Conservation status of two endangered freshwater mussel species in Bavaria, Germany: Habitat quality, threats, and implications for conservation management

1. The freshwater pearl mussel ( Margaritifera margaritifera ) and the thick-shelled river mussel ( Unio crassus ) are relatively widespread across Europe, but are strongly declining and are now protected by the European Habitats Directive. In the course of this study, 20 pearl mussel and 14 thick-shelled river mussel streams in Bavaria, Germany, were investigated. host physicochemical parameters were investigated, e.g. substratum quality, water chem-istry, redox potential, and turbidity. Furthermore, potential risks the were and assessed. 5566 0.85 at M. margaritifera sites, U. Other parameters such as redox potential or electric conductivity also more favourable habitat conditions in M. margaritifera streams. Unio crassus


| INTRODUCTION
Freshwater bivalves have colonized diverse habitats worldwide and have adapted to highly diverse habitat conditions (Bogan, 2008); however, they also belong to the most imperilled taxonomic groups worldwide (Lydeard et al., 2004). In particular, unionoid bivalves, which have a complex life cycle including an obligate host-fish stage, are highly vulnerable to habitat alterations, as these can affect the mussels directly as well as their host fish (Taeubert & Geist, 2017). In central Europe, bivalve populations have severely declined over the last decades owing to pollution, physical habitat destruction, and land-use changes (Bauer & Wächtler, 2001;Geist, 2010Geist, , 2011Lopes-Lima et al., 2017). At the same time, bivalves are considered keystone species for the functioning of freshwater ecosystems (Lummer, Auerswald, & Geist, 2016;Vaughn, 2018), as they provide important ecosystem services such as filtration, bioturbation or nutrient allocation (Atkinson, Vaughn, Kenneth, & Cooper, 2013;Vaughn, Gido, & Spooner, 2004;Vaughn & Hakenkamp, 2001).
In European member states, species and habitat types that are considered to be of European importance are protected under the Habitats Directive (Bouchet, Falkner, & Seddon, 1999 Annex II, core areas of protection need to be defined ('special areas of conservation'), which are included in the Natura 2000 network. These sites must be managed according to the ecological demands of the target species. Species in Annex IV must be strictly protected not only within but also outside of protected areas, and the exploitation of species in Annex V must not endanger their favourable conservation status. Following Article 17 of the Directive, member states must report to the European Commission on the state of the habitat types and species every 6 years based on the results of monitoring programmes.
In most cases, member states have developed national monitoring protocols for species and habitat assessments. As a result, methodologies and data quality can differ widely between the countries. For freshwater pearl mussel (Margaritifera margaritifera), the first step to overcome this problem was taken only recently, when a group of international experts from countries with existing populations of this species came together to compile a standard, following the criteria of the European Committee for Standardization (CEN) (Boon et al., 2019). CEN brings together national standardization bodies from 34 European countries for the development of voluntary standards at a European level. The CEN standard for monitoring freshwater pearl mussel streams was developed by experts from 11 countries: Austria, Finland, France, Germany, Ireland, Luxembourg, Norway, Portugal, Spain, Sweden, and the UK. Based on best practice developed and used by those experts, the standard describes approaches that individual countries have adopted for survey, data analysis, and condition assessment, and provides guidance on a consistent approach to monitoring. Among the 44 mollusc species, the freshwater pearl mussel Margaritifera margaritifera (Linnaeus, 1758) and the thick-shelled river mussel Unio crassus (Philipsson, 1778) (Denic & Geist, 2017) and with 82 thick-shelled river mussel populations a considerable proportion of German populations occur in the federal state of Bavaria.
This article presents an overview of the population status and habitat conditions in freshwater pearl mussel and thick-shelled river mussel streams in Bavaria and assesses the condition of populations and habitats based on the German assessment criteria within Natura 2000 areas.

| Study area
The study was conducted in 20 M. margaritifera and 14 U. crassus streams in the state of Bavaria, Germany, between 2012 and 2015.
The rivers with M. margaritifera included all types of M. margaritifera habitats in Bavaria as well as populations in various condition. At a national level, Bavaria holds the majority of German pearl mussel populations. For U. crassus, 14 study streams had been selected previously by the Bavarian Environment Agency by random draw for each Bavarian state district from the state database ArtenSchutzKartierung (ASK). As a consequence of a recent illegal pearl fishing event, the names of the mussel rivers are treated as confidential and are not reported here. The natural distribution area of M. margaritifera in Bavaria is limited to the Eastern part of the state, which is dominated by siliceous headwater streams (Figure 1). In contrast, U. crassus is widely distributed across the state, with the study sites representing all faunal regions that are populated by the species.

