Genetic Diversity and Clonality of the Asian Clam Corbicula Fluminea Are Reflected by Inner Shell Color Pattern

Aquatic Invasions (2020) Volume 15, Issue 4: 633–645

CORRECTED PROOF

Research Article Genetic diversity and clonality of the Asian clam Corbicula fluminea are reflected by inner shell color pattern

Te-Hua Hsu1,2, Akira Komaru3 and Jin-Chywan Gwo1,* 1Department of Aquaculture, National Taiwan Ocean University, Keelung, 20224, Taiwan 2Center of Excellence for the Oceans, National Taiwan Ocean University, Keelung, 20224, Taiwan 3Faculty of Bioresources, Mie University, Mie, 514-8507, Japan Author e-mails: [email protected] (JCG), [email protected] (THH), [email protected] (AK) *Corresponding author

Citation: Hsu T-H, Komaru A, Gwo J-C (2020) Genetic diversity and clonality of Abstract the Asian clam Corbicula fluminea are reflected by inner shell color pattern. The Asian clam Corbicula fluminea is self-compatible, hermaphroditic, androgenetic, Aquatic Invasions 15(4): 633–645, and ovoviviparous. Androgenesis is a type of asexual male reproduction; although https://doi.org/10.3391/ai.2020.15.4.06 it involves genetic recombination, the offspring retain the same genome as their male Received: 28 March 2020 parent. Despite this unique reproductive character of C. fluminea, considerable Accepted: 16 July 2020 morphological diversity and internal shell color were observed among specimens Published: 16 October 2020 collected from six locations in Taiwan. We investigated these specimens through morphological examination and amplified fragment length polymorphism (AFLP). Handling editor: Fred Wells Twenty-three AFLP genotypes were found in 48 individuals by using only one Copyright: © Hsu et al. primer combination. AFLP was applied for identifying the clonal lineage of C. fluminea This is an open access article distributed under terms successfully. Our results showed that among specimens with different genotypes, of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). external shell color could be affected by environmental factors, such as substrate, showing the same pattern, but internal shell color was determined purely by OPEN ACCESS. genetics. This relationship between the inner shell color variation and genotype in C. fluminea can be easily used to study the dispersal of this invasive species and in breeding for aquaculture.

Key words: invasive species, bivalve, morphology, AFLP, androgenesis

Introduction The Asian freshwater clam Corbicula fluminea (Müller, 1774) is a highly prolific and perniciously invasive species that is widely distributed across diverse environments (Sousa et al. 2008). Corbicula fluminea is considered one of the most invasive species in freshwater aquatic ecosystems (Sousa et al. 2008). Rapid growth, early sexual maturity, short life span, high fecundity, and association with human activities contribute to the success of C. fluminea in colonizing new environments (Sousa et al. 2008). Corbicula fluminea has a mainly native range spanning Asia (e.g. China, Korea, Japan, Taiwan, and Thailand) (Morton 1977, 1982; Araujo et al. 1993; Sousa et al. 2008; Pigneur et al. 2014). The introduced range of diverse freshwater habitats spans continental North and South America and continental Europe (Counts 1981, 1986; Ituarte 1994; Araujo et al.

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 633 Genetic diversity and clonality of Corbicula fluminea

1993; Sousa et al. 2008; Pigneur et al. 2014; Crespo et al. 2015; Gomes et al. 2016; Hunicken et al. 2019). Every year, C. fluminea invasion causes damage worth billions of dollars (Castaneda et al. 2018). Corbicula fluminea grows rapidly and clogs the water inlet pipes of nuclear power plants; furthermore, it exhibits invasive behavior in temperate regions (at temperatures of < 2 °C) by growing in artificially heated water (Bespalaya et al. 2018; Castaneda et al. 2018). Moreover, C. fluminea outcompetes endemic native clam and mussel populations, resulting in localized extinction of native species (Modesto et al. 2019). Dispersal of C. fluminea is frequently accidental (Sousa et al. 2008; Coughlan et al. 2017). The reason for the global spread of C. fluminea is its economic value as a food source, a filter in aquariums and koi ponds, and as bait for recreational fishing (Sousa et al. 2008); however, the reason for the successful establishment of C. fluminea populations in several freshwater aquatic habitats is its method of reproduction (Sousa et al. 2008; Coughlan et al. 2017). Historically, small populations of C. fluminea have spread to waterways around the world; thus, their genetic trends have exemplified the founder effect. Being a self-compatible hermaphroditic species that generates sperm and egg cells, a single individual of C. fluminea can sexually produce offspring through self-fertilization (Britton and Morton 1986; Ishibashi et al. 2003). Corbicula fluminea individuals produce double-flagellated sperm cells. Reproduction in C. fluminea occurs through androgenesis (a form of quasi-sexual reproduction in which a male is the sole source of the nuclear genetic material in the embryo) (Ishibashi et al. 2003). An androgenically produced C. fluminea individual obtains all its chromosomes from a double- flagellated sperm cell. Because all meiotic chromosomes are expelled as polar bodies, only the sperm nucleus persists in the cytoplasm and constitutes the mitotic chromosomes of the first cleavage. Ploidy in androgenetic Corbicula lineages ranges from diploidy to tetraploidy, and in all cases, ploidy is maintained throughout the life cycle by unreduced sperm (Ishibashi et al. 2003; Ishibashi and Komaru 2006). Because of self-fertilization, all androgenic C. fluminea produce androgenic offspring. Eggs are brooded in the gills, and juveniles are expelled through the exhalent siphon. Up to 400 juveniles are released by a single individual in a day, and a single hermaphroditic clam can produce up to 35,000 offspring in one breeding season (McMahon 2002; Sousa et al. 2008). However, these offspring all inherit the same genome from their single parent, which implies that mutation is the major source of increasing genetic diversity over generations in a given population of C. fluminea. For example, the introduced C. fluminea populations in North America tend to have few genotypes (mitochondrial and nuclear DNA) in most of their range (Pigneur et al. 2014; Penarrubia et al. 2017; Tiemann et al. 2017). The C. fluminea population, which acts as an invasive organism in Europe and the North Americas, exhibits only a few genotypes (Pigneur et al. 2014; Penarrubia et al. 2017; Tiemann et al.

