Distribution patterns of two co-existing oyster species in the northern Adriatic Sea: The native European flat oyster Ostrea edulis and the non-native Pacific oyster Magallana gigas

Ecological Indicators 113 (2020) 106233

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Original Articles

Distribution patterns of two co-existing oyster species in the northern Adriatic Sea: The native European flat oyster Ostrea edulis and the nonnative Pacific oyster Magallana gigas

T

Nika Stagličića, Tanja Šegvić-Bubića, , Daria Ezgeta-Balića, Dubravka Bojanić Varezića, Leon Grubišića, Luka Žuvića, Yaping Linb,c, Elizabeta Briskib ⁎

a b c

Institute of Oceanography & Fisheries, Šetalište Ivana Meštrovića 63, 21000 Split, Croatia GEOMAR-Helmholtz Centre for Ocean Research Kiel, Düsternbrooker Weg 20, 24105 Kiel, Germany Chinese Academy of Sciences, Research Center for Eco-Environmental Sciences, 18 Shuangqing Rd., Haidian District, Beijing 100085, China

ARTICLE INFO

ABSTRACT

Keywords: Crassostrea gigas Mediterranean Sea Non-indigenous species MPA Size structure Oyster population density

The Pacific oyster Magallana gigas, globally one of the most translocated marine species, has never been commercially farmed in any part of the Croatian eastern Adriatic Sea, where the native flat oyster Ostrea edulis is the only cultured oyster species. The Pacific oyster has, however, established populations on the west coast of the Istria peninsula, in the northeast Adriatic Sea. Current distribution, abundance and size structure of the flat and Pacific oysters were studied there along spatial and depth gradients in natural and artificial habitats to assess their potential for coexistence. This is the first quantitative assessment of native and non-native oyster populations on a Mediterranean-wide scale providing a baseline for determining future changes in their distribution. Both species were omnipresent, with the Pacific oyster displaying a more pronounced variability in abundance and size in relation to survey regions and depths. The population density of flat oysters was low, generally less than 1 individual/m2, with no difference among regions. The density of the Pacific oyster was significantly higher, being on average 5 individuals/m2. Dense, reef-forming aggregations of Pacific oysters were contained in Lim Bay, a nationally-important shellfish aquaculture area, where its mean density was 107 individuals/m2. Along the open coastline its densities were considerably lower and followed a latitudinal gradient. The observed abundance and size distribution patterns of the Pacific oyster suggest that Lim Bay was the introduction point, with feral settlement likely originating from short-lived experimental aquaculture trials and subsequently dispersing by prevailing local currents. The flat oysters were larger in size and settled at different depths compared to the Pacific oysters. The vertical range of Pacific oysters was mostly contained in the tidal zone, while flat oysters were present in the subtidal exclusively. Such spatial partitioning likely resulted from introduced oysters occupying a vacant ecological niche and not due to interspecific competitive exclusion. Habitat type had a strong effect on proliferation of Pacific oysters. Artificial hard substrata harboured more abundant and larger Pacific oysters than the natural rocky shore habitat. The possibility of multiple local introduction pathways in ports and marinas is also discussed. Currently, there seems to be no spatial competition between the two oyster species, but as it is hard to predict future possible impacts of non-native species, we strongly suggest regular monitoring of the Pacific oyster, prioritising sensitive and protected areas as well as areas at the edge of its distribution.

1. Introduction Oyster species are typically found in enclosed, wave-sheltered coastal and estuarine areas, settled on various types of hard intertidal and shallow subtidal substrates (Beck et al., 2011; Herbert et al., 2016). Due to their long historical importance as a food source (Günther, 1897; MacKenzie et al., 1997; Voultsiadou et al., 2010), oysters are nowadays

⁎

one of the most translocated marine species globally (Ruesink et al., 2005). The native region of the European flat oyster Ostrea edulis (Linnaeus, 1758) stretches along the European Atlantic coast from Norway to Morocco, throughout the Mediterranean, as well as the Black Sea (FAO 2004-2019). Natural beds and reefs of the flat oyster were once abundant and widespread, commonly occurring in the intertidal and subtidal coastal waters, but also in deeper waters and offshore

Corresponding author at: Institute of Oceanography & Fisheries, Šetalište Ivana Meštrovića 63, 21000 Split, Croatia. E-mail address: [email protected] (T. Šegvić-Bubić).

https://doi.org/10.1016/j.ecolind.2020.106233 Received 16 September 2019; Received in revised form 15 February 2020; Accepted 18 February 2020 1470-160X/ © 2020 Elsevier Ltd. All rights reserved.

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Fig. 1. Sampling regions and locations of flat and Pacific oysters along the western Istrian coast of the Adriatic Sea. Natural and artificial habitats are presented as full and empty dots, respectively.

