Aquatic Invasions (2014) Volume 9, Issue 2: 183–194 doi: http://dx.doi.org/10.3391/ai.2014.9.2.07 Open Access

© 2014 The Author(s). Journal compilation © 2014 REABIC

Research Article

A morphometric and genetic comparison of Sinanodonta woodiana (Lea, 1834) populations: does shape really matter?

Irene Guarneri1,2, Oana Paula Popa3, Laura Gola4, Lyudmila Kamburska1, Rosaria Lauceri1, Manuel Lopes-Lima5, Luis Ovidiu Popa3* and Nicoletta Riccardi1* 1CNR – Institute for Ecosystems Studies, Largo Tonolli 50, 28922 Verbania Pallanza (VB), Italy 2Insubria University, via Dunant 3, 21100 Varese, Italy 3NMNHGA – National Museum of Natural History “Grigore Antipa”, Bucharest, Romania 4Parco del Po e dell’Orba, Piazza Giovanni XXIII 6, 15048 Valenza (AL), Italy 5CIIMAR - Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Bragas 289, 4050-123 Porto, Portugal E-mail: [email protected] (IG), [email protected] (OPP), [email protected] (LG), [email protected] (LK), [email protected] (RL), [email protected] (ML-L), [email protected] (NR), [email protected] (LOP) *Corresponding author

Received: 17 December 2013 / Accepted: 20 May 2014 / Published online: 9 June 2014 Handling editor: Vadim Panov

Abstract

Sinanodonta woodiana (Lea, 1834) is an invasive species which has spread rapidly across Europe over the last few decades. Although it is already well established throughout Italy (both in lakes and in rivers), during a regular monitoring of the mussel communities in Northern Italy, this species was found for the first time in the fluvial natural reserve of Ghiaia Grande (Po River basin). The strong differences in shell shape between these specimens and those from Lake Maggiore prompted a genetic comparison based on DNA barcoding analyses to establish whether specimens from both sites belong to the same species. Our results confirm that the wide shell plasticity may help to explain the role of environmental factors in driving shells’ morphology. Additionally, it may also induce S. woodiana misidentification with the native Anodonta sp. This fact may have strong negative implications for freshwater conservation if on one hand, the native species distribution is falsely over represented; or on the other hand, if the invasive species has a much wider distribution than predicted. Key words: Chinese pond mussel, Po River, Lake Maggiore, Italy, invasive species, geometric morphometrics, COI, DNA barcoding

Tiber and Volturno rivers (Manganelli et al. 1998; Introduction Bodon et al. 2005; Albano 2006; Solustri and Nardi 2006; Cianfanelli et al. 2007; De Vico et al. 2007) The Chinese pond mussel Sinanodonta woodiana and in Lake Santa Rosalia in Sicily (Colomba et (Lea, 1834) (Bivalvia: Unionidae), a species al. 2013). originating from East Asia (South-eastern Russia The accidental introduction of fish specimens to Malaysia), spread rapidly across Europe over bearing glochidia (the parasite larval stage of the the last few decades (Kiss 1990; Paunovic et al. bivalve) appears to be a reasonable explanation 2006; Popa et al. 2007; Munjiu and Shubernetski for the rapid expansion of S. woodiana in most 2008; Pou-Rovira et al. 2009; Lajtner and Crnčan European countries, including Italy (Spyra et al. 2011). Its first detection in Italy (Manganelli et 2012; Gherardi et al. 2008 and Cianfanelli et al. al. 1998) was followed by an increasing number 2007). In Italy, other probable invasion pathways of records. Nowadays, it seems to cover all waters were the commercial trade (e.g. for ponds and in the Italian Peninsula including the largest aquariums) and its culture for pearls or mother- Italian Lakes such as Lake Garda (Cappelletti et of pearl production (Berni et al. 2003). al. 2009) and Lake Maggiore (Kamburska et al. S. woodiana is a host generalist, being able to 2013). The presence of S. woodiana was also parasitize several native and non-native fish species. recorded in different lakes and rivers across the This behaviour is believed to increase the basins of the Po, Adige, Piave, Reno, Arno, opportunities of reaching and establishing in new