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 634 Genetic diversity and clonality of Corbicula fluminea

2017). However, because C. fluminea are considered native to Taiwan, they are expected to present high genetic diversity in Taiwan (Chen et al. 1992). Polymorphic individuals, with a yellowish-brown external shell surface but white, light orange, or deep purple internal surfaces were identified in the same location of the Keelung River, Taiwan (Komaru and Konishi 1999). This shell color polymorphism may be determined by genetic factors because the environment was the same. In this study, we estimated the genetic variability in C. fluminea in Taiwan by using amplified restriction fragment length polymorphism (AFLP). AFLP is a cheap but powerful technique for detecting DNA polymorphisms across the entire genome at individual, population, and species levels (Ley and Hardy 2013; Vašek et al. 2017). We attempted to understand the interaction between genotype and environmental factors in a fine-scale habitat to ensure that differences in the environment did not contribute to the variation in shell color. Therefore, genetic and environmental factors were studied to identify the mechanism by which exterior and interior shell coloration in the C. fluminea are determined.

Materials and methods Sample collection and DNA extraction A total of 48 specimens were examined in this study (Figure 1 and Table 1). Live specimens were obtained from six localities in Taiwan (WA: Sijhih City, New Taipei City; WB: Gongguan Township, Miaoli County; WC: Gongliao Dist., New Taipei City; WD: Yuanli Township, Miaoli County; CA: Wanluan Township, Pingtung County; and CB: Wunshan Dist., Taipei City). From each locality, at least three individuals were obtained by raking sand. Sampling localities were selected to represent stagnant water with clay substrate (WC and WB), slow-moving water with silt substrate (WA, WD, and CB), and rapid water with sand substrate (CA). “W” means wild population and “C” means cultured population. Samples were collected in a 1 m × 1 m area for each location (detailed sampling information is provided in Figure 1). Live specimens were transferred to the laboratory for molecular and morphological examination. All the specimens were opened and small pieces of the foot muscle (2 mm × 2 mm) were excised for genomic DNA extraction. All shell specimens were photographed and maintained at room temperature. Phenotypes were identified for each sampling site. We used WA_P1, WA_P2, etc, instead of yellow, white, etc. (Figure 2 and Supplementary material Figures S1-S2). The muscle samples were preserved in 95% ethanol. Genomic DNA was extracted using the standard proteinase K/phenol method with slight modifications (Hsu et al. 2010).

AFLP procedure The procedures of AFLP analysis were based on the method developed by Vos et al. (1995) with slight modifications (Hsu et al. 2010). Initially, approximately

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 635 Genetic diversity and clonality of Corbicula fluminea

Figure 1. Sampling locations in Taiwan. Sampling localities were selected to reveal stagnant water with clay substrate (WC and WB), sluggish water (WA, WD and CB) with silt substrate and rapid water (CA) with sand substrate. “W” means wild population and “C” means cultured population. Samples were collected in a 1 m × 1 m area for each location. WA: Sijhih City, New Taipei City, 25°05′49.1″N; 121°38′33.5″E; WB: Gongguan Township, Miaoli County, 24°30′21.4″N; 120°49′01.4″E; WC: Gongliao Dist., New Taipei City, 25°00′09.4″N; 121°55′46.9″E; WD: Yuanli Township, Miaoli County, 24°26′07.2″N; 120°39′59.5″E; CA: Wanluan Township, Pingtung County, 22°35′17.7″N; 120°35′56.9″E; CB: Wunshan Dist., Taipei City, 24°58′20.2″N; 121°34′37.6″E. Photo by Te-Hua Hsu.

