A New Species of Apple Snail in the Genus Pomacea (Gastropoda: Caenogastropoda: Ampullariidae)

Zoological Studies 58: 13 (2019) doi:10.6620/ZS.2019.58-13

Open Access

Qian-Qian Yang1 and Xiao-Ping Yu1,*

1Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China. *Correspondence: Tel: +86-571-86836006. Fax: +86-571-87676267. E-mail: [email protected]. E-mail: [email protected] (Yang)

Received 10 April 2019 / Accepted 19 May 2019 / Published 2 July 2019 Communicated by Benny K.K. Chan

Pomacea canaliculata and Pomacea maculata, two species of freshwater apple snails native to South America, have been introduced to and become invasive in many tropical wetlands around the world. Among their invaded regions in mainland China, we discovered a new species (Pomacea occulta nov. sp.), which has > 5.8% inter-specific genetic divergence with its closely-related species.The present study provides a morphological description of this new species. This new species can be distinguished from the other recognized species of Pomacea by a combination of the following features: a thick shell with generally smooth, fine axial growth lines and frequent periodic growth arrests, an angulated body whorl shoulder, and a yellow-orange-red inner pallial lip; a flexible operculum, thinning dorsally towards the edge; broad kidney anterior leaflets and posterior leaflets with a deep tapering cleft; a concave rachidian base; a penis sheath with apical and medial glands, with the medial gland located in the sheath channel; and 87 to ~1000 eggs per clutch, an average egg diameter of 2.38 mm, a mean hatching height of 2.23 mm, and a mean first whorl width of 1-day-old hatchlings of 2.11 mm. Key words: Invasive species, Freshwater snail, Morphology, Taxonomy.

BACKGROUND 2013; Yang et al. 2018a). These invasive apple snails have become serious agricultural and ecological pests, Apple snails are freshwater gastropods in the severely damaging rice seedlings and aquatic and family Ampullariidae (Cowie et al. 2017). Among the semiaquatic vegetables and threatening the biodiversity nine genera of Ampullariidae, Pomacea has received the of natural wetlands because of their voracious appetite most research attention because a number of species in for vegetation and wide food spectrum (Halwart 1994; this genus have been introduced outside of their native Cowie 2002; Tamburi and Martín 2009; Qiu et al. 2011; ranges since the 1980s (Estebenet and Cazzaniga 1993; Wu et al. 2011). Moreover, they have the potential to Mochida 1991; Yu et al. 2001; Estebenet and Martín outcompete local gastropods by preying on their eggs, 2002). Pomacea are New World species native to juveniles or even adults (Kwong et al. 2009). However, South, Central and North America (Hayes et al. 2015). despite their being a well-known invasive gastropod, Some Pomacea species, mainly from South America, the true identity of invasive apple snails has long been demonstrate notable invasiveness in broad regions of confusing (Cowie et al. 2006; Rawlings et al. 2007; South and Southeast Asia and North America, as well Hayes et al. 2012). as some European and Oceanic countries (Halwart There are currently 97 valid species in the 1994; Hayes et al. 2008; Wu et al. 2010; Lv et al. genus Pomacea, amounting to more than half the

Citation: Yang QQ, Yu XP. 2019. A new species of apple snail in the genus Pomacea (Gastropoda: Caenogastropoda: Ampullariidae). Zool Stud 58:13. doi:10.6620/ZS.2019.58-13.

