# Determining the natal origin of the reintroduced allis shad (Alosa alosa) in the Rhine River using otolith microchemistry

Elodie Boussinet, Françoise Daverat, Gilles Bareille, Andreas Scharbert, Stefan Stoll

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Elodie Boussinet, Françoise Daverat, Gilles Bareille, Andreas Scharbert, Stefan Stoll. Determining the natal origin of the reintroduced allis shad (Alosa alosa) in the Rhine River using otolith microchemistry. Environmental Biology of Fishes, 2025, 108, pp.1307-1323. 10.1007/s10641-025-01724-5. hal-05190464

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# Determining the natal origin of the reintroduced allis shad (*Alosa alosa*) in the Rhine River using otolith microchemistry

Elodie Boussinet* · Françoise Daverat · Gilles Bareille · Andreas Scharbert · Stefan Stoll

Received: 4 October 2024 / Accepted: 23 June 2025 / Published online: 17 July 2025
© The Author(s) 2025

**Abstract** Locally extinct since the 1960s, the anadromous allis shad (*Alosa alosa*) was reintroduced into the Rhine system through a restocking programme beginning in 2007. The population is now showing positive signs of recovery, with natural reproduction occurring for several years and a decreasing proportion of stocked fish. These findings suggest the future establishment of a self-sustaining population. Our study aimed to identify the spawning sites in the Rhine system. We conducted a tank experiment and kept shad larvae in water from four sub-catchments of the Rhine system. We analysed trace substance concentrations in water samples and the microchemical composition of otoliths from reared larvae. Using a random forest model, we were able to correctly

attribute the larvae to the sub-catchment where they were raised based on elemental/ratio (Sr/Ca) and strontium isotopes ($^{87}$Sr/$^{86}$Sr). From the 66 allis shad caught in the Rhine system between 2017 and 2020, seven individuals (11%) were identified as being stocked. Of the 59 remaining individuals that came from natural reproduction, 37 were attributed to the Rhine, 7 to the Neckar and 4 to the Lippe sub-catchments with high certainty. We also observed allis shads dispersed in adjacent catchment areas and a homing behaviour. A total of 27% of the adults (n=9) and 8% of the juveniles (n=2) were assigned to any of the four sub-catchments included in our model, suggesting the need to expand the model and include additional sub-catchments to cover all spawning sites in the Rhine system and adjacent catchments.

**Supplementary Information** The online version contains supplementary material available at https://doi.org/10.1007/s10641-025-01724-5.

E. Boussinet (✉) · S. Stoll
Environmental Campus Birkenfeld, University of Applied Sciences Trier, Standort Birkenfeld, 55768 Hoppstädten-Weiersbach Campusallee, Germany
e-mail: e.boussinet@umwelt-campus.de

E. Boussinet · S. Stoll
Faculty of Biology, University of Duisburg-Essen, Universitätsstraße 5, 45141 Essen, Germany

F. Daverat
UMR 1224 ECOBIOP, INRAe, Université de Pau Et Des Pays de L'Adour, Saint-Pée-Sur, Nivelle, France
e-mail: francoise.daverat@inrae.fr

G. Bareille
E2S UPPA, CNRS, IPREM, MIRA, Université de Pau et des Pays de l'Adour, Pau, France
e-mail: gilles.bareille@univ-pau.fr

A. Scharbert
Rheinischer Fischereiverband von 1880 e.V., Wahnbachtalstraße 13a, 53721 Siegburg, Germany
**Keywords** Attribution model · Origin assignment · Elemental ratios · Migratory species · Reintroduction

Introduction

Fish populations in Europe and worldwide have experienced significant declines, with some species becoming extinct over the last century (Worm et al. 2006; Hooper et al. 2012; Ceballos et al. 2015; Tickner et al. 2020). Migratory fish species are particularly affected (Deinet et al. 2020). The losses include river fragmentation (Fuller et al. 2015; Seliger and Zeiringer 2018; van Puijenbroek et al. 2019), overexploitation (Costa-Dias et al. 2009), environmental pollution (Sindermann 1994; Lenhardt et al. 2006) and global warming (Lassalle et al. 2008). Stimulated by nature conservation laws and policies such as the European Water Framework Directive and other similar directives worldwide (McDonald et al. 2019), there has been some success in improving the structure and water chemistry of water bodies. In waters where the most important stressors have been successfully removed, it is now possible to reintroduce fish species that have disappeared with the aim of establishing new self-sufficient populations in their native habitats (Seddon et al. 2007; Cochran-Biederman et al. 2015; Jachowski et al. 2016). In European watersheds, many programmes of reintroduction are carried out for emblematic anadromous species such as Atlantic salmon [Salmo salar (Linnaeus, 1758)], European sturgeon [Acipenser sturio (Linnaeus, 1758)] and both twaite [Alosa fallax (Lacepède, 1803)] and allis [Alosa alosa (Linnaeus, 1758)] shads (Monnerjahn 2011; Hundt et al. 2015; van Puijenbroek et al. 2019). The success of reintroduction programmes rely on an understanding of species ecology, monitoring and management within the repopulated habitat (Sarrazin and Barbault 1996). Good knowledge of the reproduction success of newly established populations is crucial. This includes knowledge of the spawning grounds to ensure long-term ecological quality through appropriate management or protection strategies.

