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Hydrobiologia

, Volume 810, Issue 1, pp 157–175 | Cite as

Challenging exploration of troubled waters: a decade of surveys of the giant freshwater pearl mussel Margaritifera auricularia in Europe

  • Vincent PriéEmail author
  • Joaquin Soler
  • Rafael Araujo
  • Xavier Cucherat
  • Laurent Philippe
  • Nicolas Patry
  • Benjamin Adam
  • Nicolas Legrand
  • Philippe Jugé
  • Nina Richard
  • Karl M. Wantzen
FRESHWATER BIVALVES

Abstract

The critically endangered Giant Freshwater Pearl Mussel Margaritifera auricularia was presumed extinct before its rediscovery in Spain in 1985 and France in 2000. Since then, numerous surveys have been set up to search for living populations in France and Spain. This article presents an up-to-date account of species distribution based on available data, i.e. the literature, museum collections and recent field surveys, and provides unpublished molecular data for France. There are still three populations of the Giant Freshwater Pearl Mussel in the Ebro River in Spain, and eight populations in France (two in the Loire watershed, one in the Charente watershed, two in the Garonne watershed and three in the Adour watershed). The biggest population lives in the Charente River with an estimated 100,000 individuals. Recruitment is very scarce in all populations but living specimens estimated to be less than 10 years old have been found in the Ebro in Spain and in the Vienne, Charente, Dronne and Adour rivers in France. The recent rediscovery of populations in France was mainly a result of intensive dedicated surveys including scuba diving. Subsequent advances in knowledge show how large rivers and downstream ecosystems remain a terra incognita for the hydrobiologist.

Keywords

Distribution Museum collections Historical data Scuba-diving surveys Large rivers Conservation 

Introduction

Freshwater ecosystems are the most threatened ecosystems worldwide (Dudgeon et al., 2006), and freshwater bivalves rank amongst the most threatened animals in the world (Lydeard et al., 2004; Lopes-Lima et al., 2016). One of them, the Giant Freshwater Pearl Mussel Margaritifera auricularia (Spengler, 1793), figures amongst the most imperilled bivalve species. Although it was considered widespread in most of the western Europe rivers at the beginning of the twentieth century, it is now considered as critically endangered by the IUCN (Araujo & Ramos, 2001; Prié, 2010). The Giant Freshwater Pearl Mussel had become so rare during the twentieth century that it was not even considered when the European Habitat Directive species lists have been established. Indeed, the Giant Freshwater Pearl Mussel is difficult to observe: it lives downstream in large rivers, a habitat that is difficult to survey due to deepness, turbidity, current and often navigation. Hence, not surprisingly, it has been overlooked by malacologists of the twentieth century. However, it nowadays still survives as a few populations in south-west France and eastern Spain.

The species was first rediscovered in Spain in 1985 (Altaba, 1990) and in France in 2000 (Cochet, 2001). Since 1998, the biology, distribution and lifecycle of the Giant Freshwater Pearl Mussel in Spain have been described (Araujo & Ramos, 1998a, b; Araujo & Ramos, 2000a, b; Araujo et al., 2000; Grande et al, 2001; Araujo et al, 2001, 2002, 2003; Gómez & Araujo, 2008). Since then, very few reports of the species in Spain have been released in national and international congresses (e.g. Nakamura et al., 2015; Online Resource 1), but, apart from Araujo & Álvarez-Cobelas (2016), there are no new scientific results published since 2008. In France, focused surveys have led to the rediscovery of many populations since 2007, but most of these results are unpublished (but see Prié et al., 2007, 2008, 2010) or available only as grey literature (Online Resource 1).

An extensive review of all available data on Margaritifera auricularia’s distribution is provided here for the first time, together with new data from museum collections and recent field surveys. This article clarifies the past and present distributions of the species, presents the results of the last ten years’ surveys in France and Spain and discusses conservation perspectives.

Materials and methods

Bibliography review

The bibliography since 1793 (species description date) has been extensively reviewed. Local publication and the grey literature were also consulted when available. Bibliographic data were generally imprecise, but allowed figuring a broad image of the original distribution and ecology of M. auricularia (Fig. 1). Bibliography review thus provided the first indications for where to look for this species.
Fig. 1

Fossil (black crosses) and historical data (white dots for precise locations, blue lines for rivers’ names only) collected from the literature and Museum collections; and subsequent intensive field surveys locations (polygons)

Museum collections

A first review of museum collections had been performed by Araujo & Ramos (2000a) at a global scale. This review mostly aimed at large national museum collections and included also Margaritifera marocana (Pallary, 1918), a species living only in Morocco (Araujo et al., 2009a). We then inventoried all the regional museum and universities’ collections in France. Fifty-eight local natural history collections were identified. Each of them was contacted and questioned about the presence of malacological collections, freshwater bivalves and eventually M. auricularia specimens. When M. auricularia specimens were recorded in the inventories or discovered in the collection by the curator, pictures were sent to us to confirm identification. Eventually, some of the most important collections (Musée des Confluences in Lyon, Museum d’Histoire Naturelle in Bordeaux, Museum d’Histoire Naturelle in Toulouse, Museum national d’Histoire naturelle in Paris, Museum d’Histoire Naturelle in Lille, Museum d’Histoire Naturelle in Nantes, Museum d’Histoire naturelle in Orleans, University of Rennes, University of Montpellier) were visited by one of us.

Specimens collected since 2000, year of the rediscovery of the species in France, were not included in the results presented here.