down to 50–80 m depth (Pogoda, 2019). Large offshore oyster grounds were particularly extensive along the eastern Atlantic, in the North Sea, the English Channel and the Irish Sea, and were heavily exploited, especially from the industrialization onwards, owing to high demand for this delicacy (Pogoda, 2019). During the 20th century, overfishing combined with habitat degradation, water pollution and disease outbreaks resulted in dramatic declines its populations throughout its range (Airoldi and Beck, 2007; Beck et al., 2011; Helmer et al., 2019) Diseases (e.g., bonamiosis, marteiliosis) also severely affected European aquaculture sites (FAO 2004-2019). To compensate for the losses of the flat oysters, the non-native Pacific oyster Magallana gigas (formerly Crassostrea gigas; Thunberg, 1793), originating from Japan and SE Asia (FAO 2005-2019), was purposefully introduced across Europe and the Mediterranean Sea,

mostly in the second half of the 20th century (Wehrmann et al., 2000; Miossec et al., 2009). Due to its favourable culturing traits such as fast growth rate, high reproductive performance, adaptive eco-physiology and low disease susceptibility (Kennedy and Roberts, 1999), the Pacific oyster has also been introduced in many other parts of the world in attempts to revitalise the declining native oyster industries (Ruesink et al., 2005). Nowadays, the Pacific oyster is the most commonly farmed oyster, with near-global distribution (Kennedy and Roberts, 1999; Ruesink et al., 2005). Escaping from aquaculture sites, the species has established wild populations in many regions where it was introduced (Ruesink et al., 2005; Herbert et al., 2016). In Northern Europe, spreading outside aquaculture areas was initially considered unlikely due to the subtropical origin of the Pacific oyster (Reise, 1998; Drinkwaard, 1999; Diederich et al., 2005; Troost, 2010). Nevertheless, 2

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the species has been steadily expanding its range, with current populations established as far north as the southern Norwegian coast (60°N) (Wrange et al., 2010; Dolmer et al., 2014). In the Mediterranean Sea, the introduced Pacific oyster has spread extensively as conditions throughout the basin are well within the range of ecological requirements of the species (MacKenzie et al., 1997). Regarding the Adriatic Sea, a northwest arm of the Mediterranean, records of feral Pacific oyster populations come mainly from the northern part where most traditional lagoon shellfish cultivation areas occur (Mattei and Pellizzato, 1997; Occhipinti-Ambrogi, 2002). In Italy, both the native flat oyster and non-native Pacific oyster are cultured in shallow, brackish lagoons from the Po estuary to Trieste (Mattei and Pellizzato, 1997). Abundant feral Pacific oysters were sighted all over the Gulf of Trieste and neighbouring areas (Crocetta, 2011) and have also colonised the entire Slovenian coast (Lipej et al., 2012). The Pacific oyster has never been commercially farmed in the Croatian part of the Adriatic Sea where the native flat oyster is the only oyster species cultured. However, in the early 1970s, feral Pacific oysters were found in Lim Bay, a shellfish aquaculture area in the northern Adriatic Sea (Filić and Krajnović-Ozretić, 1978). The introduction may not have been deliberate, and presumably, short-lived experimental aquaculture trials resulted in Pacific oysters colonising suitable habitats in Lim Bay. After this initial observation of feral Pacific oysters in Lim Bay, their presence in Croatian waters has mostly gone unnoticed until recently. Šegvić-Bubić et al. (2016) confirmed its presence by molecular techniques, and Ezgeta-Balić et al. (2019) conducted a large-scale observational survey along the eastern Adriatic coast documenting the presence of established wild populations of the species only in the northern Adriatic, specifically the western side of the Istria Peninsula. Continuing on previous observations, the affected coastline of Istria was put under focus in this survey with the aim of investigating in detail the distribution, abundance and size structure of established Pacific oyster populations along spatial and depth gradients, and comparing them with the same population parameters assessed for native flat oysters in order to evaluate the potential for coexistence of the two oyster species under environmental conditions in the Northern Adriatic. Additionally, differences in colonisation of natural versus artificial hard substrates by Pacific oysters were also tested. Given the high adaptability and even invasiveness of the Pacific oyster in areas outside its natural range, demonstrated both globally (Ruesink et al., 2005; Troost, 2010; Herbert et al., 2016) and regionally (Crocetta, 2011), this survey provides insights regarding concerns about its impact on local ecosystems, particularly on whether the Pacific oyster may outcompete the already depleted native oyster analogue. Finally, as this survey is the first quantitative assessment of introduced and native oyster populations on a region wide scale, it establishes a reference point for monitoring future population developments.

