Current protected areas provide limited benefits for European river biodiversity

Domain: biology
Published: 2025-12-17
Editorial outcome: minor_revision

Abstract
Protected areas are a principal conservation tool for addressing biodiversity loss. Such protection is especially needed in freshwaters, given their greater biodiversity losses compared to terrestrial and marine ecosystems. However, broad-scale evaluations of protected area effectiveness for freshwater biodiversity are lacking. Here, we provide a continental-scale analysis of the relationship between protected areas and freshwater biodiversity using 1,754 river invertebrate community time series sampled between 1986 and 2022 across ten European countries. Protected areas primarily benefited poor-quality communities (indicative of higher human impacts) that were protected, or that gained protection, across a substantial proportion of their upstream catchment. Protection had little to no influence on moderate- and high-quality communities, although high-quality communities potentially provide less scope for effect. Our results reveal the overall limited effectiveness of current protected areas for freshwater biodiversity, likely because they are typically designed and managed to achieve terrestrial conservation goals. Broadly improving effectiveness for freshwater biodiversity requires catchment-scale management approaches involving larger and more continuous upstream protection, and efforts to address remaining stressors. These approaches would also benefit connected terrestrial and coastal ecosystems, thus generally helping bend the curve of global biodiversity loss.

Introduction
Biodiversity is in crisis owing to human-induced global change1–3. Extensive actions have been implemented to address these losses, including legislation and agreements to expand the cover of protected areas (PAs), such as the EU Habitats Directive (92/43/EEC) and the Kunming-Montreal Global Biodiversity Framework, which sets a target of 30% global PA coverage by 20304. PAs restrict or reduce human activity in designated locations, such as national parks, nature reserves, or marine sanctuaries, with the aim of maintaining and restoring biodiversity. Whether PAs generally achieve this aim remains unclear. Several broad-scale (i.e., global or continental) studies have investigated the effectiveness of terrestrial and marine PAs, providing insights into their potential for reducing biodiversity loss5–7, exploitation8, and habitat loss9,10. However, similar broad-scale perspectives are currently lacking for freshwaters. This knowledge gap is particularly concerning given that freshwater ecosystems harbor a disproportionate amount of global biodiversity by area, and this biodiversity is declining faster compared to terrestrial and marine ecosystems11–13. Evidence of PA effectiveness for individual freshwater ecosystems, or freshwaters in individual regions, is currently mixed14, with some PAs generally benefitting freshwater biodiversity15,16 whereas others exhibit little to no effect17–19. This variability highlights the need for research that evaluates the general effectiveness of PAs for freshwater biodiversity at broader spatial scales.

Inland (i.e., non-marine) PAs may broadly fail to protect freshwater biodiversity because their boundaries and management typically prioritize terrestrial habitats and charismatic taxa20,21, lack explicit goals for freshwaters, and neglect the needs of freshwater taxa22–24. For example, most inland PAs are small, with ~85% less than 10 square kilometers25. However, many freshwater ecosystems, particularly larger rivers, can extend across tens to hundreds of kilometers with catchments encompassing thousands of square kilometers26. Thus, local river communities can be impacted by upstream terrestrial pollutants and other inputs across broad spatial scales27–29. Small-scale protection of a river site can therefore be compromised by inputs arriving from upstream, unprotected areas30,31. Small PAs may also only succeed at protecting local habitat, while other key upstream and downstream habitats used by mobile freshwater taxa remain unprotected32–34.

Evaluating the benefits of inland PAs for freshwaters requires appropriate counterfactuals, i.e., unprotected areas, for comparison. Studies often rely on spatial comparisons of protected and unprotected sites17,19,35,36, but this may produce biased results due to spatial biases in PA placement. For example, PAs tend to be designated in less impacted, forested, higher elevation areas with little human development37,38, which may already have high and/or stable biodiversity compared to unprotected sites. These biases often cannot be fully controlled, which makes it difficult to distinguish the effects of protection from pre-existing differences between sites39. An alternative approach is to incorporate a temporal component into the spatial comparisons, specifically by comparing the rate of biodiversity change between protected and unprotected sites6. This method better evaluates PA effectiveness by using earlier years within sites as the baseline, thus helping determine whether establishing or expanding protection affected biodiversity, and whether biodiversity was lost (or gained) at a faster rate in unprotected sites. However, such temporal comparisons are hindered by the scarcity of high-resolution time-series data.

To address the need for broad-scale, temporal evaluations of PA effectiveness for freshwater biodiversity, we examine 1,754 time series of river invertebrate communities collected between 1986 and 2022 across ten European countries (Fig. 1 and Supplementary Table 1). We focus on river invertebrates because they are key components of freshwater biodiversity that provide important ecosystem functions and services40, and they exhibit consistent compositional responses to human pressures41. These taxa are therefore commonly used as bioindicators and have been monitored globally for decades, including in countries across Europe. Consequently, analysis of European, long-term river invertebrate community data can address the need for broad-scale, temporal evaluations of PA effectiveness for freshwater biodiversity.

We first quantify biodiversity change as site-specific temporal changes in invertebrate abundance, taxonomic richness, and ecological quality (a measure of human impacts based on similarity to communities in least-impacted conditions; see “Methods” section). We then determine whether the rate of biodiversity change differs between sites with and without upstream PAs (as in ref. 6 for terrestrial and marine ecosystems), under the expectation that protection would better maintain biodiversity and lead to greater increases in biodiversity through time. To compare the effects of protection close to a river site versus across its broader catchment, we investigate relationships at four progressively larger upstream distances, ranging from PAs up to 1 km upstream (i.e., the immediate vicinity of a site) 10 km, 100 km, and the full upstream catchment. Lastly, for sites with upstream PAs, we determine whether biodiversity change depends on the amount of PA cover, or the degree of PA gain, and whether it varies with river size and initial ecological quality. Regarding river size, as discussed above, larger rivers integrate inputs across longer distances, thus potentially exposing their communities to cumulative pollutants from rural and urban sources, so we expect that biodiversity in larger rivers primarily responds to PA cover that spans larger upstream scales. Regarding ecological quality, PAs tend to be designated in already less impacted areas (i.e., better initial ecological quality), and we expect that the effectiveness of such PAs differs from those established in poorer quality sites, which generally have lower biodiversity42 and thus more scope for improvement.

Here, we show that upstream PAs primarily benefit poor-quality communities where PAs encompass a larger proportion of the catchment. These communities exhibit much higher rates of biodiversity recovery than likely would have occurred in the absence of protection. In contrast, PAs have little to no effect on biodiversity in moderate- and high-quality communities, although the latter group may have been unaffected because human impacts in such rivers are generally low regardless of protection status. Our results underscore the need to broadly improve PA effectiveness in freshwaters by ensuring PA design and management explicitly consider freshwater biodiversity and integrate the needs of freshwater ecosystems.

Results
Protected and unprotected sites
Protected and unprotected sites only differed in the rate of ecological quality change (represented as the Ecological Quality Ratio; EQR), and only when protections encompass smaller upstream scales, specifically when PAs were within 1-km (based on a significant Likelihood ratio test or LRT, n=1754, L=23.4, df=1, P<0.001) and 10-km upstream distances from a river site (LRT, n=1754, L=5.97, df=1, P=0.039; Fig. 2a–c). However, these changes were always greater (i.e., better) in unprotected sites, which was the opposite of our expectations. For example, the rate of EQR change for protected sites at the 1-km upstream scale was +1.1% year−1, whereas it was +1.9% year−1 for unprotected sites (Fig. 2c). For all other metrics and upstream scales, we found no differences in biodiversity change between protected and unprotected sites (Fig. 2a–c; Supplementary Table 2), with similar proportions of sites in these groups both gaining and losing biodiversity (Supplementary Fig. 1).

For sites with upstream PAs, higher PA cover was related to greater increases in taxon richness and ecological quality, but the nature of these relationships depended on initial ecological quality and upstream scale, as evidenced by significant PA cover*ecological quality interactions from generalized additive mixed models (Supplementary Table 3), and differences in effect sizes among upstream scales. Richness primarily increased with higher PA cover close to a site (i.e., at smaller upstream scales), and primarily in initially degraded communities (i.e., lower initial ecological quality; Fig. 3). For example, considering richness at the 1-km upstream scale and an initially poor ecological quality of 0.2 (i.e., 20% similarity to reference conditions), increasing PA cover from <1% to 100% almost tripled the rate at which richness increased, from +2.8% year−1 to +8.2% year−1 (Fig. 3a). These effects weakened as the upstream scale and initial ecological quality increased (Fig. 3b–d) to the point that, at the full upstream scale and an initially high ecological quality of 0.8, increasing PA cover from <1% to 100% only increased the rate at which richness increased from +0.7% to +1.1% year−1 (Fig. 3d). Similar to richness, ecological quality also increased with higher PA cover primarily in initially poorer quality communities. However, the effect of upstream scale was the opposite to that observed for richness, with greater improvements in ecological quality when PA cover increased at larger upstream scales (Supplementary Fig. 2), indicating ecological quality primarily responded to the amount of protection across the catchment.

Increasing PA cover did not affect the rate of change in abundance at any upstream scale, and we found no evidence for effects of river size (Supplementary Table 3).