Field surveys and population sizes

Numerous field surveys aiming at freshwater mussels have been performed in France and Spain (Fig. 1, Table 1).
Table 1

Summary of the methods used in France for surveying M. auricularia

Coastal drainage

River

Year

Use of a boat

Scuba diving

Other methods

Authors involved

Somme

Somme River

2011

X

4 divers, 5 days

 

XC, VP

Seine

Seine River (downstream)

2011

X

 

Dredging (2 persons for 4 days)

XC

Seine River (upstream)

2015

  

Snorkelling (2 persons for one days)

XC

Oise River

2007–2008

X

3 divers, 20 days

 

VP, LP, XC

Aisne River

2011

 

2–4 divers, 5 days

 

XC, VP

Rhône

Saône and Doubs Rivers

2016

 

4 divers, 5 days

Wading with viewing glasses (2 persons, 5 days)

VP, LP, NL, NP, BA

Loire

Loire, Indre and Cher Rivers

2010–2011

 

2–3 divers, about 5 days

Wading with viewing glasses (2-4 persons, estimated to about 10 days altogether)

LP, VP

Vienne and Creuse Rivers

2009–2016

 

2 to 6 divers, over 20 days altogether

Wading with viewing glasses and snorkelling (2 to 6 persons, estimated to over 30 days altogether)

VP, XC, LP

Charente

Charente River

2007–2016

X

2–3 divers, about 20 days altogether

 

VP

Garonne

Dronne and Isle Rivers

2012–2014

 

2 divers, about 10 days altogether

Wading with viewing glasses (1–2 persons, about 5–10 days altogether)

VP

Dordogne River

2016

X

2 divers, 3 days altogether

Wading with viewing glasses (1 person, about 5 days)

VP

Vézère River

2016

 

3 divers, 3 days

Wading with viewing glasses and snorkelling (2 persons 3 days)

VP, LP, NP, BA, NL

Garonne River

2016

 

3 divers, 2 days

Wading with viewing glass (1 person, 30 days)

VP, NL

Save River

2009

 

2 divers, 1 day

Wading with viewing glass and snorkelling (1 person, 5 days)

VP, BA

Adour

Adour River

2012–2014

 

2 divers approx. 9 days

Wading with viewing glasses and snorkelling (2 to 6 persons, 10 days)

VP, BA, NL

Arros River

2016

 

3 divers, 3 days

Wading with viewing glasses (6 persons, 4 days)

VP, BA, NL

For Spain, survey methods were detailed and reported in Gómez & Araujo (2008) and Araujo & Álvarez-Cobelas (2016)

These dedicated surveys aimed at places most likely to host the species, i.e. places identified by the literature data, museum collection data or, for France, species habitats modelling (Prié et al., 2014). Moreover, some surveys took place into the frame of impact studies. These impact studies were triggered when M. auricularia was living—or when available data suggested that it could still be living—in an area impacted by a development project. The results of these impact studies are generally not published, consisting only in various cryptic reports (but see Prié et al., 2007, 2008; Araujo & Álvarez-Cobelas, 2016). We here summarize for the first time all the grey literature related to M. auricularia in France and Spain (Online Resource 1).

Margaritifera auricularia mainly lives in downstream ecosystems. Surveying this habitat is challenging because it is often deep, turbid, strongly flowing and navigable. In the Ebro historical channels, sampling depends on the hydraulic works made by the Confederación Hidrográfica del Ebro; it is necessary to decrease the water level in order to wade the channel bottom to find the specimens (Gómez & Araujo, 2008). In France, some populations are readily accessible, living in the banks (Vienne River) or in shallow waters (Creuse, Luy or Arros River). For those populations, snorkelling or wading with viewing glasses allowed efficient surveys. However, cumbersome methods based on a team of scuba divers were needed in most cases. For some surveys, a boat was used to shuttle the divers from a place to another. For others, divers dove from the river banks, and sampling plans were then constrained by river accessibility.

Population sizes given here were estimated based on exhaustive counts of observed living individuals (Luy, Creuse and Vienne Rivers); statistical analyses (Ebro, Arros and Charente Rivers), or in the worst case, by a subjective appreciation based on the density of specimens observed (Dronne, Adour and Save Rivers).

The Seine (downstream) and Eure Rivers could only be surveyed by dredging. The dredger used had an aperture of 50 cm, a 25-mm mesh, weighed 11 kg and was propelled by a 30-horsepower engine Zodiac by means of a 30-m-long rope. In the Eure River, different biotopes and flow facies were aimed at (mud, sand, stones, riffles, vegetation). In the Seine River, water was up to 6 m deep and too troubled for operators to see the river bed. Catches were then randomly positioned. Catches were 8–10 m long in the Eure River, and up to 40-50 m long in the Seine River. Sediment collected by the dredger was pulled up and sorted out on the boat. Wading surveys were adopted upstream the Seine River.

In the Somme River, a boat was used to shuttle divers and 82 bank-to-bank transects were sampled on a 26-km-long river stretch. In the Oise River, the divers were also transported by boat from a spot to another, but diving plans were constrained by river condition (from very strong current to muddy bottoms). Areas with very strong current were sampled combining scuba-diving and climbing techniques, with a 100-m-long static rope secured on a tree on the bank. The diver used a climbing harness and caving equipment in addition to scuba- diving gear to progress on the rope. Fins were used to go from side to side in the current, allowing to cover ca. 90-m-long cone-shaped surface on the river bottom. Altogether, 115 dives have been carried out on a 35-km-long stretch of the river, from the confluence with the Aisne River downstream to a few kilometres upstream the town of Sempigny. Upstream this stretch of river, surveys were carried out wading randomly in suitable habitats.