geomorphologic value. Owing to its particular features (i.e., sheltered, brackish and highly-productive waters), Lim Bay is one of the main shellfish production areas in Croatia (Benović, 1997). The Brijuni Islands are protected as a national park and there the spread of any nonnative species is of particular concern. In each region, four locations, separated by hundreds to thousands of metres, were randomly selected (Fig. 1), taking care they meet known ecological criteria of oysters and are similar with regards to the configuration of the shoreline, the availability of hard bottom substrate and the degree of wave exposure. The sampling methodology encompassed five replicate strip transects at each location, approximately 30–50 m apart and placed perpendicularly to the shore from the high tide level down to a depth of 6 m. The chosen depth limit was based on preliminary observations showing that hard substrata suitable for oyster settlement drops markedly beyond that and soft sediments become much more common. Both flat and Pacific oysters were recorded in situ by a pair of SCUBA divers in the subtidal zone and surface observers in the intertidal zone, counting the number of oysters up to a distance of 1 m on either side of the transect line and measuring their shell length to the nearest mm with a Vernier calliper. Oyster abundance was standardised to the number of individual/m2 for every 1-m increment of depth along each transect using the total number of individuals recorded along the transect divided by the actual area surveyed on the transect. When high numbers of oysters were encountered, an exact count was taken and random subsamples of 50 oysters per species were measured for length distribution. In order to compare the colonisation of natural and artificial habitats by Pacific oysters, four locations along natural rocky shores at the Brijuni Islands and two artificial locations in the port of Veliki Brijun, the main island of the archipelago, were surveyed. Brijuni National Park is optimal for testing the settlement on natural versus artificial hard substrata as except for the alteration of the shoreline for port construction, the environment is mostly undisturbed by other anthropogenic impacts common in more heavily urbanised ports (FatovićFerenčić, 2006). 2.2. Molecular species identification and DNA analyses During field observations, species were identified morphologically by criteria described in Poppe and Goto (2000). However, to confirm accuracy of morphological identification, seven to nine individuals per region of morphologically-identified Pacific oyster from the intertidal zone were chiselled from the substrate and collected for molecular identification. Specimens from the Brijuni Islands were not collected due to restrictions on destructive sampling. DNA extraction was conducted using the DNeasy 96 Tissue Kit (Qiagen) according to the manufacturer’s instructions. The large 16S ribosomal subunit was amplified using the 16Sar and 16Sbr primers (Kessing et al., 1989) following the PCR protocol reported in Šegvić-Bubić et al. (2016). PCR products were submitted for purification and direct sequencing to Macrogen Inc., Korea. For sequence identification, the program BLASTn (NCBI, available online) was used. Sequence alignment was carried out using the ClustalW tool in Mega7 (Kumar et al., 2016), while DnaSP 5.19 software (Librado and Rozas, 2009) was used to calculate haplotype diversity. The haplotype network was constructed in PopART by the median-joining method (http://popart.otago.ac.nz).

2. Materials and methods 2.1. Field survey and sample collection The study area of the western Istrian coast defines the eastern border of the northern Adriatic Sea, which is the northernmost biogeographic sector of the whole Mediterranean basin (Fig. 1) (Bianchi and Morri, 2000). Gently-sloping limestone rocky reefs dominate the coastline and shallow subtidal areas along the west side of the Istrian Peninsula. The tide in the area follows a semi-diurnal cycle with low amplitude of about 50 cm (Malačič et al., 2000). Surveys and sampling were conducted in May 2018 in four discrete regions: i) north open coastline ii) Lim Bay iii) south open coastline and iv) the Brijuni Islands. Region represents the largest spatial scale examined, separated by tens of kilometres (Fig. 1). Lim Bay is approximately 13 km long, narrow canyon-like embayment, which is a sunken karst valley rich in underground freshwater springs and designated as a Significant Landscape and a Special Marine Reserve because of its

2.3. Statistical analyses To test whether the abundance of flat and Pacific oysters differed along spatial and depth gradients, a four-way univariate permutational analysis of variance (PERMANOVA; Anderson, 2001) was conducted. Species (two levels: the flat oyster and the Pacific oyster), Region (four levels: north open coastline, Lim Bay, south open coastline, the Brijuni Islands) and Depth (seven levels: from the upper intertidal zone (0 m depth) down to 6 m depth in 1-m depth intervals) were fixed and 3

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orthogonal factors, while Location (four levels: four randomly selected locations per region) was a random factor nested in Region. Differences in the mean size of oysters were tested by a two-way univariate PERMANOVA, with both Species (two levels: the flat oyster and the Pacific oyster) and Region (four levels: north open coastline, Lim Bay, south open coastline, the Brijuni Islands) being fixed and orthogonal factors. Size was compared only at the scale of Region because the flat oyster was too rare to include smaller spatial scales and depths in the analysis. Size frequency data were analysed using the k-sample Anderson-Darling (AD) test, for each oyster species separately, to examine whether the oysters have common population size structure between sampling regions. Patterns of Pacific oyster colonisation on different types of habitat were investigated by applying an asymmetrical two-way univariate PERMANOVA, with Habitat (two levels: natural and artificial) as a fixed factor and Location (two levels for artificial and four for natural habitats) as a random factor nested within Habitat, both on abundance and shell length data. Size frequency distributions of Pacific oysters were compared between the habitats using a Kolomogorov-Smirnov twosample test. PERMANOVAs for all of the aforementioned analyses were based on Euclidian distance measure, and were performed using PRIMER-E software (Clarke and Gorley, 2006) with the add-on package PERMANOVA+ (Anderson et al., 2008). Significant terms of the main PERMANOVAs were further examined by appropriate post-hoc pair-wise comparisons. Statistical analyses of size frequency data were performed using the ad.test function in the kSamples package in R 3.6.0 (R Core Team, 2019).

