Zoodiversity_06_2021.indb UDC 594.3(26.04:477.74) MORPHOLOGICAL AND MOLECULAR STUDIES OF THE RAPA WHELK, RAPANA VENOSA (NEOGASTROPODA, MURICIDAE), FROM ODESA BAY H. Morhun1, 2*, M. O. Son2, O. O. Kovtun3, S. Utevsky1 1Department of Zoology and Animal Ecology, Biological Faculty, V. N. Karazin Kharkiv National University, Svobody sq., 4, Kharkiv, 61022 Ukraine 2Institute of Marine Biology, National Academy of Sciences of Ukraine, Pushkinska st., 37, Odesa, 65048 Ukraine 3Hydrobiological Station, Department of Hydrobiology and General Ecology, Mechnikov Odesa National University (ONU), Dvoryanskaya st., 2, Odesa, 65082 Ukraine *Corresponding author E-mail: halynamorhun94@gmail.com H. Morhun (http://orcid.org/0000-0002-7888-477X) M. O. Son (http://orcid.org/0000-0001-9794-4734) O. O. Kovtun (http://orcid.org/0000-0001-8820-5606) S. Utevsky (http://orcid.org/0000-0003-1290-6742) Morphological and Molecular Studies of the Rapa Whelk, Rapana venosa (Neogastropoda, Muricidae), from Odesa Bay. Morhun, H., Son, M. O., Kovtun, O. O., Utevsky, S. — Th e gastropod Rapana venosa (Valenciennes, 1846) is a successful worldwide invader occurring in the Black Sea. Th e aim of this study is to overview specifi c population features of this mollusk from Odesa Bay through integrative systematic approach by means of morphological and molecular research. For this purpose, the mollusks were collected from the Black Sea and examined using morphological methods: traditional morphometry, which employs linear parameters of shells (height, width, whorl height, whorl width, height of the last whorl) and shell weight, and geometric morphometrics of the shell shape data. For a molecular genetic test, the COI gene region was used. Among all conchological variability, the two morphotypes were defi ned: the fi rst has a “broad” shape — shells have a thick and durable last whorl and a low spire, and the second one — “extended” shape: shells are relatively slender with an elongated high-conical spire. According to the geometric morphometric data, R. venosa has statistically signifi cant diff erences between defi ned morphotypes (F = 4.12, p = 0.001); however, the shapes in males and females are not signifi cantly diff erent (F = 1.13, p = 0.318). No genetic diversity, neither novel haplotypes were revealed by the molecular analysis: in Odesa Bay, the haplotype occurring also in other regions of invasion across the world is present. K e y w o r d s : COI, geometric morphometric analysis, invasive species, phylogenetics, rapa whelk, shell morphology. Iintroduction   Rapana venosa (Valenciennes, 1846) is a well-known invasive mollusk occurring in the Black Sea for the last 70 years (Drapkin, 1953). It has a substantial infl uence on the environment as a highly eff ective predator (Mann, Harding, 2003; Bondarev, 2010; Pereladov, 2013) and signifi cantly aff ects the local shellfi sh and benthic communities by displacing native bivalve species (Chukhchin, 1984; Rubinshtein, Hizniak, 1988; Marinov, 1990; Zolotarev, 1996), thus reducing the fi ltration potential of the region (Seyhan et al., 2003; Kurakin, Govorin 2008; Govorin, Kurakin, 2011).  Zoodiversity, 55(6): 467–478, 2021 DOI 10.15407/zoo2021.06.467 468 H. Morhun, M. O. Son, O. O. Kovtun, S. Utevsky Th e Black Sea is thought to be the fi rst source of the initial introduction of the rapa  whelk and then the mollusk has spread throughout the world by various vectors including ballast waters and/or intentional introductions (Chandler et al., 2008) to the Aegean (Koutsoubas, Voultsiadou-Koukoura, 1991) and Adriatic seas (Ghisotti, 1971, 1974; Mel, 1976; Cucaz, 1983; Rinaldi, 1985), France (ICES, 2004), USA (Harding, Mann, 1999), in the Rio de la Plata between Uruguay and Argentina (Scarabino et al., 1999; Pastorino et al., 2000; Giberto, Bruno, 2014), and the Netherlands (Nieweg et al., 2005). Th e eff ective invasion is explained by some specifi c reproductive and ontogenetic features of rapa whelk: extremely high fertility (15 million eggs laid by one female per season [Harding et al., 2002; Ware, 2002]), presence of plankton larva (veliger) (Harding, Mann, 2003), fast ontogenesis and maturation (Harding, Mann, 2003; Mann et al., 2006). All those features contribute to the potential for colonization and serve to a high invasive success of the rapa whelk.  Many researchers studying the populations of this mollusk in the Black Sea have noticed high ecological plasticity of the species, which may be due to specifi c biochemical peculiarities (Alakrinskaya, 1989) and great diversity of its conchological traits (Bondarev, 2010; Kos’yan, 2013; Slynko et al., 2020). In many papers, authors describe eco-morphs, metapopulations (Bondarev, 2010) and shell color forms (ICES, 2004, Savini et al., 2004; Micu et al., 2008; Bondarev, 2010), which outline the heterogeneity of morphological features; for some morphs, separate names were coined. For instance, an extremely prolonged morphotype is known in the literature as a “tower-shaped”, and small-sized adults as “dwarf forms” (Bondarev, 2010). Th is phenotypic plasticity occurring among R. venosa is associated with some environmental conditions features of habitat (e. g. sediments type), food supply, potential prey (Shukshin, 1961; Bondarev, 2010; Kos’yan, 2013) — and their sex (Bondarev, 2010; Kovtun et al., 2014).  While high morphological variability of this species is observed, genetically very low nucleotide diversity is shown in populations from the regions of invasion (Chandler et al., 2008; Xue et al., 2018; Slynko et al., 2020). An examination from within the native range revealed high levels of genetic variation (110 haplotypes of COI and NADH gene regions), while specimens from all introduced populations — showed the complete lack of genetic diversity, and only a single haplotype was common to all introduced individuals, which occur also in Japan and Korea, especially from Jeju Island (Chandler et al., 2008). Th e low diversity is explained by an extreme genetic bottleneck occurred, while individuals from the native range were being introduced into the invasive range (Chandler et al., 2008).  