In this study, we examined the case of allis shad in the Rhine River. Allis shad populations have experienced dramatic declines and severe contraction of distribution range during the last century (Lassalle et al. 2008; Bruslé and Quignard 2013). Once the

largest population in Western and Central Europe, the Rhine population was decimated by a combination of factors including overfishing, water pollution, barrier construction and destruction of spawning habitats (Limburg and Waldman 2009; Rougier et al. 2012). Since the 1960s, the allis shad population has become nearly extinct in the Rhine, and no allis shad has been observed. Following the construction of sewage treatment plants, a reduction in fishing pressure and the upgrading of many habitat structures in the Rhine, a reintroduction project for allis shad was launched in 2007. This project was supported by the European LIFE projects (“The re-introduction of the allis shad (Alosa alosa) in the Rhine system” (LIFE06 NAT/D/000005), “Conservation and restoration of the allis shad in the Gironde and Rhine watersheds” (LIFE09 NAT/DE/000008)) and other funding sources from the Rhine riparian states of Germany, France, the Netherlands and Switzerland. These projects provided the first evidence of natural reproduction in 2013, as claimed by Hundt et al. (2015), who observed unmarked young of the year in the Rhine.

The allis shad is predominantly semelparous and has an anadromous life cycle (Baglinière et al. 2003; McDowall 2008). Adults spawn during the night in fast-flowing sections of rivers during the period of April to July, while juveniles migrate downstream to the sea where they grow for 5 to 7 years until maturation before returning into the freshwater to reproduce (Baglinière et al. 2003; Sabatié et al. 2009). Yet the actual spawning areas of allis shads in the Rhine system are not well known. Each year, returnees are observed, but it is uncertain whether they are a result of natural reproduction, stocking programmes or immigration from adjacent catchments. It must be considered that monitoring migratory fish populations in large rivers, such as the Rhine, is challenging for several reasons. The Rhine is a biological waterway under strict regulation, which means that not only are observation methods costly, but there is also a lack of meaningful representation of the entire shad population. To evaluate, manage and conserve reintroduced population, it is crucial to understand their breeding behaviours and to identify potential spawning grounds.

Homing and straying behaviours are commonly observed among diadromous fish species, such as the Atlantic salmon (Salmo salar) and American shad [Alosa sapidissima (Wilson, 1811)]. Although these
species can still exhibit straying behaviour, they are more well known for their precise homing abilities (Melvin et al. 1986; Hendricks et al. 2002; Keefer and Caudill 2014; Salmenkova 2017; Armstrong et al. 2022). In comparison, both allis and twaite shads have shown lower fidelity to the spawning site, with fidelity expressed at the watershed scale rather than at a finer scale (Martin et al. 2015; Randon et al. 2018). This lower homing precision makes it more difficult to identify and manage historic and new spawning grounds for these species. In the context of reintroduction efforts, both behaviours are of crucial importance. Homing can enhance and reinforce the reintroduced population, while straying individuals may colonise new, unoccupied systems, thereby facilitating broader population recovery (Hasselman et al. 2012; Nachón et al. 2020).

Microchemical analyses of fish otoliths have proven an effective retrospective approach for determining the natal origin of fish and for identifying spawning grounds (Kennedy et al. 2002; Heidemann et al. 2012). Certain chemical trace substances such as strontium (Sr), barium (Ba) and isotopes of strontium ($^{87}$Sr/$^{86}$Sr) are incorporated in growing calcified structures, e.g. otoliths, and are related to their concentration in the surrounding water (Brown and Severin 2009; Walther and Limburg 2012; Pouilly et al. 2014; Hüssy et al. 2021). These methods have been successfully used for allis shad (Tomás et al. 2005; Martin et al. 2015; Randon et al. 2018; Nachón et al. 2020). Natal origins can be distinguished at the catchment or sub-catchment level, provided the trace element concentrations and Sr isotope signatures in the waters differ (Tomás, et al. 2005; Barnett-Johnson et al. 2010). These relate to the geological conditions in the respective catchments (Shiao et al. 2006; Zitek et al. 2010; Walther et al. 2011). To carry out such studies, databases with trace substance concentrations of Sr, Ba, calcium (Ca) and isotopes of Sr in water samples from all areas of the catchment or possibly several catchments are required to correlate these with the respective otolith signatures. Unfortunately, such data are rare or incomplete for the Rhine system.

The aim of the present work was to evaluate the natural reproduction activity of shad in the context of an ongoing shad reintroduction project by assessing the natal origins of adult and juvenile shads caught in the Rhine catchment. A two-step approach was used. First, based on a distributed tank experiment at

four sites in the Rhine catchment, a database on the microchemical otolith signatures of allis shad associated with trace element concentrations in the water was established. Second, a random forest model was trained to attribute the microchemical otolith composition of fish in one of the four sub-catchments of the Rhine system. Finally, this model was used to determine allis shad caught as juveniles or adults in the Rhine system and to attribute their natal origins to these four sub-catchments. In this way, important sub-catchments in the Rhine system for the reproduction of the recently reintroduced allis shad population were identified to narrow down the search for productive spawning sites.

## Materials and methods

### Rhine catchment

The Rhine catchment, located in Central Europe, has a total surface area of 185,000 km² with a total length of 1230 km and crosses six countries (Görgen et al. 2010). The Upper Rhine Graben section, where the Neckar River is located, is characterised by carbonate unconsolidated quaternary sands, gravel and crystalline as well as sedimentary rocks (Preusser 2008; Frings et al. 2014; van der Perk and Vilches 2020). The Middle Rhine, where the Sieg River is located, is primarily composed of shale, schist and sandstone sediments, while the Lower Rhine Embayment, which includes the Lippe River, is mostly characterised by silicate rocks, including immature sandstones (Buhl et al. 1991; Preusser 2008).