| Mussel survey
The survey area covered the colonized area of the mussels in each stream, which was known from previous monitoring programmes, and varied between 0.2 and 18.0 km of river length. For M. margaritifera, complete census monitoring was performed where mussel numbers were expected to be smaller than 1000 individuals. In populations of more than 1000 individuals, 5-m cross-channel transects were applied every 100 m to estimate the population size.
For U. crassus, a systematic sampling approach (Strayer & Smith, 2003) was applied using cross-channel transects to estimate the population size. According to Christman (2000), systematic sampling provides good spatial coverage and is particularly useful for populations of rare species. The distance between two transects was 80 m and the length of each transect was 20 m. In all streams, visual and tactile searches were performed either by wading in an upstream direction or by snorkelling or scuba diving, drifting downstream, depending on the water depth of each stream.
In areas with at least 10 mussels per metre of stream length, at least 100 mussels were removed from the sediment to determine their length and age. The age of the mussels was assessed: (i) by counting the annual rings in the shells of U. crassus; and (ii) by measuring the total length with a caliper in M. margaritifera and determining approximate ages from pre-determined age-length relationships. As the shells of M. margaritifera grow very slowly and have a dark black colour, age determination by counting the annuli is rarely possible. Therefore, the length of the mussels was measured to distinguish between juvenile (<65 mm; Hastie, Boon, & Young, 2000) and adult mussels. All live mussels were returned to the river in the approximate locations where they were found. In populations with low numbers (<100), age structure was not determined.

| Habitat parameters
Physicochemical habitat characteristics were analysed at 290 sites (5-10 sites per stream, depending on the length of the populated stretch). The sites were randomly selected and distributed across the length of each study stream. Electrical conductivity (EC, μS cm −1 , corrected to 20 C), dissolved oxygen concentration (O 2 , mg L −1 ), pH value, and temperature ( C) were measured using a handheld Multi 3430 multiparameter meter and a pH 3110 portable pH meter (WTW GmbH, Weilheim, Germany), with one measurement taken per site. Turbidity (NTU) was measured using a turbidity meter (Turb355 T; WTW GmbH).
For water quality analysis, three replicate water samples per stream were collected from free-flowing water. A volume of 50 ml was taken and stored on ice until further processing with ion chromatography (ThermoFisher Scientific, Dreieich, Germany). A mixture of Höntzsch, Waiblingen, Germany) at 50% water depth. Substratum texture was analysed at three sites per study stream using a box sampler (Pander, Mueller, & Geist, 2015). This collects the uppermost 10 cm of the substratum; it has a rectangular opening of 16.0 cm × 12.2 cm and a length of 29.3 cm. The box sampler is equipped with an adjustable metal plate on each side to ensure sampling from a well-defined substratum depth. Its use is similar to a bulkcore sampler (Kondolf, 2000). Grain sizes were fractioned with a wet-T A B L E 1 German assessment scheme for Unio crassus streams. The worst score of a single parameter determines the assessment of each subcategory, which are population status, habitat quality, and disturbance Heavily maintained running waters with no variation in depth and width

Structure of substrate and interstitial zone (expert judgement with explanatory statement)
Stable stream bed consisting of sand or fine gravel; well perfused and unclogged interstitial Stream bed predominantly consisting of sand and fine gravel or silt, but mainly stable; restricted perfusion of interstitial spaces owing to moderate siltation Very silty substrates; stream bed only partially stable; low perfusion of interstitial spaces owing to strong siltation percentage of each grain fraction was determined, but considering the restricted grab-sample volumes, the largest fraction of >20 mm was generally excluded from further analyses, as suggested by Geist and Auerswald (2007).
Redox potential (Eh) was measured both in the free-flowing water and in the interstitial zone to determine the hydrological exchange of water (and oxygen) between the two compartments. Following the method described in Geist and Auerswald (2007), the redox potential was first measured in free-flowing water and then at a depth of 10 cm into the substratum. Values above 300 mV imply oxic conditions, whereas values below indicate anoxia (Schlesinger, 1991).
If recent data (less than 2 years old) from State Fish Monitoring by the 'Fachberatung fuer Fischerei -Bezirk Niederbayern' and the 'Fachberatung fuer Fischerei -Bezirk Oberfranken' on fish populations were unavailable, electrofishing was conducted to assess host fish availability and abundance in the streams. Fish populations were sampled with a 1.5-kW portable electrofishing backpack unit T A B L E 2 German assessment scheme for Margaritifera margaritifera streams. The worst score of a single parameter determines the assessment of each subcategory, which are population status, habitat quality, and disturbance Parameter/category A B C