During a fi eld survey of the rapa population in Odesa Bay, high conchological variability was noticed. Th e goal of the current research was to study samples of invasive rapa whelks R. venosa in order to evaluate both the genetic diversity and morphological variability by means of integrative approach.   Materials and methods   S a m p l i n g Eighty adult (2–3 years old) rapa whelks were collected by scuba divers in the Black Sea near the Hydro- biological Station Odesa, I. I. Mechnikov National University (Ukraine),  at a depth of 6–10 m in 2015. Th e habitat is characterized by mixed (shelly gravel and sand) sediments and high variability in water salinity (from 4 ‰ to 18 ‰ according to monitoring records of the Hydrobiological Station). A small amount of foot tissue from fi ve individuals were taken and preserved in 95 % ethanol until further molecular processing.   M o r p h o m e t r i c s t u d i e s Th e fi ve linear distances of the shell — its height (H), width (W), aperture height (Ha), aperture width (Wa) and height of the last whorl (Ht) — were measured with a digital caliper as shown in fi g. 1.1 and shells were weighed (Ms) with a hand scale. Th e ratios of height to width (H/W), height to width of the aperture (Ha/ Wa) and height to height of the last whorl (H/Ht) were calculated to evaluate the elongation of each shell, and Ms/H was to evaluate the thickness of a shell. Th e ratios were used as thought to be “size-independent” and already corrected for allometry. To test the signifi cant diff erences of linear measurements and ratios between morphotypes and sexes the Factorial ANOVA in Statistica v10 was performed. For further geometric morphometric analysis, photos of each shell were captured and then saved in the JPG format. While taking photographs, we used a special hand-made equipment (see fi g. 1.3) to keep all shells in the same position relative to the camera, thus avoiding the distortion error associated with the rotation. Th is equipment is aimed to fi x the object by certain points. In our study each shell was fi xed on three points: the top of the shell, the bottom edge and the extreme point of the aperture.  Aft er photos were captured, all studied shells were visually assigned to the following morphotypes: the fi rst has a thick and durable last whorl and a low spire, which makes the shell look “broad” (fi g. 1.2, a), and the second one has a relatively slender shell with an elongated high-conical spire that looks like the “extended” morphotype (fi g. 1.2, b). Subsequently, the geometric morphometric analysis based on landmarks was performed. Fift een landmarks of the shell were examined which are located as follows (fi g. 1.1): LM1 — extreme anterior point of siphonal canal;  LM2 — extreme anterior point of umbilicus;  LM3 — left side extreme point of body whorl;  469Morphological and Molecular Studies of the Rapa Whelk, Rapana venosa, from Odesa Bay LM4 — left side point on suture of 2nd whorl on spire;  LM5 — apex;  LM6 — right side point on suture of 2nd whorl on spire;  LM7 — posterior canal;  LM8 — extreme point of aperture;  LM9 — columellar fold on inner lip;  LM10 — curve on umbilicus;  LM11 — left spiral rib on body whorl; LM12 — right spiral rib on body whorl; LM13 — connection of aperture curve with body whorl;  LM14 — curve on posterior outer lip;  LM15 — curve on anterior outer lip.  Digitizing was done by using the TPSdig2 soft ware (Rholf, 2013). Th e preliminary Procrustance ANOVA showed that the interaction between morphs and centroid size was not signifi cant (F = 0.5206, p = 0.883), thus the residuals of a pooled within-group regression of shape on centroid size (accounting for 3.66 % of total variance, p = 0.006) were obtained to get a corrected for intra-specifi c allometry dataset. Th is dataset then was used in subsequent analyses.  Fig. 1. Rapana venosa aggr.: 1 — linear measurements and landmarks (LM) used for GMM analysis (1–9 — fi xed LM; 10–15 — semi LM); 2 — morphotypes; 3 — tool for fi xation; 4 — museum material: a — Japan Sea, 1877, Natural History Museum of V. N. Karazin Kharkiv National University (H: 16.3 cm); b — Japan Sea, 1983, National Museum of Natural History at the National Academy of Sciences of Ukraine, Kyiv (16.1 cm); c — Black Sea, Kerch, 1972, National Museum of Natural History at the National Academy of Sciences of Ukraine, Kyiv (7.9, 7.8, 9.2 cm). 470 H. Morhun, M. O. Son, O. O. Kovtun, S. Utevsky To test for statistical diff erences in shell shapes between morphotypes, as well as the eff ect of sex on the shape within each morphotype, we used the Procrustes ANOVA evaluated for signifi cance with the F-test (Goodall, 1991). Signifi cance testing was achieved through permutation using a residual randomisation per- mutation procedure involving 1,000 permutations (Collyer et al., 2015). Shell shape variability and intergroup diff erence were analyzed through the Principal Component Analysis in Morpho J soft ware (Klingenberg, 2011).  Th e strength of covariation between diff erent morphometric approaches — traditional morphometry (ratios of linear measurements) and geometric morphometric data (PC scores of shell shape changes) — was evaluated by the linear correlation coeffi cient (Pearson) in Paleontological Statistic program (PAST v4.03) (Hammer et al., 2001).   D N A e x t r a c t i o n a n d a m p l i f i c a t i o n Five specimens of rapa whelk from the Black sea near the Hydrobiological Station were transferred to the molecular laboratory of the Department of Zoology and Animal Ecology, V. N. Karazin Kharkiv National University (Kharkiv, Ukraine) for a molecular analysis. Small pieces of muscle tissue from the foot were used for DNA extraction. Genomic DNA was isolated using a DNA Blood and Tissue extraction kit (Qiagen).  