However, the initial continuous stacking method (pSSDM) yielded low community-level prediction success (0.36), prompting evaluation of three correction approaches. The Probability Ranking Rule (PRR) substantially improved performance (prediction.success = 0.459, sensitivity = 0.704, Jaccard = 0.465), effectively mitigating the overprediction bias inherent in stacked models. Species richness mapping identified multi-species hotspots along the southwestern Black Sea shelf, the Crimean coast, the Kerch Strait, and parts of the eastern coast, while the deep central basin exhibited the lowest richness. Variable importance ranking revealed bathymetry as the primary community-level driver (41.2%), followed by dissolved oxygen (13.8%), sea surface temperature (11.9%), and salinity (10.4%). Species-specific importance patterns confirmed ecological niche segregation, with common dolphins favouring deeper offshore waters and bottlenose dolphins and harbour porpoises associated with shallower shelf environments. The moderate richness observed in the highly productive northwestern shelf, despite high nutrient inputs, may reflect a combination of natural factors (elevated turbidity, reduced salinity) and anthropogenic pressures (fisheries bycatch, shipping, coastal development, and military activity) that limit species co-occurrence. Our findings demonstrate that PRR-corrected SSDM provides a robust framework for mapping cetacean habitat quality and identifying conservation priorities in the Black and Azov Seas, offering an evidence-based tool to inform ecosystem-based management in this ecologically unique and increasingly pressured marine region.

Premise: Rapid and accurate plant species identification is a critical challenge exacerbated by the taxonomic impediment. Although portable near-infrared (Micro NIR) spectroscopy represents a promising solution, the current absence of standardized protocols and a fundamental understanding of how critical acquisition and analysis parameters influence accuracy remain significant barriers. This study focused on the systematic optimization and validation of a comprehensive workflow designed to maximize the reliability of plant identification using this technology. To ensure methodological robustness across diverse foliar matrices, four vascular plant species were strategically selected as a representative test set to encompass morphological extremes, including significant variations in leaf thickness, pubescence, and surface texture. Methods: Using a portable spectrometer on herbarium specimens (exsiccate) of four vascular plant species, we systematically tested five spectral backgrounds, seven pre-processing methods, and four classification models. Subsequently, we optimized the number of spectral readings and evaluated the influence of the leaf scanning surface (adaxial vs. abaxial) on model accuracy. Results: The highest-performing combination was a Shiny Aluminum background, Second Derivative pre-processing, and a Random Forest model, which achieved a mean cross-validated accuracy of 99%. An average of just three spectral readings from the adaxial (upper) leaf face was sufficient to saturate model performance, proving statistically superior to other approaches (p < 0.001). Discussion: This study establishes a validated, high-accuracy protocol for plant species identification from herbarium specimens using portable NIR, offering a powerful tool for biodiversity studies. Direct applicability to fresh plants in the field requires future validation to account for the spectral influence of moisture variability.

Environmental DNA (eDNA) metabarcoding has become a powerful approach for large-scale biodiversity assessment, yet taxonomic assignment remains one of its most critical error-prone steps. Current bioinformatic pipelines rely on molecular similarity searches against reference databases, but assignment accuracy is constrained not only by short marker length and database incompleteness, but also by fundamental limitations, including recent species radiations, incomplete lineage sorting, introgression, NUMTs, and the imperfect correspondence between genetic variation and species boundaries. Here, we present TRIDENT (Taxonomic Resolution and IDentification using Environmental dNa Traces), an automated and simple protocol designed to improve taxonomic assignments in eDNA metabarcoding. Initially developed for marine vertebrates, TRIDENT may be used with any barcode and integrates three complementary sources of evidence: molecular similarity (NCBI/GenBank and BOLD), curated taxonomic information (WoRMS), and ecological plausibility derived from biogeographic occurrence data (GBIF). The workflow sequentially constructs candidate taxon lists based on sequence similarity, expands them through taxonomic hierarchies, and filters them using spatial occurrence constraints. It further identifies possible taxa lacking reference barcodes and evaluates their plausibility through CO1-based similarity if data exist in BOLD. TRIDENT has been implemented as a source-available Python tool and tested using empirical eDNA datasets from marine vertebrates as well as simulated communities. Results demonstrate that the tool produces taxonomic assignments consistent with expert manual curation while substantially reducing processing time and attention errors caused by manual processing of large datasets. By combining molecular, taxonomic, and ecological criteria within a single framework, TRIDENT improves transparency and reproducibility and provides a robust and flexible solution strengthening confidence in taxonomic identifications in eDNA-based biodiversity assessments.

