The arrival of a non-indigenous ecosystem engineer to a heavily invaded and flow-regulated estuary in Europe

Ecosystem engineering bivalves can shape aquatic ecosystems because their high filtration capacity changes water quality and their shells increase the fractal dimension of benthic ecosystems with consequent abiotic and biotic effects. The Asian date mussel Arcuatula senhousia (W. H. Benson, 1842), native to East Asia between the South China Sea and Siberia, is one such bivalve that, despite its small size, can reshape a benthic ecosystem when forming dense, continuous mats. We describe here the first detected population of this non-indigenous species in southern Portugal. The Asian date mussel was found in the middle portion of the Guadiana estuary in 2022. There, river flow has been highly regulated since the construction of the biggest European reservoir in 2002, which may have been the precursor for the establishment of numerous non-indigenous species. We also discuss if this new non-indigenous species indicates an ongoing invasion meltdown process or if it can be framed under the empty niche or niche replacement hypothesis. So far, there is only circumstantial evidence supporting the niche theory hypotheses, but the interaction of several hypotheses promoting the spread and establishment of this species is also likely. Moving forward, better-informed management and conservation efforts should rely on new empirical and experimental evidence to understand the establishment mechanisms of non-indigenous species in the Guadiana estuary.


Introduction
The ecosystem impacts of invasive species include changes in nutrient cycling, predation on native species, species extirpation, and habitat alterations (some of which are promoted by ecosystem engineering invaders) (Simberloff et al. 2013).Bivalves are important ecosystem engineers both in native and non-native areas (Carranza et al. 2009;Sousa et al. 2009;Beck et al. 2011), and invasive bivalves may display a wide array of positive effects since they can increase water quality and benthic biodiversity due to, respectively, their filtration capacity (Fahnenstiel et al. 1995;Pranovi et al. 2006;Rosa et al. 2014) or added fractal dimension with their shells (either when the organisms are dead or alive) that provide refuge, feeding and spawning areas for vertebrates and invertebrates (Burlakova et al. 2012;Novais et al. 2015;Ilarri et al. 2018).However, when they reach high abundances, invasive bivalves disrupt plankton communities and dynamics by triggering a series of cascading effects (Pace et al. 1998;Crooks 2009;Pigneur et al. 2014).For example, the Asian clam Corbicula fluminea (O.F.Müller, 1774) produces impacts at different levels, from alteration of habitat structure and competition for space to impacts on organisms at the base of the food chain due to high filtration rates (Sousa et al. 2009;Dias et al. 2014).Financially, the economic impact can be tremendous; the small but highly invasive dreissenid mussels-mainly the zebra mussel Dreissena polymorpha (Pallas, 1771) and quagga mussel Dreissena bugensis (Andrusov, 1897)-caused over $51 billion USD in accumulated loses around the world because they clog irrigation and water intake pipes of hydroelectric, nuclear, and thermal power plants (Haubrock et al. 2022).
The Asian date mussel Arcuatula senhousia (W.H. Benson, 1842)originally described as Modiola senhousia and known as Musculista senhousia until recently-is another small ecosystem engineer that grows up to 28 mm in shell length.This species may form dense, continuous mats of ovoid cocoons anchored on the sediment with a matrix of byssal threads (Morton 1974;Sousa et al. 2009).Native to East Asia between the South China Sea and Siberia (Cheung et al. 1962in Morton 1974;Kulikova 1978), the species has invaded several coastal ecosystems around the globe, including the Indian Ocean (India, Madagascar, Mauritius, Zanzibar, and the adjacent Persian Gulf) (Barash and Danin 1972;Behera et al. 2019;Yasser et al. 2023), coastal ecosystems scattered along the Eastern Atlantic Ocean (e.g., southern United Kingdom, Bay of Biscay, central Portugal, Guinea-Bissau) (Lourenço et al. 2018;Cabral et al. 2020;Watson et al. 2021;Massé et al. 2022), coastlines along the Mediterranean Sea (e.g., Spain, France, Italy, Croatia, Albania, Turkey, Israel, Tunisia, Algeria) (Hamza et al. 2022) and in the adjacent Black Sea (Zhulidov et al. 2021), off Venezuela on the Western Atlantic Ocean (Martínez-Escarbassiere et al. 2003), Eastern coastal ecosystems of the Pacific Ocean (e.g., Mexico, Colombia) (Crooks 1998;Arellano and Salgado-Barragán 2012;Osorio 2013), and in the southwestern Pacific Ocean (e.g., Australia, New Zealand) (Willan 1987;Creese et al. 1997).The Asian date mussel is an epifaunal bivalve observed in sites with salinities between 5 and 18 (Yamamuro et al. 2010;Hosozawa et al. 2020), but is able to colonize fully marine areas, as observed in the Solent (southern England) (Watson et al. 2021) and French localities along the Bay of Biscay (Massé et al. 2022).