In the Charente River, the population was estimated based on scuba-diving transects’ surveys. A boat was used to shuttle the divers from a transect to another. A 20-m-long line was settled down on the bottom of the river, and scuba divers counted every living specimen left and right of the line at a distance of 2 m. Each sample then covered 80 m2. Transects were repeated every kilometre in the river stretch where mussels were present, and then every 3 km downstream and upstream the population’s distribution limits. A total of 43 transects were repeated on a stretch of 60 km. Detection probability has been estimated at 75% using iterated observations analysed with the software MARK (White & Burnham, 1999). Geographical statistics (Anselin, 1996) were performed using GeoDa software (Anselin et al., 2006). Suitable habitat length in the whole river was delimited downstream by the limit of the mud cover due to the influence of the Saint-Savinien’s impoundment, upstream by the limit of the living population. Between these limits, the substrate and general ecological quality of the river was very homogenous. In this stretch of favourable habitat, live specimens have been observed wherever we have dived between 2010 and 2016, thus confirming that the population is uniformly distributed.

Fourteen sampling surveys were undertaken between September 2000 and June 2006 in the Ebro River, covering a total length of 25 km, wading in shallow waters and with a team of divers in the deeper parts of the river. Divers used submerged ropes to perform bank-to-bank or longitudinal transects (survey methods are detailed and reported in Gómez & Araujo, 2008; Araujo & Álvarez-Cobelas, 2016).

In the Dronne and Isle Rivers, about 100 km stretch of each river upstream their confluence was surveyed, both by wading and scuba diving from the banks. The estimation of the population size was based on author’s appreciation only, and is likely underestimated: over 50 specimens have been observed during the surveys, with a subpopulation of 30 specimens in the lower location (exhaustive count). We estimate that about half of the living individuals have been observed during surveys, which is unlikely, given the detection probability estimate in this large river.

About 60 km of the Save River was surveyed by wading and scuba diving, aiming at an exhaustive count of the few remaining specimens which were found only in the lower section of the river. Most of the sampling in the Adour River was undertaken by wading and snorkelling, with scuba divers requested only for a few deeper places. As for the Dronne River, a few specimens were found in isolated places, with the biggest subpopulation numbering about ten specimens. Population size is estimated based on experts’ appreciation only. The Arros River is highly impacted by agriculture practices. The remaining favourable habitats were found isolated between the numerous impoundments’ influences. A first survey was conducted by scuba divers, but the deepest places did not have suitable habitats. A more intensive survey was then organized by a team wading with viewing glasses. The total length of river stretches having suitable habitats was 54 km. Within this 54-km stretch, sixteen sites were sampled. On each sampled site, stretches of 100 m–1 km were exhaustively surveyed. Population size was estimated based on average densities observed during surveys, multiplied by favourable habitat’s surface. In the Luy River, divers explored the deepest pools, while most of the river can be explored by wading. The main population is found in a very shallow place, and exhaustive counts were performed three times (during the years 2010, 2011, and 2012) by five persons wading in a line, about 1 m apart, ensuring efficient scanning of every single place of the river bed. However, detection probability is never 100%. Some specimens may spend some time completely buried in the sediment and are overlooked (see below the results for the Luy River). The results of these assumed exhaustive counts are therefore likely underestimated.

The most intensive surveys took place in the Vienne and Creuse Rivers. The surveys aimed at providing exhaustive counts of all living specimens. Observers with viewing glasses and divers (depending on the depth) were lined 1 m apart and moved forward upstream, ensuring efficient scanning of every single place of the river bed. Sampling was reiterated several times between 2009 and 2016 using the same methods.

In this study, when shells only have been collected, we considered “ancient shells” those that were worn and uncomplete, with neither periostracum nor ligament remains. “Recent shells” include shells with at least periostracum and ligament remains. We consider as “juveniles” specimens with shell length lower than 11 cm, and “subadults” those specimens with lengths ranging from 11 to 14 cm. Occasionally, some adult specimens had very short shells, especially in the Charente River, but these were obviously very old given the growth lines density and shell wear.

Genetic analyses

Tissue samples have been collected from ten specimens from the Ebro River in Spain, and ten specimens from the Vienne River (Loire watershed), two specimens from the Luy River (Adour River watershed), two specimens from the Charente River and one specimen from the Save River (Garonne River watershed) in France. Foot tissue samples were snipped in the field and preserved in 90° ethanol for molecular analysis.

For Spanish specimens, DNA was extracted using CTAB protocol: tissue samples, preserved in ethanol or frozen, were ground to a powder in liquid nitrogen before adding 600 mL of CTAB lysis buffer [2% CTAB, 1.4 M NaCl, 0.2% b-mercaptoethanol, 20 mM EDTA, 0.1 M TRIS (pH 8)] and subsequently digested with proteinase K (100 mg ml−1) for 2–5 h at 60°C. Total DNA was extracted according to standard phenol/chloroform procedures (Sambrook et al. 1989). For French specimen, DNA was extracted using the Nucleospin Tissue Kit (marketed by Macherey–Nagel), according to the manufacturer’s protocol. Extractions, amplifications and sequencing were performed by Genoscreen (France).

To test genetic variability between populations, we examined fragments of two mitochondrial genes, COI and 16S, used previously by Huff et al. (2004); these showed the greatest phylogenetic resolution power for relationships amongst margaritiferids. 28S nuclear gene fragments were also amplified, but different fragments were targeted for French and Spanish specimens. The COI, 16S and 28S genes were amplified by polymerase chain reaction (PCR) using the protocol described by Prié & Puillandre (2014) for French specimens, and described by Machordom et al. (2013) and Araujo et al. (2016a, b) for Spanish specimens. The amplified fragments were purified by ethanol precipitation prior to sequencing both strands using BigDye Terminator kits (Applied Biosystems, ABI). Products were electrophoresed on an ABI 3730 genetic Analyser (Applied Biosystems). The forward and reverse DNA sequences obtained for each specimen were aligned and checked using the Sequencer program (Gene Code Corporation) after removing primer regions. Sequences were automatically aligned using ClustalW multiple alignments implemented in BioEdit 7.0.5.3 (Hall, 1999). The accuracy of automatic alignments was confirmed by eye. Genebank accession numbers are provided in Table 2.
Table 2