183 I. Guarneri et al.

Figure 1. Study area. A - Po River Basin. Lake Maggiore is highlighted in light blue, Po River in dark blue. B - Natural Reserve Ghiaia Grande. Arrows indicate water flow direction. PoUS - Po UpStream Site, PoDS – Po DownStream Site.

environments (Douda et al. 2012). The parasitic method is based on a comparative analysis with a period is generally short, with development and standard reference DNA sequence. In higher animals larvae release in 8–12 days at 20–25°C (Kiss the most common used DNA barcode sequence is 1990). Characterized by a fast growth, an early a fragment of the cytochrome oxidase I mito- maturity (i.e. in the first year of life), a high chondrial gene. This molecular approach can reproductive output and a lifespan of 10–15 years provide an accurate identification of mussel (Dudgeon and Morton 1983; Afanasjev et al. 2001; species at all life stages (Campbell et al. 2008; Kraszewski and Zdanowski 2007), the species Boyer et al. 2011; Zieritz et al. 2012). displays many features of a successful invader; The aim of the present study was to compare high dispersal capacity promotes the impressively morphological distinct populations of putative S. rapid diffusion of the species and its wide woodiana specimens using both molecular and ecological tolerance allows for the successful morphometrical approaches, in order to assess if establishment in invaded ecosystems. shell shape differences are species specific or The species ability to adapt to different habitats, more related to environmental variables. tolerating changes in chemistry and physical To this aim, freshwater bivalves from the Po parameters of water and sediments, can be a River basin and from Lake Maggiore were identified driver of the observed morphological plasticity using a Barcoding technique (COI) and these (e.g. Soroka and Zdanowski 2001; Kraszewski 2006; results were compared with a combination of Douda et al. 2012; Demayo et al. 2012). The native traditional and geometric morphometric techniques, European anodontines are also characterized by a which have been previously used with success to great phenotypic plasticity (Nagel and Badino test inter- and intraspecific variations e.g., (Zieritz 2001; Reis et al. 2013). This not only hinders the and Aldridge 2009). attribution of a specimen to a particular species, but also promotes S. woodiana misidentification with the native Anodonta species. For instance, Materials and methods the first record of S. woodiana in Lake Maggiore Study area (Kamburska et al. 2013) raised serious doubts about its correct identification (Mienis 2013). Indeed, The Po River is the longest Italian river, with a the unusually elongated shape of Lake Maggiore total length of 650 km. The river collects waters specimens suggested that they could belong to from the whole Northern Italy, the Alps, the great the native European species Anodonta anatina. sub-alpine lakes, and, passing through the Padania To verify species identification based on plain, flows into the Northern Adriatic Sea. The morphological characters, a DNA barcoding catchment covers an area of 71,000 km², collecting approach was proposed (Hebert et al. 2003). This waters from 141 tributaries. The fluvial reserve