Table 1. Summary of AFLP data, with the average similarity index (S), genetic distance (D), and Nei’s gene diversity (He) of each sampling locality. Wild Cultured Sampling locality Total WA WB WC WD CA CB Sample size 7 8 12 14 3 4 48 Genotypes 7 3 4 6 1 2 23 Total bands 54 53 49 53 45 47 57 Polymorphic bands 14 13 5 18 0 9 28 Average similarity index (S) 0.935 0.947 0.981 0.942 1 0.947 0.917 Genetic distance (D) 0.067 0.055 0.019 0.059 0 0.054 0.083 Genetic diversity (He) 0.157 0.145 0.064 0.139 0.132 0.154 0.166

100 ng of total DNA was digested in 10 μL of a solution comprising 2.5 U of Tru9I and EcoRI (Promega, USA) in 1x buffer C at 65 °C and 37 °C for 3 h, respectively. Restriction fragments were ligated to EcoRI and MseI adaptors. Ligation was followed by preamplification and selective amplification.

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 636 Genetic diversity and clonality of Corbicula fluminea

Although the typical range of scorable bands was between 50 and 100, selective amplification with primer combinations of E+3/M+3 selective bases, which can garner 9 to 30 scorable bands, was used to avoid the problems of size homoplasy and weak (unscorable) bands in this study. Selective amplification was performed using pair of primers: E-AA/M-CAA (E+2/M+3). The amplification products were electrophoresed on a 5% denaturing polyacrylamide gel (size: 280 mm (W) × 370 mm (H) × 0.35 mm (D)) by using ATTO PAGE equipment (ATTO AE-6155, Japan). Fingerprint patterns were visualized using silver staining (Hsu et al. 2010). Band sizes were estimated using a 10-bp DNA ladder (Invitrogen, USA). Polyacrylamide gels were scanned and converted to digital images by using an imaging analyzing system (HP Scanjet 5370c, USA). The digital gel images were inverted, and the contrast was increased using Photoimpact Version 12.

Data analysis The clearly detectable AFLP bands with a scoring range of 80–320 bp were scored for presence (1) or absence (0) and transformed into a 0/1 binary character matrix. To ensure credibility, only reproducible and well-defined bands were counted. Total bands, polymorphic bands, average similarity index (S), and Nei’s gene diversity (He), and standard genetic distance (D), were calculated using the Dice similarity coefficient (Dice 1945), the method reported by Lynch and Milligan (1994). Furthermore, He was calculated according to allelic frequencies (square root method) by using AFLP- SURV 1.0 (Vekemans et al. 2002). Principal coordinate analysis (PCoA) was performed using a matrix of squared Euclidean distances computed using individual binary data and by using GenAlEx 6.41 (Peakall and Smouse 2012).

Results Polymorphism of phenotypes of the internal shell and AFLP genotypes In our samples, the internal shell color exhibited a color gradient with some local pattern (Figure 2 and Figures S1-S2). Identifying phenotypes as the same type across the different sampling sites was difficult. We only considered distinguished phenotypes from the same population (location). For example, internal shell color phenotype WA_P1 (white) in WA and phenotype WD_P1 (white) in WD were not considered the same phenotype (Figure 2 and Figures S1-S2). In WA, seven clams (WA_1 to WA_7) exhibited seven AFLP genotypes (clonal lineages) (WA_G1 to WA_G7) and three internal shell color phenotypes (WA_P1: white, WA_P2: purple, and WA_P3: light orange). All the clams in WA exhibited different phenotypes. The phenotype WA_P1 corresponded to five genotypes (WA_G1 to WA_G5), the phenotype WD_P2 corresponded to the genotype (WA_G6), and the phenotype WD_P3 corresponded to the genotype WA_G7.

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 637 Genetic diversity and clonality of Corbicula fluminea

Figure 2. Summary of sample identity, sampling location, phenotype, genotype, and AFLP banding pattern. AFLP banding pattern only show polymorphic bands among all specimens.

In WB, eight clams exhibited three different AFLP genotypes. Four of the eight clams in WB exhibited the genotype WB_G1, one exhibited the genotype WB_G2, and the remaining three clams exhibited the genotype WB_G3. The phenotype WB_P1 (white) corresponded to two genotypes (WB_G1 and WB_G2), whereas the phenotype WB_P2 (brown) corresponded to the genotype WB_G3. In WC, 12 clams exhibited four AFLP genotypes. Three of 12 clams in WC exhibited the genotype WC_G1, six clams exhibited the genotype WC_G2, one clam exhibited the genotype WC_G3, and the remaining two clams exhibited the genotype WC_G4. The phenotype WC_P1 (purple) corresponded to the genotype WC_G1, the phenotype WC_P2 (white) corresponded to two genotypes (WC_G2 and WC_G3), and the phenotype WC_P3 (gray) corresponded to the genotype WC_G4. In WD, 14 clams exhibited six AFLP genotypes. Eight of the14 clams in the WD exhibited the genotype WD_G1, two exhibited the genotype WD_G2, and the remaining four clams exhibited different genotypes among WD_G3 to WD_G6. The phenotypes WD_P1 (white), WD_P2 (purple), and WD_P3 (brown) corresponded to three genotypes (WD_G1 to WD_G3), one genotype (WD_G4), and two genotypes (WD_5 and WD_G6), respectively. In CA, the phenotype CA_P1 (white) corresponded to the genotype CA_G1. In CB, four clams exhibited two AFLP genotypes. Three of four clams in CB exhibited the genotype CB_G1, whereas the remaining one clam showed the genotype CB_G2. The phenotype CB_P1 (purple) corresponded to the genotype CB_G1, whereas the phenotype CB_P2 (white) corresponded to the genotype CB_G2. Scatter plots of AFLP data based on PCoA analysis present the relationship of all the AFLP genotypes with genotypes. This result indicated a high level of genetic differentiation between AFLP genotypes and revealed low population