Sites that gained and did not gain protected areas
Similar to the protected and unprotected sites, sites that gained and did not gain PA cover only differed in the rate of ecological quality change, and only at the 1-km upstream scale (LRT, n=1754, L=7.79, df=1, P=0.021), with greater increases in sites that did not gain PA cover (Fig. 2d–f). For all other metrics and upstream scales, we found no differences in biodiversity change between sites that gained and did not gain PA cover (Fig. 2d–f; Supplementary Table 2), with similar proportions of sites in these groups both gaining and losing biodiversity (Supplementary Fig. 1).

For sites that gained upstream PAs, higher gains translated to greater increases in richness and ecological quality, primarily in initially poorer quality communities and only at larger upstream scales (richness: full only, Supplementary Fig. 3; EQRs: 10 km, 100 km, and full, Fig. 4). These increases were stronger for ecological quality and weaker for richness. For example, at the full upstream scale and an initially poor ecological quality of 0.2, increasing the rate of PA gain from <1% year−1 to the maximum observed value of 7.5% year−1 more than tripled the rate of EQR gain, from +4.1% year−1 to +14% year−1 (Fig. 4d). The rate of richness gain almost doubled under the same conditions, from +2.3% year−1 to +4.5% year−1 (Supplementary Fig. 3). Furthermore, as initial ecological quality increased, we found some instances where higher PA gains translated to slightly lower rates of increase in both ecological quality and richness. Using the full upstream scale as an example and an initially high ecological quality of 0.8, increasing the rate of PA gain from <1% to 7.5% year−1 decreased the rate of EQR gain from +0.32% to +0.19% year−1 (Fig. 4d).

Increasing PA gain did not affect the rate of change in abundance at any upstream scale, and we found no evidence for effects of river size (Supplementary Table 3).

Methods
River invertebrate biodiversity
We collated river invertebrate time series from ref. 42 and from data provided by European freshwater researchers and managers. We defined the following criteria for data inclusion: (i) time series must span a duration of ≥10 years with ≥7 individual sampling years to enable robust estimation of biodiversity change; (ii) within a time series, samples in different years must be collected using the same methods and from the same three-month season; (iii) data were available at the community-level with taxa identified to a consistent taxonomic level through time (if inconsistent levels were used then taxa were adjusted to the most temporally consistent level); and (iv) ecological quality values could be calculated for each community following methods compliant with the EU Water Framework Directive (see Supplementary Table 4). These criteria allowed the inclusion of data from ten European countries (Fig. 1). Included data encompassed 1754 sites and 24,245 individual years collected between 1986 and 2022. Included time series spanned a mean total duration of 19.7 ± 5.7 years (mean ± SD) with 13.8 ± 5.5 sampling years (Supplementary Table 1). Taxonomic resolution varied among sites, with 57% (993 sites) identified only to the family level or higher, and 43% (761 sites) identified to a mixed resolution, typically a combination of families, genera, and species, with some classified to intermediate (e.g., subfamily) or higher levels (e.g., Oligochaeta at subclass). These taxonomic differences among sites did not influence our results (see Supplementary Fig. 4). Identifications higher than species level introduce some uncertainty, given that we cannot detect potential species shifts occurring within these groups. However, such identifications still reliably reflect overall community responses to environmental change65,66 and are common in invertebrate research in which many taxa cannot be reliably identified to the species level.

We quantified biodiversity for each site and year based on three community metrics: (i) abundance (total number of individuals), (ii) richness (total number of taxa), and (iii) ecological quality, quantified as the Ecological Quality Ratio (EQR). Ecological quality is commonly used in Europe as a community-based indicator of human impacts, particularly organic pollutants and general environmental degradation67. It reflects the compositional similarity of sensitive and tolerant taxa to expected values from least-impacted reference communities, which are defined based on country-specific criteria (Supplementary Table 4). EQRs are a continuous observed-to-expected ratio of this similarity, ranging from 0 (low similarity indicating high human impacts) to 1 (equal to reference conditions indicating low human impacts), although EQRs for some communities can be above 1, reflecting conditions better than the average reference state. We chose EQRs over other compositional metrics, such as temporal β-diversity, because they provide meaningful information not just about whether communities changed, but also how they changed.

Rates of temporal change in each community metric were quantified for each site by relating site-specific abundance, richness, and EQRs to sampling year using the gls function from the nlme package in R68,69, then extracting the slope of this relationship. We included a first-order autoregressive structure in each model to control for temporal autocorrelation between successive years. All slopes were converted to percent change per year by log-transforming all metrics prior to modeling, then exponentiating the slopes, subtracting 1, and multiplying by 100. This transformation ensured all rates of biodiversity change had the same units across sites and metrics.

Protected areas and upstream scales
We obtained vectorial cartographic polygons for inland PAs from Protected Planet25. We excluded 2% of European PAs (accounting for 6% of total cover) for which the year of establishment was unknown. We further excluded all point data due to analytical errors that arise when inferring the dimensions of PAs with unknown boundaries70. The majority of point data in our included countries (1171 points out of 1247 total or 94%) were natural monuments in Sweden. These PAs have a registered area of 0 km2 because they are individual features, such as a single tree or rock formation, thus contributing marginally to total protected area cover. Of the remaining 76 excluded points, 20 were Ramsar wetlands with a total area of 296 km2, and 50 were large biosphere reserves with a total area of 94,188 km2. To fill the biosphere information gap, we used data on European biosphere boundaries from ref. 71, although we excluded the outer transition zones, which are not considered protected. Polygons for all included PAs were dissolved into a single layer, with no distinctions made between different PA types (discussed further in Supplementary Note 1).

In addition to the PA polygons, for each site, we produced upstream polygons representing four different spatial scales. The four scales included: lateral buffer zones extending up the main channel and all tributaries to (i) 1-, (ii) 10-, and (iii) 100-km longitudinal distances; and (iv) the full upstream contributing area, i.e., the upstream catchment including all terrestrial areas that drain into a site. Each upstream scale was selected to account for the potential effects of PAs at progressively greater distances from each site, with the full upstream scale also accounting for PA effects outside the lateral buffer zones. Upstream distances were delineated using the Hydrography90m river network and the hydrographr package72,73. Buffer zone widths for 1–100-km upstream scales were quantified as 100 m multiplied by the Strahler order of each segment, plus half the predicted river width based on its order from ref. 74 (see Fig. 5). We used this approach because human activities adjacent to a river typically exert the strongest influence on local communities, and 100-m lateral buffer zones effectively capture these effects75–77. Additionally, our data encompassed sites from rivers of Strahler orders 1–11, representing small streams to very large rivers, and larger lateral areas are needed to capture the larger surface and ground water inputs to higher-order rivers78. Lastly, including river width as part of the buffer zones captured PAs that encompass rivers. The full upstream contributing area was delineated using the get_upstream_catchment function from the hydrographr package.

Based on the PA and upstream polygons, we calculated the percent of each upstream scale covered by PAs to represent both the presence (>0% cover) and degree (total % cover) of protection. We also calculated the rate of temporal change in percent PA cover (% year−1) to capture biodiversity responses to PA expansion. PA cover was calculated for the year before the first and last year of each invertebrate time series, which allowed invertebrate communities ≥1 year to respond to environmental changes resulting from PA establishment. Percent PA cover was quantified as the mean percent cover between the first and last years (always ranging from 0 to 100% across sites). Temporal changes in percent PA cover (% year−1) for each site were quantified as the slope of the relationship between PA cover and year, which ranged from 0 to 9% year−1 depending on the upstream scale (1km: 0–9%; 10km: 0–8.6%; 100km: 0–7.5%; Full: 0–7.5% year−1). PA cover changes were only neutral or positive because Protected Planet data cannot represent PA cover declines79, although PA cover has generally increased globally58.

In addition to PA cover, we quantified the size (in km2) of each full upstream area to represent river size, given that larger rivers have larger upstream areas. Size was calculated based on the number of 90m by 90m pixels in the full upstream area, derived from the Hydrography90m river network. Sites primarily encompassed medium- to larger-sized rivers, with 671 sites out of 1754 total having an upstream catchment size between 10 and 100 km2 and 701 sites between 100 and 1000 km2, with the remainder comprised of very small (135 sites <10 km2) and very large rivers (247 sites >1000 km2).

Statistical analyses
We conducted two sets of analyses: (1) categorical comparisons of changes in abundance, richness, and EQRs between protected and unprotected sites (i.e., those with and without upstream PAs), and between sites that did or did not gain upstream PA cover; and (2) for sites with upstream PAs, we related the rate of biodiversity change to the amount of upstream PA cover and the rate of PA gain using regression. The first set of analyses provided a broad overview of the effects of having or gaining any upstream PA cover. The second set determined whether the degree of protection, or its rate of gain, influenced biodiversity change. We also used the second set of analyses to test the influence of river size and initial ecological quality (detailed below). These temporal trend comparisons have some strengths compared to other potential approaches, such as before-and-after comparisons or spatial comparisons of protected and environmentally similar unprotected sites. Specifically, using temporal trends of percent biodiversity change enables comparison of sites that differ in total biodiversity, and allows for variation in protection timing (e.g., sites can be already protected at the start of their time series or can become protected later). Temporal analyses also allow for changes in protection effectiveness through time, such as lagged effects, and capture the potential compounding effects of establishing multiple PAs in subsequent years.

