Freshwater mussels of the genus Sinanodonta, native to East Asia, are among the most successful invasive bivalves globally, known for their rapid reproduction and growth, broad environmental tolerances, and ability to disrupt aquatic ecosystems. Their arrival and subsequent spread in parts of the Middle East remains poorly documented. Here, we present new DNA-based evidence that confirms the invasion of Iraq’s freshwater ecosystems by two genetically distinct Sinanodonta lineages: Sinanodonta woodiana and Sinanodonta pacifica. Using COI DNA sequences, we confirm the co-occurrence of these lineages in southern Iraq, a pattern not previously reported in any Middle Eastern country. The two invasions appear to result from independent introduction pathways, both through their obligatory parasitic larvae on fish: aquaculture of East Asian carp (S. woodiana), and the ornamental fish trade or tilapia farming (S. pacifica). This discovery raises significant ecological concerns, as the dual invasion may further threaten Iraq’s already declining native mussel fauna, particularly the endemic and potentially extirpated Anodonta vescoiana. Our findings highlight Iraq as an emerging hotspot for freshwater biological invasions, likely driven by its growing and largely unregulated fish farming industry. This underscores the urgent need for targeted genetic monitoring, risk assessments, and biosecurity measures to prevent further spread and ecological impact.

Aquatic bioinvasions, Invasive freshwater mussels, Molecular identification, Sinanodonta woodiana, Sinanodonta pacifica, Tigris–Euphrates Basin

Invasive bivalves are arguably one of the most disruptive biological forces in freshwater ecosystems worldwide (Lopes-Lima et al. 2025). Their ability to tolerate broad environmental conditions, rapidly increase population sizes, and disperse effectively allows them to colonize and thrive in new habitats, often at the expense of native biodiversity (Douda et al. 2025). Invasive freshwater bivalves can profoundly alter ecosystem functions, including water filtration, sediment dynamics and nutrient cycling, and disrupt interactions of native freshwater mussels (Bivalvia: Unionida) with their obligate host fish species (Sousa et al. 2014; Zieritz et al. 2018a, 2018b).

One of the most prolific invaders of the order Unionida is Sinanodonta woodiana (Lea, 1834), commonly known as the Chinese pond mussel. Initially described from the Yangtze River basin and surrounding East Asian waterways, S. woodiana rapidly expanded its range following unintentional introductions associated with the trade of Asian carp (Cyprinus carpio) and other East Asian cyprinids (Bolotov et al. 2016; Mehler et al. 2024; Douda et al. 2025). Once established, the species thrives in both natural and artificial lentic environments as well as river systems, attaining population densities of > 60 individuals m^–2 and biomass of up to 25 kg m^–2 (Kraszewski and Zdanowski 2007). In some cases, the species has been shown to progressively displace native unionids through host competition, habitat occupation, and shell-mediated habitat alteration (Donrovich et al. 2017; Urbańska and Andrzejewski 2019; Dobler et al. 2022). Now widespread across Europe, Asia, and North Africa, S. woodiana is regarded as a significant threat to native biodiversity and a growing concern in freshwater conservation and management (Bolotov et al. 2016; Lopes-Lima et al. 2017; Douda et al. 2025). Recent molecular studies have additionally demonstrated that morphologically similar Sinanodonta lineages may represent cryptic invasions, making DNA-based identification essential for reconstructing invasion pathways and accurately assessing species distributions (Bolotov et al. 2016; Douda et al. 2025). Situated at the crossroads of major transcontinental trade routes and traversed by the Tigris–Euphrates river system, Iraq represents a strategically significant yet understudied region in the context of freshwater biological invasions. Characterised by a hot (semi-)arid climate, discharge in this river system is highly seasonal, with values being up to 10-times higher during spring (March-May) compared to summer months (July-October) (Bozkurt and Sen 2013; Issa et al. 2013). Iraq’s freshwater ecosystems face numerous anthropogenic pressures, including pollution, hydrological alterations, heating and drying (Bozkurt and Sen 2013; Issa et al. 2013; Yasser and Naser 2023; Yasser et al. 2024; Naser et al. 2025a; Boloorani et al. 2026). These pressures have resulted in a sharp decline in native mussel diversity, including the potential extirpation of the endemic freshwater mussel species Anodonta vescoiana Bourguignat, 1856 (Naser et al. 2025b). The Sinanodonta genus was first recorded in Iraq in 2021 in the Hilla River, a tributary of the Euphrates River (Bogan et al. 2021), and the mainstem of the Euphrates River near Najaf (Abdul Razak and Zwair 2021) (Fig. 1). Both these records were morphologically identified as S. woodiana, which is presumed native to the Yangtze River basin and has been designated as the “temperate invasive” lineage due to its widespread introduction across Europe and other temperate regions (Bolotov et al. 2016). No DNA sequences were published by Bogan et al. (2021), but four published cytochrome c oxidase subunit I (COI) DNA sequences (Genbank Accession Numbers LC651629, LC651630, LC656037, LC658986) appear to be associated with the Abdul Razak and Zwair (2021) record from the Euphrates River. Phylogenetic analyses of these sequences by Douda et al. (2025) placed them into Sinanodonta pacifica (Heude, 1878). This species is presumed to be native to eastern China and Taiwan and due to its widespread introduction across parts of tropical Asia, has been designated as the “tropical lineage” (Bolotov et al. 2016), although it has also been reported occasionally from other tropical and non-tropical regions (Douda et al. 2025).