species number of ampullariids; however, the actual Museum of China (NZMC), Institute of Zoology, number of Pomacea species is estimated to be no Chinese Academy of Sciences and Zhejiang Provincial more than 50 (Berthold 1991; Cowie and Thiengo Key Laboratory of Biometrology and Inspection & 2003; Cowie 2015; Cowie et al. 2017). Most of the Quarantine (BIQ), China Jiliang University. Egg previous descriptions of Pomacea have been based on clutches were kept in a greenhouse at 25 ± 1°C under the conchological characteristics of a few specimens, a relative humidity of 65 ± 5% and a 16:8 h light: dark resulting in taxonomic confusion (Cowie et al. 2006). cycle. Hatchlings of egg clutches were preserved in The shell morphology of apple snails is often similar 95% ethanol immediately after hatching. among closely related species, but exhibits substantial Foot tissue of snails and one egg or hatchling intraspecific variation (Hayes et al. 2012). Molecular from each clutch was used for DNA extraction. Species studies have provided a framework for discovering and identity was confirmed via genetic analyses of the DNA delineating apple snail species (Rawlings et al. 2007; barcode fragments of the mitochondrial COI gene using Hayes et al. 2008 2009; Yang et al. 2019). For example, the primer pair LCO1490/HCO2198 (Folmer et al. Pomacea canaliculata (Lamarck, 1822) is considered 1994), following the method described in Yang et al. the only invasive apple snail species distributed widely (2019). throughout the world and is listed among 100 of the worst invasive species in the world (Lowe et al. 2004), Morphological analysis of adults but its closely-related species, P. maculata Perry, 1810, can be distinguished from P. canaliculata by The gross anatomy of adult specimens was mitochondrial COI and rDNA sequences (Rawlings et examined under a Nikon SMZ1500 dissecting al. 2007). Similarly, a new Pomacea species lineage, microscope coupled with a Nikon DS-Fi1 camera. which was misidentified as P. maculata, was discovered The shells and opercula of adult snails were cleaned based on DNA barcoding and molecular systematics by brushing gently with toothpaste, sonicated in (Yang et al. 2019). The discovery of this new Pomacea deionized water, dried in an oven, and photographed species, which has cryptically spread to 11 provinces with a Canon 5D Mark II camera. Penial sheaths of in China, has alerted us to the need to reexamine the males were preserved in alcohol and photographed number of species of Pomacea that have invaded Asia using a Keyence VHX-6000 large-depth-of-field (Yang et al. 2019). digital microscope. Radulae were dissected from the The newly discovered invasive Pomacea species bulbus oris of five individuals; tissues were digested is nested in a clade (called the P. canaliculata group by for four hours in 500 µL of GA lysis buffer containing Hayes et al. 2009) that includes P. canaliculata and P. 400 µg of proteinase K (Tiangen, China). After being maculata (Yang et al. 2019). Detailed characteristics sonicated briefly in deionized water, radulae were air distinguishing between P. canaliculata and P. maculata, dried and secured to carbon adhesive tabs mounted on including egg morphology, shell features and internal aluminum SEM stubs. After coating the radulae with anatomy of adult snails, have been provided (Hayes et gold, photographs were taken with a Hitachi TM1000 al. 2012). In this study, we describe the morphology scanning electron microscope. of the newly discovered invasive Pomacea species, focusing on how it is distinct from P. canaliculata and P. Morphological analysis of eggs and juveniles maculata. Egg clutches were photographed using a Canon 5D Mark II camera. After air-drying, SEM photographs MATERIALS AND METHODS of shells of 1-day-old hatchlings were taken using the same method for observation of radulae. The number of Sampling, preservation, and species eggs per clutch, hatchling duration, egg diameters and identification shell height of 1-day-old hatchlings of P. occulta nov. sp. were measured. Snails and egg clutches were collected from July 2017 to August 2018 from Zhejiang and Fujian Provinces in China. The soft parts of the snails used for RESULTS morphological description were removed from intact shells using the niku-nuki method, which is a heat-shock Family Ampullariidae Gray, 1834 method described by Fukuda et al. (2008). Then, the soft Genus Pomacea Perry, 1810 parts were preserved in 75% ethanol. Type and voucher Synonym Pomacea sp.: Yang et al. 2019: 4, Fig. 1. specimens were deposited in the National Zoological