For our first set of analyses, we compared sites with upstream PAs (>0% cover; ‘protected’) versus those without (0% cover; ‘unprotected’), and those that gained upstream PAs (>0% cover year−1; ‘gain’) versus those that did not (0% cover year−1; ‘no gain’). Sites were assigned to these categories separately for each upstream scale, given that sites could change assignments across scales (e.g., an upstream PA is present within 10 km but not 1 km). We then related the continuous, site-level rates of biodiversity change to these categories using linear mixed models (LMMs) conducted in the lme4 package80. Additionally, each model included fixed continuous terms for site latitude and longitude to control for broad-scale spatial trends, a fixed continuous term for time-series length to control for slope differences among shorter to longer time series, and a random intercept term designating the provider of each dataset to control for differences in sampling methods among providers (see Supplementary Table 1). We tested the significance (P<0.05) of the fixed categorical PA term by dropping it from each model and comparing the reduced versus fuller models using Likelihood ratio tests81. We ran separate models for each upstream scale. To control for conducting multiple models using the same response variables, we corrected all P-values using the Benjamini–Hochberg false discovery rate82.

For our second set of analyses, we used generalized additive mixed models (GAMMs) to relate biodiversity change to the amount of upstream PA cover for sites with upstream PAs, and to the rate of PA gain for sites that gained upstream PAs. Models were conducted in the mgcv package83. PA cover and rates of gain were converted to proportions and square-root transformed prior to analyses to produce a more even distribution of values. To determine the influence of river size, we included an interaction between the PA term and a continuous term for the size of the full upstream area (log10-transformed km2). To determine the influence of initial ecological quality, we included an interaction between the PA term and a continuous term for the EQR averaged across the first three sampling years to represent the initial status of the community. The individual PA, river size, and quality terms were modeled as fixed smoothed terms using thin-plate regression splines, with all fixed interactions modeled using tensor product smooths. Additionally, we included the same fixed and random control variables as for the LMMs. All smoothed terms used the default basis dimensions, and we checked model diagnostics, including the need for higher basis dimensions, using the gam.check function. All GAMMs used a Gaussian distribution (identity link function). The significance (P<0.05) of interactions, and if needed the individual PA term, were determined using Wald-type tests83. We corrected all P-values as above when conducting multiple models using the same response variables.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "I can see the value of an assessment of this type – particularly building on the uses of long-term data series that have been assembled by this group of authors. The biodiversity trajectory of freshwater ecosystems – and the significant challenges of restoring or protecting what’s left – also creates an urgent need to assess the options available. Managing freshwater ecosystems – along with their riparian systems and catchments – is an important option if we can find a way to enforce it while accepting the resource exploitation that would be foregone.\nAn analysis of the potential collateral downstream benefits from terrestrial protected areas is potentially interesting, but I have some significant questions – and in some cases concerns – about this analysis and write-up as currently presented. Depending on the responses, some analytical refinement may be required.\n1) It’s germane that terrestrial protected areas (PAs) are seldom notified or designated with the intention of conferring downstream benefits, and even more infrequently with the intention protection of freshwater invertebrates. This is a point for discussion.\n2) My largest question is with respect to the ways that protected areas were categorised – and linked to the hypothesis being tested. PAs come in several forms only some of which involve biodiversity conservation. The IUCN offers an outline categorisation – in which the key groups are:\nIa Strict nature reserve\nIb Wilderness area\nII National park\nIII Natural monument or feature\nIV Habitat/species management area\nV Protected landscape or seascape\nVI Protected areas with sustainable use of natural resources\nThese categories take many different forms or legal instruments in different European nations.\n(See https://portals.iucn.org/library/sites/library/files/documents/pag-021.pdf)\nSignificantly, category V PAs are identified for landscape and/or recreational purposes – often allowing continued resource exploitation such as agriculture, tourism, commercial forestry or extractive industry – and some contain urban land complete with wastewater discharge. These are often large areas – making up a significant proportion of PAs in each nation by land cover. Yet, experience shows they often fail in biodiversity protection (eg https://www.sciencedirect.com/science/article/pii/S235198942100295X) and some (many?) would not qualify as a contribution to national 30 x 30 GBF targets. In the UK, which I know best, some of our category V PAs are sources of nutrients or other pollutants to freshwater ecosystems downstream. Although less of a concern, some PAs in the other categories are notified for geological or geomorphological reasons – and again need not emphasise contemporary biodiversity conservation.\nThe key question is how such category V PAs were treated in your analysis – and whether they would be expected to confer any downstream biodiversity benefits?\n3) Related to 2, there is good evidence that many terrestrial PAs have failed to protect designated biodiversity features in the face of external pressures (eg N deposition, climate change…) or because of weak/limited enforcement (see, for eg, Starnes et al. 2021 – the link is above). This raises a point for discussion: why would they be expected to perform any better for systems downstream? A second question may be about whether any downstream biodiversity benefits vary among national administrations with stronger protection?\n4) Freshwater ecosystems are sometimes specifically protected in their own right for example through Ramsar (not so often for rivers) or, more likely, through the Natura 2000 network. While very few of the species or features involved are invertebrates, Natura 2000 rivers are often given enhanced protection – for example through tightened discharge consenting or permits. There is a key question about whether such specific protection has brought detectable benefits that might be indicated using freshwater invertebrates. At present, a far smaller proportion of river length is protected in this way than for the proportion of terrestrial landscapes under PAs.\nOn detail, I offer the following:\nLines 8 and 9: not surprising since protected areas seldom focus on river catchments but…\nLines 9/10/11 … offers some evidence that enhanced catchment-scale protection can deliver benefits\nLines 12/13/14: these ideas could profitably be expanded in discussion: are OECMs an opportunity or do we need something more enforceable? Note that this also implies reduced exploitation of other catchment resources – for example for food production.\nFig 1: Are there particular reasons for the gaps in the extent of coverage within and among nations? Spain, for example, is represented mostly by only the Basque region. The ‘UK’ looks to be just England and excludes Wales, Scotland or N. Ireland. France appears to have gaps that correspond to roughly to the Garonne, Charente, Meuse and large parts of the Seine basin. Germany – the country of the lead authors – is missing completely. I think the rationale for site choice needs to be clearer.\nFig 1: protected area in catchment? I wondered if this could be refined to indicate the extent of protection by area or length?\nFig 1: I wondered about any biases in the types of locations that figure in national monitoring networks that might affect the purposes for which the data are used here? These are typically in higher order systems and upstream catchments of a size suited to WFD purposes.\nLine 75: aren’t larger rivers also at risk of more extensive effects from urban land, industry and intensive agriculture?\nLine 77: this might depend on the type of protected area (see point 2, above).\nLine 129: are you actually assessing river protection status? I think you’re mostly assessing the possible collateral effects of terrestrial protected areas.\nLine 138-139: how would this conclusion look if you specifically assessed protection aimed specifically at freshwater locations – for example Natura 2000 rivers? This is an important question because it helps to appraise whether specific measures aimed at freshwaters deliver better than diffuse terrestrial protection\nLine 150: this depends on the type of protected area. IUCN Category V areas – often the largest – need not imply any such benefit, and some protected landscapes in this category are in quite modified landscapes\nLine 183-184: ‘inland waters’ are now recognised in the GBF – but this is recent.\nLine 204: data are plural\nLine 234: exclusions on the basis of PA category might have been interesting (point 2 again). An area of 5km sq could be quite a large proportion of a lower-order catchment.\nLine 241: I guess you mean longitudinal distances along the channel? This could easily be read as lateral buffers up to 10 or 100km wide\nLine 241: why not calculate the proportional area of catchment protected?\nLine 257: degree of protection defined how?\nS J Ormerod Cardiff April 28th 2025", "reviewer_2": "This is a really interesting and important MS, protected areas (PA) are an important part of conservation and biodiversity protection yet how well they protect freshwaters is unknown. A major omission. The authors should be congratulated for pulling together a great dataset. The MS is well written. And modern data analysis methods have been used. I do have some question regarding the analysis and interpretation of the data, which may have a large effect on the MS major conclusion. I would want to see either an alternative analysis or an explanation that I am misunderstanding something (which is quite possible) before I would recommend publication.\n1. Biodiversity has many meanings, including genetic diversity within a species, the protection of particular (often charismatic) species to ecosystem functions. The current MS is assessing one part of biodiversity, i.e. community level structural measures, e.g. taxa richness. Additionally, patterns in freshwater invertebrate communities do not necessarily match those from other groups of organisms e.g. fish, algae. I have no problem with the MS assessing invertebrate community structure (indeed this can be seen as a strength), but I do think the MS cannot claim to be assessing freshwater biodiversity, unless you first define what you mean by freshwater biodiversity. (In my following points, I will use freshwater biodiversity to mean invertebrate community structure).