### Stocking programme in the Rhine system

A cooperation programme for the restocking of allis shad in the Rhine system has been conducted since 2007 (Jatteau et al. 2008). To date, more than 16 million larvae have been released in different German river sites in the Rhine catchment (e.g. in the Sieg, the Mid-Rhine and the Neckar). Released larvae are the progeny of the native population from the Gironde system (France). Adults are caught during their upstream migration at fish passes located at dams in the Garonne and Dordogne rivers and then transported to the Bruch Hatchery (France) for breeding. After hatching, larvae are fed with natural prey
[Artemia nauplii (Linnaeus, 1758)], followed by a mass bath with oxytetracycline (OTC: C₂₂H₂₄N₂O₉, 300 ppm/4 h) and transported at a maximum age of 15 days to the Rhine system (Jatteau 2010). We can identify recaptured fish from the stocking programme by reading the OTC mark on the otolith. First-generation larvae and returning adults caught in the Rhine are therefore easily recognisable; however, this will not be the case for the next generations. It is therefore important to be able to first, distinguish between the individuals from the stocking programme, i.e. positive OTC mark, and the individuals from natural reproduction, i.e. negative OTC mark, and second, identify the natal origin of each individual that is not from the stocking programme but potentially from its progeny.

## Setup and implementation of the tank experiment

The tank experiments were conducted at four sites along the Rhine catchment. One tank was placed in the middle section of the Rhine, near Coblence, and three others were placed on the Rhine tributaries, such as the Lippe River near the city of Wesel, the Sieg River near Siegburg and the Neckar River near Ladenburg (Fig. 1). An open water circulation tank with a capacity of 800 L (Fig. 2) was placed near the riverbank at each site. The tanks were supplied with 3800 L/h of water from the respective rivers. The water injection into the tanks recreated a circular flow pattern, which is necessary for rearing shad larvae (Clave 2011). The water was returned to the river through an outlet covered with a 0.5-mm mesh, which

<figure><img src="image_2.png" /><figcaption>**Fig. 1** Overview of the Rhine catchment. Blue squares with a fish symbol indicate the four locations of the tank experiment: the Middle Rhine, the Lippe, the Neckar and the Sieg River. Orange stars represent juveniles caught in the Rhine system between 2017 and 2020. Yellow triangles represent adults</figcaption></figure>

caught in the Rhine system between 2018 and 2020. Red bars indicate the positions of dams with fish passes where allis shad were captured (Gambsheim and Ladenburg) or video-counted (Iffenzheim) during the upstream migration

<figure><img src="image_3.png" /></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Setup of the tanks at the four experimental sites: Middle Rhine, Lippe, Neckar and Sieg rivers. An online monitoring system (Power guard®) was installed to send an alarm message in the event of a water pump or electricity failure</figcaption></figure>

was fine enough to prevent larvae from escaping but large enough to prevent the tank from overflowing due to clogging by suspended matter.

A total of 2000 larvae from the French population were obtained through the stocking programme and released equitably into each tank on July 2, 2020. The larvae fed on natural prey provided by the water entering the tanks and to ease their transition from the hatchery where they received supplementary feeding (*Artemia nauplii*) for the first 20 days. A total of 10 larvae was collected during this period each week at each site. Following a period of stable acclimation and low mortality rates, the surviving larvae were monitored, sampled once a week (minimum n=3 per sampling) until the end of the experiment and stored in pure ethanol (99.9%) for subsequent microchemical analyses of the otoliths. The total length and weight of each larva were recorded. The experiment was initially designed to last for 12 weeks, and it was reached at the Neckar site. The experiments had to be stopped before this period at the Sieg site due to the disappearance of the fish from the tank after 2 weeks without any explanation. At the Lippe site, the experiment was stopped early after 9 weeks

due to high mortality. The experiment on the Middle Rhine stopped after 10 weeks because of a failure of the water pump.

## Sampling of allis shad from the Rhine

A total of 40 juvenile allis shad were collected during the downstream migration season, from August to early November between 2017 and 2020. All individuals were caught using professional stow nets in three sampling sections of the Rhine river in 2017, 2019 and 2020 and one section of the Waal river in 2019 (Fig. 1; Table S1). The beam length was 10 m with a braided and knotted mesh. The stretched mesh size decreased every 3 m from 20 cm until 12 mm at the end of the net (Collas et al. 2021).

A total of 37 adult allis shad were collected during the spawning season from April to June at the fish pass traps at Gambsheim (Rhine River) in 2018, 2019 and 2020 and at Ladenburg fish pass (Neckar River) in 2019 and 2020 (Fig. 1; Table S1). In August 2018, two individuals were collected in the Nidda River, a tributary of the Main River, by an angler. An adult allis shad carcass was found on one of the Rhine
banks at Coblence in June 2019, while one individ-
ual was caught in the Lower Rhine near confluence
with the Lippe River during its upstream migration
by a fisherman in May 2019. Another shad carcass
was collected in the Scheldt River (Belgium) by the
Natuur-en Bosonderzoek Institute in April 2020,
while adults were captured during their upstream
migration at the entrance of the Rhine system (Har-
ingvliet dam, North Sea) and in the Rhine at Illinger
Altrhein between June and August 2020. The age
of each adult was estimated using scales and otolith
samples, following Baglinière et al. (2003).

Otolith preparation and analysis

To establish the reference database, a sub-sample of
the maximum 10 fish in each tank experiment was
prepared, starting with the oldest fish, while juve-
nile and adult otoliths collected over the years were
all selected (Table S1). From the 77 allis shad sam-
ples collected in the wild, 11 individuals were not
analysed due to individual loss, damaged otoliths or
the presence of vaterite in the otoliths (Tzeng et al.
2007), leading to a total of 66 individuals included
in this study (Table S1). All sagittal otoliths were
extracted, cleaned with ultra-pure water and stored
in a dark and dry environment for 48 h. Each otolith
was embedded in epoxy resin (Araldite 2020, Escil),
labelled and stored in a black Eppendorf tube to pro-
tect the potential fluorescent OTC mark from natural
UV light. To establish the specific signature of the
experiment sites, one sagittal otolith per fish was pre-
pared in a transversal plane section by grinding down
one side to the level of the primordium. Sr/Ca and
Ba/Ca were measured using a high-resolution induc-
tively coupled plasma quadrupole mass spectrometer
(HR-ICP-MS) coupled to a UV high-repetition-rate
femtosecond laser ablation (fs-LA) system (see more
analytic details in Martin et al. (2015) and Nachón
et al. (2020)). A linear raster ablation scan (30 µm)
was performed from approximately 100 µm before
the nucleus to the edge of the otoliths along the long-
est dorso-ventral axis. The mean Sr/Ca and Ba/Ca
values were calculated on a 50- to 60-µm basis for
each fish where no variation was recorded and after
approximately 100-µm post-primordium to exclude
any mixing with the hatchery signature as they were
put in the tank at an age of 15 days. The ⁸⁷Sr/⁸⁶Sr
ratio signature in the otoliths directly reflects the