Here, we report the first genetic confirmation of golden mussels (Limnoperna fortunei) in North America, and the subsequent development, optimization, and deployment of golden mussel eDNA monitoring procedures. Aquatic species invasions are economically costly, disrupt ecosystem functionality, and impact native aquatic communities. Early detection of new invasive species enables rapid response via implementation of effective eradication or control measures and is key for reducing harmful outcomes. Initial species detection and taxonomic identification can be aided by genetic methods that have high detection sensitivity and accuracy. Genetic methods such as environmental DNA (eDNA) sampling can be used to detect invasive species before they become established in new systems, providing an early alert system to inform resource managers. Golden mussels were first detected in North America in October 2024 near the Port of Stockton in the San Francisco Estuary (SFE). The SFE is particularly vulnerable to invasion due to the access and connectivity provided by the presence of engineering infrastructure and shipping lanes. Collaborative efforts between public agencies and academic institutions are underway to develop a coordinated detection and response plan. Early detection followed by a rapid response is the best defense against prolific invasive species, such as the golden mussel.

Soil microbial diversity is shaped by the spatial scale at which communities are sampled, yet standard sampling practices often homogenize samples, obscuring fine-scale spatial structure and diversity patterns. To better understand how sampling effort, spatial extent, and physical homogenization influence plot-level microbial richness estimates, we sampled 57 forest and grassland sites across three regions in Germany using a 14-core cross-transect design and performed 16S rRNA gene metabarcoding. We simulated sampling efforts and a range of spatial extents and compared diversity estimates to those from physically homogenized composite samples. Plot-level richness increased continuously with sampling effort and spatial extent, with no evidence of saturation. However, when sequencing depth was held constant, sampling completeness declined with increasing sampling effort, meaning that more diversity is not captured. Composite samples substantially underestimated plot-level richness and altered apparent diversity relationships between ecosystems; individual cores identified forests as richer than grasslands, whereas homogenized samples suggested the opposite relationship. These results demonstrate that sampling effort, spatial extent, and homogenization fundamentally shape soil microbial diversity estimates. Homogenized composite samples cannot substitute for individual cores when the goal is to reliably quantify plot-level richness or compare diversity across ecosystems.

The Southern Hemisphere is home to extensive forests of giant kelp (Macrocystis pyrifera), including in Argentina and the southern islands of Tierra del Fuego, which has been proposed as a potential climate refugium. This study presents the first regional time series of M. pyrifera canopy dynamics in Argentina using Landsat satellite imagery from 1985 to 2023. The forests analyzed support 247.61 km{superscript 2} of emergent canopy and are situated in the coastal waters of Argentina and a small portion of Chilean islands, with 4%, 28%, and 68% in the Chubut, Santa Cruz, and Tierra del Fuego A.e.I.A.S, respectively. The small portion of Chilean Islands are included as part of the Tierra del Fuego province analyses. Range limits were scrutinized, in part, using expert knowledge and multisatellite comparisons. Linear regression shows that between 1998 and 2023, 7.4% of kelp sites exhibited a significant trend in annual canopy area, with all observed significant trends in the positive direction. Partitioning by province boundaries, linear regression produces significant positive increases in kelp canopy area across all three provinces, although reassessment when longer temporal continuity is also warranted, where available. Observed seawater nitrate concentrations were high throughout the region (7-23 {micro}mol L-{superscript 1}), suggesting that nitrate availability was not a primary driver of canopy variability. However, positive relationships between kelp canopy and the Antarctic Oscillation suggest that regional climate variability--which alters sea surface temperature and other oceanographic conditions--may be exerting a strong influence on kelp dynamics in this region. These findings document relative stability of kelp forest area in Argentina over the most recent two and a half decades and provide preliminary evidence supporting possible increases in kelp area for the region.

Native vegetation protection is a key strategy for delivering both biodiversity and climate benefits, and protected areas have been widely adopted to keep tropical biomes standing. Yet deforestation is driven by interrelated environmental and social factors, and the effectiveness of protected areas varies considerably across space. Here, we evaluated the impact of 802 protected and conserved areas in the Brazilian Amazon on preventing vegetation loss and avoiding carbon emissions over the past 40 years using statistical matching to address the location bias of protection. We found that protected areas were effective throughout the study period, reducing the probability of deforestation per km2 by an average of 0.5 percentage points per year. While the Amazon biome lost 14% of its native vegetation between 1986 and 2024, protected areas prevented the deforestation of 290,436 km2, nine times their actual internal loss. They also stored 45,336 Mt of carbon in 2016 (61% of the Amazon stock) and prevented the emission of 7,300 Mt of CO2 by 2024. Deforestation inside the areas and remoteness reduced their impact, while areas that were initially more preserved were more effective. Area size and age had no influence over impact once we analyzed the amount of avoided deforestation per size and age. Impact also varied across Brazilian states, highlighting the role of regional context. All three protection categories (conservation units, indigenous lands, and quilombola territories) showed a positive mean impact, indicating that each, in aggregate, contributes to reducing deforestation. These findings provide robust evidence of the substantial role of Amazonian protected areas in habitat conservation and climate mitigation, while underscoring that this contribution remains undervalued. We advocate for strategically expanding protection to areas of greatest potential impact, and for securing adequate funding to ensure protected areas can fulfill that potential.