Th e mitochondrial cytochrome c oxidase subunit one (COI) fragment was chosen as considered to be a standard animal DNA barcode gene region (Hebert et al., 2003). It was amplifi ed using the standard prim- ers (Folmer et al., 1994): LCO1490, 5'-GGTCAACAAATCATAAAGATATTGG-3' and HCO2198, 5'-TA- AACTTCAGGGTGACCAAAAAATCA-3'; the amplifi cation was conducted under the following PCR proto- col: 94 °C for 3 min; 5 cycles of 30 sec at 94 °C, 1:30 min at 45 °C, and 1 min at 72 °C; 35 cycles of 30 sec at 94 °C, 1:30 min at 51 °C, and 1 min at 72 °C; 5 min of denaturation step at 72 °C (Hou et al., 2007). PCR products (5 μl) were cleaned up by SIGMA columns according to the manufacturer’s guidelines and then sequenced by Macrogen Inc. (the Netherlands) using the same primers as at the amplifi cation stage. Th e resulting se- quences were manually assembled to a uniform length by removing the remaining parts of the primer regions in FinchTV v. 1.5.0 (Geospiza Inc.) and then submitted to GenBank (Accession: OL504957–61). Th e length of COI sequences is 615 bp.   P h y l o g e n e t i c a n a l y s i s  To perform the phylogenetic analysis, previously published nucleotide sequences from NCBI database in addition to our sequences and the gene of Plicopurpura patula (Linnaeus, 1758), Rapana bezoar (Linnaeus, 1767) and Rapana rapiformis (Born, 1778) employed as an outgroup were used for inferring a phylogenetic tree. Th e COI sequences were aligned using MAFFT v7.452 (Katoh et al., 2017) with FFT-NS-i strategy selected by the “Auto” option, and examined at the amino acid level for the absence of stop codons using MEGA X (Kumar et al., 2018).  Th e evolutionary history was inferred by using maximum likelihood in IQ-TREE v1.5.5 (Nguyen et al., 2015), with branch support estimated using 1000 replicates of both the SH-like approximate likelihood-ratio test (SH-aLRT; Guindon et al., 2010) and the ultrafast bootstrapping algorithm (Minh et al., 2013). Th e ModelFinder option was used to identify the optimal partitioning scheme and substitution models (Kalyaanamoorthy et al., 2017). Best-fi t models were determined according to the Bayesian information criterion (BIC); HKY+F+I model of the COI gene was chosen. Th e tree is drawn with branch lengths measured in the number of substitutions per site.  In addition, the number of base diff erences per site (p-distances) between sequences and their standard errors were calculated. All positions containing gaps and missing data were eliminated.  Haplotypes were determined using DnaSP version 6.12.03 (Rozas et al., 2017). Th ere were a total of 615 base pairs in the fi nal dataset.    Results   M o r p h o m e t r i c s t u d i e s Th e morphometric measurements of population and the signifi cant diff erence among morphotypes and sexes are shown in table 1.  Th e results of two-way ANOVA using linear measurements and the ratios revealed signifi cant diff erences in values among the morphotypes for H, H/W, and Ht/H. Th e diff erence between the sexes was revealed for H, Ha, Wa and H/W, Ha/Wa (p < 0.05, table 1). No diff erence in linear measurements between the sexes within each morphotype was found (p > 0.05, table 1).  As a result of the geometric morphometric analysis, the signifi cant diff erence in the shape of shells between the defi ned morphotypes was revealed (F = 4.12, p = 0.001) (table 2). Yet, no signifi cant shape variation between the sexes was found (F = 1.13, p = 0.318), also no diff erence in shape between the sexes within each morphotype (F = 0.22, p = 0.989) was revelaed.  471Morphological and Molecular Studies of the Rapa Whelk, Rapana venosa, from Odesa Bay Th e further principal component analysis of geometric data was performed. In total, 26 principal components were revealed, with PC1 accounting for 26.37 % of variation and PC2, 20.55 %. A cumulative proportion of these fi rst components are 46.92 % (fi g. 2). Th e remaining PC’s each contributed around or less than 10 % of the total variation and are not discussed further. PC1 is associated with sliding of the extreme point of the whorl of the shell (see fi g. 2). All samples were scattered along the PC1 axis but did not show any inter-specifi c variation within each morphotype. Th e high and signifi cant correlation between PC1 scores and ratios from traditional morphometric measurements was not revealed (p > 0.05, table 3, supplem. T a b l e 1 . Measurements of R. venosa from Odesa Bay Morph, N Sex, N Linear Distances, mm Ms, mg Ratios H W Ha Wa Ht H/W Ha/Wa Ht/H Ms/H Extended } 13 min 64.470 59.540 50.710 29.640 56.323 32.160 1.082 1.540 0.822 0.447 36 max 78.620 69.300 59.710 37.090 67.899 55.370 1.202 1.859 0.874 0.726 average 72.663 63.691 56.578 33.082 62.298 43.148 1.142 1.714 0.857 0.592 { 23 min 68.630 58.490 51.740 30.090 59.524 27.810 1.105 1.553 0.833 0.379 max 86.640 76.430 68.470 42.190 75.027 83.240 1.261 1.745 0.896 0.982 average 77.482 66.586 60.046 36.503 66.556 52.115 1.165 1.647 0.859 0.666 Broad } 13 min 61.730 56.350 53.530 29.800 52.714 33.790 1.095 1.608 0.807 0.491 44 max 88.140 74.320 66.320 41.250 76.745 81.010 1.232 1.840 0.874 0.919 average 77.125 65.164 58.811 34.196 65.073 48.094 1.183 1.725 0.844 0.617 { 31 min 69.470 60.020 54.210 28.730 58.330 32.080 1.147 1.557 0.810 0.439 max 89.910 77.030 75.800 40.940 78.164 82.260 1.316 1.974 0.878 0.930 average 78.898 65.592 60.139 35.491 66.622 50.780 1.203 1.700 0.844 0.637 p (morph) 0.024 0.829 0.278 0.947 0.236 0.548 0.000 0.123 0.001 0.958 p (sex) 0.012 0.136 0.027 0.003 0.017 0.055 0.018 0.025 0.797 0.116 p (morph*sex) 0.236 0.267 0.318 0.164 0.259 0.297 0.850 0.301 0.906 0.370 N o t e . Coeffi cients with signifi cant p < 0.05 are highlighted in bold. Fig. 2. Morphospace defi ned by the two fi rst principal components (PC’s) of shape variance using landmark data. Shape changes associated with scores of each PC axis are shown as warped surface on a transformation grid (see text for details). 