ambient water composition with a minimal fractiona-
tion (D⁸⁷Sr/⁸⁶Sr=1 (Kennedy et al. 2000; Walther
and Thorrold 2008; Martin et al. 2013; Pouilly et al.
2014)). In this study, ⁸⁷Sr/⁸⁶Sr ratios in otoliths were
not measured directly but inferred from the water
chemistry at each site, assuming that isotopic incor-
poration is primarily determined by the environment.
However, individual differences such as size, condi-
tions or diet may introduce some variation in element
uptake.

To determine the natal origin of the fish caught
in the Rhine catchment, one sagittal otolith of each
fish was prepared as described in the method by
Martin et al. (2015). Two symmetric C-shaped abla-
tion trajectories (60 µm width), one to measure the
elemental ratios and the other to measure the Sr iso-
tope, were performed at 40 µm of the primordium to
avoid maternal influence on the natal origin signature
(Kalish 1990; Rieman et al. 1994). The Sr/Ca and Ba/
Ca ratios were measured using HR-ICP-MS while
⁸⁷Sr/⁸⁶Sr was analysed with a Nu-Plasma multicol-
lector ICP-MS, both coupled to an fs-LA ablation
system.

Water chemistry analyses

Water samples from the tanks and rivers were col-
lected once a week during the experimental period
following the protocol of Martin et al. (2013). For
each sample, 125 mL of water was filtered through a
Whatman PVDF filter with a 0.45-µm pore size and
a 25-mm diameter in a sterile plastic bottle. To sta-
bilise the sample before analysis, 2-mL concentrated
ultrapure nitric acid (HNO₃; M=63.01 g/mol) was
added to the bottle. The samples were transported
to the laboratory, in a cooling box and stored at 4 °C
until further analysis. In total, 96 samples were taken,
including 48 from the tank and 48 from the river. In
addition, as a control, one water sample was taken
from the transportation bag of the fish, i.e. water from
the fish hatchery in Bruch, France.

The elemental composition of Ca, Sr and Ba in
the otoliths was analysed using solution-based induc-
tively coupled plasma optical emission spectrometry
(ICP-OES, SPECTRO ARCOS II). To correct for the
instrumental drift, indium was used as an internal
standard. The general performance of the procedure
was checked using the certified reference freshwater
SLRS-6 (NRCC, Canada), with analytical accuracy
and precision ranged from 98 to 103% and 2 to 3%, respectively. Water $^{87}$Sr/$^{86}$Sr analysis was performed for a sample selection (one per month) using the standard-sample bracketing method on a Nu-Plasma MC-ICP-MS after Sr purification using Eichrom Sr-Spec resin following the protocol described in Martin et al. (2013). The $^{87}$Rb isobaric interference on $^{87}$Sr was deduced from the $^{85}$Rb signal (Barnett-Johnson et al. 2010), and the instrumental mass bias was corrected using the $^{86}$Sr/$^{88}$Sr invariant ratio and an exponential law (Walther and Thorrold 2008). The accuracy and precision of the measurement were monitored using the standard SRM 987 (National Institute of Standards and Technology, Gaithersburg, USA).

## Data analyses

All statistical analyses were performed using the R statistical software version 4.2.2. (R Core Team 2017).

## Otolith microchemistry and water chemistry analyses

The elemental and isotope ratios from water samples were statistically compared between the tank and the river at the specific site using a non-parametric Kruskal–Wallis test, as the data did not follow a normal distribution (non-normal distribution data; Shapiro–Wilk test; *P* < 0.05). As no differences were observed between the tank and river at the same site, both were pooled. To test for differences between the four sites for the elemental ratios in the water and otolith, a non-parametric Kruskal–Wallis test (non-normal distribution data; Shapiro–Wilk test: *P* < 0.05) was performed, followed by a post hoc pairwise Wilcoxon test with Benjamini and Hochberg adjustment (Benjamini and Hochberg 1995).

## Differentiation between fish from stocking programme and natural reproduction

The otoliths of 30 juvenile and 36 adult shad were analysed under a fluorescence microscope before microchemical analysis to determine the presence or absence of the OTC mark. This mark, characterised by a circular green ring around the nucleus, was used to differentiate between fish from the stocking programme and those from natural reproduction. Fish

with a mark were classified as stocked fish, i.e. from the Gironde system, and were therefore not used for attribution. A few fish were observed with a doubtful mark, i.e. autofluorescence of the OTC, and were nevertheless included in the attribution analyses to avoid misattribution.