The non-indigenous brown macroalga Rugulopteryx okamurae has emerged as one of the most aggressive marine invaders in European waters, deeply altering benthic communities and causing severe socioeconomic impacts. While its expansion has been extensively documented along the southern Iberian Peninsula, understanding the dynamics of its northward range expansion along the Spanish Mediterranean coast remains critical for coastal management. This study documents the first formal record of R. okamurae in Calpe (Alicante), representing its current northernmost distribution limit within the Comunitat Valenciana. Sampling was conducted through an initial opportunistic scuba diving observation along the surrounding waters of the Penyal dIfac Natural Park, followed by targeted underwater surveys and an ad hoc inspection of commercial bottom-trawling nets drying at the port of Calpe during June 2026. Morphological and anatomical identification was confirmed through cross-sections of the thallus under optical microscopy, revealing the presence of both the thick and intermediate morphotypes of the species. The collected specimens were found either entangled within a native photophilic algal canopy in shallow waters or recovered from deeper offshore fishing grounds. Given the absence of records in the area during 2023-2025 surveys, these findings suggest either a very recent front-wave colonization event or a contribution from nearby, yet undetected, established patches, driven by secondary local dispersal mechanisms such as drifting fragments and explicitly highlighting commercial fishing activities as an active vector. Furthermore, considering that the species was recorded within a marine protected area and deeper environments, these results highlight a potential ecological threat to local benthic ecosystems, emphasizing the urgent need f or competent authorities to implement spatiotemporal monitoring and public awareness campaigns to prevent the definitive establishment of this invader.

This study investigates changes in habitat connectivity and meta-ecosystem resilience between 1817 and 2022 along a 150 km section of a large European river (Danube) and its adjacent floodplains. The analysis was based on a time series of functional habitat networks (graphs) constructed from historical records and remote sensing data, integrating habitat suitability and dispersal modes of functional organism groups. The results indicate that overall habitat availability declined by about 50% since 1817, leading to the near-complete loss of functional connectivity among dynamic habitats by 1910. Less dynamic habitats persisted or expanded but became functionally less connected. Connectivity-based habitat classification further revealed four distinct functional connectivity clusters and the near-complete loss of an originally dominant, dynamic, and highly connected habitat type. Furthermore, the results indicated a fundamental loss of meta-ecosystem resilience. This development was reflected by increased spatial modularity among functionally similar habitats and by reduced layer dissimilarity and structural robustness in multilayer networks representing the spatial habitat structure and functional habitat connectivity across different functional organism groups.

Tropical dryland ecosystems are highly biodiverse and fragmented and are experiencing significant anthropogenic and climatic changes. With increasing extremes in temperature and precipitation, coupled with significant alteration, these ecosystems are at greater risk of increased exposure and vulnerability to climatic change; however, little work has quantified the climatic shifts occurring within these ecosystems globally. Here, we aim to fill this gap by using the ERA-5 reanalysis and CHIRPS precipitation data to quantify changes in essential climatic variables in tropical drylands since 2000. Overall, we find that regional pressures differ, with tropical dry forests, savannas, and shrublands becoming hotter and drier in the Neotropics and parts of the Afrotropics and Australasia. By contrast, the tropical dry forests in the Indomalayan, Oceania, and Nearctic are experiencing hotter and wetter conditions. Globally, though, these ecosystems are experiencing more change than the global average, suggesting they may be approaching tipping points in their resilience, ultimately shrinking the area where they can survive.

