First established population of the Chinese pond mussel Sinanodonta woodiana (Lea, 1834) (Bivalvia: Unionidae) in Greece

This contribution presents the first established population of the Chinese pond mussel Sinanodonta woodiana (Lea, 1834) (Bivalvia: Unionidae) in Greece. Previous single reports from rivers Axios and Evros need further investigation. Chinese pond mussels were collected in Lake Pamvotida (= Pamvotis or Ioannina) during a mussel field expedition in October 2020. This alien species was first detected in 2006 in a fishpond adjacent to the lake from where it possibly dispersed. The species is now widespread and abundant throughout the lake and is most possibly displacing a genetically unique population of Anodonta cygnea endemic to the lake. Species identification was confirmed with genetic cytochrome oxidase c subunit I (COI) gene barcoding sequences. A single COI haplotype was retrieved which is shared by the vast of majority of the specimens sequenced from the invaded range in Europe. Monitoring of S. woodiana presence and abundance should be widely conducted and special attention and collaboration with fish farms is urgently required to prevent its dispersal to other freshwater ecosystems.


Introduction
Freshwater mollusks commonly figure among introduced non-indigenous species, being easily transported accidentally or intentionally by humans (Hulme 2007;Keller et al. 2011;Yanai et al. 2017).Due to human activities, a significant increase in the occurrence of alien freshwater mollusks has been recorded in the last decades especially for bivalves (Sousa et al. 2014;Karaouzas et al. 2020).Invasive mussel species have been reported to result in declines of native species (Ozgo et al. 2020).The Chinese pond mussel Sinanodonta woodiana (Lea, 1834), native to the Yangtze River basin (Lopes-Lima et al. 2020), has spread rapidly to central Asia and Europe, mostly due to the transport of farmed cyprinid fish species (Bolotov et al. 2016).Because of its reproduction success when compared to native mussel species (Huber and Geist 2019), it spreads rapidly (mainly during the parasitic stage) and can reach high densities in many rivers and standing water bodies (e.g.Douda et al. 2012;Benkő-Kiss et al. 2013;Beran 2020).
As it's first-ever discovery in Europe in 1979 from fish farms in Romania (Popa et al. 2007), S. woodiana was first discovered in Greece in 2006 in a fishpond connected to Lake Pamvotida (Albrecht et al. 2006).Two years later, two more reports of the species were recorded from Axios and Evros rivers by gastropod experts (Reischütz et al. 2008a, b).However, these records were based solely on morphological identification, which is sometimes unreliable for S. woodiana due to its wide shell plasticity (Guarneri et al. 2014) that may lead to misidentification with other species in the genus Anodonta.Furthermore, no specimens were found during several sampling campaigns conducted by the authors in these two rivers.Overall, taxonomic misidentifications are common in tribe Anodontini, because they generally lack diagnostic hinge teeth (Lopes-Lima et al. 2017;Riccardi et al. 2020).Therefore, the existence of S. woodiana in these rivers should be further investigated and confirmed with molecular tools.
In this contribution, the first established population of S. woodiana is reported from Greece, and its population status is assessed and compared with the native mussel populations.

Materials and methods
The Chinese pond mussel (Figure 1) was found in Lake Pamvotida (= Pamvotis or Ioannina) along with Anodonta cygnea during a mussel field expedition on 20 October 2020.Formed during the late Miocene to the Pliocene period, Lake Pamvotida is located in northwestern Greece (Figure 2) on the western flank of the Pindus Mountain range, at 470 m. a.s.l.It is a shallow lake with a mean water depth of 4-5 m and occupies an area of 22.8 km 2 at the lowest point of a 475 km 2 closed basin, in a region of predominantly limestone geology with a strong karstic character.The lake has been recognized as globally significant for its endemic biodiversity and is listed in the Special Areas of Conservation (SAC) of the Natura 2000 network (Gkenas et al. 2012).However, over recent decades, Lake Pamvotida has suffered significant land-use changes, hydrological alterations, and increased pollution, going from eutrophic to hypereutrophic status (Kagalou et al. 2008), resulting in the degradation of its ecological condition (Alexakis et al. 2013).
To assess the population status of S. woodiana in the lake, and whether it has affected the native population of A. cygnea, a 30-min sampling effort was conducted at 5 locations situated around the perimeter of the lake (Figures 2, 3).Sampling was performed by wading in the lake (wearing dry-suit apparatus) by three authors of this contribution.Physicochemical parameters of the sampling locations were measured with a HI9829 Multiparameter Meter from Hanna Instruments Ltd.Due to the taxonomic uncertainties involving the Anodontini tribe, the identity of both bivalve species was verified by genetic analysis.
Whole genomic DNA was extracted from small tissue pieces preserved in 96% ethanol, using a standard high-salt protocol (Sambrook et al. 1989), from eight S. woodiana and six A. cygnea individuals.A fragment of the cytochrome oxidase c subunit I gene (COI) was amplified by PCR using universal primer modified versions, i.e., LCO22me2 and HCO700dy2 (Walker et al. 2006(Walker et al. , 2007)).PCR conditions are described in Froufe et al. (2014) with an annealing temperature of 50 °C.Amplified DNA templates were purified and sequenced (forward and reverse) by the commercial company Macrogen, using the same primers.The sequences obtained were  compared with sequences present in GenBank using BLAST (Altschup et al. 1990) and with the BOLD database (Ratnasingham and Hebert 2007).No indels and no stop codons were observed, after translating all sequences to amino acids.The identification of the newly sequenced individuals (a single haplotype: GenBank SUBMITTED) as S. woodiana was confirmed by both BOLD (99.8% match) and BLAST (99.6%). A. cygnea specimens presented three haplotypes and were also confirmed in BOLD (97.0-97.3%)and BLAST (97.0-97.3%).