## Attribution model to identify the natal origin of allis shad

Linear discriminant analysis (LDA) or quadratic discriminant analysis (QDA) is often used in the classification of otolith microchemistry (Mercier et al. 2011) with a classification accuracy of 68% to 100% (Pangle et al. 2010; Tripp et al. 2020) and generally shows high attribution accuracy to the natal catchments or rivers. The use of complex models to analyse natal origin, such as a Bayesian mixing model, coupled with otoliths and water sample microchemistry (Sr/Ca, Ba/Ca and $^{87}$Sr/$^{86}$Sr) reference values, has been used and has already shown high classification accuracy (Nachón et al. 2020). Other studies have shown that the random forest (RF) algorithm, built on a network of decision trees and used to predict a quantity or probability, may offer a more powerful alternative for data that does not follow normality nor homoscedasticity, offering greater classification accuracy, stability to small perturbations in the data, and ease of interpretation, which applies to our restrictive database (Cutler et al. 2007; Carlisle et al. 2009).

The choice of Sr/Ca and $^{87}$Sr/$^{86}$Sr to be used in the RF was based on their clear relationship between water samples and otoliths in the tank experiment, i.e. in the reference database. The Ba/Ca ratio showed no clear relationship between water samples and otoliths in our study. Additionally, a matrix of confusion was performed based on the *confusionMatrix* function (randomForest R 4.2.2. package), which shows that using the Ba/Ca ratio, there was a 0.25 probability that the river Lippe was attributed to the river Neckar and vice versa, while using only Sr/Ca and $^{87}$Sr/$^{86}$Sr, the confusion matrix showed no mismatch between the rivers (Table S2). Therefore, Ba/Ca was not used in our final model. From the reference database, each individual was attributed with a high confidence, i.e. *P* > 0.70, and allowed us to establish specific clusters of attribution from the four rivers tested as well as perform an adequate discriminant analysis. The degree of
certainty for the attribution was set according to the lower percentage of misattribution, i.e. incorrect attribution of individuals to the reared river, made by the RF model (Table S3). To increase the model testing precision, random data partitioning (75% of the data as training and 25% of the data as test) on individuals reared at the four sites was performed and run 50 times. The runs were stopped at run 50, because the fish reared in the model were input into the test model at least one time each. In total, 100 fish from the Sieg, 50 fish from the Lippe, 150 fish from the Rhine and 150 fish from the Neckar were randomly tested.

The remaining 59 individuals of unknown origin without an OTC mark, i.e. 33 adults and 26 juveniles, were tested using the previously established RF model. All individuals were reattributed either in the corresponding cluster established (high certainty of reattribution *P* > 0.70), while individuals attributed to the four sub-catchments with lower certainty than *P* < 0.70 were assumed to originate from another

sub-catchment of the Rhine or adjacent sub-catchment for which we did not train the RF model.

## Results

### Chemical differentiation of the four rivers

No significant differences were observed between the corresponding water samples from the tanks and the river for Sr/Ca, Ba/Ca and ⁸⁶Sr/⁸⁸Sr ratios (Kruskal–Wallis rank sum test: X²=0.30, df=1, *P*>0.05; Fig. 3A–C). The trace element concentration and Sr isotopic ratio in the tanks were therefore representative of the rivers in which the tanks were installed. The Sr/Ca and Ba/Ca ratios differed significantly between the four rivers tested (Sr/Ca: X²=70.70, df=3, *P*<0.05; Ba/Ca: X²=75.95, df=3, *P*<0.05; Fig. 3A, B). These ratios were lowest in the Sieg, followed by increasing ratios in the Lippe, Rhine and Neckar (pairwise Wilcoxon tests,

<figure><img src="image_5.png" /><figcaption>**Fig. 3** Representation of Sr/Ca, Ba/Ca and ⁸⁷Sr/⁸⁶Sr ratios in water (A, B, C) and juvenile otoliths (D, E) at four sites in the Rhine system. Theoretical Sr isotope in the otolith (F) based on the pooled water Sr isotope from the tank and river and literature. The literature specifies that the Sr isotope in the water</figcaption></figure>

has a low partitioning ratio in the otolith, suggesting that the Sr isotope values might be like those in the water (C). The boxes represent medians, first and third quartiles. Values greater than 1.5 times the interquartile range from the median are represented as outliers by black dots
all *P*<0.05). Additionally, the ⁸⁶Sr/⁸⁸Sr ratios differed significantly between the four rivers tested (X²=24.62, df=3, *P*<0.05; Fig. 3C). The highest ⁸⁶Sr/⁸⁸Sr ratios were found in the Sieg, while the values in the Rhine, Neckar and Lippe, listed in decreasing order, were closer to each other, but still significantly different (pairwise Wilcoxon tests, all *P*<0.05).

The Sr/Ca ratios measured in the otoliths were more variable than those measured in the water samples, but still significant differences between rivers were observed (X²=21.66, df=3, *P*<0.05; Fig. 3D). Pairwise Wilcoxon tests revealed significant differences in Sr/Ca ratios between the Sieg, Rhine and Neckar samples, increasing in order (all *P*<0.05). The Sr/Ca ratios in the otoliths from the Lippe were statistically indistinguishable from those of the Rhine and Neckar samples (pairwise Wilcoxon tests, *P*=1 and 0.06, respectively). The Ba/Ca ratios measured in the otoliths differed significantly between rivers (X²=20.55, df=3, *P*<0.05; Fig. 3E). As in the water samples, the Ba/Ca ratios were lowest in the Sieg. For the Rhine and Neckar samples, a reverse order was found in the otolith compared with the water samples (pairwise Wilcoxon tests, all *P*<0.05). The Ba/Ca ratios in the otoliths from the Lippe were statistically indifferent from Rhine and Neckar samples (pairwise Wilcoxon tests, *P*=0.68 and 0.10, respectively). Since ⁸⁷Sr/⁸⁶Sr is not fractioned between water and the otoliths, we expect the ⁸⁷Sr/⁸⁶Sr otoliths to be the same as the water values (Fig. 3F). However, caution is advised, as isotope ratio analysis in otoliths is often less precise than in water, and the differences observed between the waters of the Rhine, Lippe and Neckar Rivers may be less significant in otoliths.