\n2. Follow on from the point above, the methods state that “consistent taxonomic level through time, typically a combination of families, genera, and species”, I want to see more information on the taxonomic resolution of the data assessed. Perhaps some of the lack of benefits from PA areas is because of relatively course taxonomy, e.g. if outside protected areas a minority species within a family have persisted despite most species being expiated, IDing at family (or genus level) would underestimate the benefits of PA. This is particularly so as some families with many species e.g. chironomids are typically not ID to species.\n3. Could the design be effectively turned into a BACI (before after control impact), in that you could compare changes at sites before the introduction of protected areas to after the introduction of protected areas relative to sites over similar periods that never had PA (i.e. positive controls) and, if present sites, that had PA (or effectively had PA given they are in locations with minimum human impact) over the entire period (i.e. negative controls)? Such a change may not be practical, but if it were practical, it would improve your design as the BACI design considers changes that are occurring unconnected with PA, e.g. climate change, reductions in pollution.\n4. It seems logical that if PA enhance biodiversity once they are enacted, biodiversity gain would be great in PA than outside protected areas. However, PA are often established in areas which are relatively unimpacted by human activities (the MS acknowledges this “PAs tend to be designated in less impacted, forested, higher elevation areas with little human development”). So, how would you expect biodiversity to change in PA once the protection is legally enacted? If prior to their establishment, PA tended to have had less biodiversity loss than outside PA, then would expect less gain in biodiversity from PA, relative to non-PA, as there is less potential for recovery at PA. And this is indeed what you observe Figure 2c. Such results may not necessarily indicate that PA are not effective, rather they may indicate that prior to protection PA had less biodiversity loss than non-PA. Moreover, results in Figure 2 and 3 further support my suggestion in that in protected areas that had high – moderate EQR prior to protection there was no gain in richness or EQR following protection compared to areas with low EQR prior to protection.\n5. My point above is that such results do not necessarily indicate the PA are ineffective at protecting freshwater biodiversity, rather that biodiversity protection by PA is dependent on the loss of biodiversity before PA enactment. (If, for example, no biodiversity has been lost, then PA will not result in a gain in biodiversity). So, the major conclusion conveyed by the title “Current protected areas provide limited benefits for European river biodiversity” maybe misleading. Or am I misunderstanding something fundamental?\n6. What to do? A BACI design (see above) would go some way to solving this problem. Additionally, for locations that effectively had protection before the enactment of PA (e.g. because they are in remote or at high elevations), might they be regarded as de facto PA in your analysis. Note de facto means ‘in reality or as a matter of fact’.\n7. Alternatively comparing biodiversity (not its change per unit time) in areas that become PA to those that never become PA before protected areas are established would also check my suggestion that PA may have lost less biodiversity prior to their enactment.\nSome minor points\n8. Line 2, I would think that protected areas are to prevent or reduce biodiversity loss not to mitigate biodiversity loss.\n9. Line 25, rather than “providing valuable insights into the strengths and weaknesses of current approaches” I would rather see some brief statement as to the effectiveness (or otherwise) of PA for terrestrial and marine systems."}, "author_response": "Manuscript ID: NCOMMS-25-22756\nMS title: Current protected areas provide limited benefits for European river biodiversity\nBold comments are the reviewer comments with line numbers from the previous version of the\nmanuscript (termed ‘Original’). Author comments are in non-bold text, with line numbers from\nthe revised version of the manuscript (termed ‘Current’). Comments in italics are the relevant\nsections directly quoted from the manuscript text. We also highlighted all major edits in yellow\nboth here and in the main text to aid with the review process.\nReviewer 1\nI can see the value of an assessment of this type – particularly building on the uses of long-\nterm data series that have been assembled by this group of authors. The biodiversity\ntrajectory of freshwater ecosystems – and the significant challenges of restoring or\nprotecting what’s left – also creates an urgent need to assess the options available.\nManaging freshwater ecosystems – along with their riparian systems and catchments – is\nan important option if we can find a way to enforce it while accepting the resource\nexploitation that would be foregone.\nAn analysis of the potential collateral downstream benefits from terrestrial protected areas\nis potentially interesting, but I have some significant questions – and in some cases\nconcerns – about this analysis and write-up as currently presented. Depending on the\nresponses, some analytical refinement may be required.\n1) It’s germane that terrestrial protected areas (PAs) are seldom notified or designated with\nthe intention of conferring downstream benefits, and even more infrequently with the\nintention protection of freshwater invertebrates. This is a point for discussion.\nWe agree it is important to be clear that inland PAs are typically designed and managed to\nachieve terrestrial conservation goals, which is likely why they have generally little effect on\nfreshwater biodiversity. We now include this point in the Abstract (Current Lines 11–14) to make\nit clear for all readers that we are providing a freshwater perspective on a typically terrestrial-\nfocused conservation tool. Regarding the Discussion, we now provide more detail on the\nterrestrial focus of PA design and management (Current Lines 161–166) and we now remind\nreaders of this fact in our concluding paragraph (Current Lines 230–232).\nCurrent Lines 11–14 (added): “Our results reveal the overall limited effectiveness of current\nprotected areas for freshwater biodiversity, likely because they are typically designed and\nmanaged to achieve terrestrial conservation goals.”\nOriginal Lines 135–138: “We are not suggesting PAs are generally ineffective, particularly given\nthey can succeed in their goals for terrestrial ecosystems, such as reducing habitat loss39,40, and\nhave been effective for certain, individual freshwater ecosystems and taxonomic groups12,41.”\nCurrent Lines 161–166: “This conclusion should not be misconstrued as suggesting that PAs are\ngenerally ineffective, particularly given it is based on a subset of total freshwater biodiversity\nand does not speak to whether PAs achieved the terrestrial conservation goals they are typically\ndesigned and managed for, such as reducing habitat loss9,10. We also found that PAs helped some\nriver invertebrate communities, and other studies have shown PAs benefiting certain, individual\nfreshwater ecosystems and taxonomic groups14,44.”\nOriginal Lines 183–185: “However, freshwaters are typically overlooked in global\nconservation22, raising questions about whether current PAs are benefiting these ecosystems.”\nEdited to:\nCurrent Lines 230–232: “However, these PAs typically prioritize the needs of terrestrial habitats\nand taxa, raising questions about their benefits for freshwater biodiversity.”\n2) My largest question is with respect to the ways that protected areas were categorised –\nand linked to the hypothesis being tested. PAs come in several forms only some of which\ninvolve biodiversity conservation. The IUCN offers an outline categorisation – in which the\nkey groups are:\nIa Strict nature reserve\nIb Wilderness area\nII National park\nIII Natural monument or feature\nIV Habitat/species management area\nV Protected landscape or seascape\nVI Protected areas with sustainable use of natural resources\nThese categories take many different forms or legal instruments in different European\nnations.\n(See https://portals.iucn.org/library/sites/library/files/documents/pag-021.pdf)\nSignificantly, category V PAs are identified for landscape and/or recreational purposes –\noften allowing continued resource exploitation such as agriculture, tourism, commercial\nforestry or extractive industry – and some contain urban land complete with wastewater\ndischarge. These are often large areas – making up a significant proportion of PAs in each\nnation by land cover. Yet, experience shows they often fail in biodiversity protection (eg\nhttps://www.sciencedirect.com/science/article/pii/S235198942100295X) and some (many?)\nwould not qualify as a contribution to national 30 x 30 GBF targets. In the UK, which I\nknow best, some of our category V PAs are sources of nutrients or other pollutants to\nfreshwater ecosystems downstream. Although less of a concern, some PAs in the other\ncategories are notified for geological or geomorphological reasons – and again need not\nemphasise contemporary biodiversity conservation.\nthey would be expected to confer any downstream biodiversity benefits?\nWe thank you for raising this important question. Our initial analyses actually distinguished\nbetween different PA categories. We did not specifically isolate category V, given there is not a\nstrong reason to differentiate this category from, for example, categories IV/VI which also allow\nfor more extensive human activities. However, we did separately examine categories of ‘strict’\n(I–II), ‘multi-use’ (III–VI), and ‘other’ (those without an IUCN designation), following similar\ngroupings from Elleason et al. 2021 (10.1007/s13280-020-01426-5).\nThe problem we ran into is that there are comparatively few sites in our dataset with strict PAs.\nFor example, strict PAs alone only occur in 23 of the total 1,472 sites with upstream PAs.\nSimilarly, few sites gain strict PA cover as most gains occurred in multi-use/other PAs. Our initial\napproach still enabled some general comparisons between biodiversity responses in protected\nsites with any strict upstream PA cover versus protected sites without, for example (following the\napproach underpinning Fig. 2):\nWe again found no general differences in biodiversity responses between categories, suggesting\nlittle to no influence of the strictness of protection. However, we prefer not to include this\nanalysis in our main text due to some key problems. First, sites with any upstream strict PAs\ngenerally also have upstream multi-use/other PAs, so the ‘strict’ versus ‘not strict’ groupings\ncapture the effect of having a strict PA mixed in with other PA types, rather than the effect of\n3\nstrict protection itself. Second, the low number of sites with any upstream strict PAs (site\nnumbers shown in panels c and f) mean that many comparisons are not robust. This sample size\nissue is further exacerbated in the GAMMs, which include interactions with ecological quality\nand catchment area. Consequently, to examine the influence of gaining strict PAs in the\nGAMMs, sites with strict PAs would require a gradient of strict PA cover, a gradient of\necological quality, and a gradient of catchment area, which generally does not occur across such\na small number of sites.