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 638 Genetic diversity and clonality of Corbicula fluminea

Figure 3. Principal coordinate analysis of genotypes based on AFLP data. Genotypes from six locations are labeled and enclose in a separated area with some extent of overlap. First principal coordinate (Coord. 1) was 21.27% and the second coordinate (Coord. 2) was 19.62%.

connectivity across sampling sites. Repeated AFLP genotypes were only found from the same sampling locality (Figure 2). AFLP genotypes in WA (WA_G1 to WA_G7) and WD (WD_G1 to WD_G6) exhibited the highest genetic variation, whereas AFLP genotypes in WB (WB_G1 to G3), WC (WC_G1 to WC_G4), CA (CA_GA1), and CB (CB_GB1 and CB_GB2) exhibited relatively low genetic variation (Figure 3).

Genetic diversity and genetic differentiation among populations One primer combination (E-AA/M-CAA) was used, and 57 different DNA bands (loci), which had 28 polymorphic bands, were generated. The number of total bands and polymorphic bands, respectively, were 54 and 14 in WA, 53 and 13 in WB, 49 and 5 in WC, 53 and 18 in WD, 45 and 0 in CA, and 47 and 9 in CB (Table 1). The average pairwise genetic similarity between individuals within populations (average similarity index, S) and genetic distance (D) were 0.917 and 0.083, respectively, in all individuals. The highest S index (1) and lowest D (0) were observed in CA (Table 1), whereas the lowest S (0.935) and highest D (0.067) were observed in WA (Table 1). The He was estimated and ranged from 0.064 (WC) to 0.157 (WA). The He of all individuals was 0.166 (Table 1). Scatter plots of AFLP data based on PCoA analysis exhibit clear patterns corresponding to each sampling locality (Figure 3). Analysis of molecular variance (AMOVA) among all sampling localities indicated high differentiation

(FST = 0.518; p = 0 < 0.001) with 52% of genetic variation distributed among the sampling localities (Table 3). Sampling localities were also compared

pairwise. The pairwise FST values ranged from 0.210 (WA-WB) to 0.859 (WC- CA), which indicated a high level of genetic differentiation. All differences between sampling localities were significant (0.017 ≤ p ≤ 0.001) (Table 2).

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 639 Genetic diversity and clonality of Corbicula fluminea

Table 2. Pairwise population FST values (below diagonal) and associated p values (above diagonal) based on 999 permutations from the analysis of molecular variance. WA WB WC WD CA CB WA – 0.011* 0.001*** 0.001*** 0.017* 0.002** WB 0.210 – 0.001*** 0.001*** 0.008** 0.003** WC 0.392 0.641 – 0.001*** 0.006** 0.002** WD 0.378 0.493 0.507 – 0.001*** 0.001*** CA 0.523 0.588 0.859 0.587 – 0.041* CB 0.433 0.600 0.726 0.441 0.457 – *: 0.05 > P > 0.01; **: 0.01 > P > 0.001; ***: P ≤ 0.001; NS: not significant.

Table 3. Analysis of molecular variance (AMOVA) among all sampling localities. Source df Sum of squares Mean squares Variance % total Among sampling localities 5 94.485 18.897 2.213 52% Within sampling localities 42 86.452 2.058 2.058 48% Total 47 180.938 4.272 100%

Overall FST value = 0.518; P = 0 < 0.001.

Discussion A given phenotype of an organism is usually determined by the interaction between genotype and environmental factors, which in turn can positively or negatively affect their survival and adaptation (Pigliucci 2005). For example, the darkening of tree barks because of industrial soot pollution resulted in enhanced fitness among melanistic individuals of the peppered moth Biston betularia (Cook et al. 2012). In mollusks, color polymorphisms are particularly interesting. Mollusk shell exteriors are polymorphic not only in color but also in pattern (e.g. Chiba 1999; Richards et al. 2013; Lim et al. 2018). If polymorphisms are under genetic control, they constitute visible variations and provide a means to gain insights into the maintenance of genetic variation within a population (Schilthuizen 2013). Maintenance of polymorphic phenotypes may arise through the distribution of a species across heterogeneous environments, where different environments favor different morphs with heritable pigmentation causing natural selection by facilitating evasion of predation (e.g. Johannesson and Ekendahl 2002; Schilthuizen 2013; Mendonca et al. 2015). Mechanisms underlying the heredity of traits are complex, particularly among mollusks with atypical mechanisms of reproduction (Richards et al. 2013; Williams 2017). Mollusks generally exhibit a high degree of genetic variation (e.g. pearl oyster and freshwater mussel) (Elderkin et al. 2007; Takeuchi et al. 2020), but the hermaphroditic, self-fertilizing, androgenetic Asian C. fluminea is found to exhibit less variation (Pigneur et al. 2011, 2014; Gomes et al. 2016). However, the C. fluminea in Taiwan exhibits high variation, particularly in genotypes. A high level of genetic differentiation between clonal lineages was found in this study. These findings support that the C. fluminea is most likely native to Taiwan. In addition, similar AFLP genotypes were only found in the same sampling locality, and AFLP genotypes from the