184 A morphometric and genetic comparison of Sinanodonta populations

Ghiaia Grande with its total area of 4.62 km² is shell width (W), wing height (h) and umbo height part of the large protected area of the Po and (H) were used to compute the L/W, L/h, L/H, Orba systems, with a total surface area of about W/h and W/H ratios. The normal distribution of 140 km² between Piedmont and Lombardy the measured variables, in each population, was (Figure 1-A). During October 2012, a survey was calculated using two tests: the Shapiro-Wilk test, performed on the Ghiaia Grande reserve to optimized for small sample sizes (N<50) and/or a monitor the populations of native unionids (Unio Chi-squared test. T (for equality of means) and F sp., Anodonta sp.). Structured and recruiting (for equality of variance) tests were performed to Unio sp. populations were found, but no native assess whether the measurements from each of Anodonta specimens were sampled. Instead, various the three groups of samples (PoUS, PoDS, LM) S. woodiana individuals were caught. These are significantly different. The multivariate norma- specimens were located along a secondary river lity of the log-transformed ratios was checked by branch (Figure 1-B). The entire area is characterized computing Mardia's multivariate skewness and by the presence of meanders, a slow water flow kurtosis, with tests based on chi-squared and fine sediments deposition. The upstream (skewness) and normal (kurtosis) distributions. A (PoUS, 45°09'05''N, 8°19'41''E) and downstream MANOVA analysis was performed with the sites (PoDS 45°09'35''N, 8°19'24''E) are defined populations’ location designation as independent relatively to the intake of reserve water in the variables and the five above mentioned ratios as secondary branch. Lake Maggiore is the second dependent variables. A Bonferroni correction for deepest and largest subalpine lake in Italy. Samples multiple testing was applied for p-values. from Lake Maggiore (LM) were collected at a The growth patterns of the studied populations sampling point (45°50'09.0"N, 8°37'25.5"E) (allometric or isometric) were analyzed through located along the southeast coast of the lake during a curve fitting procedure. This was done with a the summer of 2012 (Figure 1-A; Kamburska et linear regression of the log-transformed values al. 2013). This sampling site is characterized by of our measurements, fitting the points to the a moderate wave intensity and a high sediment allometric equation: patchiness. However, S. woodiana was found y=bxa only in soft sediments. For this purpose, the shell length vs. shell height Studied samples (L-H, L-h), shell width vs. shell length (W-L) and shell width vs. shell height (W-H, W-h) as A total of 23 S. woodiana specimens (11 PoUS, well as all four linear measurements (L, W, h, H) 12 PoDS) were collected by visual and tactile vs. body weight (BW) were analyzed for all Po search from the secondary branch of the Po river samples. The growth patterns were considered River, while 22 specimens were collected from to be isometric if the slope (a) of the regression Lake Maggiore. The abundance of mussels was line was not statistically different from 1 (3, determined by collecting and counting individuals 2 when compared to BW), negative allometric if within randomly selected quadrats of 0.25 m . a<1 (3, when compared to BW), and positive allometric if a>1 (3, when compared to BW). A Linear morphometrics bootstrapped 95 % confidence interval for a was computed with 2000 replicates. All analyses Sampled organisms were measured with a digital were performed with the software package PAST calliper to the nearest 0.01 mm, defining the (Hammer et al. 2001). following four morphometric variables: shell length (L), the maximum antero-posterior dimension of Geometric morphometrics the shell; shell width (W), the maximum left- right dimension with both valves compressed; Specimens were classified in four length classes umbo height (H), the dorsal-ventral dimension of and assigned to the respective age groups the shell measured perpendicular to the length according to (Afanasjev et al. 2001) and (Spyra measured at the level of the umbo; wing height et al. 2012): i) up to 5 cm (young), ii) from 5 to (h), the maximum dorsal-ventral dimension of 10 cm (small), iii) from 10 to 15 cm (medium), the shell measured perpendicular to the length and iv) above 15 cm (large) (Appendix 1a, 1b, (Figure 2). Live animals (only from the River Po) 1c). The age of the individuals was determined were weighed in triplicate to get the individual from shell length (L), since the annual growth weight (BW, g). The measured shell length (L), rings may not faithfully represent the actual age

185 I. Guarneri et al.

Figure 2. Sinanodonta woodiana shell measurements and selected landmarks (numbered from 1 to 15): L – total length, h – wing/ventral edge height, H umbo/ventral edge height, W – width.