three regions sampled (22 individuals in the north and south open coastlines and Lim Bay, Fig. 2) and corresponded to the COI haplotype C (Boudry et al., 2003), which is the most common haplotype of the Pacific oyster in non-native regions (Huvet et al., 2000). Haplotype 2 was found only in the north open coastline and haplotype 3 only in Lim Bay. Distribution of oyster abundances differed significantly depending on the species, sampling region and depth (Table 1, significant Species × Region × Depth interaction, Fig. 3). Most of the spatial variation occurred along a north-south gradient of sampling regions (scale of tens of kilometres), with very little additional variation seen at smaller spatial scales (locations and transects, scale of tens to thousands of metres). Both species co-existed in all investigated regions and locations, but were spatially segregated in relation to depth. No flat oyster specimens were observed in the intertidal zone, whereas the majority of Pacific oysters were found there. Below the intertidal zone, the Pacific oyster was only present sporadically in the shallowest subtidal zone (i.e. the first meter of depth). At the depth of one metre, where both species were present, their abundance was similar in all sampling regions along the north-south gradient (mean density of 0.4 ± 0.1 and 0.5 ± 0.1 individual/m2 for the flat and Pacific oyster, respectively; Table 1, pairwise for Species). Population density of flat oysters was low and generally < 1 individual/m2, with a mean density of 0.1 ± 0.02 individuals/m2 and a maximum of 4 individuals/m2. There was no significant change in flat oyster densities along the spatial north-south gradient of sampling regions (Table 1, pair-wise for Region). Distribution according to depth differed significantly (Table 1, pair-wise for Depth), with the species being significantly more abundant in the shallowest subtidal zone up to 1 m depth. In contrast, the mean density of the Pacific oyster (5.1 ± 1.0 individuals/m2) was significantly higher than that of the flat oyster, and its distribution significantly differed on the regional scale (Table 1, pair-wise for Region). It was particularly abundant and widespread in Lim Bay where it often formed dense clumps. Its highest density in this region was 248.3 individuals/m2, with a mean density of 107.5 ± 13.6 individuals/m2. In the other regions, its density dropped markedly and rarely exceeded 25 individuals/m2; slightly higher densities of the Pacific oyster were found in the north rather than the south

3. Results Overall, 643 individuals of flat oyster and 6991 of Pacific oyster were observed over 6465 m2 of seafloor along 80 transects surveyed. Molecular identification confirmed morphological identification, with all 26 individuals being identified as the Pacific oyster (GenBank accession numbers MT090161-MT090163). Three haplotypes of the Pacific oyster, with low haplotype diversity (h = 0.16), were determined. The most abundant haplotype (haplotype 1) was found in all

Fig. 2. 16S haplotype network based on median-joining analysis (A) and geographical distribution of haplotypes (B) for the Pacific oyster. Short black bars on panel A correspond to the number of mutation steps between haplotypes. Pie charts on panel B display haplotype frequencies in each locality. 4

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Table 1 Four-way PERMANOVA testing for differences in the abundance of oyster species along the western Istrian coast. Species (two levels: the flat oyster and the Pacific oyster), Region (four levels: north open coastline, Lim Bay, south open coastline, the Brijuni Islands) and Depth (seven levels: from upper intertidal zone (0 m depth) down to 6 m depth in 1-m intervals) were fixed and orthogonal factors, while Location (four levels: four randomly selected locations per region) was a random factor nested in Region. Source of variability

df

MS

F

Species Region Depth Location = Location(Region) Species × Regiion Species × Depth Reregion × Deepth Species × Loocation(Region) Location(Region) × Depth Species × Region × Depth Species × Location(Region) Residual

1 3 6 12 3 6 18 12 72 18 72 896

7026.8 3525.1 7090 97.6 3472 7210.9 3369.5 94.8 96.6 3377.1 97.2 76.2

74.1*** 36.1*** 73.4*** 1.3 36.7*** 74.2*** 34.9*** 1.2 1.3 34.7*** 1.2

Pair wise for Species × Region × Depth Species, 1 m depth: the Region the the the Depth the the the

flat oyster = the Pacific oyster: Pacific oyster: 0 m: Pacific oyster: 1 m: flat oyster: all depths: Pacific oyster: 0 m > 1 m: Pacific oyster: 0 m = 1 m: flat oyster, in all regions:

in all four regions (north and south open coastlines, Lim Bay and Brijuni Islands Lim Bay > north open coastline > south open coastline > the Brijuni Islands Lim Bay > north open coastline = south open coastline = the Brijuni Islands Lim Bay = north open coastline = south open coastline = the Brijuni Islands north open coastline, Lim Bay south open coastline, the Brijuni Islands 1m > 2m=3m=4m=5m=6m > 0m

*** P ≤ 0.001.

open coastline, with the lowest density in the Brijuni Islands (Table 1; Fig. 3). In regions with denser Pacific oyster populations, such as Lim Bay and the north open coastline, the species was significantly more abundant in the intertidal zone than at a depth of 1 m. No difference in species abundance in the two depth zones was observed in regions where density of Pacific oysters was relatively low, such as the south open coastline and the Brijuni Islands (Table 1; Fig. 3). The flat oyster was significantly bigger than the Pacific oyster in all four regions surveyed (Table 2; Fig. 4). The smallest and largest flat oysters were 14 and 123 mm, respectively, with a mean shell length of 58.0 ± 0.8 mm, while those of the Pacific oyster were 11 and 89 mm, with a mean length of 43.4 ± 0.3 mm. The difference in size between larger flat oysters and smaller Pacific oysters was least pronounced in Lim Bay where the Pacific oyster was larger on average (Fig. 4). While the Pacific oyster was significantly larger in Lim Bay than in the other three regions, the largest size of flat oyster, on average, was found in the Brijuni Islands followed by the south open coastline (Table 2, pairwise for Region). For both species, significant differences were found also regarding their size frequency distribution among the regions (Anderson-Darling test for the flat oyster: AD = 20.1, p < 0.001; for the Pacific oyster: AD = 139.1, p < 0.001). The size distribution of the Pacific oyster differed between Lim Bay and the other three regions. In Lim Bay, where the greatest abundance of this species was observed, populations had a wider size range. Smaller size ranges and smaller proportions of larger individuals (> 60 mm shell length) were observed in the north open coastline, south open coastline and Brijuni Islands. The flat oyster showed an opposing size distribution pattern, with Lim Bay having a size structure similar to the north and south open coastlines; while in the Brijuni Islands a polymodal size structure with a good representation of large individuals, typical of protected areas, was present. The comparison of colonisation of natural versus artificial habitats by the Pacific oyster revealed that both the abundance and size of the species were significantly higher and larger in artificial habitats (Table 3; Figs. 5 and 6). The mean density of the species in artificial habitats was 50.1 ± 5.7 individuals/m2, while in natural habitats it was 1.31 ± 0.6 individuals/m2; the mean shell lengths were 45.5 ± 0.5 mm and 39.9 ± 1.5 mm, respectively. The size frequency distributions were also different between the two types of habitats