Genes and Genbank accession numbers of French specimens used for DNA analyses

Coastal drainage

River

Specimen voucher number

Genbank accession number

COI

16S

28S

Charente

Charente

MNHN-IM-2009-12596

MF494673

MF494681

MF494677

MNHN-IM-2009-12597

MF494674

MF494682

MF494678

Garonne

Save

MNHN-IM-2009-12601

MF494675

MF494683

MF494679

Adour

Luy

MNHN-IM-2009-12662

MF494671

MF494696

MF494676

MNHN-IM-2009-12663

MF494672

MF494697

 

Loire

Vienne

Maur91

MF494670

MF494695

MF494680

MNHN-IM-2009-12611

MF494661

MF494684

 

MNHN-IM-2009-12615

MF494662

MF494685

 

Maur70

MF494663

MF494686

 

Maur72

MF494664

MF494687

 

Maur74

MF494665

MF494688

 

Maur76

MF494666

MF494689

 

Maur77

MF494667

MF494690

 

Maur78

MF494668

MF494691

 

Maur85

MF494669

MF494693

 

Maur79

 

MF494692

 

Maur88

 

MF494694

 

Results

Bibliography

Available literature provided valuable data, albeit generally with neither precise location nor date. Nevertheless, a first historical distribution map could be drawn from the ancient literature data. Margaritifera auricularia is known from the Netherland, England and Germany from fossil records only. However, some shells collected in the Unstrut River in Germany are very well preserved and perhaps might date back to historical times, at least until the early Middle Ages (Bössneck et al., 2006). Fossil data in Spain includes a Mediterranean Quaternary river in Yecla (Murcia) with 129,000–140,000-year-old specimens (Andrés & Ortuño, 2014) and many other Atlantic rivers with 5,000-year-old specimens (Araujo & Moreno, 1999). In France, fossil data near Marseille (coming from archaeological excavation) and in Massif Central (found amongst fossils collected in a cave) were presumably a result of human transportation.

According to historical data collected, Margaritifera auricularia was only found in large rivers, in a calcareous substrate, in France, Spain and Italy. In France, historical data mainly come from the Atlantic and Channel sea watersheds, with only one occurrence in the Mediterranean drainages, in the Saône River (Rhône tributary). In Italy and Spain, the species is historically known from two Mediterranean watersheds, the Po and Ebro Rivers (Araujo & Ramos, 2000a). In Spain, M. auricularia lived in two historical channels from the Ebro River, the Canal Imperial and the Canal de Tauste, where there were about 5,000 live specimens. The more recent data published about these Spanish populations were recorded in Araujo & Ramos (2000b), Gómez & Araujo (2008) and Araujo & Álvarez-Cobelas (2016).

Museum collections

The Museum collections have been examined first by Araujo & Ramos (2001) at a wide scale, focusing mainly on national museums worldwide. Prié et al. (unpublished data, Online Resource 1) have focused on French regional collections only. Out of the 58 collections identified, 25 had at least one specimen of M. auricularia (Fig. 2A): Musée du Château in Annecy, Musée des Confluences in Lyon, Museum of Perpignan, Musée zoologique of Strasbourg, Muséum—Aquarium of Nancy, Museum of Auxerre, Muséum d’histoire naturelle in Bordeaux, Muséum d’histoire naturelle in Bourges, Muséum d’histoire naturelle in Grenoble, Museum d’Histoire Naturelle in Nantes, Muséum d’Histoire Naturelle in Toulouse, Museum d’Histoire Naturelle Victor Brun in Montauban, Museum d’Histoires Naturelles in Colmar, Muséum of Orléans, Muséum of Dijon, Muséum Lecoq in Clermont-Ferrand, Muséum national d’Histoire naturelle in Paris, Muséum national d’histoire naturelle in Lille, Paraclet centre of ONEMA in Boves, Pôle muséal of Troyes, Université of Bourgogne in Dijon, Université of Montpellier I, Université of Rennes I, Museum d’histoire naturelle in la Rochelle, Museum of Cherbourg-Octeville. Part of the data from Museum collections were fossil specimens. A total of 400 non-fossil specimens were found in Museum collections, including the 37 specimens already found by Araujo & Ramos (2001). Amongst them, 332 were localized at a river drainage scale. One-third of the specimens came from the Garonne watershed, 19% from the Saône River (half of them coming from a single batch collected by Coutagne in 1879) and 17% from the Ebro River (Fig. 2B). Other watersheds represented less than 30% of the Museum collections specimens. About 80% of the specimens dated were collected before the beginning of the twentieth century.
Fig. 2

A Location of main museum collection investigated (dot size according to number of M. auricularia specimens). B number of specimens held in Museum collections per main watersheds (the Saône River is actually a tributary of the Rhône, but all the specimens are located in the Saône and none elsewhere in the Rhône)

Field surveys and populations sizes

A total of 2,500 km of rivers has been surveyed for M. auricularia in France and Spain for the last 10 years (see bibliography and Online Resource 1 for details). These surveys covered most of the river stretches for which the literature or museum collections data were available. Eleven populations could be identified, eight in France and three in Spain, plus a single individual found recently in the Ebro River (pers. comm. from R. Álvarez-Halcón to R. Araujo) upstream Zaragoza (Fig. 3, Table 3). In Spain, the main population, with 5,000 live specimens, lives in the Canal Imperial in Aragón. Although there have been some recent mortalities, some young specimens probably less than 10 years old have been observed during the last years (pers. comm. from J. Guerrero to R. Araujo). A new population was recently discovered in the Quinto Ditch, with 25 live specimens, including some subadults (Nakamura, com. pers.). The populations on the Canal de Tauste still host several live specimens and juveniles. The population of the lower Ebro River is today practically testimonial (pers. com. of the Generalitat of Catalonia to R. Araujo). See Gómez & Araujo (2008), Araujo (2012) and Araujo & Álvarez-Cobelas (2016) for more information.
Fig. 3