472 H. Morhun, M. O. Son, O. O. Kovtun, S. Utevsky fi g. 1). PC2 relates with elongation of shell shape: with negative PC2 scores shell appeared to be broad, while with positive PC2 scores shells are prolonged (fi g. 2). All specimens showed inter-specifi c variation being scattered about the PC2 axis within each morphotype. Th ere was a signifi cant correlation between PC2 scores and the H/W and Ht/H ratios values re- vealed (r = 0.58, р < 0.05 and r = –0.68, р < 0.05 respectively, table 3, supplem. fi g. 1). M o l e c u l a r s t u d i e s Th e phylogenetic analysis of our samples and GenBank data revealed the evolutionary history of R. venosa, which is illustrated using the resulting phylogenetic tree (fi g. 3). It was found that all 5 specimens from Odesa Bay are identical and shared the same haplotype; this haplotype is identical to sequences previously published in GenBank, including samples from the native range — Mikawa Bay (Japan), Jeju-do (Korea) — and from the invasive range — the Black Sea, Adriatic Sea, Quiberon Bay in France, the Netherlands, and Chesapeake Bay in the USA (sequences beginning with the codes EU and MH in fi g. 3) (Chandler et al., 2008). Particularly in the Black Sea, this haplotype currently known from Anatolia (sequences with code KP and KU), Crimea (sequences were not deposited in a Genbank by Slynko et al., 2020; personal communication), and the north-eastern Black Sea (sequences with the code EU) (Chandler et al., 2008).  Th e number of base substitutions per site from averaging over all sequence pairs between groups and within groups are shown in table 4. T a b l e 3 . Correlation coeffi cient of linear measurements and PC scores (under diagonal) and p-value aft er permutation test (above diagonal) H W Ha Wa Ht Ms H/W Ha/Wa Ht/H PC1 PC2 H 0.000 0.000 0.000 0.000 0.000 0.002 0.240 0.555 0.732 0.030 W 0.865 0.000 0.000 0.000 0.000 0.122 0.004 0.001 0.495 0.618 Ha 0.881 0.837 0.000 0.000 0.000 0.176 0.458 0.003 0.423 0.851 Wa 0.781 0.852 0.846 0.000 0.000 0.560 0.000 0.005 0.129 0.141 h 0.959 0.913 0.921 0.822 0.000 0.130 0.185 0.002 0.488 0.727 Ms 0.822 0.870 0.857 0.802 0.889 0.773 0.056 0.000 0.336 0.870 H/W 0.341 –0.174 0.153 –0.066 0.171 –0.033 0.002 0.000 0.528 0.000 Ha/Wa –0.133 –0.322 –0.084 –0.599 –0.150 -0.214 0.339 0.422 0.090 0.009 Ht/H 0.067 0.353 0.329 0.310 0.343 0.395 –0.527 –0.091 0.168 0.000 PC1 0.039 0.077 0.091 0.171 0.079 0.109 –0.072 –0.191 0.155 1.000 PC2 0.242 –0.057 –0.021 –0.166 0.040 –0.019 0.579 0.290 –0.677 0.000 N o t e . Coeffi cients with signifi cant p < 0.05 are highlighted in bold. T a b l e 2 . Procrustes ANOVA evaluating variation in shape between morphotypes and between sexes within each morphotype Df SS MS Rsq F Z Pr (> F) CS 1 0.009725 0.0097254 0.04838 4.0784 3.1615 0.001 Morph 1 0.009909 0.0099092 0.04929 4.1555 3.2605 0.001 Sex 1 0.002692 0.0026916 0.01339 1.1288 0.5135 0.320 CS:Morph 1 0.001241 0.0012413 0.00617 0.5206 –1.2089 0.882 CS:Sex 1 0.001986 0.0019859 0.00988 0.8328 –0.1443 0.544 Morph:Sex 1 0.000770 0.0007702 0.00383 0.3230 –2.2098 0.989 CS:Morph:Sex 1 0.003027 0.0030274 0.01506 1.2696 0.7102 0.242 Residuals 72 0.171691 0.0023846 0.85401 Total 79 0.201042 N o t e . P-values based on 1,000 random residual permutations. 473Morphological and Molecular Studies of the Rapa Whelk, Rapana venosa, from Odesa Bay Fig. 3. Phylogenetic relationships between major groups of Rapana genus obtained by Maximum-Likelihood method and based on COI sequences (ultrafast bootstrap values are shown for clades; the tree is rooted at Plicopurpura patula). Sequences from the current research are highlighted in bold. T a b l e 4 . Estimates of evolutionary divergence over sequence pairs of R. venosa dataset. Th e number of base diff erences per site (based on p-distances) between groups and within each group are shown Group Between group Within group 1 2 3 4 5 p-dist S. E. 1. P. paluta 0.150 0.134 0.110 0.105 0.001 0.001 2. R. bezoar 0.083 0.064 0.072 0.072 0.000 0.000 3. R. rapiformes 0.076 0.058 0.134 0.132 0.000 0.001 4. R. venosa Odesa 0.076 0.064 0.083 0.003 0.000 0.000 5. R. venosa 0.074 0.064 0.082 0.004 0.003 0.002 N o t e . Standard error estimates are shown in italic. 474 H. Morhun, M. O. Son, O. O. Kovtun, S. Utevsky Discussion Studies of the morphological diversity of R. venosa from diff erent parts of the northern Black Sea region have shown high capability of this mollusk to vary in its shell conchology under the infl uence of environmental conditions (Bondarev, 2010; Snigirov et al., 2013; Kos’yan, 2013). All systematic features of shells, including the general shape and color, the thickness of walls, the presence and development of axial and spiral ribs and grooves, spines and other sculptural surface elements of the shell, are variable. Th e most common and main driver for changes is assumed to be diff erent trophic conditions in habitats (Bondarev, 2010; Kovtun et al., 2014). Depletion of food sources causes a slowdown in growth, a decrease in the size of individuals and also decrease in the size when maturity occurs (Chukhchin, 1961). Th e results of our geometric morphometric analysis revealed signifi cant diff erences in shell shape between the defi ned morphotypes in the spire elongation: shells vary from a tall (extended) to a squatted one (fi g. 2). Such a high variability of shell habitus might be explained based on previously published studies in which it is associated with habitat where the food objects occur: a narrow and long shell is more capable for moving on sandy seabed and hunting for mollusks burrowing into sand (Bondarev, 2010). Th us, high diversity of potential food objects in Odesa Bay, which R. venosa hunts for, is assumed to result in the high morphological heterogeneity of molluscs which we observed. Although the shells vary in the thickness of their walls from thick-walled to thin-walled, the thickness is not a sign of either sexual dimorphism or morphotype (table 1, Ms/H, p > 0.05). It is believed that food abundance can aff ect on thickness: if the amount of nutrients is enough, the growth is more or less constant and the carbonate layer is lied on the inner surface of the shell evenly in a certain unit of time; however, if food is in short supply, the growth of the soft body (its weight and size) slows down signifi cantly, but the shell itself continues to become thicker (Kos’yan, 2013).  