## Identification of allis shad from natural reproduction

The age of the adults ranged from 4 to 6 years. The adults were therefore considered to belong to the juvenile cohorts of 2013, 2014, 2015 and 2016. The OTC mark was observed on one otolith from an adult caught in 2018 at Gambsheim and on otoliths from another adult caught in 2019 at Gambsheim and Ladenburg, respectively. Hence, approx. 8% of the adult population that returned for spawning between 2018 and 2020 (3 out of 36 adults that were analysed in total) can be expected to come from the stocking programme. The OTC mark was observed on the otolith

of one juvenile caught in 2019 in the River Waal (Netherlands) and three juveniles caught in the Rhine at Kalkar Grieth in the same year. Juveniles from the stocking programme can therefore be estimated to represent 13% (4 out of 30 individuals) of the total number of juveniles analysed migrating downstream the Rhine system.

## Natal origin using the Random forest model

The RF-based attribution model correctly attributed 100% of allis shad with a known origin from the tank experiment with high certainty to the four sub-catchments covered in the model. Applying the trained attribution model on all 59 caught individuals from natural reproduction based on their otolith Sr/Ca and ⁸⁷Sr/⁸⁶Sr ratios, 81% (*n*=48) were attributed with a high degree of certainty to one of the four sub-catchments tested, while 19% of individuals (*n*=11) were attributed with only a low certainty to any of the four sub-catchments. None of the individuals, neither juvenile nor adult, was attributed with high certainty to the Sieg.

From the 26 juveniles among these 59 allis shad, 92% (*n*=24) were attributed with high certainty, and only 8% (*n*=2) were attributed with low certainty to any of the four sub-catchments (Fig. 4A). Among the juveniles caught at Kalkar Grieth (*n*=21), 86% (*n*=18) were attributed over 0.70 certainty to the Rhine and 10% (*n*=2) to the Lippe. One individual was not attributed to any sub-catchments tested. All juveniles caught at Rees (*n*=3) were reattributed with a high degree of certainty to the Rhine. Finally, one of the individuals caught at Tiel (*n*=2) was attributed with a high degree of certainty to the Rhine and one to any of the four sub-catchments.

Among the 33 adults from natural reproduction, 73% (*n*=24) were attributed with high certainty (*P*>0.7) to one of the rivers tested in the Rhine system (Fig. 4B). Consequently, the origin was unknown for 27% (*n*=9) of the individuals. Most of the individuals were caught at Gambsheim (Table S1). At this location, 39% (*n*=9) of the individuals were reassigned with a high degree of certainty to the Rhine, followed by the Neckar (26%; *n*=6) and the Lippe (4%; *n*=1). The remaining seven individuals (30%) could not be attributed to any of the four sub-catchments. The individuals caught in Ladenburg made the second most important contribution to the
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>
► **Fig. 4** Attribution of natal origin of juvenile (A) and adult (B) allis shad caught in the Rhine system between 2017 and 2020. Pie charts indicate the proportion of individuals from a catch site that were assigned to each of the four sub-catchments in the RF model, i.e. Rhine, Lippe, Neckar and Sieg. The size of the pie charts represents the number of individuals per catch location (small: less than 20 individuals; big: more than 20 individuals)

study. Half of the individuals with a high certainty of attribution (n=2) were each attributed to the Rhine and Neckar rivers. From the two individuals caught in the Nidda River, one was attributed to the Rhine River, while the other could not be attributed with a high certainty to any of the four sub-catchments. The two individuals caught at the Haringvliet Dam (Netherlands), one individual from the Lower Rhine and the Illiger Altrhein site (near Iffenzheim Dam) were all attributed with high certainty to the Rhine. The one adult individual found at Avelgem (Belgium) was highly attributed to the Lippe. The individual found at the middle Rhine near Coblence could not be attributed to any of the four sub-catchments.

## Discussion

### Towards a population recovery

After more than 10 years of reintroduction program and more than 16 million larvae released into the Rhine system, the allis shad population has shown good signs of recovery. Reintroduction studies have often reported low to moderate survival rates of released juvenile fish, particularly due to environmental conditions (Hause et al. 2022), delayed adaptation to natural food (Brown and Day 2002), habitat quality (Anderson et al. 2014) and predation pressures (Donnelly and Whoriskey 1991; Beckmann et al. 2006). Nevertheless, the considerable recovery of allis shad suggests that survival rates of stocked larvae may be favourable in the Rhine system. Our results corroborate the findings of Hundt et al. (2015) who showed the first evidence of natural reproduction in the Rhine system after half a century of no records. This positive outcome contrasts with other reintroduced fish species in Europe, such as the Atlantic salmon, which often encounter significant challenges despite the observation of wild returnees (Aprahamian et al. 2003; Lennox et al. 2021; van Rijssel et al. 2024).

Natural reproduction is an essential criterion for considering a population as self-sufficient (IUCN 2013). Our study shows that most recent migrating allis shad come from natural reproduction, with stocking measures contributing only to a small proportion of migrating adults and juveniles. Although specific numeric thresholds for self-sufficiency are not always given, some studies suggest that a 3–5% return rate of stocked individuals, combined with increasing wild populations or high habitat occupancy (> 70%), may be considered evidence of self-sufficiency (Borcherding et al. 2010; Schneider 2011; van Rijssel et al. 2024). In addition, demonstrating sustainable natural reproduction over several generations (3+ years) and maintaining genetic diversity, with an effective population size of at least 500 individuals, are essential for long-term self-sufficiency (Fraser 2008; Schneider 2011). Given that 10% of the stocked allis shad was recaptured during our study, it would therefore be premature to consider the Rhine population as fully self-sufficient at this time, indicating that ongoing stocking is still necessary. Nevertheless, this positive trend suggests that the Rhine population is moving towards greater self-sufficiency.

