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

Abstract

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.

BACKGROUND

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

There are currently 97 valid species in the genus Pomacea, amounting to more than half the species number of ampullariids; however, the actual number of Pomacea species is estimated to be no more than 50 (Berthold 1991; Cowie and Thiengo 2003; Cowie 2015; Cowie et al. 2017). Most of the previous descriptions of Pomacea have been based on the conchological characteristics of a few specimens, resulting in taxonomic confusion (Cowie et al. 2006). The shell morphology of apple snails is often similar among closely related species, but exhibits substantial intraspecific variation (Hayes et al. 2012). Molecular studies have provided a framework for discovering and delineating apple snail species (Rawlings et al. 2007; Hayes et al. 2008 2009; Yang et al. 2019). For example, Pomacea canaliculata (Lamarck, 1822) is considered the only invasive apple snail species distributed widely throughout the world and is listed among 100 of the worst invasive species in the world (Lowe et al. 2004), but its closely-related species, P. maculata Perry, 1810, can be distinguished from P. canaliculata by mitochondrial COI and rDNA sequences (Rawlings et al. 2007). Similarly, a new Pomacea species lineage, which was misidentified as P. maculata, was discovered based on DNA barcoding and molecular systematics (Yang et al. 2019). The discovery of this new Pomacea species, which has cryptically spread to 11 provinces in China, has alerted us to the need to reexamine the number of species of Pomacea that have invaded Asia (Yang et al. 2019).

The newly discovered invasive Pomacea species is nested in a clade (called the P. canaliculata group by Hayes et al. 2009) that includes P. canaliculata and P. maculata (Yang et al. 2019). Detailed characteristics distinguishing between P. canaliculata and P. maculata, including egg morphology, shell features and internal anatomy of adult snails, have been provided (Hayes et al. 2012). In this study, we describe the morphology of the newly discovered invasive Pomacea species, focusing on how it is distinct from P. canaliculata and P. maculata.

MATERIALS AND METHODS

Sampling, preservation, and species identification

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 morphological description were removed from intact shells using the niku-nuki method, which is a heat-shock method described by Fukuda et al. (2008). Then, the soft parts were preserved in 75% ethanol. Type and voucher specimens were deposited in the National Zoological Museum of China (NZMC), Institute of Zoology, Chinese Academy of Sciences and Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine (BIQ), China Jiliang University. Egg clutches were kept in a greenhouse at 25 ± 1°C under a relative humidity of 65 ± 5% and a 16:8 h light: dark cycle. Hatchlings of egg clutches were preserved in 95% ethanol immediately after hatching.

Foot tissue of snails and one egg or hatchling from each clutch was used for DNA extraction. Species identity was confirmed via genetic analyses of the DNA barcode fragments of the mitochondrial COI gene using the primer pair LCO1490/HCO2198 (Folmer et al. 1994), following the method described in Yang et al. (2019).

Morphological analysis of adults

The gross anatomy of adult specimens was examined under a Nikon SMZ1500 dissecting microscope coupled with a Nikon DS-Fi1 camera. The shells and opercula of adult snails were cleaned by brushing gently with toothpaste, sonicated in deionized water, dried in an oven, and photographed with a Canon 5D Mark II camera. Penial sheaths of males were preserved in alcohol and photographed using a Keyence VHX-6000 large-depth-of-field digital microscope. Radulae were dissected from the bulbus oris of five individuals; tissues were digested for four hours in 500 μL of GA lysis buffer containing 400 μg of proteinase K (Tiangen, China). After being sonicated briefly in deionized water, radulae were air dried and secured to carbon adhesive tabs mounted on aluminum SEM stubs. After coating the radulae with gold, photographs were taken with a Hitachi TM1000 scanning electron microscope.

Morphological analysis of eggs and juveniles

Egg clutches were photographed using a Canon 5D Mark II camera. After air-drying, SEM photographs of shells of 1-day-old hatchlings were taken using the same method for observation of radulae. The number of eggs per clutch, hatchling duration, egg diameters and shell height of 1-day-old hatchlings of P. occulta nov. sp. were measured.

RESULTS

Family Ampullariidae Gray, 1834

Genus Pomacea Perry, 1810

Synonym Pomacea sp.: Yang et al. 2019: 4, Fig. 1.

Pomacea occulta nov. sp.

(Fig. 1)

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Holotype: FG 609800 in NZMC, male, collected from a cultivated Zizania latifolia (wild rice) field, Hemudu town, Yuyao city, Zhejiang Province, 121°10'22"E, 30°02'35"N, 29 August 2018. Collectors: Qian-Qian Yang, Xiao-Ping Yu, and Ai-Zhong Qian.