Results and discussion
The physicochemical characteristics of sampling localities are presented in Table 1.Molecular analysis confirmed the presence of both S. woodiana  Reischütz and Reischütz (2002) and Frogley and Preece (2004) also reported Anodonta anatina from the lake, but we did not find this species during our survey, thus assuming that A. anatina is most possibly a misidentification.However, alternative explanations for our failure in detecting the presence of A. anatina in our survey can be attributed to the limited number of sequenced specimens (and difficulty in separating them from A. cygnea) or to its rare or fragmented presence in the lake.Sinanodonta woodiana has established an increasing population since its invasion into the lake, probably sometime after 2006 when it was first discovered in a fish pond connected to the lake.The highest relative abundances were found in sites 1, 2 and 3 (Figure 2, Table 2) where it has almost displaced the native A. cygnea (100% in site 1; 93% and 95% in sites 2 and 3, respectively).We tentatively presume that the distribution patterns show that the introduction of S. woodiana may have been on the northeast side of the lake along with vector fish from the fishpond.The abundance of S. woodiana is such, that in our opinion, this invasive alien species may eliminate A. cygnea from Lake Pamvotida in the near future.However, we cannot know whether A. cygnea populations have declined due to the significant pollution and other anthropogenic pressures that the lake has endured in the last decades, and S. woodiana has just taken the "empty" niche, or due to competition with S. woodiana.Several studies have shown that S. woodiana can have a direct impact on native mussel populations.For example, S. woodiana has almost completely replaced A. anatina in some channels in North Italy (Fabbri and Landi 1999) and Venetian area (Niero 2003).Identical trends have also been documented in Lake Balaton (Benkő-Kiss et al. 2013) and in several locations of the Danube in Serbia (Paunovic et al. 2006), where the Chinese pond mussel has rapidly reached high abundance and biomass and has significantly replaced both A. anatina and A. cygnea populations.Furthermore, recent studies by Urbańska et al. (2019) and Benedict and Geist (2021) showed that S. woodiana can expand into colder regions and establish new populations.
As fishponds have been the main habitat of S. woodiana in many invaded areas and are often sources for its further dispersal, as has been the case for Pamvotida Lake, it is critical to adopt programs and measures on fish farms to prevent dispersal from the source (rather from natural environments which is far more difficult).In addition, awareness should be brought to both the local authorities responsible for water and biodiversity management and to fish farmers to protect their fish stocks from infestation by S. woodiana glochidia, thus preventing both losses in fish yield and further expansion to other water bodies.
Before the current study, the known genetic diversity of A. cygnea was extremely low and populations across most of the distribution presented very low genetic differentiation (less than 1%) (Riccardi et al. 2020).The sequenced specimens from Lake Pamvotida represent a unique and divergent population that should be protected because it greatly expands the species' genetic pool.We recommend that a wide-ranging survey and periodical monitoring of this species should be implemented as a first step to understand the population trends in Lake Pamvotida.More thorough studies should be undertaken to understand its habitat requirements and relevant biological characteristics, e.g.growth, lifespan, host fish range and fertility.This might help in the implementation of future conservation measures and, therefore, help this native species survive in the new conditions created by the Chinese pond mussel invasion.

Figure 2 .
Figure 2. Map showing the five sampling sites of S. woodiana and A. cygnea in Lake Pamvotida (Greece) and their relative abundances.

Table 1 .
Mean physicochemical parameters of S. woodiana and A. cygnea collection sites.
and A. cygnea in the lake.All eight S. woodiana individuals share the same haplotype as in the invaded range of S. woodiana in Europe (e.g.Italy, France, Spain, Poland and Russia; Lopes-Lima et al. 2020), while the three distinct haplotypes of A. cygnea are an unreported unique lineage of Anodonta cygnea with less than 97.3% of sequence identity in BLAST, GenBank database.