Th e results of our geometric morphometric analysis also showed no diff erences in shape between the sexes within the population of Odesa Bay. Yet, according to linear morphological measurements (H, Wa, Ha) and some ratios (H/W and Ht/H), signifi cant diff erences between sexes were revealed. Th us, the approaches we applied contributed to each other making the results more informative: although we detected signifi cant diff erences between males and females in shell sizes (linear measurements), the sexes did not diff er in their shell shape (geometric morphometrics).  Kovtun et al. (2014) also showed that males have larger shell sizes than females do. Th is fact is explained by the need for the latter to spend additional energy on reproduction of off spring. According to Bondarev (2010), the sex of individual can be determined based on both size and conchological traits of shell, especially shape: males have a higher and nar- rower shell than females. We also obtained signifi cant diff erences in H/W and Ht/H ratios between sexes, but a more sensitive approach (geometric morphometrics) shows that this trend in shape morphology is absent for the Odesa Bay population.  Summarizing the results of morphological examination, the following features of population were revealed: no sexual dimorphism either in shell shape or in thickness was found, and the diff erence in size between males and females is caused by the need for the latter to reproduce. Th e diversity in elongation of individuals might be a result of a high variety of food objects in Odesa Bay. Th e second essential part of our research was devoted to a molecular study of R. venosa from Odesa Bay. Our analysis revealed phylogenetic relationships and nucleotide diversity of the population. In particular, all specimens shared the same haplotype known from pre- vious publications as the only one occurring in the regions of invasion around the world (Chandler et al., 2008). Our results are consistent with previous publications on the mo- lecular diversity of R. venosa from the northern Black Sea region, especially from Crimea (Slynko et al., 2020).  475Morphological and Molecular Studies of the Rapa Whelk, Rapana venosa, from Odesa Bay Th e observed low genetic diversity can be interpreted as the evidence of an one-time invasion of the rapa whelk into the Black Sea and the further dispersal to other regions. Moreover, it can be assumed that the worldwide success of R. venosa invasion is resulted from this one specifi c haplotype, and the low genetic variation may be the consequence of successful adaptation to new environmental conditions aff ecting evolutionary rescue in the process if invasion aimed to establish the invader in a new region (Estoup et al., 2016).  Th e results obtained using the integrative approach (combining genetics and morphology) imply high morphological diversity and, at the same time, low genetic variability. Th is can be considered in a context of the “Genetic Paradox of Biological Invasions” concept (Estoup et al., 2016). Th us, a single-time introduction of a species (as was with rapa whelks in the 1940s) accompanied by the bottleneck eff ect usually leads to a depletion of the genetic variation and, accordingly, the reduced phenotypic diversity of an introduced population in general. Th is is because a small part of the population is introduced, and it does not carry all the genetic diversity of its species. However, in case of the rapa whelks from the Black Sea, we observe rather a high heterogeneity of morphological characteristics: high phenotypic plasticity, the emergence of new morphs in biotopes with diff erent ecological characteristics, which suggests high adaptability in general; and all this is along with the complete absence of nucleotide diversity for both the COI gene (this study) and nad2 (Chandler et al., 2008). Less conservative markers could be used in future as an attempt to reveal higher nucleotide diversity in the population. To explore the phenomenon of phenotypic plasticity of the rapa whelks from the Odesa Bay in a comparative way, some available museum material from its native region (Japan) and from the Kerch Strait sampled in 1972 (about 25 years aft er invasion) were examined to compare conchological characters of those populations (fi g. 1.4).  Firstly, a size diff erence is seen: the Odesa mollusks are smaller than the native ones — the average H is 70–80 mm, while shells from the Sea of Japan are 161 and 163 mm (fi g. 1.4, a, b). Secondly, a diff erence in the sculpture and massiveness of the shells are observed: specimens from the native area (fi g. 1.4, a, b) have more pronounced spines and thicker-walled shells than shells from Odesa Bay. Th e fact of reducing the shell size in the Black Sea rapa whelk population was previously recorded by other researchers (Ivanov, 1961; Bondarev, 2010). Yet, the studied shells from the Kerch sampled in 1972 have dimensions similar to the Odesa population — 79–92 mm. It can be assumed that the mollusks became fi ner rapidly aft er the invasion, and this reduction in size is related not only to the available amount of food. Th e populations of mussels and other bivalves in those years in the Kerch Strait were at a high level (both free-living populations and existing mussel farms) and there was a rather suffi cient amount of food (Ivanov, 1987; Ivanov, Synegub, 2008). Th us, reduction in shell size can be a result of the infl uence of unknown environmental factors and / or the