In their native range, the Asian date mussel may reach densities of up to 2500 ind.m -2 (Morton 1974).However, in invaded areas, it has reached densities of 10,000 ind.m -2 in Italy (Mistri et al. 2004), 16,000 ind.m -2 in New Zealand (Creese et al. 1997), and 170,000 ind.m -2 in Mission Bay (California, United States of America) which is among the highest ever recorded for marine bivalves (Crooks and Soulé 1999).As an ecosystem engineer, the Asian date mussel also increases habitat complexity, which may result in a increase in species richness and density, particularly of amphipods, gastropods, and polychaetes (Crooks 1998;Crooks and Khim 1999).The water filtration capacity of the species accelerates the transfer of suspended sediment and organic matter from the pelagic compartment into the benthic compartment through the production of faeces and pseudo-faeces.This process alters the nutrient balance of sediment and reduces the redox potential discontinuity layer, which can prevent certain organisms from living below the byssal mats (Morton 1974;Creese et al. 1997;Crooks 1998Crooks , 2009)).
Our study focuses on the Guadiana estuary (southwest Iberian Peninsula), which has been suffering from ecological impacts for over two decades due to the construction of the biggest water reservoir in Europe-the Alqueva dam-that began operating in February 2002 (Morais 2008).The five most noticeable changes in the Guadiana estuary are the (1) increased salinization of the middle and upper portions of the estuary (Chícharo et al. 2006;Barbosa et al. 2010;Encarnação et al. 2013), (2) change in the phytoplankton community due to a shift from a light-limited ecosystem towards a more nutrient-limited one (Barbosa et al. 2010), (3) decrease in the diversity of zooplankton and increase of annual blooms of invasive jellyfish species (Muha et al. 2012), (4) shift from freshwater-associated fish species in the upper portions of the estuary, such as barbell species, to short-lived fish such as Atherina and Pomatoschistus species (Chícharo et al. 2006), and (5) appearance of numerous non-indigenous species (Chícharo et al. 2009a;Morais et al. 2009aMorais et al. , 2019;;Morais and Teodósio 2016;Cruz et al. 2017;Gonçalves et al. 2017;Seyer et al. 2017;Nuño et al. 2018).The Guadiana estuary is now a hotspot for non-indigenous species, including the Asian clam Corbicula fluminea, weakfish Cynoscion regalis (Bloch & Schneider, 1801), and Atlantic blue crab Callinectes sapidus Rathbun, 1896, just to cite the most prominent (Morais et al. 2009a(Morais et al. , 2017(Morais et al. , 2019;;Morais and Teodósio 2016;Encarnação et al. 2021).
Considering the recent history of biological invasions in the Guadiana estuary, the discovery of the Asian date mussel Arcuatula senhousia in October of 2022 is alarming.Therefore, we intend to describe the most recent non-indigenous species detected in the Guadiana estuary and present hypotheses on why so many non-indigenous species have established in this estuary since the construction of the Alqueva dam.

Study area
The Guadiana River basin is the fourth largest in the Iberian Peninsula and is characterized by a Mediterranean climate (i.e., periods of extended droughts and infrequent heavy floods) (Morais 2008).River flow is heavily controlled by dams, with the biggest European water reservoir-the Alqueva dam, located in the river mainstem approximately 170 km from the river mouthregulating the majority of water reaching the estuary (Morais 2008;Morais et al. 2009b) and resulting in pronounced salinity intrusion into the upper estuary (Chícharo et al. 2006;Barbosa et al. 2010;Encarnação et al. 2013).This estuary can be divided into the upper portion of the estuary, with salinity closer to zero but still with tidal influence, middle portion of the estuary, characterized as the mixing zone of brackish water (salinity 0.5-25), and the lower portion of the estuary with salinity above 25 (Chícharo et al. 2009b), although inflow regularization is reshaping such boundaries with the increasing salinization of the estuary.