\nTherefore, what we can say with our data is that the strictness of protection does not appear to\naffect biodiversity outcomes, although a more robust dataset is needed to really tackle this\nquestion. Such a conclusion is also supported by the terrestrial literature and individual examples\nfrom freshwater ecosystems. To outline these points for readers, we added a new section to the\nsupplementary that discusses the potential effects of PA strictness and the limitations of our\ndataset in addressing this question (now Supplementary information 9), and we added text to the\nMethods that directs readers to this section (Current Lines 305–307). Additionally, we now\noutline the equivocal evidence from the literature that protection strictness influences\nconservation outcomes (Current Lines 214–221).\nCurrent Lines 214–221 (added): “An additional solution to improving PA effectiveness for\nfreshwaters could be to further limit human activities within current PA boundaries, given that\nmany still allow for continued human use58, such as land development and resource extraction.\nDesignating stricter PAs that do not allow such activities may reduce human impacts46, thus\npotentially benefiting downstream freshwaters. However, evidence that the strictness of a PA’s\ndesignation determines its conservation benefits is equivocal59, including in freshwater\necosystems16,44. Stricter protection can also counterintuitively lead to worse conservation\noutcomes by disenfranchising local communities and promoting illegal use of protected\nresources60.”\nCurrent Lines 305–307 (added): “Polygons for all included PAs were dissolved into a single\nlayer, with no distinctions made between different PA types (discussed further in Supplementary\nFigs. 5 and 6).”\n3) Related to 2, there is good evidence that many terrestrial PAs have failed to protect\ndesignated biodiversity features in the face of external pressures (eg N deposition, climate\nchange…) or because of weak/limited enforcement (see, for eg, Starnes et al. 2021 – the link\nis above). This raises a point for discussion: why would they be expected to perform any\nbetter for systems downstream? A second question may be about whether any downstream\nbiodiversity benefits vary among national administrations with stronger protection?\nWe now do a better job in the Introduction of highlighting that terrestrial and marine PAs have a\nproven potential for reducing biodiversity and habitat loss (Current Lines 26–29), thus clarifying\nour expectation that inland PAs may generally confer similar benefits to connected freshwater\necosystems. However, we also agree that many PAs fail in this regard due to a combination of\nfactors, such as unaddressed external pressures, a lack of funding, and ineffective management,\nall of which undoubtedly vary among national administrations. We now underscore the key\nimportance of effective management and funding in the final sentences of the newly added\nOriginal Lines 23–25: “Several broad-scale (i.e., global or continental) studies have investigated\nthe effectiveness of terrestrial and marine PAs5–8, providing valuable insights into the strengths\nand weaknesses of current approaches.”\nEdited to:\nCurrent Lines 26–29: “Several broad-scale (i.e., global or continental) studies have investigated\nthe effectiveness of terrestrial and marine PAs, providing insights into their potential for\nreducing biodiversity loss5–7, exploitation8, and habitat loss9,10.”\nCurrent Lines 221–228 (added): “Integrating terrestrial with freshwater approaches to PA design\nand management may be an alternative approach for improving freshwater conservation\noutcomes14,50. Freshwater-focused PAs (e.g., Ramsar wetlands or river PAs61) can be designed\nbased on the distribution of both terrestrial and freshwater biodiversity while accounting for\nhabitat connectivity and downstream impacts22,30,50. Effective, adequately funded, and co-\nproduced management is also key to PA effectiveness14,60. We therefore advocate that freshwater\necosystems would further benefit from inclusion in PA management priorities that integrate the\nfreshwater needs of local communities and stakeholders.”\n4) Freshwater ecosystems are sometimes specifically protected in their own right for\nexample through Ramsar (not so often for rivers) or, more likely, through the Natura 2000\nnetwork. While very few of the species or features involved are invertebrates, Natura 2000\nrivers are often given enhanced protection – for example through tightened discharge\nconsenting or permits. There is a key question about whether such specific protection has\nbrought detectable benefits that might be indicated using freshwater invertebrates. At\npresent, a far smaller proportion of river length is protected in this way than for the\nproportion of terrestrial landscapes under PAs.\nThis is an interesting question, and is again linked to similar points above regarding the need for\nPA design and management to consider freshwater ecosystems. There are only ~600 Ramsar sites\nacross Europe designated in our download of the Protected Planet database (out of ~60,000 total\nPAs), and as you suggest not many of these are captured by our river-focused dataset so we\ncannot speak to their specific effectiveness. As for Natura 2000, the effectiveness of these PAs is\ngenerally unknown (see the 2020 EU report on management effectiveness; 10.2800/717133) and\nthere is currently no way for us to identify river-focused Natura 2000 sites based solely on the\navailable information in the Protected Planet database. We could make some selections using\nexpert advice and educated guesses based on PA design (e.g., some are clearly designed to a river\nshape), but this would require an involved follow-up study beyond our principal objectives.\nTo address your concern, we incorporated some text into the new paragraph we added to the\nDiscussion to acknowledge the value of freshwater-focused PAs, including Ramsar wetlands and\nriver-focused PAs (which would include Natura 2000 river PAs), and the need for effective PA\ndesign and management (edits shown in our response to your point #3).\nLines 8 and 9: not surprising since protected areas seldom focus on river catchments but…\nLines 9/10/11 … offers some evidence that enhanced catchment-scale protection can deliver\nbenefits\nThankfully we found some evidence of benefits, otherwise the entire manuscript would be just\ndoom and gloom!\nLines 12/13/14: these ideas could profitably be expanded in discussion: are OECMs an\nopportunity or do we need something more enforceable? Note that this also implies reduced\nexploitation of other catchment resources – for example for food production.\nRather than recommending a particular type of official or unofficial PA, we now better highlight\nthe need for effective design and management regardless of the type of PA being implemented.\nOur edits first outline the pros and cons of stricter protection (see response to your point #2). We\nthen discuss the need to incorporate local communities and stakeholders in management, which\naligns with the purpose of OECMs while acknowledging that management still needs to be\neffective (see response to your point #3).\nFig 1: Are there particular reasons for the gaps in the extent of coverage within and among\nnations? Spain, for example, is represented mostly by only the Basque region. The ‘UK’\nlooks to be just England and excludes Wales, Scotland or N. Ireland. France appears to\nhave gaps that correspond to roughly to the Garonne, Charente, Meuse and large parts of\nthe Seine basin. Germany – the country of the lead authors – is missing completely. I think\nthe rationale for site choice needs to be clearer.\nGaps were determined by data availability and our inclusion criteria. For example, we have little\nto no suitable data from many countries (e.g., our German data primarily comes from a single\nriver system, the Rhine, and so was excluded). Rivers in many countries also lack suitable time\nseries, such as France and Spain where rivers often only have enough data collected to meet the\nminimum reporting requirements of the EU WFD, which may mean only a few years of data\nwith samples collected 3–6 years apart in most regions, making them unsuitable for quantifying\nrobust temporal trends. Additionally, our analysis required ecological quality values for any\nincluded site, and such data are not available for some countries (e.g., Wales, Ireland, and\nScotland). We revised our Methods to clarify these points for readers (Current Lines 251–262).\nOriginal Lines 201–207: “We defined the following inclusion criteria: (i) time series must span a\nduration of ≥ 10 years with ≥ 7 individual years of data; (ii) within a time series, samples in\ndifferent years must be collected using the same methods and from the same three-month season;\nand (iii) data was available at the community-level, and taxa were identified to a consistent\ntaxonomic level through time, typically a combination of families, genera, and species. These\ncriteria resulted in 24,245 individual years of data for 1,754 sites collected between 1986 and\n2022 across ten European countries (Fig. 1).”\nCurrent Lines 251–262: “We defined the following criteria for data inclusion: (i) time series must\nspan a duration of ≥ 10 years with ≥ 7 individual sampling years to enable robust estimation of\nbiodiversity change; (ii) within a time series, samples in different years must be collected using\nthe same methods and from the same three-month season; (iii) data were available at the\ncommunity-level with taxa identified to a consistent taxonomic level through time (if inconsistent\nlevels were used then taxa were adjusted to the most temporally consistent level); and (iv)\necological quality values could be calculated for each community following methods compliant\nwith the EU Water Framework Directive (see Supplementary Table 4). These criteria allowed for\nthe inclusion of data from ten European countries (Fig. 1). Included data encompassed 1,754\nsites and 24,245 individual years collected between 1986 and 2022. Included time series\nspanned a mean total duration of 19.7 ± 5.7 years (mean ± SD) with an average of 13.8 ± 5.5\nsampling years (see Supplementary Table 1).”\nFig 1: protected area in catchment? I wondered if this could be refined to indicate the\nextent of protection by area or length?\nWe modified this figure as suggested to have points sized by the proportion of the full upstream\ncatchment overlapped by protected areas.