Pomacea occulta nov. sp. row with one central tooth, one lateral tooth and two (Fig. 1) marginal teeth (tooth type: 2·1·1·1·2). Central tooth urn:lsid:zoobank.org:act:95B8BA21-C410-4135-B743- elongated, approximately 2 times broader than tall, FC1C8698D6F2 with a main trapezoidal cusp. Rachidian base is slightly concave. Three smaller cones present on each side of Holotype: FG 609800 in NZMC, male, collected central cone. Width of first cusp of central tooth 0.44- from a cultivated Zizania latifolia (wild rice) field, 0.52 mm. Lateral tooth with sharp central cusp of Hemudu town, Yuyao city, Zhejiang Province, straight edges located on each side of central tooth, 121°10'22"E, 30°02'35"N, 29 August 2018. Collectors: flanked by three small cones. One of these small Qian-Qian Yang, Xiao-Ping Yu, and Ai-Zhong Qian. cones faces central tooth. The other two small cones Paratypes: 5 females, FG 609801, YYHMD face marginal teeth, with outer one very small and 18082920, FJFZ 18082007, TZWL 17070922, and nonobvious. Two elongated triangular marginal teeth TZWL 17070924; 5 males, YYHMD 18082903, are located on each side of lateral teeth, ending in two YYHMD 18082917, YYHMD 18082918, HMD19, sharp tips each. Outer marginal teeth approximately one and FJFZ 18082009. The FG and YYHMD specimens third the breadth of inner marginal teeth. had the same collection data as the holotype. The FJFZ Kidney: Kidney consisting of larger posterior specimens were collected from lotus ponds in Fuzhou chamber and smaller anterior chamber. Posterior city, Fujian Province (119°14'E, 26°05'N), by Bao- chamber broad semicircle shaped, interconnected with Zhen Tang and Cheng-Jun Ma on 20 August 2018. The anterior chamber on straighter edge at nearly two-thirds TZWL specimens were collected from a paddy field in on the right. Left tip of the straight edge forms narrow Wenling city, Zhejiang Province (121°21'E, 28°26'N), intersection angle. Arc-wise edge with narrow cleft on by Su-Wen Liu and Qian-Qian Yang. FG 609801 is right side. Anterior chamber triangular. stored in NZMC, and all other paratypes are stored in Penis sheath: Penis sheath tapering from base. BIQ. Apical and medial sheath glands present. Apical Seven egg clutches with the same collection data gland large, comprising distal half of sheath dorsal as the holotype were examined. surface, with top elliptical protuberance and stratified Mitochondrial COI sequences for the type folds below. Medial sheath channel starts at base and specimen and hatchlings from each of the clutches terminates at base of apical gland on dorsal surface. deposited into GenBank under the accession numbers Medial gland ovoid, located in medial sheath channel MK756260-MK756277. between base and tip. Description: The following description is modeled Eggs: Eggs are laid individually and form a clutch on the descriptions of P. maculata and P. canaliculata of above the water line. The eggs are bright pink-reddish Hayes et al. (2012) to facilitate comparison of P. occulta with calcareous shells, which harden after a couple nov. sp. with those two species. of hours. The color eventually turns pale pink before Adult shell: Thick, generally smooth with an hatching. It takes ~14 days from oviposition to hatching uneven surface of channeled sutures and spiral, lined, at 25 ± 1°C and a relative humidity of 65 ± 5% under a shallow malleation, axial growth lines and frequent 16:8 h light: dark cycle. Egg number per clutch varies periodic growth arrests, adult shell height ranging from from 87 to as many as ~1000, with an individual egg 54 to 63 mm, width from 45 to 58 mm. Periostracum diameter of 2.38 mm on average based on measurement thick, variable in color from reddish to dark brown. of 60 individuals. The height of one-day-old hatchlings Spiral bands thick with interphase colors of greenish is ~2.23 mm; the first whorl ~2.11 mm in diameter. and brown or reddish. Shoulder angulate. Spire height Etymology: The specific epithet occulta is a Latin low, with ratios of spire height to overall shell height adjective meaning hidden, concealed, or secret, which ranging from 0.08 to 0.12. Aperture ovoid to kidney- was chosen in reference to the misidentification and shaped. Inside of pallial lip pigmented with variable cryptic invasion of this species in the past. colors from yellow to reddish. Distribution: Presently widely distributed in the Operculum: Corneous, moderately thick, flexible, southern provinces of China. However, as an invasive thinning towards the parietal edge. Color variable from species, the native distribution range of P. occulta nov. yellow to dark brown, lightening from the nucleus to sp. in South America remains unknown. the edge. Sexually dimorphic, uniformly concave in females, concave at nucleus and convex towards the margins in males. DISCUSSION Radula: 8.8-12.8 mm long, 3.4 mm wide, with ~23-33 rows of teeth (mean = 28; N = 6 adults). Each A molecular phylogenetic analysis based on

mitochondrial COI and 16S rDNA, and nuclear H3, 18S showed that P. occulta nov. sp. is a member of this P. rDNA, and EF1α sequences showed that several species, canaliculata group (Yang et al. 2018b 2019). However, including P. canaliculata, P. dolioides, P. lineata, P. the divergence in COI sequence between P. occulta nov. maculata, P. megastoma, and P. paludosa, are clustered sp. and other species in the P. canaliculata group ranges in a clade called the P. canaliculata group (Hayes et from 5.8% to 17.2%, much larger than the intraspecific al. 2009). More recent phylogenetic studies based on divergence (0.1% to 0.16%), which justifies the COI sequences and whole mitochondrial genomes recognition of this new species. Nevertheless, P. occulta