Sampling methods
Asian date mussels were collected during two sampling campaigns, under the scope of the ATLAZUL project, to evaluate the state of fishing resources and distribution of non-indigenous species in the Guadiana estuary during the spring (April) and fall (October) of 2022.Samples were collected with a bottom beam trawl (mouth opening: 4.2 m wide × 0.5 m tall; total length: 8.0 m) with a variable mesh size, decreasing from 20 mm in the aperture to 10 mm in the cod end.Sampling areas included the lower, middle, and upper portions of the Guadiana estuary.
In 2022, 14 trawls were made in the lower and upper estuary and 15 in the middle estuary.The choice of sampling locations within each estuary section followed previous studies (e.g., Chícharo et al. 2001Chícharo et al. , 2006;;Morais et al. 2009b).Trawls were performed in a boat equipped with an 80 hp engine at a constant speed-between 2 and 5 knots (depending on flow conditions)-during ebb tides and along the same direction of water flow.The number of trawls (minimum of three) and duration varied between sampling locations (shorter distances in sites with boulders or biogenic reefs, longer in areas with muddy or sandy bottoms) and abundance of organisms.Trawl duration varied between 2 and 10 minutes and lasted 7.1 ± 1.8 min on average.Trawl distance varied between 106.1 and 719.19 m, averaging 481.9 ± 131.5 m, while the average sampled area was 2024.1 ± 552.5 m 2 .
After collection, specimens were measured (total shell length and width, ± 0.1 cm), weighed (total fresh weight, ± 0.01 g), and identified in the laboratory.According to descriptions available in the literature, specimens were identified as Asian date mussels Arcuatula senhousia (Morton 1974;Hoenselaar and Hoenselaar 1989;Faasse 2018;Lourenço et al. 2018).

Results
Twelve specimens of a previously unknown mussel species for the study area were collected in October 2022 close to Foz de Odeleite in the middle  portion of the Guadiana estuary (Figure 1).Specimens were visually identified as Arcuatula senhousia due to the following key morphological features: a thin shell, radiating lines posteriorly and concentric lines in the middle, several riblets at the anterior end, with the periostracum showing a shiny and pale greenish colour and irregular brownish-reddish markings, as well as radiating reddish lines on the posterior area (Faasse 2018;Lourenço et al. 2018).
The Guadiana estuary specimens were collected in three of the seven trawls made in October 2022, representing 7% of the trawls made in 2022 (3 out of 43 trawls).In October 2022, the average water temperature, salinity, and depth in the sites where Asian date mussels were collected were 22.3 ± 0.3 °C, 26.8 ± 1.9, and 6.6 ± 1.5 m, respectively.On average, the total length and width of Asian date mussels were 12.1 ± 3.0 mm and 6.9 ± 1.6 mm, respectively, and the total weight was 0.14 ± 0.09 g (Table 1).Density in each trawl was 0.046 (trawl 2), 0.178 (trawl 3), and 0.504 ind.100m -2 (trawl 1).

