Not in the Least Concern: Anthropogenic Influences on a South-East Asian Apple Snail Pila Scutata (Ampullariidae)

Not in the Least Concern: anthropogenic influences on a South-east Asian apple snail Pila scutata (Ampullariidae)

T ING H UI N G ,SIONG K IAT T AN,AMIRRUDIN A HMAD,DO V AN T U R AVINDRA C. JOSHI,WENDY Y. WANG,HEOK H UI T AN and D ARREN C.J. YEO

Abstract South-east Asian apple snails, Pila spp., have been Keywords Cryptogenic species, freshwater snails, human declining since the introduction of globally invasive, confa- introductions, Peninsular Malaysia, Pila scutata,Singapore, milial South American Pomacea spp., yet Pila ecology re- threat status mains poorly studied, with most occurrence records Supplementary material for this article is available at unconfirmed. Pila scutata, a previously widespread species, https://doi.org/./S presumed native to the Malay peninsula and assessed as Least Concern in the IUCN Red List, was formerly harvested for food, and may have experienced anthropogenic translocations. We surveyed the Malay peninsula (specifically Introduction Peninsular Malaysia and Singapore) to investigate the current distribution and genetic diversity of P. scutata. Six po- lthough invertebrates comprise % of known species, pulations were found in Singapore, but only one in the conservation status of , % is known (Collen et al.,  A Peninsular Malaysia. Mitochondrial COI and S sequen- ). Amongst invertebrates the freshwater gastropods are cing revealed that the Malaysian population shared a single most at risk: at least % of assessed species are considered haplotype of both genes with the Singapore populations threatened, with the potential threat level ranging from %  ( km distant). This low genetic diversity could stem (assuming no Data Deficient species are threatened) to % from a recent anthropogenic introduction, which brings (assuming all Data Deficient species are threatened; Collen into question the true native range of P. scutata and, coupled et al., ). Hotspots of freshwater gastropod diversity in- with poorly resolved taxonomy of the genus, necessitates a clude the ancient lakes of Sulawesi, Indonesia and the reassessment of its IUCN Red List status. Introduced popu- Mekong basin (Strong et al., ). Despite the high diver- lations pose a dilemma, and the lack of genetic diversity is of sity of freshwater molluscs in South-east Asia, research has concern in light of Pila decline throughout South-east Asia. mostly focussed on the zoonotic parasites they host, with lit- Our results highlight that conservation management of tle other emphasis beyond inventories of the molluscan P. scutata and its congeners must therefore be better in- hosts (e.g. Palmieri et al., ; Burch & Lohachit, ; formed by greater taxonomic resolution and more compre- Attwood, ). hensive investigations of their ecology, both in native and Among the known parasite hosts are Ampullariidae, the introduced ranges. largest freshwater snails in South-east Asia (Komalamisra et al., ; Hayes et al., ). Up to  ampullariid species of the genus Pila, and multiple synonyms, have been recorded from the region (Cowie, ). The confused tax- TING HUI NG* and DARREN C.J. YEO (Corresponding author) Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, onomy has complicated the understanding of species Singapore 117543, Republic of Singapore. E-mail [email protected] distributions, and other than a few important studies in SIONG KIAT TAN,WENDY Y. WANG and HEOK HUI TAN Lee Kong Chian Natural the s and s (Keawjam, , a,b; Pagulayan & History Museum, National University of Singapore, Singapore, Republic of Remigio, ), the current status of Pila in South-east Asia Singapore remains largely unknown. In contrast, the introduced South AMIRRUDIN AHMAD School of Marine and Environmental Sciences, Universiti Malaysia Terengganu, Terengganu, Malaysia American confamilials, Pomacea spp., have been well studied because of their impacts in paddy fields (Isnaningsih & DO VAN TU Department of Aquatic Ecology and Environment, Institute of Ecology and Biological Resources, Viet Nam Academy of Science and Marwoto, ; Nghiem et al., ; Joshi et al., ). There Technology, Ha Noi, Viet Nam have been numerous anecdotal accounts of the decline of RAVINDRA C. JOSHI† Pampanga State Agricultural University, Magalang, Pila species since the introduction of Pomacea spp. (e.g. Pampanga, Philippines Halwart, ; Marwoto et al., ), but the neglect of *Present address: Department of Biology, Faculty of Science, Chulalongkorn Pila taxonomy has made it difficult to ascertain the status University, Pathumwan, Bangkok, Thailand †Also at: University of South Pacific, Suva, Fiji of the species. Although population trends of South-east Received  August . Revision requested  November . Asian Pila species are unknown, seven species have been as- Accepted  March . First published online  November . sessed as Least Concern (IUCN, ).