POPULATION STATUS
Estimated total population size >10 000 1000-10 000 <1000 Age structure and estimated proportion of juvenile mussels Proportion of juvenile mussels (<10 years) is >20% of all live individuals Proportion of juvenile mussels (<10 years) is ≤20% of all live individuals No juvenile mussels found

HABITAT QUALITY
Overall habitat quality (expert judgement with explanatory statement) Well structured, natural streams with clear oxygen-rich water and a strong variation in depth and width Maintained streams with clear water, near-natural ditches; partially strong variation in depth and width Heavily maintained running waters with no variation in depth and width

Structure of substrate and interstitial zone (expert judgement with explanatory statement)
Stable substrate consisting of fine gravel to boulders; well perfused and unclogged interstitial zone Stable stream bed predominantly consisting of fine gravel and pebbles; restricted perfusion of interstitial spaces owing to moderate siltation Partially stable stream bed consisting of sand and silt; low perfusion of interstitial zone owing to strong siltation  5 and 10% of the length of the river stretch that was populated by the mussels. The stunned fish were collected with a dip net and kept in plastic tanks with a permanent oxygen supply. After species determination and length measurements, all fish were released into the same stretch of stream from which they were sampled. Species richness and host fish density were calculated for each stream.

| Threat assessment
To identify the main threats to mussel populations, a qualitative assessment was conducted in each section between two transects, evaluating the following criteria: land use in the catchment area, diffuse input, pollution/water quality, indicators of predation, river maintenance, weirs, and pearl fishing. The extent of each threat was categorized in the following three classes: (i) not observed; (ii) weak, but detectable impact; and (iii) severe impact.

| Data analysis
The monitoring results were used to classify the mussel populations and their habitats. Quantitative and qualitative parameters were recorded in an evaluation matrix (Tables 1 and 2), following the national assessment system (Sachteleben & Fartmann, 2009

| Mussel populations
Margaritifera margaritifera population size ranged between <100 and 35 000 individuals (Figure 2). In three streams (15%) with previously known populations, no living individuals were detected. The mean population size of all pearl mussel streams was 3 517 ± 8 500 individuals (mean ± standard deviation). In 90% of the M. margaritifera populations, the population size was smaller than 10 000 individuals.
Size estimates for U. crassus populations ranged between <100 and 40 000 individuals, with an overall mean population size of 5566 ± 10 800 individuals. In 92% of the studied U. crassus populations, the population size was smaller than 10 000 individuals.
In nine M. margaritifera populations, sufficient numbers of individuals were obtained for the compilation of age profiles. The percentage of juvenile mussels (<65 mm) in the populations was very low (mean 2.2%, Figure 3), indicating that there has been no recruitment for at least 25 years. In contrast, the proportion of juvenile mussels in U. crassus populations was considerably higher, with a mean of 41.4%.
In one population, the proportion of juvenile mussels exceeded 90%, which results from a lack of adult age classes.
F I G U R E 4 Pie charts categorizing mussel population status, habitat quality, and disturbance, according to Sachteleben and Fartmann (2009) Figure 4).

| Habitat quality
Habitat quality in M. margaritifera streams was assessed as moderate (category B) in 24% of the streams and as bad (category C) in 76% of the streams. In the case of U. crassus, 7% of the streams were assessed as good, 33% as moderate, and 60% as bad in terms of habitat quality.
Fish species richness was higher in U. crassus streams (total number of species, n = 28) compared with M. margaritifera streams (total number of species, n = 23). For both mussel species, overall fish species richness was higher in streams with mussel recruitment     which decreased to 4.7 ± 1.9 mg L −1 in category-C habitats. In U. crassus streams the TOC was higher and increased in habitats from category A to C, with 8.1 ± 2.3, 8.1 ± 3.0, and 11.5 ± 4.6 mg L −1 , respectively.