(Kolomogorov-Smirnov test: KS = 2.3, p < 0.001). Smaller size classes, up to 30 mm shell length, were more represented in natural habitats and larger size classes (45–60 mm) in artificial habitats. The largest individuals, with shell length > 75 mm, were observed only in artificial habitats. 4. Discussion 4.1. Competition between flat and Pacific oysters Both oyster species were omnipresent along the western Istrian coastline in the north Adriatic Sea, with spatially-variable abundance and size in relation to survey regions and depths. Variability was more pronounced for the Pacific oyster which was highly dominant in Lim Bay where it often formed spatially-complex three-dimensional reef structures, while it had rather low densities in regions of open coastline. Populations of flat oysters were sparse and had similar abundances in all sampling regions, corresponding to low densities recorded elsewhere in the Adriatic (Peharda, 2000, 2003; Lotze et al., 2011; Mautner et al., 2018), Mediterranean (Cano and Rocamora, 1996) and north-east Atlantic (Kennedy and Roberts, 1999; Thorngren et al., 2017; Helmer et al., 2019; Pogoda, 2019) confirming the current general rarity of the species. Although once widespread and plentiful, flat oyster beds and reefs are now practically non-existent (Riesen and Reise, 1982; Beck et al., 2011; Lotze et al., 2011; Thurstan et al., 2013; Mautner et al., 2018; Helmer et al., 2019; Pogoda, 2019). Unlike Pacific oysters, rarely anywhere today does the flat oyster reach densities of > 5 individuals/ m2 categorised as shellfish reef (Christianen et al., 2018). In addition to the differences in population densities, depth segregation of Pacific and flat oysters is another conspicuous field characteristic observed across the study area of the west Istria coast. The vertical range of Pacific oysters is mostly contained in the tidal zone, while flat oysters are present in the subtidal exclusively. This is consistent with many previous studies reporting subtidal preferences of flat oysters and predominantly intertidal occurrence of Pacific oysters (Reise, 1998; Laugen et al., 2015; Herbert et al., 2016 and references therein). However, the view of no overlap in flat and Pacific oyster distribution is challenged by recent surveys that found the two oyster species co-occurring, both intertidally (Ireland, Zwerschke et al., 2017) 5

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Fig. 3. Abundance of flat and Pacific oysters in four different regions along the western Istrian coast of the Adriatic Sea at different water depths.

and subtidally (Dutch North Sea, Christianen et al., 2018). At present, knowledge of the extent of habitat overlap is limited as ecological distribution patterns of species is known to considerably vary geographically, and most studies are from Europe and have focused on

different intertidal habitats, whereas sublittoral ones are largely underrepresented (Herbert et al., 2016). Observations of subtidal Pacific oyster settlements (Fey et al., 2010; Hollander et al., 2015; Mortensen et al., 2017; Smyth et al., 2017) and

Table 2 Two-way univariate PERMANOVA testing for differences in the size of oysters along the western Istrian coast, with Species (two levels: the flat oyster and the Pacific oyster) and Region (four levels: north open coastline, Lim Bay, south open coastline, the Brijuni Islands) being fixed and orthogonal factors. Source of variability

df

MS

F

Species Region Species × Region Residual

1 3 3 2995

93,794 3616.9 6362.7 209.7

447.2 *** 17.2 *** 30.3 ***

Pair wise for Species × Region Species: the flat oyster > the Pacific oyster: Region: the flat oyster: the Pacific oyster:

in all four regions (north and south open coastlines, Lim Bay and Brijuni Islands) the Brijuni Islands = south open coastilne > north open coastline = Lim Bay Lim Bay > south open coastline = the Brijuni Islands = north open coastline

*** P ≤ 0.001. 6

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Fig. 4. Mean shell length (A) and shell length frequencies (B) of the flat and Pacific oysters in four different regions (N = north open coastline, L = Lim Bay, S = south open coastline, BI = the Brijuni Islands) along the western Istrian coast of the Adriatic Sea.

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to the spread of Pacific oysters when devastated intertidal flat oyster beds in the Wadden Sea were replaced by mussel banks (Riesen and Reise, 1982).