Figure 1. 

Verified occurrences of Sinanodonta spp. in the Tigris–Euphrates Basin of Iraq. Records for which species have been identified by DNA barcoding in red (S. pacifica) and black (S. woodiana), respectively; white records are not DNA barcoded and are considered Sinanodonta sp. For each record, the name of the water body and the reference of the relevant publication are provided.

More recently, additional records of Sinanodonta have been reported from several Iraqi localities, including the Euphrates River, Ptera Dam, Adua Marsh, Al-Majer, Al-Sweap River, Al-Gharraf River and Al-Ezz River (Naser et al. 2024, 2025b; Mashjel and Al-Taher 2025). However, because many of these records were based primarily on morphology, the identity and distribution of invasive Sinanodonta lineages in Iraq remain incompletely resolved.

Given Iraq’s geographic position at the intersection of temperate and tropical Sinanodonta invasion pathways, accurate identification of invasive unionid species and mitochondrial lineages is essential to reconstruct introduction routes, assess lineage-specific ecological risks, and design effective conservation and management strategies for native mussel assemblages (Douda et al. 2025; Naser et al. 2025b). In this study, we aimed to (1) determine the identities and current distributions of genetically distinct Sinanodonta lineages in Iraq, (2) discuss their likely invasion pathways and vectors, and (3) provide recommendations for managing their spread and impacts. By generating new DNA sequence data and comparing them with previously published COI sequences, we clarify the occurrence and distribution of invasive Sinanodonta lineages in Iraq and highlight the Tigris–Euphrates Basin as a potential contact zone for cryptic co-invasion in Southwest Asia.

The specimens analysed in this study originated from previously published Iraqi records of Sinanodonta from the Tigris and Euphrates river systems (Naser et al. 2024, 2025b). Specimens were collected during field surveys conducted in 2022 from the Tigris River at the Ptera Dam and from the lower Euphrates River (sites 3 and 4 in Fig. 1, respectively). Geographic coordinates and sampling localities are provided in Fig. 1 and associated references. The original Iraqi records from the Euphrates River near Najaf were morphologically identified as S. woodiana but were subsequently shown by molecular analyses to belong to S. pacifica; this issue is discussed further in the Discussion section. DNA sequences were obtained from two Sinanodonta specimens from each of the two sampled sites. The specimens were sampled in 2022 and preserved in 96% ethanol. A total of 16 specimens were processed for DNA extraction; however, only four yielded successful COI amplification, while the remaining samples failed to amplify, likely due to DNA degradation associated with poor preservation. DNA was extracted from foot tissue samples using the DNeasy® Blood and Tissue Kit following the manufacturer’s protocol. A 658 base pair (bp) fragment of the COI region was amplified using standard primers LCO1490 and HCO2198 (Folmer et al. 1994) in 10 μl reactions containing 5.0 μL 10X Green Dye PCR Buffer, 0.5 μL 10 mmol L⁻¹ of each primer, 1.0 μL DNA template and 3.0 μL nuclease free water. Polymerase chain reaction (PCR) cycle parameters were initial denaturation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C (50 s), annealing at 52 °C (50 s) and extension at 72 °C (50 s), and a final extension at 72 °C for 5 min. Amplified and purified DNA fragments were sequenced by the commercial company Eurofins Genomics (Ebersberg, Germany) and deposited in the National Center for Biotechnology Information (NCBI) GenBank database under Accession Numbers [PZ392808, PZ392809, PZ392810 and PZ392811].