of the Chinese pond mussel (Kiss 1995). The age elongated morphotypes as summarized in Table 1. of the mussels was estimated from the length-age All pictures were scaled and the differences in relationship reported by Dudgeon and Morton shape and dimensions were analyzed. (1983). By making the assumption of a similar growth rate of S. woodiana in its native range DNA isolation, amplification and sequencing and in colonized areas, mussel age classification was also performed following Spyra et al. (2012). For the genetic analysis small samples from the The geometric morphometrics and statistical mantle were excised from 26 S. woodiana from analyses were performed with software MorphoJ the three sites: Lake Maggiore LM. (n=16), Po (Klingenberg 2011). A dataset comprising 45 River Downstream (PoDS) (n=6) and Po River pictures (11 from PoUS, 12 from PoDS and 22 Upstream (PoUS) (n=4). DNA isolation was from LM) was analysed with MorphoJ after a performed with a NucleoSpin® Tissue kit previous elaboration with Tps series of morpho- (Macherey–Nagel GmbH and Co. KG, Düren, metric programs (Rohlf 2008), where 15 landmarks Germany), according to the company specifications. were defined (5 groups of 3 landmarks each: For each DNA sample, a 650 bp fragment of umbo, wing, ventral edge, anterior and posterior the mitochondrial gene for cytochrome oxidase c edges, numbered dots on Figure 2). This software subunit I (COI) was amplified, using the primers provides a platform for a wide range of analyses LCO1490 and HCO2198 as described by Folmer in geometric morphometrics. Landmark data allow et al. (1994). for the visualization of the difference between The amplification was performed in 25 μl of a population means or the deviation of an individual solution containing 10 mM Tris–HCl (pH 8.8 at from its population mean. The major variation 25°C), 50 mM KCl, 0.08% (v/v) Nonidet P40, patterns of size and shape were explored using a 2.5 mM MgCl2, each dNTP at 0.1 mM, each principal component analysis (PCA) and centroid primer at 0.1 μM, 1 unit of Taq DNA polymerase size to scale for the configuration of selected (Fermentas UAB, Vilnius, Lithuania), and approxi- landmarks. Landmarks were selected to enlighten mately 10 ng of DNA template. The temperature visible differences between the rounded and the profile of the polymerase chain reaction for the

186 A morphometric and genetic comparison of Sinanodonta populations

Table 1. Landmarks position along Sinanodonta woodiana shell (Figure 2) and relative shell shape characters analyzed.

Landmark position Shell shape character Anterior edge (1), top (2) and posterior edge (3) of umbo relative height and width of umbo Tip (4) and two equidistant points (5, 6) on the edge of wing degree of wing development Posterior margin: point at the maximum distance from the anterior margin (8) and two points degree of posterior margin convexity (7, 9) at the beginning of curvature Ventral margin: point at the maximum distance from the top of umbo (10) and two degree of ventral margin convexity equidistant points (11, 12) along the maximum curvature Anterior margin: point at the maximum distance from the posterior margin (14) and two degree of anterior margin convexity points (13, 15) at the beginning of curvature

COI marker consisted of initial denaturation at to an age of 4–5 years. On the other hand, the 94°C for 30 s, followed by 5 cycles at 93°C for downstream population included older animals, 30 s, 45°C for 45 s, 72°C for 45 s, followed by with a maximum length up to 194.08 mm probably 30 cycles at 93°C for 30 s, 55°C for 45 s, 72°C over 10 years of age. The length of animals from for 45s, and a final extension step performed at Lake Maggiore ranged from 91.27 mm to 164.96 72° for 10 min. The PCR products were separated with a mean value of 130.47 ± 22.89 mm (Table on 1% agarose gel, and the fragments of interest 2, supplementary Table 3S). purified using the GeneJET™ Gel Extraction Kit (Fermentas UAB, Vilnius, Lithuania). Uni- Univariate T and F-tests directional sequencing reactions were performed by Macrogen Europe (1105 AZ, Amsterdam). All measurements data exhibited a normal distri- Sequences were aligned using CodonCode bution. A comparison between linear measurements Aligner 3.7.1. (CodonCode Corporation, Dedham, and between length/wing height (L/h) and length MA, USA) and verified manually. The consensus /width (L/W) ratios of the specimens in our sequence was used as a query to the BOLD samples, performed with univariate T and F tests, SYSTEMS Identification Request (http://www.bold suggested that all collected animals manifested a systems.org). similar relationship among their measurements at different size classes (Table 3). The p-values of these tests are presented in Table 4. The relation Results between length and umbo height (L/H) and between length and fresh total weight (L/BW) are Morphometric analysis (linear, geometric) represented graphically (Figure 3). The mean density of S. woodiana in PoUS and in -2 PoDS was of 4.4 ± 1.3 ind m and 9.6 ± 2.2 ind Multivariate Analysis of Variance (MANOVA) m-2, respectively. Although in the upstream station both S. woodiana and the native Unio The log-transformed data were tested for normality elongatulus were found, downstream only S. with a Mardia test. The p-values for skewness woodiana was present. Additionally, a different and kurtosis exhibited values above 0.05 in both shell size range was also detected in these two cases. The MANOVA test (after Bonferroni river sections The younger animals, aged from correction for multiple comparisons) showed a three to five years (mean length 89.42 ± 18.69 mm) significant difference of the multivariate means were collected upstream (Table 2, supplementary between the sample from Lake Maggiore and Table 1S), whereas the downstream population was each of the samples from the Po River. However, represented by animals belonging to different size no differentiation was observed between both Po classes, with a mean length of 132.44±36.24 mm river samples (Table 5). (Table 2, supplementary Table 2S). The smallest collected specimens measured 64.28 mm on the Curve fitting analysis upstream site and 73.73 mm downstream. The upstream site was apparently colonized by a The a values from the allometric equation, younger population, where the maximum length calculated for each of the three samples, are of the organisms was 115.19 mm, corresponding presented in Table 6.