Results of last ten years’ field surveys and known past and actual distribution of M. auricularia. Fossil data (black crosses), historical data (white dots), shells collected in the last 10 years (orange dots) and still living populations (red dots)

Table 3

Summary of literature data, museum collections (fossil data are not considered) and field surveys

Country

Coastal drainage

 

Literature

Museum collections (number of specimens)

Dedicated field surveys (references with an * refer to grey literature, summarized in Online Resource 1)

Recent surveys results

Estimated population size

France

Charente

Charente

X

9

Prié et al. (2007)*, Prie (2010), Prié & Mouton (2016)*

Live specimens and juveniles

100. 000

Garonne

Garonne (mainstream)

X

125

Prié et al. (2016)*

Ancien shells

 

Isle

X

0

Prié (2012)*

Recent shells

 

Dronne

X

0

Prié (2012)*; Prié (2013)*

Live specimens and juveniles

> 100

Save

 

0

Prié (2012)*

Few live specimens, declining population

< 30

Adour

Adour (mainstream)

X

9

Prié (2012)*

Live specimens and juveniles

> 300

Arros

 

6

Prié & Néri (2016)*

Live specimens and subadults

200

Luy

 

0

Prié (2012)*

Live specimens and subadults

150

Loire

Loire (mainstream)

X

1

 

Recent shells

 

Vienne

 

0

Cochet (2006)*, Philippe et al. (2009)*, Philippe et al. (2010*, 2011*, 2012*)

Live specimens and juveniles

 > 100

Indre

 

0

Dohogne (2008)*; Philippe et al. (2009)*

Recent shells

 

Creuse

 

0

Philippe et al. (2012)*; Philippe et al. (2013*, 2014*, 2015*, 2016*)

Live specimens and juveniles

> 150 

Cher

 

1

Prié et al. (2011)*, Prié et al. (2016)*

Nothing

 

Seine

Seine (mainstream)

X

12

Cucherat et al. (2011)*

Ancien shells

 

Oise

 

0

Prié et al. (2007)*

Recent shells

 

Aube

X

1

Cucherat et al. (2011)*

Ancien shells

 

Aisne

X

6

Philippe et al. (2009)*, Cucherat et al. (2011)*

Ancien shells

 

Escaut

Escaut

X

2

 

No dedicated survey

 

Somme

Somme

X

1

Cucherat & Prié (2011)*

Nothing

 

Rhône

Saône

X

65

Prié et al. (2016)*

Ancien shells

 

Italy

Po

Po

X

15

 

No dedicated survey

 

Spain

Ebro

Upper Ebro

X

 

Araujo et al. (2009b)*, Araujo & Álvarez-Cobelas (2016); pers. comm. from R. Álvarez-Halcón to R. Araujo

Live specimen (at Gallur)

1

Ribera alta

  

Nakamura & Guerrero (2008), Araujo & Álvarez-Cobelas (2016)

Used to be 38–40 live specimens, today likely extirpated

 

Canal Imperial de Aragon

X

55

Gómez & Araujo (2008), Araujo et al. (2009a, b), Araujo & Álvarez-Cobelas (2016); pers. comm. from J. Guerrero to R. Araujo

Live specimens and juveniles

4,000

Canal de Tauste

X

 

Araujo et al. (2009b)*; pers. comm. from J. Guerrero to R. Araujo

Live specimens and juveniles

200

Quinto ditch

  

Gómez & Araujo (2008), Nakamura et al. (2017)

Live specimens and subadults

25

Lower Ebro

X

 

Araujo et al. (2009b)*; Araujo & Álvarez-Cobelas (2016)

Used to be 70 live specimens, today extirpated

 

Tajo

Tajo

X

1

Villasante et al. (2016)

Nothing

 

Fossil data

U.K.

Thames

Thames

 

17

 

No dedicated survey

 

Germany

Rhine

Rhine

X

3

 

No dedicated survey

 

Netherland

Rhine

Rhine

X

  

No dedicated survey

 

In France, field surveys allowed finding ancient shells in the Seine, in the Vesle and in the Aisne Rivers; in the Saône River (Rhône watershed) near Pontailler-sur-Saône and in the Garonne River near Agen, findings which corroborate historical data. We believe the species was extirpated long time ago in those rivers. In the Oise River (Seine watershed), very recent shells have been found in 2007 and 2008, some of them still embedded in their natural position, suggesting that the species became extirpated very little time before the surveys took place.

The populations of the Creuse and Vienne Rivers (Loire watershed) are the most studied in France. They live in shallow and clear water, allowing regular surveys using viewing glasses or snorkelling. Although these populations are rather small (about 250 specimens altogether), over 40 juveniles were found in the Vienne and Creuse Rivers, which represent about 15% of the population.