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.

Paratypes: 5 females, FG 609801, YYHMD 18082920, FJFZ 18082007, TZWL 17070922, and TZWL 17070924; 5 males, YYHMD 18082903, YYHMD 18082917, YYHMD 18082918, HMD19, and FJFZ 18082009. The FG and YYHMD specimens had the same collection data as the holotype. The FJFZ specimens were collected from lotus ponds in Fuzhou city, Fujian Province (119°14'E, 26°05'N), by Bao-Zhen Tang and Cheng-Jun Ma on 20 August 2018. The TZWL specimens were collected from a paddy field in Wenling city, Zhejiang Province (121°21'E, 28°26'N), by Su-Wen Liu and Qian-Qian Yang. FG 609801 is stored in NZMC, and all other paratypes are stored in BIQ.

Seven egg clutches with the same collection data as the holotype were examined.

Mitochondrial COI sequences for the type specimen and hatchlings from each of the clutches deposited into GenBank under the accession numbers MK756260-MK756277.

Description: The following description is modeled on the descriptions of P. maculata and P. canaliculata of Hayes et al. (2012) to facilitate comparison of P. occulta nov. sp. with those two species.

Adult shell: Thick, generally smooth with an uneven surface of channeled sutures and spiral, lined, shallow malleation, axial growth lines and frequent periodic growth arrests, adult shell height ranging from 54 to 63 mm, width from 45 to 58 mm. Periostracum thick, variable in color from reddish to dark brown. Spiral bands thick with interphase colors of greenish and brown or reddish. Shoulder angulate. Spire height low, with ratios of spire height to overall shell height ranging from 0.08 to 0.12. Aperture ovoid to kidney-shaped. Inside of pallial lip pigmented with variable colors from yellow to reddish.

Operculum: Corneous, moderately thick, flexible, thinning towards the parietal edge. Color variable from yellow to dark brown, lightening from the nucleus to the edge. Sexually dimorphic, uniformly concave in females, concave at nucleus and convex towards the margins in males.

Radula: 8.8-12.8 mm long, 3.4 mm wide, with ~23-33 rows of teeth (mean = 28; N = 6 adults). Each row with one central tooth, one lateral tooth and two marginal teeth (tooth type: 2·1·1·1·2). Central tooth elongated, approximately 2 times broader than tall, with a main trapezoidal cusp. Rachidian base is slightly concave. Three smaller cones present on each side of central cone. Width of first cusp of central tooth 0.44-0.52 mm. Lateral tooth with sharp central cusp of straight edges located on each side of central tooth, flanked by three small cones. One of these small cones faces central tooth. The other two small cones face marginal teeth, with outer one very small and nonobvious. Two elongated triangular marginal teeth are located on each side of lateral teeth, ending in two sharp tips each. Outer marginal teeth approximately one third the breadth of inner marginal teeth.

Kidney: Kidney consisting of larger posterior chamber and smaller anterior chamber. Posterior chamber broad semicircle shaped, interconnected with anterior chamber on straighter edge at nearly two-thirds on the right. Left tip of the straight edge forms narrow intersection angle. Arc-wise edge with narrow cleft on right side. Anterior chamber triangular.

Penis sheath: Penis sheath tapering from base. Apical and medial sheath glands present. Apical gland large, comprising distal half of sheath dorsal surface, with top elliptical protuberance and stratified folds below. Medial sheath channel starts at base and terminates at base of apical gland on dorsal surface. Medial gland ovoid, located in medial sheath channel between base and tip.

Eggs: Eggs are laid individually and form a clutch above the water line. The eggs are bright pink-reddish with calcareous shells, which harden after a couple of hours. The color eventually turns pale pink before hatching. It takes ~14 days from oviposition to hatching at 25 ± 1°C and a relative humidity of 65 ± 5% under a 16:8 h light: dark cycle. Egg number per clutch varies from 87 to as many as ~1000, with an individual egg diameter of 2.38 mm on average based on measurement of 60 individuals. The height of one-day-old hatchlings is ~2.23 mm; the first whorl ~2.11 mm in diameter.

Etymology: The specific epithet occulta is a Latin adjective meaning hidden, concealed, or secret, which was chosen in reference to the misidentification and cryptic invasion of this species in the past.

Distribution: Presently widely distributed in the southern provinces of China. However, as an invasive species, the native distribution range of P. occulta nov. sp. in South America remains unknown.