187 I. Guarneri et al.

Figure 3. Regression analysis of selected parameters: A- Linear regression relation between L/H (length and umbo height in mm); B- Curvilinear regression relation between L/BW (length (mm) and total fresh weight (g)); Po River UpStream Site (PoUS ) and DownStream Site (PoDS ).

Table 2. Morphometric measurements of Sinanodonta woodiana collected in River Po and in Lake Maggiore. PoUS-Po River, UpStream Site; PoDS- Po River, DownStream Site; LM-Lake Maggiore Site.

Length Wing height Umbo height Width Body Weight Ratio (L, mm) (h, mm) (H,mm) (W, mm) (BW, g) L/H mean 89.42 60.66 57.37 32.56 83.41 1.56 ±std 18.69 11.85 11.92 7.81 48.79 0.07 PoUS Min. 64.28 44.01 41.50 21.28 26.52 1.47 Max. 115.19 80.72 77.53 43.88 160.44 1.72 mean 132.44 95.69 91.84 51.74 341.16 1.45 ±std 36.24 26.96 26.24 15.10 275.66 0.06 PoDS Min. 73.73 50.64 50.18 28.36 45.21 1.38 Max. 194.08 141.70 137.90 77.62 918.85 1.57 mean 130.47 80.77 75.63 47.56 1.73 LM ±std 22.89 13.11 12.86 6.33 0.13 Min. 91.27 61.47 54.90 36.42 1.58 Max. 164.96 107.88 100.43 57.68 2.16

A linear correlation was found between length respectively, of the total variability among the and height, and an exponential relation between landmarks, on all analysed specimens (Figure 4). length and weight for all sampled animals. The The population from the Po River comprised two analogous trend could suggest that the growth of overlapping groups representing the populations individuals sampled from two sites was similar. from down and upstream. The population from While fitting of regressions were higher for the Lake Maggiore is well separated on the right. It downstream population (R2=0.983), data of the was evident that animals from Po River were upstream population were slightly more dispersed more rounded in respect to specimens collected around regressions, but nonetheless with a high in Lake Maggiore. R2 value (0.961). Younger animals appeared to be more similar than older ones, among all populations. Indeed, Geometric morphometrics analysis using a classifier that labelled organisms with sampling site and the proper length (L), a clear To find out if other differences among animals grouping by size (age) could be distinguished by occurred, pictures with landmarks were analysed the PCA (Figure 5). The first relative warp clearly with MorphoJ software. The principal component distinguished individuals by size, highlighting a analysis revealed that the first two components wider shape variation among large (> 15 mm)

Pc1 and Pc2 explained 54.29% and 13.48%, than among small animals. While the younger

188 A morphometric and genetic comparison of Sinanodonta populations

Figure 4. Principal component analysis of geometric morphometric of Po River UpStream Site (PoUS in blue) and DownStream Site (PoDS in green) populations compared with Lake Maggiore population (LM in red). Confidence ellipses p=0.9. Labels of dots refer to length of organisms in mm. ANOVA test of null hypothesis: organisms are different in shape due to change, p<0.0001.