### Geochemical differentiation and otolith microchemistry in the Rhine system

The results of our tank experiments demonstrate that the geochemical differences between sub-catchments within the Rhine system are sufficient to discriminate the origins of individuals. The combination of Sr/Ca and ⁸⁷Sr/⁸⁶Sr ratios revealed distinct patterns that allowed discrimination of otoliths from the Sieg, Lippe, Rhine and Neckar sub-catchments. Variations in trace element concentrations and isotopic ratios of the water in our study area reflect these differences. These variations can be attributed to the geological heterogeneity of the region, as well as to the influence of groundwater from its respective drainage (Pangle et al. 2010; Pracheil et al. 2014; Strohm et al. 2017). The more heterogeneous the geological background, the more distinguishable variations in water can be observed, even in small study areas (Veinott and Porter 2005; Strohm et al. 2017).

In our study, the Sr/Ca ratios in the otoliths and water samples were closely related, which is consistent with previous observations (Brown and Severin 2009). In contrast, no covariation was found for the
Ba/Ca ratios between the water samples and the otoliths reared in the respective tanks, especially due to the lower Ba/Ca ratios observed in the otoliths, e.g. at the Neckar. Therefore, Ba/Ca ratios seemed to not be suited to attribute natal origins to fish in our study. This may have resulted from the poor incorporation of Ba into the otoliths due to physiological processes caused by abrupt changes in habitats during the transfer of larvae from the French hatchery to the tanks of the Rhine system (Tabouret et al. 2011). Alternatively, this may be related to the differences in growth between the four experimental sites. Individuals from the Lippe grew slower than those from the Neckar at the same age. Finally, water temperature affects otolith microchemistry; however, contradictory effects have been reported in the literature. Temperature can have an indirect effect on otolith chemistry by affecting food uptake (Bath et al. 2000) and somatic growth (Tripp et al. 2020), which is related to the incorporation of Ba/Ca in otoliths (Walther et al. 2010; Miller 2011; Sturrock et al. 2014; Hüssy et al. 2021). Additionally, positive effects of temperature on the incorporation of Ba in otoliths were observed in some studies (Elsdon and Gillanders 2002; 2004), whereas no effect was recorded in others (Bath et al. 2000; Bath and Thorrold 2005; Martin and Wuenschel 2006). Finally, Miller (2009) showed species-specific temperature responses to Ba incorporation into otoliths. In order to clarify the variation in Ba incorporation in the otoliths, multivariate models could be used, taking into account different variables such as temperature, stomach content, growth rate, river discharge, and other trace element concentrations in the water (e.g., Manganese).

### Spawning locations, homing patterns and dispersal patterns of the allis shad

Compared to the study of Strohm et al. (2017), which showed individual assignation from their experimental tributaries with 91% accuracy using a random forest model and distinguished between fish from the wild to the origin hatchery with 100% accuracy, our classification model showed 100% reattribution to each river tested. A potential limitation of our RF model is that for our trained model, the isotope ratios were transferred directly from the water samples to the otoliths based on the findings of previous studies (Kennedy et al. 2000; Walther and Thorrold 2008;

Martin et al. 2013; Pouilly et al. 2014). Nonetheless, measurements of the 87Sr/86Sr ratios in the otoliths were not available in our study, and this could slightly influence the precision of the attribution, especially the differentiation between the Rhine, Lippe and Neckar sub-catchments, which have very similar 87Sr/86Sr ratios in water.

In the period considered, i.e. 2017 to 2020, natural reproduction in the middle Rhine section contributed the most to the recruitment of the reintroduced allis shad population into the Rhine system. However, targeting specific spawning locations in the middle Rhine section with the aim of protecting them is challenging, as allis shads spawn at night and are often distant from the banks. Additionally, recovering fish at barriers is not possible in the lower and Middle Rhine because the Rhine River is free flowing in these sections. The first physical barrier that returning shads encounter is located over 700 km from the North Sea entrance at Iffenzheim in the Upper Rhine. Furthermore, homogenous chemical signatures regarding elemental ratios and Sr isotopes (Buhl et al. 1991) prevent a more finely resolved attribution of natal origin in the middle Rhine section.

The Neckar River was identified as another important sub-catchment for the reproduction of allis shads in the Rhine system, with 21% of the adults attributed with high certainty to this river. This result is consistent with the increased observation of returning allis shad, which are occasionally caught during monitoring operations at the Ladenburg fish pass on the Neckar River. Individuals attributed to the Neckar were recaptured far upstream in the Rhine system upon their return to the Rhine as spawners either at the dam in Gambsheim in the Upper Rhine section or in the Neckar at the dam in Ladenburg. These migration patterns point to some degree of homing behaviour in allis shad. For a long time, the idea of homing had been raised for the allis shad (Baglinière et al. 2003). In recent years, a few studies have shown that allis shads can perform a high degree of homing at the catchment, sub-catchment or even site level, while other studies have shown that many individuals return to spawn in adjacent catchments (Martin et al. 2015; Randon et al. 2018). Nachón et al. (2020) investigated the potential for dispersion of sub-adult shads at sea and exposed two hypothetic behaviours. First, the resident gregarious behaviour, consisting of a group of individuals that remain close to the natal catchment,

<figure><img src="image_9.png" /></figure>
and second, dispersive gregarious behaviour consist-
ing of a group of individuals with an identical natal
origin but migrating further distances to spawn. Rulif-
son and Dadswell (2020) observed distinct seasonal
variations among the alewife [Alosa pseudoharengus
(Wilson, 1811)] and blueback herring [Alosa aesti-
valis (Mitchill, 1814)] populations captured in inner
Bay of Fundy, Canada, intertidal weirs, indicating
that different migratory groups converge at sea. While
their study focused on North American Alosa spe-
cies, the results suggest that European allis shad may
exhibit similar variations in marine behaviour and
stock structure.