Oryx, 2019, 53(2), 230–238 © 2018 Fauna & Flora International doi:10.1017/S0030605318000443 Downloaded from https://www.cambridge.org/core. IP address: 170.106.35.76, on 29 Sep 2021 at 05:53:08, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0030605318000443 South-east Asian apple snail 231

One such species is Pila scutata (Sri-Aroon & Richter, Methods ), with which various synonyms have been associated (Low et al., ; Ng et al., ; Cowie, ). It is reported Surveys and data collection to be widely distributed across South-east Asia (Low et al., ), but some occurrences are unconfirmed and others Records of P. scutata and other congeners from Singapore are probably misidentifications. For example, Keawjam and Peninsular Malaysia (collectively referred to here as () concluded from extensive surveys of Thailand am- the Malay peninsula) were obtained from available literature  pullariids that previous records (Brandt, )ofP. scutata (Supplementary Table ). Specimens in the Zoological were Pila gracilis that lacked bands. Hayes et al. (b) re- Reference Collection (ZRC) of the Lee Kong Chian Natural ported on a previous misidentification of P. scutata (as Pila History Museum at the National University of Singapore conica) from Viet Nam. Further south, the distribution of were examined. Records and photographs of type material  P. scutata in Peninsular Malaysia has not been mapped, of P. scutata and its synonyms (following Cowie, ), but it has been reported to be common across the peninsula and of other Pila species, were also obtained from the follow- (van Benthem Jutting, ; Berry, ). In Singapore, ing collections: the Australian Museum, Sydney (AMS), the P. scutata was formerly widespread and common in ponds Academy of Natural Sciences, Philadelphia (ANSP), the and ditches (e.g. Ponniah, ; Johnson, ; H.E. Ng, Field Museum, Chicago (FMNH), the Forest Research Insti- pers. comm.), but by the early s the species remained tute Malaysia, Kuala Lumpur, Malaysia (FRIM), the Natural extant in only a small number of localities (Clements History Museum, London (NHMUK), the Zoological Survey et al., ). It was also reportedly declining in Peninsular of India, National Zoological Collection India, Kolkata Malaysian rice fields (Joshi et al., ). (NZSI), the Forschungsinstitut und Naturmuseum Sencken- Early records of P. scutata were from streams and rivers berg, Frankfurt (SMF), the University of Michigan Museum (e.g. de Morgan, ,asAmpullaria wellesleyensis), but later of Zoology, Ann Arbor (UMMZ), and the Zoologisches surveys failed to find it in natural, undisturbed habitats (Tan Museum Hamburg (ZMH). et al., ; D.S. Johnson, unpubl. manuscript, based on work Based on records from these sources, we surveyed known in the s and early s). Records from the s indi- ampullariid localities in the Malay peninsula from August   cated that P. scutata was associated with anthropogenic ha- to March . Opportunistic sampling was also carried bitats, such as fishponds, throughout the Malay peninsula out at other potentially suitable habitats, including water bod- (Johnson, ). Up to the mid th century the species ies around limestone outcrops, ditches, rice fields and ponds. was a common food item for local communities, and for live- Samples from one additional population in Singapore were stock feed (Ponniah, ; Lim et al., ). Its popularity as a obtained from Daniel J.J. Ng. Comparative material from protein source suggests that it was either naturally wide- Java in Indonesia was obtained from the collection of the spread (and abundant) or widely translocated by people, or Zoologisches Museum Berlin, Germany (the type locality of  both. This species has also been introduced to Pacific islands P. scutata is Pardana in Java; Mousson, ), and other ma- (Cowie, , ; Smith, ) purportedly as food, via the terial was collected from the Philippines and Viet Nam (re- Philippines (Cowie, ; Tran et al., ). The introduced gions where P. scutata has previously been recorded;   population in Hawaii has very low mitochondrial DNA di- Pagulayan & Remigio, ; Jørgensen et al., ). versity (Tran et al., ), as has been recorded for many non-native taxa that experience a genetic bottleneck during DNA analysis the introduction stage (Dlugosch & Parker, ). Despite being considered indigenous to Singapore (Ng, We extracted total genomic DNA from the foot tissue of at ; Chan, ; Tan et al., ), considering the near- least  individuals of each P. scutata population, and at exclusive occurrence of P. scutata in man-made or disturbed least one individual of other Pila species obtained. We habitats, it is possible that its current distribution reflects added  µl CTAB buffer (. M Tris pH ; . M NaCl; past anthropogenic translocations. Such history would com- . M EDTA;  g/l CTAB) and  µl Proteinase K plicate efforts to resolve the taxonomy and biogeography of (Invitrogen) to tubes containing the tissue samples. The Pila in South-east Asia. With the apparent decline of Pila samples were then incubated for  hat °C. We extracted populations throughout the region, it is imperative that the DNA using  :  :  phenol : chloroform : isoamyl alco- any anthropogenic influence be uncovered and considered hol (Biozol), and precipitated with % ethanol. After when examining their biogeography. Thus, we aimed to washing the DNA pellets in % ethanol, we dissolved the document the current distribution of P. scutata in the pellets in  µL of water. Mitochondrial COI and S rRNA Malay peninsula (specifically Peninsular Malaysia and genes were amplified in polymerase chain reactions (PCR) Singapore) and to provide an assessment of the status of with a total volume of – µl PCR reaction mixture (. µl this cryptogenic species (assumed native, but possibly intro- of BioReady rTaq  × buffer,  mM dNTPs,  µl each of duced) in Singapore.  µM primers, . µl of BioReady rTaq DNA Polymerase