Three sites with a few tens of live specimens were discovered in the Dronne River, including one juvenile of about ten cm. Additionally, some isolated individuals were also observed, suggesting the population is scarce but relatively widespread. In the Save River, only five live specimens were observed. Sampling conditions are difficult, with variable depth and current strength, and very low visibility. We can therefore suppose that our detection probability is low. But based on survey results, we estimate that the population should not exceed a few tens of living individuals. It is likely rapidly declining given the bad condition of the river and the large number of recent shells collected compared to the very few living specimens observed. The Adour drainage rivers were known to host M. auricularia from both the literature and Museum collections data. In the Adour mainstream, the population is now highly fragmented, with only three sites identified, where live specimens could be found. One of them, the most upstream, is now extirpated (Prié et al., 2010). The total population is estimated to be about 300 specimens in the total length of the Adour mainstream, but we still need a better estimation based on an appropriate sampling protocol. On the Luy tributary, a population of about 150 specimens is found in a very small stretch of river. Interestingly, although this River is very shallow (from 30 cm to 1.5 m), clear and easy to survey (hence detection probability is optimal), successive counts of 2010, 2011 and 2012 lead to, respectively, 110, 96 and 145 specimens. We suppose that a significant part of the population lives buried in the sediment, which biases the results of the counts. The Arros River had been overlooked by the literature review and field surveys up to 2016. Following the findings in Museum collections, dedicated field surveys were conducted in 2016, allowing the rediscovery of a living population. This population’s size was estimated to be about 200 individuals on the 54 km stretch of favourable habitat. The Charente River was known from the ancient literature to host an important population of M. auricularia (Bonnemère, 1901). Shell fragments and very few live specimens had been found by naturalists since 2003 (Nienhuis, 2003; P. Jourde pers. com.). Intensive field surveys performed in 2007, 2010 and 2016 led to the discovery of the largest population worldwide. Geographical statistics based on scuba-diving transects showed that the population was not aggregated. Hence, the total population size could be estimated by multiplying the average density by the total surface of suitable habitat in the stretch of river inhabited by M. auricularia. The average population size in the Charente River was estimated to be about 100,000 (range 80,000–120,000) individuals, between the towns of Cognac upstream to Port-d’Envaux downstream.

Genetic diversity

Margaritifera auricularia is genetically remarkably homogenous. The specimens from France and Spain all shared the same 16S and COI haplotypes, but two specimens from Spain: specimens vouchered with FW1238-14 and FW1238-12; for COI T → A in position 37, T → A in position 50 and G → C in position 73; and for 16S T → C in position 176. The French and Spanish specimens could not be compared for 28S as different gene fragments were amplified. However, within France, all specimens shared the same haplotype and within Spain, all specimens shared the same haplotype.

Discussion

Historical and actual data

The number of specimens found in the various regional museum collections was unexpected. Margaritifera auricularia is a large species that retained collector’s attention. Most data from museum collections corresponded to the literature data, excepted those from the Arros and Vezere Rivers in France. Surprisingly, most French specimens came from the Garonne and Saône Rivers, where the species is now believed to be extirpated or very rare. In contrast, very few specimens came from the Charente River, where the largest population is found nowadays, and where industrial fisheries were established to make nacre shirt buttons (Bonnemère, 1901). Similarly, museum collections host no specimen from the Vienne or Creuse Rivers, where healthy populations live in shallow and clear waters. In the Seine watershed, most shells came from upstream and the Aisne tributary, while the Oise tributary seems to have host the last population.

The historical review confirmed that M. auricularia was once present as far as the Thames in England and Netherlands and Germany where fossil specimens have been found and studied (Araujo & Ramos, 2001). On historical times, we found museum records (recent shells) from the Rhine in France or Germany (precise location being unknown), the Seine and the Rhône in France, the Pô in Italy and the Tajo in Spain, where the species is now believed to be extirpated (Araujo & Ramos, 2001). Today, Margaritifera auricularia is considered restricted to five watersheds: from north to south the Loire watershed (two close populations in the Vienne and Creuse Rivers), the Charente watershed, the Garonne watershed (two very isolated populations, in the Dronne and Save Rivers), the Adour watershed (at least three isolated populations, one in the Adour itself, one in the Luy and one in the Arros) and the Ebro River (two populations, three in channels and a small one remaining in the Ebro itself). As has been previously estimated (Prié et al., 2014), Margaritifera auricularia’s range contraction has probably reached about 90% in the last two centuries.

Surveying downstream ecosystems

Large rivers are amongst the most difficult ecosystems to sample. Deepness, turbidity and water current are challenging conditions. In addition, large rivers are subject to navigation, which makes scuba diving potentially hazardous. Nevertheless, scuba diving appears to be the most efficient way to produce data for species such as M. auricularia: despite malacological surveys undertaken with canoes and dredging, only a few shell fragments had been collected in the Charente River before scuba- diving sampling had been set up. Scuba divers met hundreds of shells and living specimens there. Similarly, scuba divers collected the few living specimens that are probably dead by now, in the main Ebro River in Spain (Araujo & Álvarez-Cobelas, 2016). In the Oise River, a few ancient shell fragments had been collected on the banks by amateur malacologists, but scuba diving allowed finding numerous shells in most of the river stretches investigated. In the Garonne River mainstream, a malacologist spent about 20 days wading and searching for shells on the gravelled banks. In 2 days, a team of three divers found four shell fragments.

While bivalve surveys have been conducted in the Saône River (ex. Mouthon & Daufresne, 2006), no shell fragments had ever been collected before 2016’s scuba-diving prospections. The advances in the distribution knowledge of M. auricularia in France and Spain are directly linked to new investigation methods and scuba diving is so far the most efficient mean of survey for this species.

Conservation and further perspectives

Main threats

While overfishing may have contributed to the species decline in the past (Bonnemère, 1901; Prié et al., 2011; Araujo & Álvarez-Cobelas, 2016), it is obviously river management and agriculture impacts that nowadays cause the most important threats to the Giant Freshwater Pearl Mussel. Both causes are linked together, at least in the southern part of the species distribution area: river management aims at providing freshwater for corn culture, especially in summer. Hence, numerous dams are built, even in small rivers, to maintain pools for pumping water in the dry season. These dams produce lotic and silty conditions unsuitable for the Giant Freshwater Pearl Mussel. Altogether, these small dams can affect about than 70% of a given rivers stretch. In the Dronne, Arros and Save Rivers in France for example, the Giant Pearl Freshwater Mussel populations survive in the form of dashed lines, only in riffles (shallow parts of streams where water flows brokenly) with gravel or stony bottoms, between long portions of lotic conditions. Moreover, these dams constitute obstacles for potential fish hosts. The presumed natural host fish of the Giant Freshwater Pearl Mussel, the European Sturgeon Acipenser sturio, has been extirpated from almost all European rivers mainly because of dams (Lepage & Rochard, 1995; Gesner et al., 2010). River management has been an important threat in Spain too. Water regulation and the replacement of natural bottoms with concrete have been responsible for a massive death of Naïads. Recently, there has been an unusual high mortality of adults in the Imperial Channel (pers. com. from the Diputación General de Aragón to R. Araujo), but the causes are unknown.