Figure 5. PCA of centroid size and Pc1 classified by age (Young=S in blue, Adult=M in yellow and Old=L in black) and sites (Po Upstream=PoUS (full circle), Confidence ellipse (dotted line in light blue) p=0.9. Po Downstream=PoDS (full quadrat), Confidence ellipse (dotted line in red) p=0.9. Lake Maggiore=LM (full triangle) Confidence ellipse (dotted line in green) p=0.9. Labels of dots refer to length of organisms in mm.

189 I. Guarneri et al.

Table 3. Relation of morphometric measurements of Sinanodonta woodiana. PoUS-Po River, UpStream Site; PoDS- Po River, DownStream Site; LM-Lake Maggiore Site.

Sampling Site L/H L/h L/W H/W (h-H)/H % PoUS 1.56±0.07 1.47±0.06 2.77±0.21 1.78±0.15 5.60±2.02 PoDS 1.45±0.06 1.39±0.05 2.58±0.19 1.79±0.14 3.98±2.38 LM 1.73±0.13 1.61±0.11 2.75±0.36 1.59±0.13 6.45±2.48

Table 4. Statistical significance of univariate F and T test of variance, respectively mean equality between our investigated populations. pvar-p value for the F test of variance equality; pmean-p value for the T test of mean equality. Shaded cells show comparisons where at least one of the tested values (mean, variance) is statistically different. PoUS-Po River, UpStream Site; PoDS- Po River, DownStream Site; LM-Lake Maggiore Site.

LM-PoDS LM-PoUS PoDS-PoUS

h pvar=0.004 pvar=0.766 pvar=0.015 pmean=0.033 pmean<0.001 pmean=0.001 H pvar=0.005 pvar=0.835 pvar=0.019 pmean=0.020 pmean<0.001 pmean<0.001 L pvar=0.068 pvar=0.516 pvar=0.046 pmean=0.847 pmean<0.001 pmean=0.002 W pvar<0.001 pvar=0.047 pvar=0.401 pmean=0.257 pmean<0.001 pmean<0.001 BW - - pvar<0.001 pmean=0.001 L/H pvar=0.005 pvar=0.030 pvar=0.555 pmean<0.001 pmean<0.001 pmean<0.001 L/h pvar=0.013 pvar=0.042 pvar=0.703 pmean<0.001 pmean<0.001 pmean=0.002 L/W pvar=0.033 pvar=0.087 pvar=0.721 pmean=0.148 pmean=0.842 pmean=0.034 H/W pvar=0.300 pvar=0.982 pvar=0.373 pmean=0.003 pmean=0.003 pmean=0.726 (H-h)/H pvar=0.921 pvar=0.507 pvar=0.610 pmean=0.009 pmean=0.334 pmean=0.094

analysed. Only one haplotype was identified in Table 5. MANOVA analysis. p-values uncorrected above the three sampling sites (GenBank accession diagonal; p-values Bonferroni corrected below diagonal. PoUS- numbers KF731775-KF731777). The BOLD SYSTEMS Po River, UpStream Site; PoDS- Po River, DownStream Site; LM-Lake Maggiore Site. Identification request query returned a species match with Sinanodonta woodiana at similarity 0 LM PoDS PoUS levels between 99.84% and 100% (9 matches). LM 0 4,69E-07 0,00060 PoDS 1,41E-06 0 0,01998 Discussion PoUS 0,00181 0,05994 0 The two populations of S. woodiana analysed in the present study are morphologically distinct, individuals (3–4 years old; < 10 mm) are closely with specimens from the Po River being more grouped, regardless of the sampling site, the similar in shape to the typical S. woodiana. In distribution of the larger specimens seems to be contrast, the unusual elongated shape of Lake affected by their origin. In fact, there is an Maggiore (Kamburska et al. 2013) and Lake Garda increasingly distinctive grouping by site, with Po (Cappelletti et al. 2009) specimens lead some River sites on the left and Lake Maggiore on the authors to question whether they were indeed S. right of the graph, along with size increase. woodiana (Mienis 2013). Other authors consider that S. woodiana could easily be confused with other species in the genus Anodonta (Bogan et al. Genetic analysis 2011). As a consequence, our first aim was to A 636 bp COI fragment was successfully establish if the populations reported in the present sequenced from the 26 specimens of S. woodiana study belong indeed to S. woodiana. In order to

190 A morphometric and genetic comparison of Sinanodonta populations

Table 6. Curve fitting analysis. The values in the table represent a values with the bootstrapped 95 percent confidence interval from 2000 replicates (in parantheses). Shaded cells show where an allometric growth pattern was identified. PoUS-Po River, UpStream Site; PoDS- Po River, DownStream Site; LM-Lake Maggiore Site.