The Asian date mussel and its putative impacts
The constant low-flow conditions set by the Alqueva dam over numerous years, rarely disrupted by heavy rain or floods, has reshaped the ecosystem functioning of the Guadiana estuary, which coincided with the establishment of multiple non-indigenous species.The brackish zone is now a hotspot for non-indigenous species, where they occupy the pelagic and benthic compartments while exploiting different resources (Chícharo et al. 2009a;Morais et al. 2009aMorais et al. , 2019;;Encarnação et al. 2013;Morais and Teodósio 2016;Seyer et al. 2017).
The Asian date mussel is the most recent non-indigenous bivalve collected from the Guadiana estuary since 2000.Back then, the Asian clam Corbicula fluminea was caught in the upper portion of the estuary, and in 2003 it was already established along the middle and upper portions of the estuary, including several tributaries (Morais et al. 2009a).While the infaunal Asian clam inhabits freshwater and brackish ecosystems (salinities up to 15 but tolerating salinity up to 20) (Ferreira-Rodríguez and Pardo 2016; Bertrand et al. 2017), the Asian date mussel is an epifaunal bivalve commonly observed in environments with salinities between 5 and 18, but can also be found in fully marine zones (Yamamuro et al. 2010;Hosozawa et al. 2020;Watson et al. 2021;Massé et al. 2022).In the Guadiana estuary, we found Asian date mussels in an area with a salinity of 26 in the middle portion of the Guadiana estuary (~ 20 km upstream from the river mouth) during early fall; in this location, salinity can drop to 0 during higher inflow regimes (Morais et al. 2009b).The increased salinity intrusion in the Guadiana estuary, observed since the construction of the Alqueva dam (Chícharo et al. 2006;Morais et al. 2009b;Barbosa et al. 2010;Encarnação et al. 2013), may continue to facilitate the expansion of the Asian date mussel to more upstream locations, considering the salinity ranges previously reported for the species.
The detection of the Asian date mussel in three trawls conducted closely together, out of 43 trawls made along the entirety of the estuary in 2022, suggests that the species was recently introduced and it is not widespread for now.However it is possible that its low density precluded its detection during previous sampling campaigns.In Portugal, the species was found in two estuaries on the western coast-in 2015 in the Sado estuary and 2018 in the Tagus estuary (Cabral et al. 2020).These estuaries are located in the western coast of the country, and the entry to the Guadiana estuary is 370 km of coastline away in the southeastern tip of Portugal.Furthermore, no connecting aquatic corridors or water transfers, which could have transported larvae to the study region, are known between the Tagus/Sado and Guadiana river systems.In Spain, the presence of the Asian date mussel dates back to 2006 in the Bidasoa estuary on the Atlantic border with France (Bachelet et al. 2009) and in the Mediterranean coast in 2014 in the Ebro delta (Soriano and Salgado 2014) and 2016 in a Barcelona's marina (Ulman et al. 2017).It is also unlikely that the species reached the Guadiana estuary by natural dispersion from any of these locations, so additional primary or secondary introduction events must have occurred.Additional molecular genetics work could be used in the future to shed light on how these populations may be connected to one another and possible vectors that transported, and continue to transport, the species to new locations.
The impact of the Asian date mussel will depend on its invasiveness, but it has the potential to become a serious ecosystem engineer (Crooks 1998(Crooks , 2009;;Crooks and Soulé 1999).Such impacts can be divided into two categories-autogenic and allogenic engineering (Jones et al. 1994).The shells of the Asian date mussel are considered an autogenic engineering by creating three-dimensional habitats for small organisms such as amphipods, gastropods, and polychaetes (Crooks 1998;Crooks and Khim 1999).In the worst-case scenario, the dense mats that Asian date mussels often form could impair the survival of other bivalves, such as the invasive Asian clam in the middle and upper portions of the estuary and native bivalves in the middle and lower portions of the estuary.The most notable native infaunal bivalves in the middle and/or lower portions of the Guadiana estuary are Scrobicularia plana (da Costa, 1778), Ruditapes decussatus (Linnaeus, 1758), and Cerastoderma edule (Linnaeus, 1758) (Conde et al. 2013), besides oyster reefs in the middle portions of the estuary and mussel beds in artificial substrates in the lower portions of the estuary (Morais unpublished data).In New Zealand, dense mats of the Asian date mussel significantly impacted other infaunal suspension-feeding bivalves, such as the endemic pipi Paphies australis (Gmelin, 1791), cockle Austrovenus stutchburyi (W.Wood, 1828), and black mussel Xenostrobus pulex (Lamarck, 1819) (Creese et al. 1997).Allogenic engineering involves the change of the physical state of the ecosystem via, for example, mechanical or chemical means (Jones et al. 1994).At higher densities, the filtration capacity of the Asian date mussel may increase water clarity by removing suspended materials from the water column and transferring them to the benthic compartment (Crooks 2009).This may increase the euphotic zone and enable the growth of macroalgae and seagrass (Strayer et al. 1999;Nielsen et al. 2002;Wall et al. 2008).In the Guadiana estuary, in tandem with the presence of the Asian clam (Morais et al. 2009a) and the saline intrusion into upper areas of the estuary (Chícharo et al. 2006;Barbosa et al. 2010;Encarnação et al. 2013), a reduction in turbidity may result in an alteration of benthic communities, with a higher presence of algae, or even seagrasses that are mainly found in the lower portions of the estuary (Cunha et al. 2013).
The Asian date mussel may also serve as a new food source for other species, including other non-indigenous species.The Atlantic blue crab C. sapidus was detected for the first time in the Guadiana estuary in 2017 (Morais et al. 2019); since then, its abundance and distribution has increased significantly (Encarnação et al. 2021).Bivalves are a common food source for the Atlantic blue crab in the native range, including mussels, oysters, and clams (Laughlin 1982), but also other non-indigenous species, such as Corbicula spp. in the Ebro River in Spain (Ventura et al. 2018).Thus, the Asian date mussel, with its small size and relatively fragile shell (Hoenselaar and Hoenselaar 1989;Massé et al. 2022), may become an additional food source for the Atlantic blue crab in the Guadiana estuary.Maintaining healthy populations of the green crab Carcinus maenas (Linnaeus, 1758), a native estuarine omnivore species (Baeta et al. 2006;Young and Elliott 2019), can also contribute to controlling this new invader, as the closely related Carcinus aestuarii (Nardo, 1847) is an effective predator of the Asian date mussel in Italy (Mistri et al. 2004;Cabiddu et al. 2023).
The ecosystem engineering capabilities of the Asian date mussel, the high densities it may attain, and their tolerance to a broad range of environmental variables suggest that this species may become a problematic invasive species in the southern Iberian Peninsula.The numerous estuarine ecosystems adjacent to the Guadiana estuary along the Gulf of Cadiz present an opportunity for this species to colonize new habitats and highlight the need to increase transboundary monitoring efforts for the early detection of non-indigenous species.