Despite being a probable cause of recruitment failure, moderate levels of pollution and eutrophication have not demonstrated to be a significant threat to adult specimens. Some populations survive in highly human-impacted waters. For example, one of the highest Giant Freshwater Pearl Mussel densities spot lies just downstream the Saintes sewage system in the Charente River. The same kind of conditions occurs at the Canal Imperial in Aragón with the water coming from the Ebro River, which is highly polluted. Overall, the species survives in rivers that are highly impacted by agriculture and domestic effluents. But we still don’t know how these eutrophic and polluted waters may impact juvenile survival (Augspurger et al., 2007; Strayer & Malcom, 2012; Archambault et al., 2014).

Invasive species probably add to the threats M. auricularia is facing. Widespread invasive species such as Corbicula fluminea probably affect the freshwater mussels of Europe like it has been demonstrated for other species in North America (e.g. Soussa et al., 2014). However, no clear impacts have been described for M. auricularia, and the healthiest populations survive in rivers largely colonized by Corbicula. The zebra mussel Dreissena polymorpha attaches to the valves of M. auricularia in the Ebro, probably affecting filtration efficiency. This phenomenon has not been observed in France, where the zebra mussel remains at low densities in the rivers of the Atlantic coast.

Habitat management

Contrarily to the Freshwater Pearl Mussel M. margaritifera, for which experiments of habitat managements have proved to be successful (Altmüller & Dettmer, 2006), the Giant Freshwater Pearl Mussel lives in downstream ecosystems. Attempts to implement broad scale watersheds management are therefore unrealistic. However, some realistic management objectives can be achieved to improve the habitat quality locally, in a short or middle term. The deconstruction of the numerous impoundments (many of them being disused) seems the most efficient way to restore suitable riverbed conditions for the Giant Freshwater Pearl Mussel. Although the negative impacts of pollution and eutrophication are not clearly known, they are for sure not needed for the species survival. Improving water quality through reasonable agricultural practices, with buffer strips or grass strips along waterways, should be a medium-term objective.

Farming projects

Breeding farms have been established for many endangered mussel species. In Europe, there is an abundant literature dealing with M. margaritifera breeding farms. Some trials are also ongoing for U. crassus and for various Unio species in Spain (Araujo et al., 2015). Regarding the Giant Freshwater Pearl Mussel, attempts of ex situ breeding have been performed in Spain (Nakamura et al., 2015), and a LIFE project is ongoing in France to artificially breed the species in controlled conditions. Juveniles have been successfully produced (Nakamura et al., 2015), but we still face obstacles in the rearing of these juveniles (although some juveniles are still alive, Nakamura com. pers.).

Genetic diversity

The very low genetic diversity for the mitochondrial genes studied was unexpected as (i) the Giant Freshwater Pearl Mussel populations are geographically isolated for a long time; and (ii) strong morphological differences are found between populations (Fig. 4). (i) The populations from France belong to the Atlantic drainage and the population from Spain to the Mediterranean drainage, two geographically isolated bioregions. Strong genetic divergences are observed for other freshwater mussel species from the Iberian Peninsula: U. delphinus from the “pictorum” lineage and U. tumidiformis from the “crassus” lineage were recently considered as distinct species based on molecular divergences (Reis & Araujo, 2009; Araujo et al., 2009b). But on the other hand, some species do not show significant genetic divergences (ex. U. mancus, Prié et al., 2012; Potomida littoralis, Araujo et al., 2016a, b; Froufe et al., 2016). (ii) The different populations known today have obvious morphological differences in shell size and shape (Fig. 4). The specimens from the Charente River population have a peculiarly small and conspicuously ear-like shell shape, contrasting to the Vienne and Dordogne Rivers’ populations, which are larger and more elongated; and to the Arros and Save Rivers populations, which are remarkable with their huge sizes. Some populations live in deep coastal rivers (ex. Ebro, Vienne and Charente populations) while others seem to be confined to shallow riffle sections of the upstream rivers (ex Save and Adour populations), but these ecological traits are not linked to shell morphological differences.
Fig. 4

Morphological variability of Margaritifera auricularia. A Vienne River; BC Charente River; D Dronne River; E Save River; F Luy River; G Arros River, H Ebro River

Margaritiferidae are known to have very low mitochondrial DNA evolution rates (Araujo et al., 2016a, b; Bolotov et al., 2016). Population genetics based on microsatellites allowed to differentiate evolutionary units within the related species Margaritifera margaritifera (Geist et al., 2010; Stoeckle et al., 2016) and M. marocana (Sousa et al., 2016). But first studies using microsatellites based on M. margaritifera primers have failed to reveal any population structure in France (Prié, unpublished data). If the ex situ breeding projects are successful, the population genetics question will become unavoidable.

The fish host issue

The known host fish of Margaritifera auricularia are sturgeon species Acipenser sturio, A. nacari and A. baeri, the River Blenny Salaria fluviatilis and the Eastern Mosquitofish Gambusia holbrooki (Araujo & Ramos, 1998b; Araujo et al., 2000, 2001; Altaba & Lopez, 2001; Lopez & Altaba, 2005; Lopez et al., 2007).