Allis shad dispersal and potential exchange of
individuals between catchments is also visible in
our results. Fish originating from the Lippe and
Neckar returned to the upper Rhine, and one indi-
vidual originating with high certainty from the Lippe
was recorded to have returned to a different catch-
ment, the nearby Scheldt system. Strong dispersal
and the establishment of new populations, covering
over 5000 km along the coastline, have also been
observed for American shad, which were introduced
into two Pacific Northwest coastal rivers (Hasselman
et al. 2012). This strong spread is not observed in the
native range of American shad from the East coast
of North America, where more than 90% of the indi-
viduals return to their natal rivers, as demonstrated in
tagging studies (Melvin et al. 1986; Hendricks et al.
2002). The allis shad population in the Rhine system
has been newly reintroduced, and even though the
Rhine historically belonged to the distribution area
of this species, this reintroduction programme could
contribute to an increased tendency of this population
to spread to nearby catchments and sub-catchments.
In line with this hypothesis, recent observations have
shown a recent and unexpected increase in allis shad
populations in British channel rivers, e.g. the Vire
River (France) and the Seine River (France), located
500 to 600 km from the Rhine catchment (Belliard
et al. 2009; Martin et al. 2015). Nevertheless, it can-
not be excluded that other adjacent watersheds to
the Rhine system may also contribute to the repopu-
lation of allis shads in the Rhine system, as 27% of
the returning adults could not be attributed with high
certainty to any of the four studied Rhine sub-catch-
ments. Additionally, only 8% of juveniles migrating
downstream of the Rhine were not attributed either
with high certainty to any of the four studied Rhine

sub-catchments. As shads are known for their gregar-
ous behaviour (Nachón et al. 2020), individuals from
small populations in neighbouring catchments could
be attracted to the Rhine system by the larger allis
shad swarms of the growing Rhine population. Alter-
natively, or in conjunction with this social attraction,
some individuals may return to their natal grounds
through genetically programmed homing behaviour.

Future research and conservation strategies for allis
shad in the Rhine system

The 8% of unassignable juveniles in this study also
clearly indicate that there are other active spawning
grounds for allis shad in other sub-catchments of the
Rhine system. Juveniles caught during downstream
migration must have been born in the Rhine catch-
ment. Based on our successful proof of concept that
allis shad can be attributed to different sub-catch-
ments of the Rhine system, we will expand our model
and include further geochemically unique sub-catch-
ments. The first candidate rivers suitable for repro-
duction for allis shad are the Mosel, Main, Nahe and
Ill rivers.

The findings of this study also provide evidence
that active spawning sites should be present at the
Neckar River, as homing behaviour has been observed
in this sub-catchment. Further nocturnal monitor-
ing at locations with suitable hydrological and mor-
phological conditions should be pursued, although
this approach has not yet been successful in directly
observing spawning in the Neckar. Furthermore, an
extension of the attribution model using water and
otolith samples from other rivers in the British Chan-
nel is planned. These include the Vire and Seine
catchments (both in France) and the Severn catch-
ment (in the UK). This will facilitate an investigation
into the potential exchange of individuals between
these catchments, which is hypothesised in this study.
Finally, future studies should investigate whether the
composition of migrating aggregations becomes more
homogeneous the further upstream they migrate. This
would provide strong support for the idea that all shad
homing occurs at the sub-catchment level.

**Acknowledgements** We are thankful to the MIGADO association (Migrateurs Garonne Dordogne Charente Seudre—France) for providing allis shad fish larvae. We are grateful to Dr. Christophe Pecheyran, Dr. Hélène Tabouret, Fanny Claverie, and Gaelle Barbotin from the University of
Pau and Pays de l'Adour CNRS, IPREM—France who performed and provided support with the LA-ICP-MS analyses. We thank Prof. Dr. Jost Borcherding, Josephine Voß and Finja Laureen Kollorz from the University of Cologne, Germany, as well as Christopher Schmidt from the University of Applied Sciences Trier—Environmental Campus Birkenfeld, Germany, for their help during the experimental setup and laboratory work.

**Funding** Open Access funding enabled and organized by Projekt DEAL. This study was funded by many supporters of the project for allis shad reintroduction into the Rhine system: the State Fishing Association of Baden-Württemberg; the Ministry for Climate Protection, Environment, Energy and Mobility for the State of Rhineland-Palatinate; the Hessian Ministry for the Environment, Climate Protection, Agriculture and Consumer Protection; the Ministry for the Environment, Agriculture, Nature Conservation and Consumer Protection of the State of North Rhine-Westphalia; the Rhine Fishing Cooperative from North Rhine-Westphalia; the Sportfishing of the Netherlands; the Association of Hessian Fishermen e.V.; the District government of Düsseldorf and the Swiss Federal Office for the Environment (BAFU). The HIT Umwelt- und Naturschutz Stiftungs GmbH financially supported the collection and analyses of the water samples.

Umwelt- und Naturschutz Stiftungs-GmbH, Landesfischereiverband Baden-Württemberg e.V., Ministerium für Umwelt, Energie, Ernährung und Forsten Rheinland Pfalz, Rheinfischereigenossen schaft, Sportvisserij Nederland, Verband Hessischer Fischer e.V., Bezirksregierung Düsseldorf, Hessisches Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Ministerium für Klimaschutz, Umwelt, Landwirtschaft, Natur- und Verbraucherschutz des Landes Nordrhein-Westfalen

**Data availability** The authors declare that the data supporting the findings of this study are available from the corresponding author on reasonable request.

## Declarations

**Ethical statement** The study followed international and national guidelines for ethical treatment.

**Conflict of interest** The authors declare no competing interests.

**Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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