\nFig 1: I wondered about any biases in the types of locations that figure in national\nmonitoring networks that might affect the purposes for which the data are used here?\nThese are typically in higher order systems and upstream catchments of a size suited to\nWFD purposes.\nOur dataset is biased towards medium to larger sized rivers, with 701 sites out of 1,754 total\nhaving a full upstream area between 100–1,000 km2. However, we do have 135 sites coming\nfrom very small rivers (<10 km2), and 671 sites from smaller to medium sized rivers (10–100\nkm2), so smaller waterbodies are represented. We added some text to the Methods to provide this\ndetail.\nCurrent Lines 337–343 (added): “In addition to PA cover, we quantified the size (in km2) of each\nfull upstream area to represent river size, given that larger rivers have larger upstream areas.\nSize was calculated based on the number of 90 m by 90 m pixels in the full upstream area,\nderived from the Hydrography90m river network. Sites primarily encompassed medium to larger\nsized rivers, with 671 sites out of 1,754 total having an upstream catchment size between 10–100\nkm2 and 701 sites between 100–1,000 km2, with the remainder comprised of very small (135 sites\n<10 km2) and very large rivers (247 sites >1,000 km2).”\nLine 75: aren’t larger rivers also at risk of more extensive effects from urban land, industry\nand intensive agriculture?\nAgreed. We added some text to this effect in this section.\nOriginal Lines 74–76: “Regarding river size, as discussed above, larger rivers integrate inputs\n7\nacross longer distances, so we expected that biodiversity in larger rivers would respond to PA\ncover across larger upstream scales.”\nEdited to:\nCurrent Lines 77–80: “Regarding river size, as discussed above, larger rivers integrate inputs\nacross longer distances, thus potentially exposing their communities to cumulative pollutants\nfrom rural and urban sources, so we expected that biodiversity in larger rivers would respond to\nPA cover across larger upstream scales.”\nLine 77: this might depend on the type of protected area (see point 2, above).\nOur edits detailed in our response to your point #2 above should address this concern.\nLine 129: are you actually assessing river protection status? I think you’re mostly assessing\nthe possible collateral effects of terrestrial protected areas.\nWe re-worded this text to avoid implying these are river-focused protected areas.\nOriginal Lines 128–129: “Our results show that, broadly speaking, the same biodiversity\nchanges occurred regardless of river protection status…”\nEdited to:\nCurrent Lines 150–152: “Our results show that, broadly speaking, the same changes in river\ninvertebrate biodiversity occurred regardless of the presence or degree of upstream protection\n…”\nLine 138-139: how would this conclusion look if you specifically assessed protection aimed\nspecifically at freshwater locations – for example Natura 2000 rivers? This is an important\nquestion because it helps to appraise whether specific measures aimed at freshwaters\ndeliver better than diffuse terrestrial protection\nOur response above to your point #4 should address this concern. The overall message we now\nbetter convey is that it may not be so important what particular type of PA is designated (e.g.,\ncategory I/II versus V/VI, OECM, etc.) because all types can be effective or ineffective,\nincluding freshwater-focused PAs such as Ramsar wetlands or Natura 2000 rivers (see human\nimpacts in Ramsar wetlands discussed in Acreman et al. 2020; 10.1111/conl.12684). What likely\nmatters more is whether freshwater biodiversity is incorporated into PA design and management,\nand that management is effective and adequately funded.\nLine 150: this depends on the type of protected area. IUCN Category V areas – often the\nlargest – need not imply any such benefit, and some protected landscapes in this category\nare in quite modified landscapes\nOur response to your point #2 shows the edits made to outline the mixed evidence for the greater\n8\neffectiveness of stricter protection. Category V protected areas have not consistently been found\nto be any less effective than, for example, category IV/VI or sometimes even categories I/II.\nElleason et al. 2021 provides a somewhat recent review of this topic (10.1007/s13280-020-\n01426-5), with Dudley et al. 2016 (10.3390/land5040038) providing a similar overview specific\nto category V.\nLine 183-184: ‘inland waters’ are now recognised in the GBF – but this is recent.\nWe edited this section to clarify that it is the protected areas that tend to overlook freshwater\necosystems.\nOriginal Lines 183–185: “However, freshwaters are typically overlooked in global\nconservation22, raising questions about whether current PAs are benefiting these ecosystems.”\nEdited to:\nCurrent Lines 230–232: “However, these PAs typically prioritize the needs of terrestrial habitats\nand taxa, raising questions about their benefits for freshwater biodiversity.”\nLine 204: data are plural\nFixed from “data is” to “data are”.\nLine 234: exclusions on the basis of PA category might have been interesting (point 2\nagain). An area of 5km sq could be quite a large proportion of a lower-order catchment.\nWe edited this section to provide further detail on these exclusions (Current Lines 297–304). The\n5 km2 value was for all natural monuments in the Protected Planet point data, but the vast\nmajority of these do not occur in any countries used in our analyses (e.g., many are in Estonia or\nSlovakia). Those that do are primarily in Sweden, but all of them are natural monuments with a\nregistered area of 0 km2 because they are extremely small, encompassing individual features\nsuch as a single tree or rock formation. These protected areas effectively contribute nothing to\ntotal coverage, even for very small rivers, and there is no method to include them anyway (e.g.,\ncreating a buffer zone based on the PA area) because they have area values of 0.\nOriginal Lines 232–235: “We excluded point data due to difficulties in delineating their PA\nboundaries. Most point data represent small natural monuments that contribute little to PA cover\n(area typically < 5 km2), but some are large biosphere reserves. To fill this information gap…”\nEdited to:\nCurrent Lines 297–304: “We further excluded all point data due to analytical errors that arise\nwhen inferring the dimensions of PAs with unknown boundaries70. The majority of point data in\nour included countries (1,171 points out of 1,247 total or 94%) were natural monuments in\nSweden. These PAs have a registered area of 0 km2 because they are individual features, such as\na single tree or rock formation, thus contributing marginally to total protected area cover. Of the\n9\nremaining 76 excluded points, 20 were Ramsar wetlands with a total area of 296 km2, and 50\nwere large biosphere reserves with a total area of 94,188 km2. To fill the biosphere information\ngap…”\nLine 241: I guess you mean longitudinal distances along the channel? This could easily be\nread as lateral buffers up to 10 or 100km wide\nWe now clarify that these are longitudinal distances.\nLine 241: why not calculate the proportional area of catchment protected?\nLine 257: degree of protection defined how?\nWe addressed these two comments by clarifying that we did quantify the proportional (i.e.,\npercent) area of each upstream scale that was protected, including the full upstream catchment,\nand that this percent cover is the ‘degree of protection’.\nOriginal Lines 239–240: “For each site, we quantified the proportion of four different upstream\nscales covered by PAs.”\nThe opening sentence of this paragraph may have been confusing because it mentions\nquantifying PA proportions in a section focused on the delineation of the four upstream scales.\nWe edited this for simplicity by now focusing solely on how the different upstream scales were\ncreated:\nCurrent Lines 308–309: “In addition to the PA polygons, for each site we produced upstream\npolygons representing four different spatial scales.”\nOriginal Lines 256–263: “For each site and upstream scale, we calculated the amount of PA\ncover to represent whether a site was protected or not and its degree of protection. We also\nquantified the rate of PA temporal change to capture biodiversity responses to PA expansion. PA\ncover was calculated for the year before the first and last year of each invertebrate time series,\nwhich allowed invertebrate communities ≥ 1 year to respond to environmental changes resulting\nfrom PA establishment. The amount of PA cover was quantified as the mean percent cover\nbetween the first and last years (always ranging from 0–100% across sites). Temporal changes in\nPA cover (% year-1) for each site were quantified as the slope of the relationship between PA\ncover and year…”\nIn this section, we edited to clarify how percent PA cover was calculated for each upstream scale,\nand to define the ‘degree of protection’:\nCurrent Lines 325–333: “Based on the PA and upstream polygons, we calculated the percent of\neach upstream scale covered by PAs to represent both the presence (> 0% cover) and degree\n(total % cover) of protection. We also calculated the rate of temporal change in percent PA cover\nto capture biodiversity responses to PA expansion. PA cover was calculated for the year before\nthe first and last year of each invertebrate time series, which allowed invertebrate communities ≥\n10\n1 year to respond to environmental changes resulting from PA establishment. Percent PA cover\nwas quantified as the mean percent cover between the first and last years (always ranging from\n0–100% across sites). Temporal changes in percent PA cover (% year-1) for each site were\nquantified as the slope of the relationship between PA cover and year…”\nThis is a really interesting and important MS, protected areas (PA) are an important part\nof conservation and biodiversity protection yet how well they protect freshwaters is\nunknown. A major omission. The authors should be congratulated for pulling together a\ngreat dataset. The MS is well written. And modern data analysis methods have been used. I\ndo have some question regarding the analysis and interpretation of the data, which may\nhave a large effect on the MS major conclusion. I would want to see either an alternative\nanalysis or an explanation that I am misunderstanding something (which is quite possible)\nbefore I would recommend publication.\n1. Biodiversity has many meanings, including genetic diversity within a species, the\nprotection of particular (often charismatic) species to ecosystem functions. The current MS\nis assessing one part of biodiversity, i.e. community level structural measures, e.g. taxa\nrichness. Additionally, patterns in freshwater invertebrate communities do not necessarily\nmatch those from other groups of organisms e.g. fish, algae. I have no problem with the MS\nassessing invertebrate community structure (indeed this can be seen as a strength), but I do\nthink the MS cannot claim to be assessing freshwater biodiversity, unless you first define\nwhat you mean by freshwater biodiversity. (In my following points, I will use freshwater\nbiodiversity to mean invertebrate community structure).