DISCUSSION

A molecular phylogenetic analysis based on mitochondrial COI and 16S rDNA, and nuclear H3, 18S rDNA, and EF1α sequences showed that several species, including P. canaliculata, P. dolioides, P. lineata, P. maculata, P. megastoma, and P. paludosa, are clustered in a clade called the P. canaliculata group (Hayes et al. 2009). More recent phylogenetic studies based on COI sequences and whole mitochondrial genomes showed that P. occulta nov. sp. is a member of this P. canaliculata group (Yang et al. 2018b 2019). However, the divergence in COI sequence between P. occulta nov. sp. and other species in the P. canaliculata group ranges from 5.8% to 17.2%, much larger than the intraspecific divergence (0.1% to 0.16%), which justifies the recognition of this new species. Nevertheless, P. occulta nov. sp. has not been formally described prior to this study.

Among the species in the P. canaliculata group, P. occulta nov. sp. is most closely related to P. canaliculata and P. maculata morphologically. Pomacea occulta nov. sp. shares some morphological features with the two species, but there are some notable differences. The adult shell of P. occulta nov. sp. is very similar to that of P. maculata, both of which are thick and have a generally smooth surface with fine axial growth lines and periodic growth arrests; the body whorl shoulder is angulated, and the inner pallial lip is yellow to reddish in color. However, most of the P. occulta nov. sp. shells examined are distinct from those of P. maculata in that most specimens have fine-channeled sutures and spiral, lined, shallow malleation on the surface, which makes the shell appear unsmooth. Nevertheless, shell surface features such as smoothness may be affected by the condition of the snail and may vary depending on the living environment of the specimen. The operculum of P. occulta nov. sp., similar to that of P. canaliculata, is flexible and thins dorsally towards the edge, while that of P. maculata differs in being inflexible and thick throughout. The eggs of P. occulta nov. sp. are more similar to those of P. canaliculata than to those of P. maculata, with fewer individuals per clutch but larger in size. Statistically, the egg diameter and height and first whorl width of 1-day-old hatchlings of P. occulta nov. sp. are approximately 15–20% smaller than those of P. canaliculata on average, but clearly (> 80%) larger than those of P. maculata (Table 1). The kidney chambers of P. occulta nov. sp. are also more similar to those of P. canaliculata, with an apparent broad anterior chamber and deep tapering cleft in the posterior chamber. Radular morphology is similar among apple snail species. The angle of extended lines on the central teeth’s first cusp has been reported to be less than 90° in P. maculata but greater than 90° in P. canaliculata (Ye et al. 2017). Moreover, a discrete difference was found on the rachidian base, which is convex in P. canaliculata but concave in P. maculata (Hayes et al. 2012). In P. occulta nov. sp., the angle of extended lines of the central tooth’s first cusp might be close to, less than, or greater than 90°, and the rachidian base is concave. The penis sheath shows the most apparent differences between P. canaliculata and P. maculata; P. maculata has an apical and a basal gland, whereas P. canaliculata has an apical and a medial gland (Hayes et al. 2012). Pomacea occulta nov. sp., similar to P. canaliculata, has an apical and a medial gland. However, the location of the medial gland of P. occulta nov. sp. is in the sheath channel, while the medial gland of P. canaliculata is located to the right of the sheath channel. The position of the penis sheath glands can be used as the most reliable character to distinguish P. canaliculata, P. maculata, and P. occulta nov. sp.

Table 1.

Morphological comparison of Pomacea occulta n. 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 lines and frequent periodic growth arrests	Smooth	General smooth, fine axial growth lines and frequent periodic growth 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 edge	Flexible, thinning dorsally towards 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, medial gland located right of the sheath channel and extending into channel	Apical gland and medial gland, medial gland located in the sheath 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

An integrated approach employing a combination of molecular and systematic morphological features helps resolve the taxonomic confusion and recovering new species lineages in Pomacea (Cowie et al. 2017). In addition to the P. occulta nov. sp., a number of unidentified cryptic species have been discovered in South America, including at least four belonging to the P. canaliculata group (Hayes 2009). However, the taxonomy of Pomacea will remain obscure until natural history collections in museums around the world are extensively examined. A DNA sample of P. occulta nov. sp. was collected from one specimen from a population in La Horqueta, Arroyo del Medio, Argentina, but the shell and whole soft body were not preserved (personal communication, K. Matsukura). Therefore, no known specimens of P. occulta sp. have been collected from South America. Considering the great economic importance of agricultural and natural wetlands and the threat that P. occulta poses to them, it is necessary to clarify the species' identity, as this study does, before surveying the species in its native range.

In summary, although the morphological 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 sheath morphology). The unique combination of adult and egg characters makes it possible to distinguish P. occulta nov. sp. from P. canaliculata and P. maculata. Identifying P. occulta nov. sp. will facilitate studies of the distribution patterns, invasion mechanisms, and management strategies for this and other species of the P. canaliculata group.