bottleneck eff ect occurred as a consequence of invasion. Th e size of the rapa whelk can also be determined by a potential prey: if small Chamelea gallina (Linnaeus, 1758), Anadara kagoshimensis (Tokunaga, 1906) and Mytilus spp. are predominant in the diet, then the rapa whelk has a large size (Kos’yan, 2010, 2013). Th is is explained by the fact that the mollusk prefers the prey of particular size, thus compensating the energy for foraging (the opening of the shell valves of the prey) (Savini, Occhipinti- Ambrogi, 2006; Kos’yan, 2009, 2010, 2013). Comparing the sizes of the rapa whelks in previous studies, the maximum size of the shell of R. venosa from the native range is 212.3 mm (Pisor, 2005); Bondarev (2010) found that shells from the Sea of Japan vary from 75 to 168.7 mm. In the non-native regions, the following shell lengths are observed: in the Chesapeake Bay (1998–1999) from 67 mm to 160 mm (ICES, 2004); in the estuary of the Rio de la Plata — 28–120 mm (Giberto et al., 2006); in the Mediterranean Sea near Venice — 78–139 mm (Cesari, Pellizzato, 1985); off the coast of Romania — from 50 to 95 mm (Micu et al., 2008; Sereanu et al., 2016); off the coast of Turkey — from 58 to 102 mm (Seyhan et al., 2003). Th e specimens from our 476 H. Morhun, M. O. Son, O. O. Kovtun, S. Utevsky sampling had approximately the same size (table 1) with no extremes and are similar to those from the previous studies within invasive regions.  Th e tendency to decrease in size in comparison with the native area are seen in general; however, the aforementioned assumption about the infl uence of the bottleneck eff ect as one of the reasons should be further verifi ed by carrying out appropriate molecular studies accompanied with morphological studies based on more extensive sampling (museum col- lections and fresh specimens).  Along with the genetic and statistical analyses, this work was aimed to test the applicabil- ity of such an advanced and sensitive method as geometric morphometrics on rapa whelks. Our research can be a starting point for a more detailed survey of morphological diversity among multiple morphotypes, eco-forms and forms of R. venosa inhabiting the Black Sea.  One of those morphs named “dwarf” is of particular interest as it has a size abnormal- ity: a sexually mature mollusk is 4 times smaller in size than a regular adult one (Bondarev, 2010: fi g. 5, A). Th ese individuals are rare to fi nd and rather unique in samples, but are the key to understand the ecology of the rapa whelk: the mature “dwarf” can maintain juvenile feeding strategy — drilling through the shell of its prey (Kingsley-Smith et al., 2003) — while large adults tend to open bivalve mollusks (Chukhchin, 1970; Savini et al., 2002, 2006; Bondarev, 2010). It is believed that, due to having this feeding strategy, rapa dwarfs can switch to another ecological niche, which may result in their genetic isolation (Bondarev, 2010). Similarly, by keeping this feeding strategy throughout a life it can impact on the shell shape in general; this hypothesis of the shape phenotypic plasticity of regular and dwarf forms will need specifi cally designed studies to be properly tested. It is also reasonable to expand a study for detailed examination of R. venosa shape changes towards ecological abiotic factors (salinity, temperature, illumination, soil, depth); this could be done using Partial least squares analysis (Rohlf, Corti, 2000; Fruciano et al., 2011) or Rarefaction analysis of morphospace volumes (Foote, 1992; McClain et al., 2004). Similarly, to explore the contribution of heredity into the shell shape it is possible to perform through the Mantel test (Liu et al., 1996; Lynch, Walsh, 1998; Klingenberg, Leamy, 2001) but this requires a fairly large array of data.  In general, the methodology used in this paper (landmarks and photographing equipment) can be used for a future morphometric study of R. venosa. Th e applied geometric morphometric approach appeared to be a useful tool to visualize the morphological heterogeneity of the population of Odesa Bay in general, as well as to test and evaluate it statistically. We thank Nina Petrenko from National Museum of Natural History of the NAS of Ukraine for assistance in collecting photos of museum materials. We thank the Malacological Society of London for a travel grant for HM to attend a workshop on geometric morphometric analysis. Th is research was supported by grant no. 0117U0048360 from the Ministry Education and Science of Ukraine. References Alakrinskaya, J. O. 1989. Physiological and biochemical reasons for stability of Rapana thomasiana. Gidrobiologicheskii Zhurnal, 6, 83–87 [In Russian]. Bondarev, I. P. 2010. Shell morphogenesis and intraspecifi c diff erenciation of Rapana venosa (Valenciennes, 1846). Ruthenica, 20 (2), 69–90. Cesari P., Pellizzato M. 1985. Insediamento nella Laguna di Venecia e distribuzione Adriatica di Rapana venosa (Valenciennes) (Gastropoda, Th aididae). Lavori – Societa Veneziana Scienze Naturale, 10, 3–16. Chandler, E. A., McDowell, J. R., Graves, J. E. 2008. Genetically monomorphic invasive populations of the rapa whelk, Rapana venosa. Molecular Ecology, 17, 4079–4091. Chukhchin, V. D. 1961. The Growth of Rapa (Rapana bezoar L.) in Sevastopol’ Bay. Trudy Sevastopolskoi Biologicheskoi Stancii, 14, 169–177 [In Russian]. Chukhchin, V. D. 1970. Functional morphology of Rapana venosa. Naukova Dumka, Kiev, 1–138 [In Russian]. Chukhchin, V. D. 1984. Ecology of gastropod molluscs of the Black Sea. Naukova Dumka, Kiev, 1–176. Collyer, M. L., Sekora, D. J., Adams, D. C. 2015. A method for analysis of phenotypic change for phenotypes described by high-dimensional data. Heredity, 115, 357–365. Cucaz, M. 1983. Rapana venosa (Valenciennes, 1846) vivente nel Golfo di Trieste. Bolletino Malacologico, Milano, 19, 261–262. 