(A) (D) (F)

(G)

(B)

(E)

(C)

Fig. 1. Key morphological characteristics of Pomacea occulta nov. sp. (A) Shell morphology of FG 609800 (upper) and FG 609801 (lower). (B) Opercula external and internal surfaces of FG 609800 (left) and FG 609801 (right). (C) Radular morphology of FG 609800. (D) Male reproductive morphology of YYHMD 18082917. (E) Outline of kidney morphology. (F) Egg morphology of two clutches. (G) Apertural view, apical view, and embryonic whorl close-up of the shells of 1-day-old hatchlings. Abbreviations: apsg, apical penis sheath gland; mpsg, medial penis sheath gland; ak, anterior kidney; pk, posterior kidney. Scale bars: A, B = 1 cm; C = 100 µm; D = 1 mm; E = 2 mm; F = 5 mm; G = 300 µm.

nov. sp. has not been formally described prior to this that of P. maculata differs in being inflexible and thick study. throughout. The eggs of P. occulta nov. sp. are more Among the species in the P. canaliculata group, P. similar to those of P. canaliculata than to those of P. occulta nov. sp. is most closely related to P. canaliculata maculata, with fewer individuals per clutch but larger in and P. maculata morphologically. Pomacea occulta size. Statistically, the egg diameter and height and first nov. sp. shares some morphological features with the whorl width of 1-day-old hatchlings of P. occulta nov. two species, but there are some notable differences. sp. are approximately 15–20% smaller than those of P. The adult shell of P. occulta nov. sp. is very similar to canaliculata on average, but clearly (> 80%) larger than that of P. maculata, both of which are thick and have a those of P. maculata (Table 1). The kidney chambers of generally smooth surface with fine axial growth lines P. occulta nov. sp. are also more similar to those of P. and periodic growth arrests; the body whorl shoulder is canaliculata, with an apparent broad anterior chamber angulated, and the inner pallial lip is yellow to reddish and deep tapering cleft in the posterior chamber. Radular in color. However, most of the P. occulta nov. sp. shells morphology is similar among apple snail species. The examined are distinct from those of P. maculata in that angle of extended lines on the central teeth’s first cusp most specimens have fine-channeled sutures and spiral, has been reported to be less than 90° in P. maculata lined, shallow malleation on the surface, which makes but greater than 90° in P. canaliculata (Ye et al. 2017). the shell appear unsmooth. Nevertheless, shell surface Moreover, a discrete difference was found on the features such as smoothness may be affected by the rachidian base, which is convex in P. canaliculata but condition of the snail and may vary depending on the concave in P. maculata (Hayes et al. 2012). In P. occulta living environment of the specimen. The operculum of nov. sp., the angle of extended lines of the central P. occulta nov. sp., similar to that of P. canaliculata, tooth’s first cusp might be close to, less than, or greater is flexible and thins dorsally towards the edge, while than 90°, and the rachidian base is concave. The penis

Table 1. Morphological comparison of Pomacea occulta nov. sp. with Pomacea maculata and Pomacea canaliculata. Typical characteristics of Pomacea maculata and Pomacea canaliculata are from Hayes et al. (2012). Columns in gray are features shared by two species

Pomacea maculata Pomacea canaliculata Pomacea occulta nov. sp.