Table 3 Results of two-way univariate PERMANOVA based on Euclidian distance for effects of habitat (natural vs. artificial) on abundance and size of the Pacific oyster. Source of variability

Abundance df

Habitat Location Residual

1 4 24

MS 15,836 255.4 85.2

Size F 62.0 2.9

**

4.2. Size of the Pacific oyster

df

MS

F

1 4 556

1891.8 233.1 153.9

10.2 1.5

The Pacific oyster is a fast-growing, long-lived species capable of attaining sizes of over 300 mm shell length (Reise et al., 2017). Its highest growth rate is in the first year after settlement, when the species typically reaches 20–40 mm (Schmidt et al., 2008; Fey et al., 2010; Reise et al., 2017). In our study, the Pacific oyster was characterised by populations mainly composed of small to medium-sized individuals and most likely had recent recruitment events because Pacific oysters ≤ 40 mm in length were regularly encountered at all surveyed regions and comprised almost 44% of the overall sample. The largest Pacific oysters found did not exceed 90 mm shell length giving a narrow size distribution for the Adriatic compared to recent studies in other European regions, for example Scotland (Cook et al., 2014), Ireland (Guy and Roberts, 2010; Kochmann et al., 2013; Zwerschke et al., 2017) and the Wadden Sea (Fey at al., 2010; Reise et al., 2017) where the largest Pacific oysters are regularly over 140 mm shell length. At the moment, it is unclear why Pacific oysters tend to be smaller in the Adriatic where ecological conditions for their recruitment (Ezgeta-Balić et al., 2020) and growth (MacKenzie et al., 1997) are favourable. Several studies conducted on the cultured Pacific oyster in the Mediterranean Sea and South Africa determined that environmental factors, such as food availability, fouling, salinity and temperature were important for the growth rate of the species, but none of them reported the average size of the populations (Sara and Mazzola, 1997; Gangnery et al., 2003; Pieterse et al., 2012). Consequently, additional studies are necessary to determine the factors affecting the growth of the Pacific oyster along the western Istrian coast as well as globally, so as to distinguish if environmental conditions prevent the Pacific oyster from reaching bigger sizes in some areas, or if there is an inherent size difference among populations established in different areas of Europe and globally.

**

** P < 0.01.

intertidal native oysters (Kennedy and Roberts, 1999; Smyth and Roberts, 2010; Smyth et al., 2017) are as yet few. In the northern Adriatic, besides the shallow subtidal, Pacific oysters seem to be absent thus far also from deeper waters (20–40 m) as the only oyster species found in beam trawl catches is the flat oyster (Ezgeta-Balić et al., 2019). In their native range, Pacific oysters are commonly found subtidally, to a depth of about 40 m (FAO 2005-2019; Poppe and Goto, 2000). Further colonisation of the subtidal and extensions of depth range are expected in regions where they are introduced (Diederich et al., 2005; Smaal et al., 2009). On the other hand, flat oysters, which are currently considerably contained in subtidal habitats, most likely were common in the intertidal zone in the past (Fey et al., 2010; Lotze et al., 2011; Reise et al, 2017; Helmer et al., 2019). Unlike records of subtidal flat oyster reef devastation (Beck et al., 2011; Herbert et al., 2016), the history of change in the intertidal flat oyster distribution is largely undocumented, and suffers from shifting baseline syndrome (Beck et al., 2011; Plumeridge and Roberts, 2017). Shifting baseline or “environmental generational amnesia” (Pogoda, 2019 and references therein), the tendency for younger generations to lose the past as a benchmark for interpreting the present, can be illustrated also for Lim Bay area. Only a few old, retired shellfish farmers remember past conditions of flat oyster common and abundant existence along the rocky intertidal shoreline, while the younger generation is mostly unaware of the loss (Šošić, pers. comm.). Ease of access to the intertidal zone facilitated the eradication of flat oysters, long before the harvesting of less-accessible subtidal habitats and readily-available documentation or scientific studies (FariñasFranco et al., 2018). Human collection and fishing are the most likely factors shaping the current distribution of flat oysters, with a notable absence from easily-accessible intertidal zones (Smyth et al., 2009). The introduction of Pacific oysters likely followed, rather than caused, the decline of native flat oyster populations (Occhipinti-Ambrogi, 2002) and their displacement from the intertidal zone. To summarise, at present in the Adriatic, the native flat and introduced Pacific oysters coexist with distributions that, to a large extent, do not overlap. Their spatial partitioning with regards to depth likely resulted from introduced oysters filling an empty ecological niche and not due to interspecific competitive exclusion. A similar regime shift occurred prior

4.3. Possible vectors of Pacific oyster introduction and spread The aquaculture and shipping industries are the two strongest human-mediated vectors for introduction of species to marine habitats (Molnar et al., 2008). On the western Istrian coast, an order of magnitude higher abundance together with the different population size structure found in Lim Bay, suggest that Lim Bay is the introduction point. Current dense wild settlements probably originated from shortlived aquaculture attempts. In most cases establishment of feral nonindigenous Pacific oyster populations is facilitated by repeated introductions (Herbert et al., 2016; Reise et al., 2017). Nevertheless, the species can be introduced after a singular event. Besides the Adriatic, abandoned aquaculture trials and one-time

Fig. 5. Abundance of the Pacific oyster on natural and artificial habitats in the Brijuni Islands. 8

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Fig. 6. Shell length (A) and shell length frequencies (B) of the Pacific oyster on natural and artificial habitats in the Brijuni Islands.