| DISCUSSION
In this study, a total of 34 freshwater mussel populations and their habitats were assessed using the national evaluation methodology European CEN standard to provide guidance on monitoring a single species (Boon et al., 2019). The species has been characterized as a highly specialized inhabitant of oligotrophic gravel-bed streams with high water quality and a low content of fine sediment (Bauer, 1988;Denic & Geist, 2015;Geist, 2010;Geist & Auerswald, 2007;Hastie et al., 2000). The clogging of macropores in the interstitial zone by fine sediments was identified as the main reason for declining populations, resulting in recruitment failure, which is clearly supported by the results of this study. In most of the samples, the proportion of fine sediments exceeded the thresholds of 20% for particles <1 mm and turbidity levels of 0.96 NTU, identified from investigations of intact habitats by Geist and Auerswald (2007) and Österling, Arvidsson, and Greenberg (2010), respectively. At the same time, we observed a strong decline of redox potential from the freeflowing water to the interstitial zone, which leads to a lack of oxygen in juvenile habitats, thus explaining the over-aged pearl mussel populations in the Bavarian study streams. Moreover, some water quality parameters, such as EC or nitrate, were higher in several streams, but as this was also the case in some of the streams with recruitment, these parameters do not seem to have as great an impact as substratum quality.
In contrast to M. margaritifera, research activities targeting U. crassus were rare until a few years ago. Until then, the habitat needs of both species were assumed to be similar. However, recent studies indicated pronounced differences in their ecological requirements: for example, in their sensitivity to fine sediments or in their general substratum preferences (Denic, Stoeckl, Gum, & Geist, 2014;Inoue, Stoeckl, & Geist, 2017;Stoeckl & Geist, 2016). Thus, in this study, recruitment of U. crassus was observed in streams with a maximal proportion of particles <0.85 mm of 99%. It is clear that there is no correlation between substratum structure and recruitment success in the Bavarian study streams (Figure 5). At the same time, even higher deltas of redox potentials were detected between free-flowing water and the interstitial zone in functional U. crassus habitats than in non-  (Stoeckl, Taeubert, & Geist, 2015). In contrast, host fish abundance is sufficiently high in most of the M. margaritifera streams investigated, and higher densities of fish in these situations can even be indicative of eutrophication and poor habitat quality for juvenile mussels (Geist, Porkka, & Kuehn, 2006). An additional threat for U. crassus is its occurrence in nutrient-enriched and often heavily modified habitats, or even in man-made ditches located in intensively used catchments (Inoue et al., 2017 preferences and resilience against adverse habitat conditions. As a result, deducing the ecological requirements of one species based on another is probably rarely possible, and exact knowledge about ecological needs, population status, genetic structure, and the causes for decline is indispensable for the development of effective conservation strategies. Despite this, there is a lack of such basic information for the other native European mussel species. With respect to the important ecosystem services that freshwater mussel species provide (Vaughn, 2018) and the continuing loss of biodiversity, it is urgent to gather the missing information on species so far neglected through fieldwork and research. This will allow the design of species-specific conservation actions, as has been achieved for M. margaritifera, where several conservation projects around Europe apply a combination of artificial breeding and habitat restoration in prioritized target streams. Unfortunately, the annexes of the European Habitats Directive that list the species for special protection are not updated; hence, monitoring and conservation action for other endangered mussel species that are not listed on the annexes are rarely conducted because of an absence of supporting legislation. This may even result in the decline of tolerant and widely distributed species, with drastic effects on ecosystem functioning and the provision of ecosystem services.
Furthermore, the conservation of habitats of those species already protected may also gain from legislative measures. Dobler, Geist, Stoeckl, and Inoue (2019) showed that designated sites such as the special areas of conservation (SACs) selected for mussel F I G U R E 5 Triangle plots of grain size distribution in Unio crassus (left) and Margaritifera margaritifera streams (right) species in Bavaria are frequently too small to be effective, as in many cases the SACs do not cover the catchment areas of the streams. This means that critical factors such as nutrient and sediment loads originating from the wider catchment cannot be reduced using the legal instruments of the Habitats Directive. The limited capacity of the Natura 2000 network to protect freshwater biodiversity seems to be true for other countries in the European Union: by assessing the coverage of protected areas on the Iberian Peninsula for more than 90 freshwater species, Hermoso, Filipe, Segurado, and Beja (2015) showed that the current areas of protection fail to provide sufficient coverage of freshwater biodiversity, with less than 20% of the range of species covered on average. We therefore recommend supplementing conservation projects with an expansion of nature reserves, wherever possible.