Adult shell Thickness Thick Thin Thick Surface General smooth, fine axial growth Smooth General smooth, fine axial growth lines and frequent periodic growth lines and frequent periodic growth arrests arrests, channeled sutures and spiral lined shallow malleation Body whorl shoulder Angulated Rounded Angulated Inner pallial lip Pigmented yellow-orange-red Unpigmented Pigmented yellow-orange-red Operculum Inflexible, evenly thick Flexible, thinning dorsally towards Flexible, thinning dorsally towards edge edge Anatomical characters Kidney Anterior leaflets Narrow Broad Broad Posterior leaflets Shallow cleft with arc top Deep tapering cleft Deep tapering cleft Radula Rachidian base Concave Convex Concave Penis sheath glands Apical gland and basal gland Apical gland and medial gland, Apical gland and medial gland, medial gland located right of the medial gland located in the sheath sheath channel and extending into channel channel Eggs Eggs per clutch A few hundred to > 4500 12 to ~1000 87 to ~1000 Average egg diameter < 2.00 mm > 3.00 mm 2.38 mm One-day hatchlings Mean hatching height 1.249 mm 2.754 mm 2.23 mm Mean first whorl width of hatchling 0.812 mm 2.406 mm 2.11 mm

sheath shows the most apparent differences between H. Cowie for his valuable comments to this manuscript. P. canaliculata and P. maculata; P. maculata has an We thank Dr. Kenneth A. Hayes for his professional apical and a basal gland, whereas P. canaliculata has an instruction in specimen preparation and dissection of apical and a medial gland (Hayes et al. 2012). Pomacea apple snails. We thank both Dr. Robert H. Cowie and Dr. occulta nov. sp., similar to P. canaliculata, has an apical Kenneth A. Hayes for their contributions in developing and a medial gland. However, the location of the medial the idea of species delineation of Pomacea. We thank gland of P. occulta nov. sp. is in the sheath channel, Bio-ultrastructure Analysis Laboratory of Analysis while the medial gland of P. canaliculata is located to Center of Agribiology and Environmental Sciences, the right of the sheath channel. The position of the penis Zhejiang University for taking SEM photographs. We sheath glands can be used as the most reliable character thank Dr. Xiao-Gui Zhou from Tea Research Institute, to distinguish P. canaliculata, P. maculata, and P. Chinese Academy of Agricultural Sciences for his occulta nov. sp. assistance in taking photos using the large depth-of-field An integrated approach employing a combination digital microscope. We thank Ai-Zhong Qian, Chun- of molecular and systematic morphological features Long Zheng, Chang-Huan Fu, and Li-Qing Weng for helps resolve the taxonomic confusion and recovering their assistance in sample collection. Special thanks new species lineages in Pomacea (Cowie et al. 2017). to the anonymous reviewer whose comments greatly In addition to the P. occulta nov. sp., a number of improved the manuscript. unidentified cryptic species have been discovered in South America, including at least four belonging to Authors’ contributions: QQY and XPY collected the P. canaliculata group (Hayes 2009). However, the the material and wrote the manuscript. QQY dissected taxonomy of Pomacea will remain obscure until natural the specimens and carried out the molecular experiment. history collections in museums around the world are QQY and XPY arranged for the morphological images extensively examined. A DNA sample of P. occulta nov. to be made. sp. was collected from one specimen from a population in La Horqueta, Arroyo del Medio, Argentina, but the Competing interests: QQY and XPY declare that shell and whole soft body were not preserved (personal they have no conflict of interest. communication, K. Matsukura). Therefore, no known specimens of P. occulta sp. have been collected from Availability of data and materials: Available type South America. Considering the great economic material of the new species is described in the text. importance of agricultural and natural wetlands and the threat that P. occulta poses to them, it is necessary to Consent for publication: Not applicable. clarify the species' identity, as this study does, before surveying the species in its native range. Ethics approval consent to participate: Not In summary, although the morphological applicable. characters of P. occulta nov. sp. are intermediate between those of P. canaliculata and P. maculata, there are still characteristics unique to each species (i.e., penis REFERENCES sheath morphology). The unique combination of adult and egg characters makes it possible to distinguish P. Berthold T. 1991. Vergleichende anatomie, phylogenie und historische occulta nov. sp. from P. canaliculata and P. maculata. biogeographie der ampullariidae (Mollusca, Gastropoda). Abhandlungen des Naturwissenschaftlichen Vereins in Hamburg Identifying P. occulta nov. sp. will facilitate studies of (NF) 29:1–256. the distribution patterns, invasion mechanisms, and Bian Q, Li X, Fang Y, Jia Y, Mu X. 2015. Molecular identification management strategies for this and other species of the P. of Pomacea canaliculata and P. insularum from rice paddy canaliculata group. in different origins in China using mitochondrial adenosine triphosphate subunit 6 gene. Mitochondrial DNA 26:11–14. doi: 10.3109/19401736.2013.830302. Acknowledgments: This work and the new species Cowie RH. 2002. Apple snails (Ampullariidae) as agricultural pests: name have been registered with ZooBank under their biology, impacts and management. In: Barker GM (ed) urn:lsid:zoobank.org:pub:D5FE4E42-EFD2-408D- Molluscs as crop pests. CABI, Wallingford, pp. 145–192. B8EB-34A9C52D4E1A. This work was supported by doi:10.1079/9780851993201.0145. grants from National Key Research and Development Cowie RH. 2015. The recent apple snails of Africa and Asia (Mollusca: Gastropoda: Ampullariidae: Afropomus, Forbesopomus, Programs of China (No. 2018YFC0809200), National Lanistes, Pila, Saulea): A nomenclatural and type catalogue. The Natural Science Foundation of China (No. 31800462), apple snails of the Americas: addenda and corrigenda. Zootaxa and National Key Research and Development Programs 3940:1–92. doi:10.11646/zootaxa.3940.1.1. of China (No. 2017YFF0210200). We thank Dr. Robert Cowie RH, Hayes KA, Strong EE, Thiengo SC. 2017. Non-native