recruitments have also been attributed as sources of feral Pacific oyster populations along the coasts of Argentina (Escapa et al., 2004), Sweden, Denmark (Laugen et al., 2015) and Scotland (Smith et al., 2015). The development and containment of dense Pacific oyster aggregations within Lim Bay is supported by its features—highly favorable habitat characteristics in an enclosed body of water, thus ensuring a high degree of larval retention (Benović, 1997). The occurrence of the Pacific oyster in the Port of Pula (Ezgeta-Balić et al., 2019), situated in the southern area of the western Istrian coast, suggest that commercial shipping may also be a vector because whole communities from viruses and bacteria to macroinvertebrates are found inside ballast tanks and attached to ship hulls (Sylvester et al., 2011; Briski et al., 2012, 2013, 2014). Spatial distribution patterns of Pacific oysters along open coastlines showed a latitudinal gradient with higher abundances in the north than in the south, and the lowest abundance in the Brijuni islands, likely reflecting dispersal via prevailing local currents. The North Adriatic surface circulation is primarily thermohaline and cyclonic with one of the main branches flowing northwards along the western Istrian coast (McKinney, 2007). Hence, local hydrodynamic conditions typically support larval dispersal northwards. However, on occasion maestrale, a strong north-west summer wind, or the Istrian Coastal Countercurrent, a departure from the general cyclonic gyre (Supić et al., 2000), may enhance the transfer of oyster larvae in a southerly direction. Current and wind-induced water-borne transport has been demonstrated as a key factor regulating the dispersal of a number of benthic species, oysters included (Smyth et al., 2016 and references therein). To conclusively resolve the question of origin and connectivity of established Pacific oyster populations in the Adriatic, future studies using

population genetic approaches are needed. 4.4. Establishment of the Pacific oyster on natural versus artificial habitats Anthropogenic changes to the natural shoreline and its replacement with artificial surfaces such as seawalls and pilings often modify native species assemblages and create windows of opportunity for non-native species to colonise habitat that might have otherwise been inaccessible (Bulleri and Chapman, 2004; Lam et al., 2009; Dafforn et al., 2012). Intrinsic differences in physical features, such as topography and orientation, as well as different hydrodynamic regimes are some of the factors involved in explaining the distinct recruitment and assemblage development in natural versus artificial habitats (Bulleri, 2005; Bulleri et al., 2005). In the Brijuni Islands, where the distribution of Pacific oysters in different habitat types was surveyed, much more abundant and larger individuals were found attached on artificial as compared to natural substrata. The same pattern of artificial substrates supporting greater abundances of oysters has been reported from south-east England (McKnight and Chudleigh, 2015) and south-east Australia (Scanes et al., 2016). Anderson and Underwood (1994) have suggested that oyster abundance and size are enhanced by the ease of settlement and growth on the uniform, relatively-smooth surface of built walls compared to the irregular and creviced rocky shore found in nature. In Brijuni Port on the north-east coast of Veliki Brijun Island, alternative pathways of introduction might have contributed to stronger proliferation of Pacific oyster population on artificial substrata. Apart from larval dispersal by currents and winds on nearby rocky shore, wild oyster settlement on port walls could also occur as a result of boat traffic. Numerous yachts and leisure boats reside in Brijuni National 9

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Park, especially during the summer season, which coincides with the spawning period of Pacific oysters (Miossec et al., 2009; Ezgeta-Balić et al., 2020) and fouling adult individuals could potentially enhance larval supply to Brijuni port. Although no direct evidence for such introduction pathway exists, Pacific oysters are often found attached to the hulls of ships (Miossec et al., 2009; Wrange et al., 2010), and on a number of occasions, this vector has been suspected of introducing the non-native Pacific oyster to new locations, especially in popular recreational boating areas distant from shellfish aquaculture sites (Smith et al., 2015; Herbert et al., 2016; Anglès d’Auriac et al., 2017). Due to the presence of many ports and marinas and a highly developed recreational boating fleet in the Northern Adriatic, Occhipinti-Ambrogi (2002) suggested that this vector may play an important role in the secondary spreading of a number of non-indigenous species. In fact, recreational boaters are considered to pose a higher risk of transporting Pacific oysters and other non-native species than commercial vessels, because they spend more time in ports and marinas where there are often favourable spawning and settlement conditions (Herbert et al., 2012).

rocky substrata and is likely to be more successful at early stages of spread while densities are low (Guy and Roberts, 2010). This method is non-intrusive, but it can be labour intensive (Wijsman et al., 2007; McKnight and Chudleigh, 2015). Maximum achievable outcomes, longterm commitment and cost-effectiveness of the activity can be ensured by involving non-profit associations and volunteer schemes (McKnight and Chudleigh, 2015). Nevertheless, manually eradicating oysters from rocky shores is feasible only for small, easily-accessible areas (Herbert et al., 2016). Hence, such mitigation of the alien oyster species should prioritise sensitive and protected areas such as Brijuni National Park in order to preserve the undisturbed biodiversity and habitat structure as well as the areas at the edge of distribution, currently the tip of Istria peninsula, to prevent further spread and proliferation. CRediT authorship contribution statement Nika Stagličić: Conceptualization, Methodology, Investigation, Formal analysis, Writing - original draft, Writing - review & editing. Tanja Šegvić-Bubić: Methodology, Investigation, Writing - original draft, Writing - review & editing. Daria Ezgeta-Balić: Methodology, Investigation, Writing - original draft, Writing - review & editing. Dubravka Bojanić Varezić: Methodology, Investigation, Writing original draft, Writing - review & editing. Leon Grubišić: Investigation. Luka Žuvić: Investigation. Yaping Lin: Writing - original draft. Elizabeta Briski: Investigation, Writing - original draft.