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 640 Genetic diversity and clonality of Corbicula fluminea

same sampling sites were genetically similar (Figures 2, 3). The findings indicate that because of low motility, individuals in the same region are genetically similar. Different genotypes may occasionally arise because of mutations (Figure 3; Table 2). We also found some individuals with the same genotype, particularly in sampling localities with slow-moving water (WA and WD) and in the cultured population (CA and CB). In Taiwan, where there are typhoons every year, C. fluminea is easily dispersed by floods, especially in rapid water. These findings indicate that the C. fluminea produces a large number of offspring in a short period and resides in the same location without accumulating mutations. Corbicula fluminea was studied in Europe and America as an invasive species and was found to have low genetic diversity after analysis of the mitochondrial COI gene, nuclear 28S ribosomal DNA, and microsatellites (Pigneur et al. 2014; Penarrubia et al. 2015; 2017). In addition, some studies have associated phenotypes (forms A, B, and D) with genotypes and considered these phenotypes to be characteristic of these specific genotypes (Tiemann et al. 2017, 2018). In this study, we used the AFLP technique, which can be used to identify mutations on the genome simply and quickly. This type of molecular mark is considered neutral, and most of the loci are not affected by natural selection (Bensch and Akesson 2005). We found several AFLP genotypes from a single phenotype in this study (Figure 2; Table 1). Furthermore, we clarified associations between phenotype and genotype. For a given population (e.g. WB) of C. fluminea in Taiwan, internal shell color phenotypes have the following associations: (1) individuals with identical genotypes always have identical internal shell color phenotypes; (2) individuals with different internal shell color phenotypes do not exhibit identical genotypes; (3) individuals with different genotypes may or may not share the same internal shell color phenotypes; and (4) individuals with the same internal shell color phenotypes may or may not exhibit identical genotypes (Figure 2). Although the sample size in this study is relatively small (48 individuals from six locations), it includes 23 genotypes. Pigneur et al. (2014) reported 124 individuals from Europe (from 20 locations) had only 4 genotypes (using 10 microsatellite loci). Our results may be limited but available to other invasion areas. We found that even individuals with identical genotypes were highly susceptible to the effects of microhabitats on the outer shell color (broken, color and pattern gradient), but the inner shell color was highly stable (Figure 2 and Figures S1-S2). The outer shell color does not appear to be affected only by genetics, but the inner shell color is completely affected by genetics (Figure 2). Both heritable factors and environmental effects have been reported to control molluscan shell color variation (Luttikhuizen and Drent 2008; Williams 2017; Wang et al. 2018). Luttikhuizen and Drent (2008) reported a genetic basis for color polymorphism of the bivalve Macoma balthica, as the animals grow, the color of the outside and inside

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 641 Genetic diversity and clonality of Corbicula fluminea

of the shells starts to diverge; in most older animals, the inside of the shell continues to display bright colors, while the outside increasingly becomes chalky white (Luttikhuizen and Drent 2008). Environmental factors are encoded in shells of bivalves in the form of geochemical properties, shell microstructure and shell growth rate. External shell surface (ESS) of bivalve is known to adsorb various dissolved metals, the content of metals recorded in shells can often be higher than in soft tissues, and metals in shells have longer biological half-lives (Zuykov et al. 2012; Mancuso et al. 2019). Water characteristics, such as pH and sulfide ion concentration, may change the color of the periostracum (Williams 2017). It appears that individuals with lighter colors of periostracum tend to be found in areas of lighter colored substrate, whereas individuals with darker colors of periostracum are found in areas of the darker substrate to avail of the advantage of camouflage against potential predators (Johannesson and Ekendahl 2002; Cook 2017). Therefore, when mutations occur, mutants may be easily spotted by predators. However, mutations that affect the inner shell color of the individual do not affect its survival; consequently, variation in internal shell color has persisted (Luttikhuizen and Drent 2008; Cook 2017; Williams 2017). The Asian clam C. fluminea exhibits special reproductive mechanisms (being hermaphroditic, self-fertilizing, androgenetic, and ovoviviparous) and has various ecological effects (invasions). This study indicates the relationship between the inner shell color variation and genotype in C. fluminea. This relationship has an application in studies on the dispersal of this invasive species. In addition, we highlighted that AFLP is applied for identifying the clonal lineage of C. fluminea successfully. It could be a useful tool for assessing the genetic diversity or clonality in C. fluminea accurately. Studies on commercial food production (Chang et al. 2017), and in functional food (clam extract) (Chijimatsu et al. 2008) could further consider genetic diversity or clonality in C. fluminea accurately.