Aims: Species' distributions are determined by the interplay between ecological niche and dispersal ability, constrained by biogeographical barriers. Bald-headed uakaris (Cacajao spp.) are highly specialized primates often associated with seasonally flooded forests. In this study, we used ecological niche models to assess changes in habitat suitability and geographic distribution of uakari species under future scenarios. Location: Western Amazonia. Methods: We integrated ecological niche models, current deforestation data, and dispersal ability to estimate habitat suitability under two Shared Socioeconomic Pathway (SSP) scenarios: intermediate (SSP2-4.5) and very high (SSP5-8.5) greenhouse gas (GHG) emissions. Results: Our models project shifts in suitable conditions for all species. Three of the five species are projected to experience substantial reductions ([&ge;]62%) in suitable habitat conditions within their current ranges by 2050 under both future scenarios. Across the western Amazonia, up to 219,189 km2 and 211,276 km2 of land are projected to be unsuitable within the uakari ranges under the intermediate and very high emissions scenarios, respectively. This is particularly relevant for C. calvus, C. rubicundus, and C. ucayalii. At the species level, the uakaris may lose between 343 km2 and 84,531 km2 of their ranges in the intermediate scenario and 858 km2 and 76,216 km2 in the very high scenario. Shifts in suitability due to climate change are expected to vary from 6 to 191 km in the intermediate scenario and from 5 to 168 km in the very high scenario. Furthermore, the uakaris may lose between 0.5% and 8% of their current ranges due to deforestation in all scenarios. Main conclusions: Our findings reveal a high sensitivity of the uakaris to climate change impacts. It is projected that all species may experience contractions in the suitable areas and spatial suitability within their ranges by 2050, underscoring climate change as a relevant threat to these taxa.

Tropical dry evergreen forests (TDEFs) are a unique and highly threatened forest type of the dry tropics. Their restoration could be strengthened if native species demonstrate carbon sequestration comparable to widely used non-native trees. We assessed biodiversity and carbon sequestration in a restored TDEF in India, developed over 50 years from a largely barren landscape. The site now supports high woody-plant diversity, with 91 native species across 34 families. Aboveground biomass (AGB) averaged 66.91 +/- 41.2 Mg/ha comparable to seasonally dry tropical forests globally. Although native species were planted more recently and are shorter than non-natives, they contributed 23.86 +/- 23.4 Mg/ha to AGB and show potential for future increases in basal area. Given their comparable wood densities and capacity to attain similar heights, native species are predicted to sequester carbon at levels similar to non-natives in the long term. AGB was unrelated to species diversity. Overall, native TDEF species can achieve carbon storage while maintaining ecological integrity.

The biodiversity crisis is exacerbated by persistent gaps in taxonomic, geographic, and conservation knowledge. This study provides a comprehensive assessment of Linnean, Wallacean, and conservation shortfalls in Rivulidae (Cyprinodontiformes), one of the most diverse families of Neotropical freshwater fishes. To this end, an extensive dataset was compiled, comprising 494 valid species, 51 synonyms, 3,419 occurrence records, and information on life cycle, distribution, and conservation status. The Linnean shortfall remains open: after 1975, the rate of species description increased sharply, and estimates indicate that 103 species remain undescribed (95% CI: 53-212). The year of species description was influenced by detectability and accessibility factors, with larger species, more widely distributed species, and species occurring in more densely populated areas being described earlier. The Wallacean shortfall was broad and spatially uneven: only 5.34% of the region was considered adequately sampled. The conservation shortfall was also substantial: 179 species are threatened with extinction, 69 species remain Not Evaluated, and 62 are Data Deficient. The mean time between taxonomic description and first IUCN extinction-risk assessment was 25.7 years. Moreover, 59.9% of species have no records within protected areas, including 69.3% of threatened species. These findings synthesize an urgent challenge: biodiversity knowledge and conservation shortfalls must be overcome simultaneously to protect species that are still being discovered, remain poorly documented spatially, and are restricted to habitats under intense anthropogenic pressure. The conservation of Rivulidae cannot wait for complete knowledge; action amid uncertainty is necessary to prevent both known and unknown species from disappearing.

A majority of stream restoration efforts in central Europe focus on streams that are less than five meters wide. Restoration aims to increase structural complexity, thereby enhancing habitat heterogeneity, promoting biodiversity, and reestablishing aquatic-terrestrial linkages that can drive responses in adjacent terrestrial communities. However, the effects of small stream restoration on terrestrial biodiversity remain poorly understood because research focuses mainly on large rivers. Here, we investigated the effects of restoration on the structural complexity of the stream channel as well as the terrestrial habitat and plant diversity on a local and landscape scale across 55 small streams in an agricultural landscape. We compared restored stream sections with non-restored ones that were similar to the conditions before restoration. We also assessed how restored sections changed over time since restoration. Restored stream sections showed a higher stream structural complexity and habitat diversity, both of which are targets of active restoration measures. Restoration also increased riparian plant diversity, both directly and indirectly through structural complexity and habitat diversity. Although time since restoration did not influence structural complexity, it drove successional changes in plant communities that became increasingly associated with wetland habitat conditions. Our results demonstrate that small stream restoration effectively increases floodplain habitat and plant diversity in agricultural landscapes, primarily by enhancing water availability in the floodplain. Restoration actions on small streams support biodiversity if they improve stream channel complexity, connect the stream with its floodplain, and create floodplain habitats.