4.5. Possible impact on local communities and management strategies Currently, it seems that there is no spatial competition between native flat and introduced Pacific oysters. High-density Pacific oyster populations raise, however, further concerns about ecological impacts such as alterations of native biodiversity, assemblage structure, habitats and associated ecosystem functioning (Ruesink et al., 2005; Laugen et al., 2015; Herbert et al., 2016), genetic pollution of local oyster species (Gaffney and Allen, 1992, 1993) and transfer of parasites, diseases and pest species (Ford and Smolowitz, 2007). The directionality (beneficial or detrimental) and intensity of impacts are difficult to predict as they are highly dependent on context (Green and Crowe, 2014; Zwerschke et al., 2018). Habitat type is one of the crucial factors determining the ecological impact of introduced oysters on invaded ecosystems (Padilla, 2010; Zwerschke et al., 2018). In sediment habitats the Pacific oyster can be a strong ecosystem engineer fundamentally changing the substratum from soft and flat to solid complex reef with profound consequences for existing communities (Kochmann et al., 2008; Troost, 2010; Lejart and Hily, 2011; Zwerschke et al., 2018). On hard substrata, the Pacific oyster does not substantially change the character of its new environment and abundances tend to remain relatively low (Ruesink, 2007) with recorded high-density populations being patchy and localised. Wild Pacific oyster aggregations of similar high densities as observed in Lim Bay have been described on rocky coastlines of Argentina (Orensanz et al., 2002; Escapa et al., 2004) and south-east England (McKnight and Chudleigh, 2015; Herbert et al., 2016). On rocky shores in Scandinavia (Laugen et al., 2015), South Africa (Robinson et al., 2005), Ireland (Guy and Roberts, 2010; Zwerschke et al., 2017) and Scotland (Cook et al., 2014; Smith et al., 2015), Pacific oyster settlements occur in low densities similar to those along the Istrian open coastline. As ecological conditions for growth and reproduction of Pacific oysters are favourable all along the eastern Adriatic (MacKenzie et al., 1997; Ezgeta-Balić et al., 2020), population expansions are expected to occur. Given the high variability of impact and demonstrated potential for invasiveness (Ruesink et al., 2005; Streftaris and Zenetos, 2006; Herbert et al., 2016), we strongly suggest regular monitoring of this species. A robust, coherent monitoring scheme is an essential prerequisite not only for identifying the population distribution of Pacific oysters and the effects they have on the ecosystem, but also for developing scientifically-based management strategies to mitigate impacts and prevent further spread (Mortensen et al., 2017). Rocky habitats pose the greatest challenge in terms of management and containment due to the difficulty in physically removing oysters (Herbert et al., 2016). Manual removal is the method of choice for

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was fully supported by the Unity through Knowledge Fund (UKF) project “Competition between native Ostrea edulis and invasive Crassostrea gigas oysters in the Adriatic Sea – effects on the ecosystem, fisheries and aquaculture “(COCOA) under UKF Grant Agreement no. 2/17. EB was additionally supported by the Alexander von Humboldt Sofja Kovalevskaja Award. Authors are thankful to Luka Čulić for technical assistance in the field. References Airoldi, L., Beck, M.W., 2007. Loss, status and trends for coastal marine habitats of Europe. Oceanogr. Mar. Biol. Annu. Rev. 45, 345–405. Anderson, M.J., 2001. Permutation tests for univariate or multivariate analysis of variance and regression. Can. J. Fish. Aquat. Sci. 58, 629–636. Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVA+for PRIMER: Guide to Software and Statistical Methods. PRIMER-E, Plymouth. Anderson, M., Underwood, A., 1994. Effects of substratum on the recruitment and development of an intertidal estuarine fouling assemblage. J. Exp. Mar. Biol. Ecol. 184, 217–236. https://doi.org/10.1016/0022-0981(94)90006-X. Anglès d’Auriac, M.B., Rinde, E., Norling, P., Lapègue, S., Staalstrøm, A., Hjermann, D.Ø., Thaulow, J., 2017. Rapid expansion of the invasive oyster Crassostrea gigas at its northern distribution limit in Europe: naturally dispersed or introduced? PLoS ONE 12 (5), e0177481. https://doi.org/10.1371/journal.pone.0177481. Beck, M.W., Brumbaugh, R.D., Airoldi, L., Carranza, A., Coen, L.D., Crawford, C., Defeo, O., Edgar, G.J., Hancock, B., Kay, M.C., Lenihan, H.S., Luckenbach, M.W., Toropova, C.L., Zhang, G., Guo, X., 2011. Oyster reefs at risk and recommendations for conservation, restoration, and management. Bioscience 61, 107–116. https://doi.org/10. 1525/bio.2011.61.2.5. Benović, A., 1997. The history, present condition, and future of the molluscan fisheries of Croatia. In: MacKenzie Jr CL, Burrell Jr VG, Rosenfield A, Hobart WL (eds), The History, Present Condition, and Future of the Molluscan Fisheries of North and Central America and Europe: Volume 3, Europe. U.S. Department of Commerce, NOAA Tech. Rep. NMFS 129, Seattle, Washington, pp. 217–226. Bianchi, C.N., Morri, C., 2000. Marine biodiversity of the Mediterranean Sea: situation, problems and prospects for future research. Mar. Pollut. Bull. 5, 367–376. Boudry, P., Heurtebise, S., Lapègue, S., 2003. Mitochondrial and nuclear DNA sequence variation of presumed Crassostrea gigas and C. angulata specimens: a new oyster species in Hong Kong? Aquaculture 228, 15–25.

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