Acknowledgements

We are grateful to the editor and anonymous reviewers for their valuable comments. This work was supported by grants from the Center of Excellence for the Oceans (National Taiwan Ocean University), which were financially supported by the Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education, ROC (Taiwan).

References

Araujo R, Moreno D, Ramos MA (1993) The asiatic clam Corbicula fluminea (Müller, 1774) (Bivalvia: Corbiculidae) in Europe. American Malacological Bulletin 10: 39-49 Bensch S, Akesson M (2005) Ten years of AFLP in ecology and evolution: why so few animals? Molecular Ecology 14: 2899–2914, https://doi.org/10.1111/j.1365-294X.2005.02655.x Bespalaya YV, Bolotov IN, Aksenova OV, Kondakov AV, Gofarov MY, Laenko TM, Sokolova SE, Shevchenko AR, Travina OV (2018) Aliens are moving to the Arctic frontiers: an integrative approach reveals selective expansion of androgenic hybrid Corbicula lineages towards the North of Russia. Biological Invasions 20: 2227–2243, https://doi.org/10.1007/s10530-018-1698-z

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 642 Genetic diversity and clonality of Corbicula fluminea

Britton JC, Morton B (1986) Polymorphism in Corbicula fluminea (Bivalvia Corbiculoidea) from North America. Malacological Review 19: 1–44 Castaneda RA, Cvetanovska E, Hamelin KM, Simard MA, Ricciardi A (2018) Distribution, abundance and condition of an invasive bivalve (Corbicula fluminea) along an artificial thermal gradient in the St. Lawrence River. Aquatic Invasions 13: 379–392, https://doi.org/10. 3391/ai.2018.13.3.06 Chiba S (1999) Accelerated evolution of land snails Mandarina in the oceanic Bonin Islands: evidence from mitochondrial DNA sequences. Evolution 53: 460–471, https://doi.org/10.1111/ j.1558-5646.1999.tb03781.x Chijimatsu T, Tatsuguchi I, Abe K, Oda H, Mochizuki S (2008) A freshwater clam (Corbicula fluminea) extract improves cholesterol metabolism in rats fed on a high-cholesterol diet. Bioscience Biotechnology and Biochemistry 72: 2566–2571, https://doi.org/10.1271/bbb.80257 Chang PH, Chang WT, Shih CH, Liu DF, Lee YC (2017) A study of the growth and burrowing ability for the environmentally friendly cultured freshwater clam Corbicula fluminea. Aquaculture Research 48: 3004–3012, https://doi.org/10.1111/are.13133 Chen TC, Liao KY, Wu WL (1992) The research and evaluation on Corbicula fluminea in Taiwan (Bivalvia: Corbiculidae). Bulletin of Malacology R.O.C. 17: 37–49 Cook LM, Grant BS, Saccheri IJ, Mallet J (2012) Selective bird predation on the peppered moth: the last experiment of Michael Majerus. Biology Letters 8: 609–612, https://doi.org/ 10.1098/rsbl.2011.1136 Cook LM (2017) Reflections on molluscan shell polymorphisms. Biological Journal of the Linnean Society 121: 717–730, https://doi.org/10.1093/biolinnean/blx033 Coughlan NE, Stevens AL, Kelly TC, Dick JTA, Jansen MAK (2017) Zoochorous dispersal of freshwater bivalves: an overlooked vector in biological invasions? Knowledge and Management of Aquatic Ecosystems 418: 42, https://doi.org/10.1051/kmae/2017037 Counts CL (1981) Corbicula fluminea (Bivalvia: Corbiculidea) in British Columbia. Nautilus 95: 12–13 Counts CL (1986) The zoogeography and history of invasion of the United States by Corbicula fluminea (Bivalvia: Corbiculidae). American Malacological Bulletin Special Edition 2: 7–39 Crespo D, Dolbeth M, Leston S, Sousa R, Pardal MA (2015) Distribution of Corbicula fluminea (Muller, 1774) in the invaded range: a geographic approach with notes on species traits variability. Biological Invasions 17: 2087–2101, https://doi.org/10.1007/s10530-015-0862-y Dice LR (1945) Measures of the Amount of Ecologic Association Between Species. Ecology 26: 297–302, https://doi.org/10.2307/1932409 Elderkin CL, Christian AD, Vaughn CC, Metcalfe-Smith JL, Berg DJ (2007) Population genetics of the freshwater mussel, Amblema plicata (Say 1817) (Bivalvia: Unionidae): Evidence of high dispersal and post-glacial colonization. Conservation Genetics 8: 355–372, https://doi.org/10.1007/s10592-006-9175-0 Gomes C, Sousa R, Mendes T, Borges R, Vilares P, Vasconcelos V, Guilhermino L, Antunes A (2016) Low Genetic Diversity and High Invasion Success of Corbicula fluminea (Bivalvia, Corbiculidae) (Muller, 1774) in Portugal. PLoS ONE 11: e0158108, https://doi.org/10.1371/ journal.pone.0158108 Hunicken LA, Abrameto M, Bonel N (2019) Corbicula at its southernmost invasion front in Patagonia: unusual low density and asymmetric trait responses to varying environmental conditions. Journal of Molluscan Studies 85: 143–153, https://doi.org/10.1093/mollus/eyy058 Hsu TH, Wang ZY, Takata K, Onozato H, Hara T, Gwo JC (2010) Use of microsatellite DNA and amplified fragment length polymorphism for cherry salmon (Oncorhynchus masou) complex identification. Aquaculture Research 41: e316–e325, https://doi.org/10.1111/j.1365- 2109.2010.02533.x Ishibashi R, Komaru A (2006) Abortive second meiosis detected in cytochalasin-treated eggs in androgenetic diploid Corbicula fluminea. Development Growth & Differentiation 48: 277– 282, https://doi.org/10.1111/j.1440-169X.2006.00862.x Ishibashi R, Ookubo K, Aoki M, Utaki M, Komaru A, Kawamura K (2003) Androgenetic reproduction in a freshwater diploid clam Corbicula fluminea (Bivalvia: Corbiculidae). Zoological Science 20: 727–732, https://doi.org/10.2108/zsj.20.727 Ituarte CF (1994) Corbicula and Neocorbicula (Bivalvia: Corbiculidae) in the Paraná, Uruguay, and Rio de la Plata Basins. Nautilus 107: 129–135 Johannesson K, Ekendahl A (2002) Selective predation favouring cryptic individuals of marine snails (Littorina). Biological Journal of the Linnean Society 76: 137–144, https://doi.org/10. 1111/j.1095-8312.2002.tb01720.x Komaru A, Konishi K (1999) Non-reductional spermatozoa in three shell color types of the freshwater clam Corbicula fluminea in Taiwan. Zoological Science 16: 105–108, https://doi.org/10.2108/zsj.16.105 Ley AC, Hardy OJ (2013) Improving AFLP analysis of large-scale patterns of genetic variation- a case study with the Central African lianas Haumania spp. (Marantaceae) showing interspecific gene flow. Molecular Ecology 22: 1984–1997, https://doi.org/10.1111/ mec.12214