A hotspot for non-indigenous species -framing hypotheses
We hypothesize that the growing number of non-indigenous species in the middle and upper portions of the Guadiana estuary can be framed either by the invasion meltdown hypothesis (Simberloff and Von Holle 1999) or niche-based hypotheses (Ricciardi et al. 2013).On the one hand, the invasion meltdown hypothesis proposes that a community becomes more and more vulnerable to biological invasions as the number of attempted introductions or established introductions increases (Simberloff and Von Holle 1999).Regarding only the non-indigenous species that our research group has detected in the Guadiana estuary, the numbers have been increasing over the years -Corbicula fluminea in 2000 (Morais et al. 2009a);Blackfordia virginica Mayer, 1910, Palaemon macrodactylus Rathbun, 1902, and Acartia tonsa Dana, 1849in 2008(Chícharo et al. 2009a;Cruz et al. 2017); Cordylophora caspia (Pallas, 1771) in 2015 (Seyer et al. 2017); Cynoscion regalis in 2016 (Morais and Teodósio 2016); Callinectes sapidus in 2017 (Morais et al. 2019); and Arcuatula senhousia in 2022 (this study).On the other hand, the niche-based hypotheses propose that either invasive species use unexplored resources (empty niche hypothesis) or are more competitive than native species to exploit the same limited resource (niche replacement hypothesis) (Ricciardi et al. 2013).Currently, we only have circumstantial evidence supporting the niche-based hypotheses.For example, the Atlantic blue crab and the Ponto-Caspian hydroid C. caspia are likely occupying empty niches in the middle portion of the estuary due to the increased salinization of the estuary (Chícharo et al. 2006;Morais et al. 2009b;Barbosa et al. 2010;Encarnação et al. 2013), while the weakfish C. regalis is a successful invader because it may outcompete congeners (Cerveira et al. 2021).The Asian date mussel is, to our best knowledge, the only soft sediment epifaunal bivalve in the Guadiana estuary, as other native and invasive bivalves prefer and/or require harder substrate.Therefore, both hypotheses may help explain the presence and putative expansion of the Asian date mussel, which may benefit from a heavily disturbed ecosystem due to anthropogenic impacts and biotic disturbance from previous invasions but also from empty niches that can be promptly colonized.
The differences in a community's competitive ability/resistance towards non-indigenous species are vital in shaping community structure and succession (Belyea and Lancaster 1999;Young et al. 2001).So, the priority effects hypothesis, a broader ecological theory, is also intertwined with the abovementioned hypotheses since the succession of disturbed communities relies heavily on which species were established first (Belyea and Lancaster 1999;Young et al. 2001).Moving forward, empirical and experimental approaches to understand the establishment mechanisms of non-indigenous species in the Guadiana estuary should be conducted; first, to assess if an invasion meltdown is ongoing and, second, to weigh the prevalence of which niche-theory hypothesis prevails according to species traits.Such an approach will better inform management and conservation efforts aiming at tackling non-indigenous species in the Guadiana estuary and other estuaries along the Gulf of Cadiz, hopefully preventing the establishment of new species.

Figure 1 .
Figure 1.(A) Photo of an Asian date mussel Arcuatula senhousia (W.H. Benson, 1842) specimen retrieved from the middle portion of the Guadiana estuary on October 7 th , 2022.(B) Location of the bottom beam trawls performed in April and October 2022 in the middle portion of the Guadiana estuary.The number of specimens and density of the Asian date mussels collected are shown for the three trawls in which specimens were collected in October 2022.

Table 1 .
Morphometric details of the 12 Asian date mussel Arcuatula senhousia (W.H. Benson, 1842) specimens collected in the middle portion of the Guadiana estuary on October 7 th , 2022.