apple snails: systematics, distribution, invasion history and specialist group (ISSG) a specialist group of the species survival reasons for introduction. In: Joshi RC, Cowie RH, Sebastian commission (SSC) of the world conservation union (IUCN). LS (ed) Biology and management of invasive apple snails. First published as special lift-out in aliens 12 December 2000. Philippine Rice Research Institute, Muñoz, pp. 3–32. Updated and reprinted version: November 2004. No. 12. Cowie RH, Hayes KA, Thiengo SC. 2006. What are apple snails? Lv S, Zhang Y, Liu H X, Hu L, Liu Q, Wei FR, Guo YH, Steinmann Confused taxonomy and some preliminary resolution. In: P, Hu W, Zhou XN, Utzinger J. 2013. Phylogenetic evidence Joshi RC, Sebastian LC (ed) Global advances in ecology and for multiple and secondary introductions of invasive management of golden apple snails. Philippine Rice Research snails: Pomacea species in the People’s Republic of China. Institute, Muñoz, pp. 3–23. Diversity and Distributions 19:147–156. doi:10.1111/j.1472- Cowie RH, Thiengo SC. 2003. The apple snails of the Americas 4642.2012.00924.x. (Mollusca: Gastropoda: Ampullariidae: Asolene, Felipponea, Mochida O. 1991. Spread of freshwater Pomacea snails (Pilidae, Marisa, Pomacea, Pomella): a nomenclatural and type catalog. Mollusca) from Argentina to Asia. Micronesica Suppl. 3:51–62. Malacologia 45:41–100. Qiu JW, Chan MT, Kwong KL, Sun J. 2011. Consumption, Estebenet AL, Cazzaniga NJ. 1993. Egg variability and the survival and growth in the invasive freshwater snail Pomacea reproductive strategy of Pomacea canaliculata (Gastropoda: canaliculata: does food freshness matter? J Mollus Stud 77:189– Ampullariidae). APEX 8:129–138. 195. doi:10.1093/mollus/eyr005. Estebenet AL, Martín PR. 2002. Pomacea canaliculata (Gastropoda: Rawlings TA, Hayes KA, Cowie RH, Collins TM. 2007. The Ampullariidae): life-history traits and their plasticity. Biocell identity, distribution, and impacts of non-native apple snails 26:83–89. in the continental United States. BMC Evol Biol 7:97. Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R. 1994. DNA doi:10.1186/1471-2148-7-97. primers for amplification of mitochondrial cytochrome c oxidase Tamburi NE, Martín PR. 2009. Feeding rates and food conversion subunit I from diverse metazoan invertebrates. Mol Mar Biol efficiencies in the apple snail Pomacea canaliculata Biotechnol 3:294–299. (Caenogastropoda: Ampullariidae). Malacologia 51:221–233. Fukuda H, Haga T, Tatara Y. 2008. Niku-nuki: A useful method doi:10.4002/040.051.0201. for anatomical and DNA studies on shell-bearing molluscs. Wu JY, Meng PJ, Liu MY, Chiu YW, Liu LL. 2010. A high incidence Zoosymposia 1:15–38. doi:10.11646/zoosymposia.1.1.5. of imposex in Pomacea apple snails in Taiwan: a decade after Halwart M. 1994. The golden apple snail Pomacea canaliculata triphenyltin was banned. Zool Stud 49:85–93. in Asian rice farming systems: present impact and future Wu JY, Wu YT, Li MC, Chiu YW, Liu MY, Liu LL. 2011. threat. International