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 643 Genetic diversity and clonality of Corbicula fluminea

Lim JY, Tay TS, Lim CS, Lee SSC, Teo SLM, Tan KS (2018) Mytella strigata (Bivalvia: Mytilidae): an alien mussel recently introduced to Singapore and spreading rapidly. Molluscan Research 38: 170–186, https://doi.org/10.1080/13235818.2018.1423858 Luttikhuizen PC, Drent J (2008) Inheritance of predominantly hidden shell colours in Macoma balthica (L.) (Bivalvia: Tellinidae). Journal of Molluscan Studies 74: 363–371, https://doi.org/10.1093/mollus/eyn026 Lynch M, Milligan BG (1994) Analysis of population genetic structure with RAPD markers. Molecular Ecology 3: 91–99, https://doi.org/10.1111/j.1365-294X.1994.tb00109.x Mancuso A, Stagioni M, Prada F, Scarponi D, Piccinetti C, Goffredo S (2019) Environmental influence on calcification of the bivalve Chamelea gallina along a latitudinal gradient in the Adriatic Sea. Scientific Reports 9: 11198, https://doi.org/10.1038/s41598-019-47538-1 McMahon RF (2002) volutionary and physiological adaptations of aquatic invasive animals: r selection versus resistance. Canadian Journal of Fisheries and Aquatic Sciences 59: 1235– 1244, https://doi.org/10.1139/f02-105 Mendonca V, Vinagre C, Cabral H, Silva ACF (2015) Habitat use of inter-tidal chitons - role of colour polymorphism. Marine Ecology 36: 1098–1106, https://doi.org/10.1111/maec.12205 Modesto V, Castro P, Lopes-Lima M, Antunes C, Ilarri M, Sousa R (2019) Potential impacts of the invasive species Corbicula fluminea on the survival of glochidia. Science of the Total Environment 673: 157–164, https://doi.org/10.1016/j.scitotenv.2019.04.043 Morton B (1977) The population dynamics of Corbicula fluminea (Bivalvia: Corbiculacea) in Plover Cove Reservoir, Hong Kong. Journal of Zoology 181: 21–42, https://doi.org/10.1111/ j.1469-7998.1977.tb04568.x Morton B (1982) Some aspects of the population structure and sexual strategy of Corbicula cf. fluminalis (Bivalvia: Corbiculacea) from the Pearl River, People’s Republic of China. Journal of Molluscan Studies 48: 1–23 Peakall R, Smouse PE (2012) GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research–an update. Bioinformatics 28: 2537–2539, https://doi.org/ 10.1093/bioinformatics/bts460 Penarrubia L, Araguas RM, Pla C, Sanz N, Vinas J, Vidal OO (2015) Identification of 246 microsatellites in the Asiatic clam (Corbicula fluminea). Conservation Genetics Resources 7: 393–395, https://doi.org/10.1007/s12686-014-0378-2 Penarrubia L, Araguas RM, Vidal O, Pla C, Vinas J, Sanz N (2017) Genetic characterization of the Asian clam species complex (Corbicula) invasion in the Iberian Peninsula. Hydrobiologia 784: 349–365, https://doi.org/10.1007/s10750-016-2888-2 Pigliucci M (2005) Evolution of phenotypic plasticity: where are we going now? Trends in Ecology & Evolution 20: 481–486, https://doi.org/10.1016/j.tree.2005.06.001 Pigneur LM, Marescaux J, Roland K, Etoundi E, Descy JP, Doninck KV (2011) Phylogeny and androgenesis in the invasive Corbicula clams (Bivalvia, Corbiculidae) in Western Europe. BMC Evolutionary Biology 11: 147, https://doi.org/10.1186/1471-2148-11-147 Pigneur LM, Etoundi E, Aldridge DC, Marescaux J, Yasuda N, Van Doninck K (2014) Genetic uniformity and long-distance clonal dispersal in the invasive