\nGood point! We made some edits to our Discussion to clarify that our results are specific to river\ninvertebrate biodiversity, while also better highlighting that even if our results are limited to this\naspect of biodiversity, river invertebrates are key indicators of overall water and habitat quality\n(Current Lines 150–161).\nOriginal Lines 128–135: “Our results show that, broadly speaking, the same biodiversity\nchanges occurred regardless of river protection status, although PAs improved biodiversity\noutcomes in a subset of highly impacted rivers that had or gained a higher amount of PA cover.\nAdditionally, some rivers lost invertebrate biodiversity during our 1986–2022 study period,\nwhich occurred in a comparable proportion of protected and unprotected sites (see Fig. S1).\nCurrent inland PAs have therefore generally not benefited biodiversity in European rivers,\nproviding continental-scale support for similar results reported for individual freshwater\necosystems and regions16,17,29.”\nEdited to:\nCurrent Lines 150–161: “Our results show that, broadly speaking, the same changes in river\ninvertebrate biodiversity occurred regardless of the presence or degree of upstream protection,\nalthough PAs improved biodiversity outcomes in a subset of poor-quality communities that had\nor gained PA cover across a larger proportion of their upstream catchment. Additionally, some\nrivers lost invertebrate biodiversity during our 1986–2022 study period, which occurred in a\ncomparable proportion of protected and unprotected sites. We therefore found no consistent\nevidence that inland PAs have generally benefited European river invertebrate biodiversity,\nsuggesting that PAs may have also not generally benefited water and habitat quality given that\ninvertebrates are key indicators of both43. These findings provide continental-scale support for\n12\nsimilar results reported from individual freshwater ecosystems and specific regions for\ninvertebrates18,31, other taxonomic groups (e.g., fish17–19), and water quality18.”\n2. Follow on from the point above, the methods state that “consistent taxonomic level\nthrough time, typically a combination of families, genera, and species”, I want to see more\ninformation on the taxonomic resolution of the data assessed. Perhaps some of the lack of\nbenefits from PA areas is because of relatively course taxonomy, e.g. if outside protected\nareas a minority species within a family have persisted despite most species being expiated,\nIDing at family (or genus level) would underestimate the benefits of PA. This is particularly\nso as some families with many species e.g. chironomids are typically not ID to species.\nWe added some detail to our Methods to provide more information on the taxonomic resolution\nof our data, and we added a supplementary analysis (now Supplementary information 8) to\ndetermine whether our results change if we do or do not include sites with only higher-level IDs\n(i.e., only family level or higher). You are also correct that there could be community changes\noccurring within higher-level identifications that are not detected by our dataset and we now note\nthis for readers. We further highlight that higher-level identifications still reveal\nmacroinvertebrate community responses to changes in anthropogenic impacts.\nCurrent Lines 262–271 (added): “Taxonomic resolution varied among sites, with 57% (993 sites)\nidentified only to the family level or higher, and 43% (761 sites) identified to a mixed resolution,\ntypically a combination of families, genera, and species, with some classified to intermediate\n(e.g., subfamily) or higher levels (e.g., Oligochaeta at subclass). These taxonomic differences\namong sites did not influence our results (see Supplementary information 9). Identifications\nhigher than species level introduce some uncertainty given we cannot detect potential species\nshifts occurring within these groups. However, such identifications still reliably reflect overall\ncommunity responses to environmental change65,66 and are common in invertebrate research\nwhere many taxa cannot be reliably identified to the species level.”\n3. Could the design be effectively turned into a BACI (before after control impact), in that\nyou could compare changes at sites before the introduction of protected areas to after the\nintroduction of protected areas relative to sites over similar periods that never had PA (i.e.\npositive controls) and, if present sites, that had PA (or effectively had PA given they are in\nlocations with minimum human impact) over the entire period (i.e. negative controls)?\nSuch a change may not be practical, but if it were practical, it would improve your design\nas the BACI design considers changes that are occurring unconnected with PA, e.g.\nclimate change, reductions in pollution.\nWe agree a BACI analysis can be powerful approach, but there are several factors that make our\ndataset unsuited to this design and better suited for the approach we adopted:\n1. The BA component requires a pre/post intervention period, but out of our 1,472 protected\nsites only 311 (20%) were not protected by the beginning of the collection of each\ninvertebrate time series. Additionally, analyzing this 20% would require splitting each\ntime series into a pre/post period, resulting in many sites having insufficient data for\n13\nquantifying robust pre- and post-temporal trends (our requirements are for min. 10 years\nin duration and min. 7 sampling years).\n2. The CI component also requires that the timing of sampling matches between the\nimpacted and control sites, and in our case both would need to come from the same river\ntype and geographic region. In our dataset, there is wide variation both in timing and\nriver characteristics between the protected and unprotected sites, making it infeasible to\nfind appropriate unprotected matches for each of the above 311 protected sites.\n3. Furthermore, BACI requires a single intervention, however many of our protected sites\nexperience multiple interventions because multiple PAs are often established in\nsubsequent years. For example, only about 311 sites were unprotected at the start of their\ntime series, but 1,070 sites gain PA cover through time, meaning most of the gains are\ndriven by further PA establishment in already protected sites.\nOur approach addresses the above problems because it does not require that each site has a\npre/post intervention period, it does not require exactly matching time series for the protected\nand unprotected sites, and temporal trend analyses can capture potential lagged effects and\ncompounding effects of multiple interventions. To better highlight the strengths of our approach,\nwe added some of the above detail to the section of our Methods that discusses our analyses.\nCurrent Lines 354–361 (added): “These temporal trend comparisons have some strengths\ncompared to other possible approaches, such as before-after comparisons or spatial\ncomparisons of protected and environmentally similar unprotected sites. First, using temporal\ntrends of percent biodiversity change allows for comparing sites that differ in total biodiversity,\nand allows for variation in protection timing (e.g., sites can be already protected by the start of\ntheir time series or can become protected later). Second, temporal analyses allow for changes in\nprotection effectiveness through time, such as lagged effects, and capture the potential\ncompounding effects of establishing multiple PAs in subsequent years.”\n4. It seems logical that if PA enhance biodiversity once they are enacted, biodiversity gain\nwould be great in PA than outside protected areas. However, PA are often established in\nareas which are relatively unimpacted by human activities (the MS acknowledges this “PAs\ntend to be designated in less impacted, forested, higher elevation areas with little human\ndevelopment”). So, how would you expect biodiversity to change in PA once the protection is\nlegally enacted? If prior to their establishment, PA tended to have had less biodiversity\nloss than outside PA, then would expect less gain in biodiversity from PA, relative to non-\nPA, as there is less potential for recovery at PA. And this is indeed what you observe Figure\n2c. Such results may not necessarily indicate that PA are not effective, rather they may\nindicate that prior to protection PA had less biodiversity loss than non-PA. Moreover,\nresults in Figure 2 and 3 further support my suggestion in that in protected areas that had\nhigh – moderate EQR prior to protection there was no gain in richness or EQR following\nprotection compared to areas with low EQR prior to protection.\nYou raise a very important point about our need to clarify the conclusions drawn from our\nresults, and what we can and cannot say using our analytical approach. These clarifications are\nparticularly important for ensuring readers do not misconstrue our text as implying that PAs\nprovide no benefits.\nprotection did not alter biodiversity outcomes because protected high-quality sites exhibited the\nsame biodiversity outcomes as unprotected high-quality sites (e.g., the dark blue lines in Figs. 3\n& 4). This also means PAs did not maintain biodiversity that would otherwise have been lost\nwithout protection. You are, however, correct that we cannot be certain why this occurred. It\ncould be that PAs were ineffective, but high-quality communities may also generally experience\nlow human impacts whether they are protected or not, thus providing PAs with a limited scope\nfor effect. The latter explanation is consistent both with why biodiversity change did not differ\nmuch between high-quality protected versus unprotected sites, and why biodiversity gain was\neven sometimes slightly lower with protection.\nHowever, our results and analytical approach still support a conclusion of generally limited PA\neffectiveness. This is partly based on the various panels of Fig. 2, but also on reduced (or no) PA\neffectiveness across a variety of moderate-quality communities (e.g., initial EQR of 0.4–0.6 in\nFig. 4). These communities have higher initial biodiversity compared to poor-quality\ncommunities, but not high enough that there is limited scope for further improvement as they still\ndiffer substantially from reference conditions.\nTo address your concerns, we made a variety of edits throughout our text to clarify our results\nand conclusions across the gradient of poor to moderate to high quality communities. These edits\nalso somewhat soften the implication that PAs had no effect by more frequently leading with the\npositive benefits we found in poor-quality communities. The edits were as follows:\nAbstract\nOriginal Lines 7–12: “Contrary to our expectations, site-level rates of biodiversity change were\ngenerally unaffected by upstream protection. The only exceptions were some highly impacted\nsites that had, or gained, protection across a substantial proportion of their catchments, which\nwas associated with a two to three times higher rate of biodiversity gain. To broadly improve the\nfreshwater effectiveness of protected areas…”\nEdited to provide more nuanced results and conclusions across the ecological quality gradient.