LM PoDS PoUS Combined L-h 0.896 (0.756-1.023) 1.042 (0.969-1.098) 0.917 (0.810-1.006) 0.968 (0.877-1.053) L-H 0.942 (0.795-1.077) 1.030 (0.979-1.123) 0.968 (0.848-1.063) 0.990 (0.890-1.094) W-L 1.302 (1.138-1.548) 0.927 (0.792-1.052) 0.886 (0.717-1.110) 0.964 (0.896-1.054) W-H 1.227 (0.994-1.489) 0.955 (0.811-1.108) 0.858 (0.664-1.105) 0.955 (0.861-1.052) W-h 1.167 (0.915-1.475) 0.966 (0.831-1.065) 0.813 (0.630-1.062) 0.933 (0.846-1.026) L-BW 3.171 (3.008-3.506) 2.945 (2.575-3.288) 3.173 (3.043-3.313) W-BW 2.941 (2.662-3.280) 2.610 (2.303-3.016) 2.805 (2.622-2.941) h-BW 3.043 (2.853-3.331) 3.211 (2.800-3.776) 2.993 (2.838-3.128) H-BW 3.078 (2.950-3.317) 3.041 (2.686-3.536) 2.928 (2.802-3.056)

answer this question, a DNA barcoding analysis (Afanasjev et al. 2001; Soroka and Zdanowski 2001; was performed with specimens from Po River, Kraszewski 2006; Demayo et al. 2012; Urbanska but also with elongated specimens collected from et al. 2013), allometry explains most of the intra- Lake Maggiore. All 26 analyzed specimens population shell shape variation (Demayo et al. exhibited the same mitochondrial haplotype, 2012). which returned a species match with S. woodiana Several unionid species are reported to display from the BOLD SYSTEMS Identification Request. a high intraspecific morphological variability All these matching sequences were obtained from which can reflect an adaptive phenotypic response specimens collected in Europe. We consider this to habitat factors. For instance, shell shape is finding as a confirmation that the specimens influenced by hydrological features and substrate reported in this study, but also those from composition, i.e., parameters which are mutually Kamburska et al. (2013), represent different related and apparently induce typical adaptive morphotypes of the species S. woodiana. modifications (e.g. Hinch and Bailey 1988; Zieritz The Lake Maggiore specimens are significantly and Aldridge 2009; Hornback et al. 2010). more elongated (i.e. higher length/ height -wing However, attempts to identify a specific habitat and umbo- ratios) and with less pronounced parameter which determines a specific change in wings than specimens from both locations of the shell shape gave contradictory results. As an Po River. Shape differentiation increased with example, more elongated shell shapes were reported specimen’s size: while younger animals were to reflect adaptation to strong wave action in lakes much closer, bigger animals are more dispersed (Hinch and Bailey 1988) but also to characterize along the first axis of PCA. populations of slow flowing habitats in rivers (e.g. Collection of specimens with a wide size Zieritz and Aldridge 2009). According to Hornback range allowed for considerations about the type et al. (2010), shell shape reflects the degree of of growth. hydraulic variability rather than flow intensity, While younger animals are similar in shape, and slender morphotypes have advantages under an increased shape differentiation occurs with high hydraulic disturbance (i.e. hydraulically flashy ageing. Factors like habitat physico-chemical environments). Variations in growth determined conditions and food availability are likely to by ontogenetic factors (e.g., feeding and/or meta- drive shell shape on the animals’ development bolic shifts) or parasitic infestation by bucephalid and growth. This is also suggested by the fact trematodes (Rhipidocotyle sp.) can also significantly that growth patterns, either isometric or allometric, alter sagittal and lateral shell shape of unionids are not exactly the same in all populations (see (Zieritz and Aldridge 2011). Table 6), which could imply that the species In the present study, river specimens collected growth pattern is not controlled only genetically, from areas with very slow currents and soft but environmental factors are likely involved. sediments, presented more rounded shells and While habitat characteristics are considered the more pronounced wings than lake specimens, most likely driving factors of morphological which were collected from a lake area with a divergence between S. woodiana populations moderate degree of wave exposure. Enlargement