477Morphological and Molecular Studies of the Rapa Whelk, Rapana venosa, from Odesa Bay Drapkin, E. I. 1953. A New Mollusk in the Black Sea. Priroda, 9, 92–95 [In Russian]. Estoup, A., Ravigné , V., Hufbauer, R., Vitalis, R., Gautier, M., Facon, B. 2016. Is there a genetic paradox of biological invasion? Annual Review of Ecolology, Evolution and Systimatics, S 47, 51–72. Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. 1994. DNA primers for amplifi cation of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299. Foote, M. 1992. Rarefaction analysis of morphological and taxonomic diversity. Paleobiology, 18, 1–16. Fruciano, C., Tigano, C., Ferrito, V. 2011. Traditional and geometric morphometrics detect morphological variation of lower pharyngeal jaw in Coris julis (Teleostei, Labridae). Italian Journal of Zoology, 78 (3), 320–327. Ghisotti, F. 1971. Rapana thomasiana Crosse, 1861 (Gastropoda, Muricidae) nel Mar Nero. Conchiglie, Milano, 7, 55–58. Ghisotti, F. 1974. Rapana venosa (Valenciennes), nuova ospite Adriatica? Conchiglie, Milano, 10, 125–126. Giberto, D. A., Bremec, C. S., Schejter, L., Schiariti, A., Acha, E. M. 2006. Th e invasive Rapa whelk Rapana ve- nosa (Valenciennes, 1846): Status and potential impact in the Rio de la Plata estuary, Argentina–Uruguay. Journal of Shellfi sh Research, 25 (3), 1–6. Giberto, D. A. Bruno, L. I. 2014. Recent records of the exotic gastropod Rapana venosa (Valenciennes, 1846) along the Argentine coastline: is the invasion progressing southwards? Pan-American Journal of Aquatic Sciences, 9 (4), 324–330. Goodall, C. 1991. Procrustes methods in the statistical analysis of shape. Journal of the Royal Statistical Society. Series B (Methodological), 53 (2), 285–339. Govorin, I. A., Kurakin, A. P. 2011. Otsenka vlyianyia khyshchnoho briukhonohoho molliuska Rapana venosa (Valenciennes 1846) na fyltratsyonnyi potentsyal mydyinykh poselenyi. Ekolohichna bezpeka pryberezh- noi ta shelfovoi zon ta kompleksne vykorystannia resursiv shelfu, 25, 435–442 [In Russian]. Hammer, Ø., Harper, D., Ryan, P. 2001. PAST: Paleontological Statistics Soft ware: Package for Education and Data Analysis. Palaeontologia Electronica, 4, 1–9. Harding, J. M., Mann, R. 1999. Observations on the Biology of the veined rapa whelk, Rapana venosa (Valenci- ennes, 1846) in the Chesapeake Bay. Journal of Shellfi sh Research, 18, 9–17. Harding, J. M., Mann, R. 2003. Current status and potential establishment range for veined rapa whelks Rapana venosa on the US East coast. Aquatic Invaders: Th e Digest of National Aquatic Nuisance Species Clearinghouse, 14, 1–7. Harding, J. M., Clark, V. P., Mann, R. 2002. Rundown on the Rapa. Virginia Institute of Marine Science: Gloucester Point, Virginia. VSG-02-19, VIMS-ES-51 10/2002. Hebert, P. D., Cywinska, A., Ball, S. L., Dewaard, J. R. 2003. Biological identifi cations through DNA barcodes. Proceedings of the Royal Society B: Biological Sciences, 270, 313–321. Hou, Z. G., Fu, J. H., Li, S. Q. 2007. A molecular phylogeny of the genus Gammarus (Crustacea: Amphipoda) based on mitochondrial and nuclear gene sequences. Molecular Phylogenetics and Evolution, 45, 596–611. ICES (International Council for the Exploration of the Sea). 2004. Alien species alert: Rapana venosa (veined whelk). Mann, R., Occhipinti, A., Harding, J. M., eds). ICES Cooperative Research Report N 264, 1–14. Ivanov, A. I. 1961. Nekotorye dannye o kolychestvennom raspredelenyy rapany (Rapana bezoar L.) v vostochnoi chasty Cher- noho Moria y Kerchenskom Prolyve y ob umenshenyy ejo razmerov. Doklady AN SSSR, 141 (2), 467–468 [In Russian] Ivanov, A. I. 1987. Raspredelenye y zapasy mydyi v Kerchenskom prolyve. Oceanologia, 27 (5), 850–854 [In Russian]. Ivanov, D. A., Sinegub, I. A. 2008. Transformatsyia byotsenozov Kerchenskoho prolyva posle vselenyia khyshch- noho molliuska Rapana thomasiana y dvustvorchatykh Mya arenaria y Sunearca cornea. In: Materials of III International Conference Current problems of the Azov-Black Sea Region ecology (10–11 October 2007, Kerch, YugNIRO, Ukraine). YugNIRO Publishers’, Kerch, 45–51 [In Russian]. Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A., Jermiin, L. S. 2017. ModelFinder: fast model selection for accurate phylogenetic estimates. Nature Methods, 14 (6), 587–589. Katoh, K., Rozewicki, J., Yamada, K. D. 2017. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefi ngs in Bioinformatics, 20, 1160–1166. Klingenberg, C. P. Leamy, L. J. 2001. Quantitative genetics of geometric shape in the mouse mandible. Evolu- tion, 55, 2342–2352. Klingenberg, C. P. 2011. Morphoj: an integrated soft ware package for Geometric Morphometrics. Molecular Ecology Resources, 11, 353–357. Kos’yan, A. R. 2009. Muricid Rapana venosa (Valenciennes, 1846) in the Black Sea. In: Proceedings of the Ninth International Conference on the Mediterranean Coastal Environment, MEDCOAST 09 (10–14 November 2009, Sochi, Russia). Ankara: Middle East Technical Univ., 305–315. Kos’yan, A. R. 2010. Ecological state of Rapana venosa populations in the northern part of the Black Sea. Nau- kovi zapiski Ternopilskogo natcional’nogo ped. universitetu, Seriaya Biologia, 3, 44 [In Russian] Kos’yan, A. R. 2013. Comparative analysis of Rapana venosa (Valenciennes, 1846) from diff erent biotopes of the Black Sea based on its morphological characteristics. Oceanology, 53, 47–53. Koutsoubas, D., Voultsiadou-Koukoura, E. 1991. Th e occurrence of Rapana venosa (Valenciennes, 1846) (Gas- tropoda, Th aididae) in the Aegean Sea. Bolletino Malacogico, 26 (10–12), 201–204. Kovtun, O. О., Toptikov, V. A., Totsky, V. M. 2014. Comparative morphological characteristic of Rapana ve- nosa (Gastropoda: Muricidae, Rapaninae) from diff erent water areas of the Northern part of the Black Sea. Vistnik ONU, 19 (1), 68–80 [In Russian]. 478 H. Morhun, M. O. Son, O. O. Kovtun, S. Utevsky Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. 