\nWe also now start this section with the positive results for poor-quality communities, rather than\nleading with the negative results, placing more emphasis on the benefits PAs provided to certain\nfreshwater ecosystems:\nCurrent Lines 8–14: “Protected areas primarily benefited poor-quality communities (indicative\nof higher human impacts) that were protected, or that gained protection, across a substantial\nproportion of their upstream catchment. Protection had little to no influence on moderate- and\nhigh-quality communities, although high-quality communities potentially provide less scope for\neffect. Our results reveal the overall limited effectiveness of current protected areas for\nfreshwater biodiversity, likely because they are typically designed and managed to achieve\nterrestrial conservation goals. Broadly improving effectiveness for freshwater biodiversity…”\nNature Communications requires a summary of the results at the end of the Introduction, which\nhas now been added in this revision round. We’ve taken the opportunity through these additions\nto further remind readers of how our results varied across the ecological quality gradient, and to\nlead with the positive results:\nCurrent Lines 84–91 (added): “Here, we show that upstream PAs primarily benefited poor-\nquality communities where PAs encompass a larger proportion of the catchment. These\ncommunities exhibit much higher rates of biodiversity recovery than likely would have occurred\nin the absence of protection. In contrast, PAs had little to no effect on biodiversity in moderate-\nand high-quality communities, although the latter group may have been unaffected because\nhuman impacts in such rivers are generally low regardless of protection status. Our results\nunderscore the need to broadly improve PA effectiveness in freshwaters by ensuring PA design\nand management explicitly consider freshwater biodiversity and integrate the needs of\nfreshwater ecosystems.”\nResults\nWe added two small sections to the results that specifically detail the effects of protection in\nhigh-quality sites (i.e., sometimes slight improvement but sometimes even a reduction in\nbiodiversity gain with protection). These results are then referred to specifically in a modified\nsection of the Discussion (detailed below) that outlines what may have happened in the the high-\nquality communities:\nOriginal Lines 101–102: “These effects weakened as the upstream scale and initial ecological\nquality increased (Fig. 3b–d).”\nEdited to better detail the effects of protection in high quality sites:\nCurrent Lines 114–117: “These effects weakened as the upstream scale and initial ecological\nquality increased (Fig. 3b–d) to the point that, at the full upstream scale and an initially high\necological quality of 0.8, increasing PA cover from < 1% to 100% only increased the rate at\nwhich richness increased from +0.7% to +1.1% year-1 (Fig. 3d).”\nCurrent Lines 141–145 (added): “Furthermore, as initial ecological quality increased, we found\nsome instances where higher PA gains actually translated to slightly lower rates of increase in\nboth ecological quality and richness. Using the full upstream scale as an example and an\ninitially high ecological quality of 0.8, increasing the rate of PA gain from < 1% to 7.5% year-1\ndecreased the rate of EQR gain from +0.32% to +0.19% year-1 (Fig. 4d).”\nWe made some modifications to our first Discussion paragraph (Current Lines 161–167) to\nclarify for readers that we are not suggesting PAs are ineffective, and to better highlight the\nbenefits we found.\nOriginal Lines 135–139: “We are not suggesting PAs are generally ineffective, particularly given\nthey can succeed in their goals for terrestrial ecosystems, such as reducing habitat loss39,40, and\nhave been effective for certain, individual freshwater ecosystems and taxonomic groups12,41. Our\nfindings do, however, highlight the urgent need to broadly improve the capacity of PAs to support\nfreshwater biodiversity.”\nEdited to:\nCurrent Lines 161–167: “This conclusion should not be misconstrued as suggesting that PAs are\ngenerally ineffective, particularly given it is based on a subset of total freshwater biodiversity\nand does not speak to whether PAs achieved the terrestrial conservation goals they are typically\ndesigned and managed for, such as reducing habitat loss9,10. We also found that PAs helped some\nriver invertebrate communities, and other studies have shown PAs benefiting certain, individual\nfreshwater ecosystems and taxonomic groups14,44. Our findings do, however, highlight the need to\nbroadly improve the capacity of inland PAs to support freshwater biodiversity.”\nAdditionally, we expanded the section of the Discussion that deals with the reduced effect of\nprotection in higher quality communities. We are now clear that the lack of protection\neffectiveness in high quality sites could be attributed to their low human impacts and initially\nhigh biodiversity, but this explanation likely does not apply to the moderate-quality communities\n(Current Lines 170–179). Furthermore, we edited our concluding paragraph, which previously\nfocused too strongly on the overall lack of PA effect. We re-worded this section (Current Lines\n232–239) to provide a more nuanced overview of our results across the different river types:\nOriginal Lines 142–146: “That comparable protection did not affect biodiversity in higher\nquality rivers could reflect their communities’ lower breadth for improvement. However, this\nexplanation is unlikely because protection was less effective even in moderately impacted rivers\n(e.g., initial EQR around 0.4–0.6) where biodiversity is considerably below reference levels. A\nmore likely explanation is that…”\nEdited to:\nCurrent Lines 170–179: “The lesser influence of protection on higher quality communities\npotentially reflects the already low human impacts in these sites, thus biodiversity remained high\nand stable regardless of protection status. This explanation fits with our results showing low PA\neffectiveness in high-quality communities (e.g., initial EQR around 0.8) where biodiversity was\nlikely already high, and may explain why protection was sometimes associated with lower\nbiodiversity gains, which may occur if PAs are placed in areas with a lower scope for\nimprovement (e.g., remote, forested catchments37,38). However, it does not explain why PAs\nexhibited reduced effectiveness in moderate-quality communities (e.g., initial EQR around 0.4–\n17\n0.6), which have considerable potential for further improvement. A more likely explanation for\nthese communities…”\nOriginal Lines 185–188: “Our findings show generally poor effectiveness of PAs for freshwater\nbiodiversity, with benefits limited primarily to highly impacted rivers where PAs encompass a\nlarger proportion of the catchment. Improving overall effectiveness requires PA management\nstrategies to explicitly consider the needs of freshwater ecosystems…”\nEdited to:\nCurrent Lines 232–239: “Our findings, based on European river invertebrate communities, show\nthat PAs have benefited certain freshwater communities, specifically poor-quality communities\nwhere protection encompassed a larger proportion of the upstream catchment. All other\ncommunities exhibited more limited (or no) effects of protection, although the lack of effect in\nhigh-quality communities may have occurred because these communities are less impacted\nregardless of whether they are protected or not. Improving overall PA effectiveness, particularly\nin impacted rivers, requires design and management strategies that explicitly integrate the needs\nof freshwater ecosystems…”\n5. My point above is that such results do not necessarily indicate the PA are ineffective at\nprotecting freshwater biodiversity, rather that biodiversity protection by PA is dependent\non the loss of biodiversity before PA enactment. (If, for example, no biodiversity has been\nlost, then PA will not result in a gain in biodiversity). So, the major conclusion conveyed by\nthe title “Current protected areas provide limited benefits for European river biodiversity”\nmaybe misleading. Or am I misunderstanding something fundamental?\nOur edits to your point #4 above should address this concern by improving the clarity of our\nconclusions, and demonstrating that our approach can speak to the more nuanced effects of\nprotection across the gradient of poor-, moderate-, and high-quality sites.\n6. What to do? A BACI design (see above) would go some way to solving this problem.\nAdditionally, for locations that effectively had protection before the enactment of PA (e.g.\nbecause they are in remote or at high elevations), might they be regarded as de facto PA in\nyour analysis. Note de facto means ‘in reality or as a matter of fact’.\n7. Alternatively comparing biodiversity (not its change per unit time) in areas that become\nPA to those that never become PA before protected areas are established would also check\nmy suggestion that PA may have lost less biodiversity prior to their enactment.\nWe grouped these two points together because one follows from the other. Our above responses\noutline the unsuitability of our dataset for a BACI-type analysis, and how our approach and\nresults allow us to determine the effects of protection (or lack thereof) across the quality\ngradient.\n8. Line 2, I would think that protected areas are to prevent or reduce biodiversity loss not\nto mitigate biodiversity loss.\nWe adjusted this wording to “addressing”.\n9. Line 25, rather than “providing valuable insights into the strengths and weaknesses of\ncurrent approaches” I would rather see some brief statement as to the effectiveness (or\notherwise) of PA for terrestrial and marine systems.\nWe modified the wording to better highlight the positive potential of protected areas in these\necosystems.\nOriginal Lines 23–25: “Several broad-scale (i.e., global or continental) studies have investigated\nthe effectiveness of terrestrial and marine PAs5–8, providing valuable insights into the strengths\nand weaknesses of current approaches.”\nEdited to:\nCurrent Lines 26–29: “Several broad-scale (i.e., global or continental) studies have investigated\nthe effectiveness of terrestrial and marine PAs, providing insights into their potential for\nreducing biodiversity loss5–7, exploitation8, and habitat loss9,10.”\n19"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I apologise for the lateness of my review - reflecting the effects of annual leave and the scientific conference season.\nIn general, the authors have expended considerable effort in addressing the reviewers' comments, modifying the manuscript accordingly. I don't have any further comments that are sufficiently serious to hinder publication.\nI find this an interesting and valuable contribution that should add to debate and prompt action.\nS J Ormerod, Cardiff September 8th 2025", "reviewer_2": "The authors have addressed the previous comments I made and I am happy for this MS to be published."}, "author_response": ""}]

Decision letter