The only native Acipenser species in the area of occurrence of Margaritifera auricularia is the European sturgeon A. sturio. This species became extirpated from most European Rivers during the twentieth century. Nowadays, it is almost extinct, with the last documented case of natural reproduction dating back to 1994 in the Garonne River. The River Blenny is a Mediterranean species whose range does not overlap with the French populations of M. auricularia. The Eastern Mosquitofish, an introduced species, lives in shallow and standing to slow-flowing waters. It is not usually found in places favoured by Margaritifera auricularia. Reported success as host fish for M. auricularia glochidia was questionable. Experiments with other common fish species that occur within the distribution range of M. auricularia (Anguilla Anguilla, Barbus graellsii, Barbus haasi, Parachondrostoma toxostoma, Cobitis paludicola, Liza aurata, Mugil cephalus, Alburnus alburnus, Carassius auratus, Cyprinus carpio, Gobio gobio, Scardinus erythrophthalmus and Tinca tinca) failed to produce juveniles (Araujo et al., 2001; Lopez & Altaba, 2005).

The actual knowledge on the M. auricularia host fish cannot explain the recruitment observed recently in the Atlantic watersheds. We therefore suspect an overlooked host fish species. For example, the Alosa species, which are migratory fish and still breed in the watersheds where M. auricularia produces juveniles, are good candidates (Llorente et al., 2015). But there must be another fish host in order to explain recruitment in the Dronne and Charente Rivers, which are isolated from the sea by impoundments, or in very upstream populations such as those of the Arros or Aisne Rivers, where migratory fishes do not breed. The other hypothesis could be that reproduction occurs in France periodically, taking advantage of accidental releases of A. baeri, a common species on French fish farms (but not in Spain). We have recently succeeded in completing the full cycle on the three-spined stickleback Gasterosteus aculeatus in controlled conditions (Soler et al., in prep.). As this species is widespread within the range of M. auricularia and tolerant to brackish waters, it could also be a good candidate as a natural fish host. Finding the natural host fish species of M. auricularia in France is now vital for the survival and conservation of this freshwater bivalve.

Conclusion

Margaritifera auricularia has become very rare in the twentieth century, with an estimated range contraction of 90%. Only three populations were known worldwide before 2007. Intensive surveys in the last decade allowed for the rediscovery of nine more. Given the magnitude of the efforts allocated to surveying the species in its historical range, we now believe that there are very few chances to rediscover unnoticed populations (except maybe in north-east France).

Although some juveniles were found recently, they remain very scarce, and most extant populations seem to live on borrowed time. Within the time lapse of this study, some populations already became extirpated in the Ebro and Adour Rivers. The status of the species therefore remains worrying. Priority populations for conservation are the Charente River’s population, because it is by far the largest worldwide; the Vienne and Creuse population, because it has the highest level of natural recruitment; the Adour watershed populations, because they form an important and unique metapopulation; and the Ebro population because it is now the only remaining one in the Mediterranean drainage system. Conservation challenges for the next years are (i) appropriate management of the rivers which host the priority populations; (ii) the development of farming projects, in order to reinforce existing populations; (iii) research on fish hosts for better comprehension of threats to the species, ecological requirements, to understand what those habitat factors are which drive the species’ success with recruitment, population genetics to plan conservation efforts according to the genetic diversity of the remaining populations; and (iv) wide scale development of modern survey methods such as scuba diving and environmental DNA in order to discover the unnoticed populations which potentially remain.

Despite these efforts, we may fail to save the Giant Freshwater Pearl Mussel from extinction. However, current research helps to shed light on the obscure downstream river ecosystems’ ecological functions and the threats to it, as well as to develop exploration methods for this challenging environment.

Notes

Acknowledgements

This work was conducted within the scope of the LIFE project “Life13BIOFR001162 Conservation of the Giant Pearl Mussel in Europe”. We thank Dominique Tesseyre from the Adour-Garonne Water Agency; Julie Marcinkowsky and Gérard Tardivo from the DREAL Centre as well as the DREAL Picardie for providing financial support for large-scale surveys of M. auricularia in France; Elodie Hugues, Guillaume Métayer (Conseil Général de Charente Maritime), David Bécart (Voies Navigables de France) and Amandine Szurpicki (COSEA), Frédérique Moinot and Olivier Guerri (EPIDOR) for financing focus surveys in the Charente, Seine, Vienne and Garonne Rivers; and for Spain, the Government of Arag ón, FMC Foret S.A., Enagas, Gas Natural, INYPSA, Hidroeléctrica La Zaida, Edison Mission Energy and EID Consultores.

Supplementary material

10750_2017_3456_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 18 kb)

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2017

Authors and Affiliations

  • Vincent Prié
    • 1
    • 2
    Email author
  • Joaquin Soler
    • 3
  • Rafael Araujo
    • 4
  • Xavier Cucherat
    • 5
  • Laurent Philippe
    • 1
  • Nicolas Patry
    • 1
  • Benjamin Adam
    • 1
  • Nicolas Legrand
    • 1
  • Philippe Jugé
    • 6
  • Nina Richard
    • 6
  • Karl M. Wantzen
    • 3
    • 7
  1. 1.Biotope, Service International, Diversification, InnovationMèzeFrance
  2. 2.Institut de Systématique, Évolution, Biodiversité ISYEB – UMR 7205 – CNRS, MNHN, UPMC, EPHE, Muséum national d’Histoire naturelle, Sorbonne UniversitésParisFrance
  3. 3.Université François Rabelais, UMR 7324 – CITERESTours Cedex 03France
  4. 4.Museo Nacional de Ciencias Naturales - C.S.I.CMadridSpain
  5. 5.GondecourtFrance
  6. 6.Université François-Rabelais, CETU Elmis IngénieriesChinonFrance
  7. 7.UNESCO River Culture – Fleuves et Patrimoines ChairUniversité François Rabelais, UMR 7324 – CITERESTours Cedex 03France

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