2018. MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution, 35, 1547–1549. Kurakin, A. P., Govorin, I. A. Rapana venosa as one of the factors of decrease of fi ltration potential of mussel population. In: Ukrainian scientifi c and practical conference “Ecology of cities and recreational areas” (17– 18 April 2008, Odessa, Ukraine). Innovative-Information Center “INVAZ”, Odessa 245–249 [In Russian]. Lynch, M., Walsh, B. 1998. Genetics and analysis of quantitative traits (Vol. 1, 535–557). Sunderland, MA: Sinauer. Liu, J., Mercer, J. M., Stam, L. F., Gibson, G. C., Zeng, Z. B., Laurie, C. C. 1996. Genetic analysis of a morphological shape diff erence in the male genitalia of Drosophila simulans and D. mauritiana. Genetics, 142 (4), 1129–1145. Mann, R., Harding, J. M. 2003. Salinity tolerance of larval Rapana venosa: Implications for dispersal and establishment of an invading predatory gastro- pod on the North American Atlantic coast. Biological Bulletin, 204, 96–103. Mann, R., Harding, J. M., Westcott, E. 2006. Occurrence of imposex and seasonal patterns of gametogenesis in the invad- ing veined rapa whelk Rapana venosa from Chesapeake Bay, USA. Marine Ecology Progress Series, 310, 129–138. Marinov, T. M. 1990. Th e Zoobenthos from the Bulgarian Sector of the Black Sea. Bulgarian Academy of Science Publishing, Sofi a, 1–195 [In Bulgarian]. McClain, C. R., Johnson, N. A., Rex, A. M. 2004. Morphological disparity as a biodiversity metric in lower bathyaland abyssal gastropod assemblages. Evolution, 58 (2), 338–348. Mel, P. 1976. Sulla presenza di Rapana venosa (Valenciennes) e di Charonia variegate sequenza (AR & BEN.) nell’Atlo Adriatico. Conchiglie, Milano, 12, 129–132 [In Italian]. Micu, S., Kelemen, B., Mustata, G. 2008. Current distribution and shell morphotypes of Rapana venosa (Valenciennes, 1846) in the Agigea 4 m littoral. Analele Stiintifi ce ale Universiatii “Al. I. Cuza” Iasi, s. Biologie animala, 54, 185–189. Minh, B. Q., Nguyen, M. A. T., von Haeseler, A. 2013. Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution, 30 (5), 1188–1195. Nguyen, L. T., Schmidt, H. A., von Haeseler, A., Minh, B. Q. 2015. IQ-TREE: a fast and eff ective stochastic al- gorithm for estimating maximum likelihood phylogenies. Molecular Biology and Evolution, 32, 268–274. Nieweg, D. C., Post, J. J. N., Vink, R. J. 2005. Rapana venosa (Gastropoda: Muricidae): a new invasive species in the North Sea. Deinsea, 11, 169–174. Pastorino, G., Penchaszedeh, P. E., Schejter, L., Bremec, C. 2000. Rapana venosa (Valenciennes, 1846) (Mol- lusca: Muricidae): a new gastropod in south Atlantic waters. Journal of Shellfi sh Research, 19, 897–899. Pereladov, M. V. 2013. Th e current state of the population and the biology of the Rapana venosa in the north- eastern Black Sea. Trudy VNIRO, 150, 8–20 [In Russian]. Pisor, D. L. 2005. Registry of World Record Size Shells. ConchBooks, Hackenheim, 1–171. Rinaldi, E. 1985. Rapana venosa (Valenciennes) spiaggiata in notevole quantità sulla spiaggia di Rimini (Fo). Bolletino Malacologico, Milano, 21, 318 [In Italian]. Rohlf, F. J., Corti, M. 2000. Use of two-block partial least-squares to study covariation in shape. Systematic biology, 49 (4), 740–753. Rozas, J., Ferrer-Mata, A., Snchez-DelBarrio, J. C., Guirao-Rico, S., Librado, P., Ramos-Onsins, S. E., Snchez- Gracia, A. 2017. DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution, 34, 3299–3302. Rubinshtein, I. G., Hizniak V. I. 1988. Stocks of Rapana thomusiana in the Kerch Strait. Rybnoye Khoz (Mos- cow), 1, 39–41 [In Russian]. Savini, D., Castellazzi, M., Favruzzo, M., Occhipinti, A. 2004. Th e alien mollusk Rapana venosa in the northern Adriatic Sea: population structure and shell morphology. Chemical Ecology, 20, 411–424. Savini, D., Occhipinti-Ambrogi, A. 2006. Consumption rates and prey preference of the invasive gastropod Rapana venosa in the Northern Adriatic Sea. Helgoland Marine Research, 60, 153–159. Scarabino, F., Menafra, R., Etchegaray, P., 1999. Presence of Rapana venosa (Valenciennes, 1846) (Gastropoda: Muricidae) in the Rio de la Plata. Boletí n de la Sociedad Zooló gica de Uruguay, 11, 40. Sereanu, V., Meghea, I., Vasile, G., Simion, M., Mihai, M. 2016. Morphology and chemical composition relation of Rapana thomasiana shell sampled from the Romanian Coast of the Black Sea. Continental Shelf Research, 126, 27–35. Seyhan, K., Mazlum, E. R., Emiral, H., Engin, S., Demirhan, S. 2003. Diel feeding periodicity, gastric emptying, and estimated daily food consumption of whelk (Rapana venosa) in the south eastern Black Sea (Turkey) marine ecosystem. Indian Journal of Marine Sciences, 32 (3), 249–251. Snigirov, S., Medinets, V., Chichkin, V., Sylantyev, S. 2013. Rapa whelk controls demersal community structure off Zmiinyi Island, Black Sea. Aquatic Invasions, 8 (3), 289–297. Slynko, Е. Е., Slynko, Y. V., Rabushko, V. I. 2020. Adaptive strategy of Rapana venosa (Gastropoda, Muricidae) in the invasive population of the Black Sea. Biosystems Diversity, 28 (1), 48–52. Ware, C. 2002. Temporal and spatial variation in reproductive output of the veined rapa whelk (Rapana venosa) in the Chesapeake Bay. MS Th esis, College of William and Mary, Williamsburg, Virginia. Xue, D.-X., Graves, J., Carranza, A., Sylantyev, S., Snigirov, S., Zhang, T., Liu, J.-X. 2018. Successful worldwide invasion of the veined rapa whelk, Rapana venosa, despite a dramatic genetic bottleneck. Biological Invasions, 20, 3297–3314. Zolotarev, V. 1996. Th e Black Sea ecosystem changes related to the introduction of new mollusc species. Marine Ecology, 17, 227–236. Received 9 April 2021 Accepted 3 November 2021 << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.0000 /ColorConversionStrategy /CMYK /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams false /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments true /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile () /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV (Za stvaranje Adobe PDF dokumenata najpogodnijih za visokokvalitetni ispis prije tiskanja koristite ove postavke. 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