Journal of Pest Management 40:199–206. Reproduction and juvenile growth of the invasive apple doi:10.1080/09670879409371882. snails Pomacea canaliculata and P. scalaris (Gastropoda: Hayes KA, Joshi RC, Thiengo SC, Cowie RH. 2008. Out of Ampullariidae) in Taiwan. Zool Stud 50:61–68. South America: multiple origins of non-native apple snails Yang Q, Liu S, He C, Cowie RH, Yu X, Hayes KA. 2019. Invisible in Asia. Divers Distrib 14:701–712. doi:10.1111/j.1472- apple snail invasions: importance of continued vigilance and 4642.2008.00483.x. rigorous taxonomic assessments. Pest Manag Sci 75:1277–1286. Hayes KA. 2009. Evolution, molecular systematics and invasion doi:10.1002/ps.5241. biology of Ampullariidae. PhD dissertation, University of Yang QQ, Liu SW, He C, Yu XP. 2018a. Distribution and the origin Hawaii. of invasive apple snails, Pomacea canaliculata and P. maculata Hayes KA, Burks RL, Castro-Vazquez A, Darby PC, Heras H, Martín (Gastropoda: Ampullariidae) in China. Sci Rep 8:1185. PR et al. 2015. Insights from an integrated view of the biology doi:10.1038/s41598-017-19000-7. of apple snails (Caenogastropoda: Ampullariidae). Malacologia Yang QQ, Liu SW, Ru WD, Liu GF, Yu XP. 2016. Molecular 58:245–302. doi:10.4002/040.058.0209. identification of invasive golden apple snails in Zhejiang Hayes KA, Cowie RH, Thiengo SC. 2009. A global phylogeny of Province based on DNA barcoding. Biodiv Sci 24:341–350. (in apple snails: Gondwanan origin, generic relationships, and the Chinese). doi:10.17520/biods.2015260. influence of outgroup choice (Caenogastropoda: Ampullariidae). Yang QQ Liu SW, Song F, Liu GF, Yu XP. 2018b. Comparative Biol J Linn Soc 98:61–76. doi:10.1111/j.1095-8312.2009.01246. mitogenome analysis on species of four apple snails x. (Ampullariidae: Pomacea). Int J Biol Macromol 118:525–533. Hayes KA, Cowie RH, Thiengo SC, Strong EE. 2012. Comparing doi:10.1016/j.ijbiomac.2018.06.092. apples with apples: clarifying the identities of two highly Ye M, Fan T, Chen L, Chen Y, Zhu S, Wu J, Chen G, Xu H, Liu invasive Neotropical Ampullariidae (Caenogastropoda). Zool J Y. 2017. Comparative study on morphology of radula of Linn Soc 166:723–753. doi:10.1111/j.1096-3642.2012.00867.x. Pomacea canaliculata, P. maculata and Cipangopaludina Kwong KL, Chan RK, Qiu JW. 2009. The potential of the invasive chinensis. Chinese J Zool 52:97–107. (in Chinese). doi:10.1016/ snail Pomacea canaliculata as a predator of various life-stages j.ijbiomac.2018.06.092. of five species of freshwater snails. Malacologia 51:343–357. Yu X, Wada T, Li Z, Lu Z, Sun L, Zhu Y, Chen J, Zheng X, Xu H. doi:10.4002/040.051.0208. 2001. Occurrence of golden apple snail, Pomacea canaliculata Lowe S, Browne M, Boudjelas S, Poorter MD. 2004. 100 of the (Lamarck), in paddy fields and its management. Acta Agric world’s worst invasive alien species a selection from the global Zhejiangensis 13:247–252. (in Chinese). doi:10.3969/ invasive species database. Published by the invasive species j.issn.1004-1524.2001.05.002.