MAFFT v7.304 (Katoh and Standley 2013) was used to assemble the COI dataset, which included the four sequences produced in this study, plus all previously published Sinanodonta COI sequences (n = 553), including those in the recent review by Douda et al. (2025) and additional sequences published since then (i.e. Mashjel and Al-Taher 2025; LC833751, LC833752) (see Suppl. material 1). To ensure that there was minimal or no bias in phylogenetic inference, third-party COI sequences were examined for potential diagnostic terminal artefacts (clusters of end-confined substitutions and/or deletions). This included sequences LC833751 and LC833752 (Mashjel and Al-Taher 2025). Publicly available sequences of the freshwater mussels Margaritifera laevis (F. Haas, 1910), Beringiana japonica (Clessin, 1874), and Beringiana beringiana (Middendorff, 1851) were included as outgroup taxa (Suppl. material 1).

The COI dataset was collapsed into unique haplotypes (see Suppl. material 1 for haplotype codes) and analysed using Maximum Likelihood (ML) and Bayesian Inference (BI). ML analyses were performed in IQ-TREE v2.2.0 (Minh et al. 2020), and BI analyses were conducted in MrBayes v3.2.7a (Ronquist et al. 2012). PartitionFinder 2 (Lanfear et al. 2016) was used to determine the best-fit substitution models and partitioning schemes under the Bayesian Information Criterion for BI analyses. Bayesian analyses were initiated from random trees using four Markov chains with default incremental heating. Two independent runs of 20 × 10⁶ generations were performed, sampling every 1,000 generations to produce 20,000 trees. Convergence and burn-in were assessed using Tracer v1.7.1 (Rambaut et al. 2018). For ML analyses, the best-fit models and partitioning schemes were identified with ModelFinder (Kalyaanamoorthy et al. 2017). Phylogenetic inference was then performed in IQ-TREE with an initial tree search followed by 10 independent runs and 10,000 ultrafast bootstrap replicates.

Our data provide new DNA-based confirmation of the presence of S. woodiana in the lower Tigris–Euphrates Basin, specifically from the Tigris River at Ptera Dam and the lower reaches of the Euphrates River. This complements a previous DNA-based record of S. pacifica from the middle Euphrates River (Fig. 1), confirming that both of the invasive Sinanodonta lineages occur in Iraq but currently exhibit apparently distinct distributions. Together, these findings support the presence of cryptic co-invasion by two genetically distinct Sinanodonta lineages within the Tigris–Euphrates Basin.

Our analyses additionally revealed that the two previously published DNA sequences from the Al-Gharraf River (LC833751, LC833752) contained terminal sequencing artefacts. After conservative end-trimming, both sequences matched the invasive S. woodiana haplotype SWA01 exactly; they were therefore included in the haplotype dataset but excluded from phylogenetic reconstruction. Both ML and BI analyses of the Sinanodonta COI dataset produced congruent topologies, recovering eleven clades corresponding to the species delimitations previously reported by Douda et al. (2025) (Fig. 2). The four newly sequenced specimens from Iraq (haplotype SWA01) clustered within the Sinanodonta woodiana sensu stricto clade, and were identical to the widespread invasive haplotype recorded across Europe and Central Asia (Fig. 2, Suppl. material 1). In contrast, the Iraqi sequences already available in GenBank (haplotype SWB02) grouped within the Sinanodonta pacifica clade and were identical to sequences from Peninsular Malaysia (Fig. 2; Suppl. material 1). Representative shell morphology of the two lineages is shown in Fig. 3.

Figure 2. 

Bayesian Inference (BI) and Maximum Likelihood (ML) phylogenetic tree inferred from the cytochrome c oxidase subunit I (COI) gene of Sinanodonta species. Support values above the branches are percent posterior probabilities/ultrafast bootstraps. Support values > 95% for both phylogenetic analyses are represented by an asterisk; support values within each species clade were erased for clarity. Haplotypes including Iraqi sequences were highlighted in red.

Figure 3. 

Shell morphology of invasive Sinanodonta species recorded in Iraq. A. Sinanodonta woodiana from the Tigris River (Ptera Dam); B. Sinanodonta pacifica from the Euphrates River. Photo Murtada Naser.

Sample collections: MN, AY. Sample design and methodology: MN, AY, AZ, ML. Data analysis and interpretation: MN, AY, ML, AZ. writing: original draft: MN, AZ, AY, ML, JS, B AG, JD. DB, FE. writing - review & editing: MN, AZ, AY, ML, JS, BAG, JD. DB, FE.

We appreciate the reviewers’ valuable comments, which helped improve the manuscript.

This research was funded in whole or in part by the Austrian Science Fund (FWF) [10.55776/I5825]. For open access purposes, the author has applied a CC BY public copyright license to any author accepted manuscript version arising from this submission.

Regarding the use of AI in the preparation of this manuscript, the authors declare the following: GPT

Used for: Language, style and writing.

All of the data that support the findings of this study are available in the main text or Supplementary material.