androgenetic Corbicula clams. Molecular Ecology 23: 5102–5116, https://doi.org/10.1111/mec.12912 Richards PM, Liu MM, Lowe N, Davey JW, Blaxter ML, Davison A (2013) RAD-Seq derived markers flank the shell colour and banding loci of the Cepaea nemoralis supergene. Molecular Ecology 22: 3077–3089, https://doi.org/10.1111/mec.12262 Schilthuizen M (2013) Rapid, habitat-related evolution of land snail colour morphs on reclaimed land. Heredity 110: 247–252, https://doi.org/10.1038/hdy.2012.74 Sousa R, Antunes C, Guilhermino L (2008) Ecology of the invasive Asian clam Corbicula fluminea (Muller, 1774) in aquatic ecosystems: an overview. Annales De Limnologie- International Journal of Limnology 44: 85–94, https://doi.org/10.1051/limn:2008017 Takeuchi T, Masaoka T, Aoki H, Koyanagi R, Fujie M, Satoh N (2020) Divergent northern and southern populations and demographic history of the pearl oyster in the western Pacific revealed with genomic SNPs. Evolutionary Applications 13: 837–853, https://doi.org/10. 1111/eva.12905 Tiemann JS, Haponski AE, Douglass SA, Lee T, Cummings KS, Davis MA, Foighil DO (2017) First record of a putative novel invasive Corbicula lineage discovered in the Illinois River, Illinois, USA. Bioinvasions Records 6: 159–166, https://doi.org/10.3391/bir.2017.6.2.12 Tiemann J, Lawlis C, Douglass S (2018) First occurrence of a novel Corbicula (Bivalvia: Corbiculidae) Form D lineage in the Ohio River, USA. Nautilus 132: 30–32 Vašek J, Čepková PH, Viehmannová I, Ocelák M, Huansi DC, Vejl P (2017) Dealing with AFLP genotyping errors to reveal genetic structure in Plukenetia volubilis (Euphorbiaceae) in the Peruvian Amazon. PLoS ONE 12: e0184259, https://doi.org/10.1371/journal.pone.0184259 Vekemans X, Beauwens T, Lemaire M, Roldçn-Ruiz I (2002) Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size. Molecular Ecology 11: 139–151, https://doi.org/10.1046/j.0962-1083.2001.01415.x

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 644 Genetic diversity and clonality of Corbicula fluminea

Vos P, Hogers R, Bleeker M, Reijans M, Vandelee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP - a new technique for DNA fingerprinting. Nucleic Acids Research 23: 4407–4414, https://doi.org/10.1093/nar/23.21.4407 Wang J, Li Q, Zhong X, Song J, Kong L, Yu H (2018) An integrated genetic map based on EST-SNPs and QTL analysis of shell color traits in Pacific oyster Crassostrea gigas. Aquaculture 492: 226–236, https://doi.org/10.1016/j.aquaculture.2018.04.018 Williams ST (2017) Molluscan shell colour. Biological Reviews 92: 1039–1058, https://doi.org/ 10.1111/brv.12268 Zuykov M, Pelletier E, Saint-Louis R, Checa A, Demers S (2012) Biosorption of thorium on the external shell surface of bivalve mollusks: The role of shell surface microtopography. Chemosphere 86: 680–683, https://doi.org/10.1016/j.chemosphere.2011.11.023

Supplementary material The following supplementary material is available for this article: Figure S1. Phenotype of specimens (WA, WB, WC and CA). Figure S2. Phenotype of specimens (WD and CB). This material is available as part of online article from: http://www.reabic.net/aquaticinvasions/2020/Supplements/AI_2020_Hsu_etal_SupplementaryFigures.pdf

Hsu et al. (2020), Aquatic Invasions 15(4): 633–645, https://doi.org/10.3391/ai.2020.15.4.06 645