191 I. Guarneri et al. of the wings has been associated with an increase Acknowledgements in stability when on soft or unstable substrates to O.P.P. and L.O.P. were supported by the National Council of prevent sinking (Watters 1994). This pattern has Scientific Research from Romania (Grant PNII RU-PD been observed in Anodonta anatina specimens no.36/2013) Financial support was provided for M.L.L by from slow flowing areas on the River Thames Portuguese Foundation for Science and Technology (FCT) under (Zieritz and Aldridge 2009). Under this view, the project PTDC/AAC-AMB/117688/2010. Financial support for the assessment of alien invasive bivalves distribution in Lake very low water turbulence and the presence of Maggiore and Po River was provided by the 6 Rotary Clubs very soft and unstable sediments in both Po (Pallanza-Stresa, Varese Verbano, Alto Verbano, Borgomanero- River locations could explain the presence of Arona, Sesto Calende-Angera, Locarno) and the three Rotary “rounded” and “winged” specimens. However, Districts present in the Lake Maggiore catchment basin: with a joint Swiss-Italian Project (One Lake, Three Districs, Only One while the Po River specimens present a more Rotary). We thank the divers team (Associazione Sub Verbania) typical S. woodiana shape, those from Lake who greatly helped us during sampling. We also thank the Maggiore are “unusually” elongated, indicating anonymous reviewers for their critical reading of this manuscript that other factors than the physical habitat and the editors Frances Lucy and Vadim Panov for fruitful characteristics may be involved. For instance, discussions and helpful comments. parasitic infestation by Rhipidocotyle sp., which was recently found in Lake Maggiore (Riccardi, References unpublished data), could have contributed to Afanasjev SA, Zdanowski B, Kraszewski A (2001) Growth and alter the shape of lake’s S. woodiana specimens, population structure of the mussel Anodonta woodiana (Lea, as demonstrated to occur in parasitized A. anatina 1834) (Bivalvia, Unionidae) in the heated Konin lake system. populations (Zieritz and Aldridge 2009). Archives of Polish Fisheries 9(1): 123–134 Albano PG (2006) Ritrovamenti presso la spiaggia di Palo Laziale Although a specific factor associated with this (Roma). Notiziario della Società Italiana di Malacologia change in shell shape could not be identified, 24(1–4): 21–22 this study evidences that such differences in shape Berni P, Bitossi S, Salvato M, Orlandi M, Salviati J, Silvestri M, could induce species misidentification. This is a Orlandi P, Billard R (2003) Valorizzazione del territorio attraverso produzioni alternative di perle di acqua dolce di common problem in field surveys of freshwater elevata qualità, con tecniche di policoltura eco-sostenibile. mussels and could potentially bias estimates of Convegno Nazionale "Parliamo di allevamenti alternativi e population status and trends (Shea et al. 2011). valorizzazione del territorio", Cuneo, pp 179–182 Addressing this issue is crucial for the assessment Bodon M, Cianfanelli S, Manganelli G, Castagnolo L, Pezzoli E, Giusti F (2005) Mollusca Bivalvia. In: Checklist e of alien bivalve species distribution. Indeed, if distribuzione della fauna italiana. 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Supplementary material

The following supplementary material is available for this article.

Table 1S. Morphometric measurements of Sinanodonta woodiana collected in River Po (PoUS). Table 2S. Morphometric measurements of Sinanodonta woodiana collected in River Po (PoDS). Table 3S. Morphometric measurements of Sinanodonta woodiana collected in Lake Maggiore.

This material is available as part of online article from: http://www.aquaticinvasions.net/2014/Supplements/AI_2014_Guarneri_etal_Supplement.xls

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