(Bulldog Bio), and DNase-free sterile water), at  °C for ). Pila scutata, however, was widely distributed prior  min,  cycles of  °C for  s, – °C for  s, and to the s, and found in fishponds and reservoirs through-  °C for  s, and a final extension of  °C for  min. out Singapore (Fig. ; Supplementary Table ). In Peninsular Amplicons of each gene were obtained using different Malaysia P. scutata was similarly recorded from human- pairs of primers depending on the quality of tissue obtained disturbed habitats such as artificial ponds and paddy fields (Supplementary Table ). The primer pairs LCO/ (Fig. ; Supplementary Table ). These records of P. scutata HCO and dg_LCO/dg_HCO amplified ap- from the peninsula were from the north, close to the border proximately  base pairs (bp) in the COI barcode region, with Thailand (e.g. Kangar, Perlis, ANSP: Sow-Yan and mlCOintF/jgHCO amplified a shorter fragment Chan, pers. comm.; Machang, Kelantan: Lim et al., ; ( bp) in the ′ region of the COI. The latter pair was Boo Liat Lim, pers. comm.), and along the western coast used in specimens that failed to amplify the standard bar- of the peninsula. The southernmost record for the peninsula code region. The size of S fragments amplified ranged was from a stream in central Johor (ZRC.MOL.), from  to  bp. The PCR products were checked visually c.  km from Singapore. on a % agarose gel. Post PCR clean-ups were performed on successfully amplified products using SureClean reagent (Bioline Inc., London, UK) following the manufacturer’s re- Current distribution of P. scutata in the Malay peninsula commendations. The purified products were sequenced We collected P. scutata from six localities in Singapore: with BigDye Terminator reactions and analysed on the Kranji, Tampines, Sarimbun, Seletar, Pandan and Jurong ABI PRISM XL sequencer (Applied Biosystems, Foster (Fig. , Plate ). Pomacea spp. were syntopic with P. scutata City, USA) at the DNA Sequencing Laboratory of the at four of these sites (Kranji, Tampines, Sarimbun, Seletar). National University of Singapore. Only one population of P. scutata was found in Peninsular We visually inspected and trimmed sequences using Malaysia, in Ipoh (Fig. , Plate ). In contrast, Pomacea spp. Sequencher . (Genecodes, Ann Arbor, USA). The COI and were found at multiple sites where Pila spp. were previously S genes were aligned using MAFFT v.  (Katoh & Standley, recorded, although not at the Ipoh site where P. scutata was ) with default settings. Aligned COI sequences were found (Fig. ). checked for translatability into amino acids and were gap free. DNA sequences were inspected using objective clustering     in SpeciesIdentifier v. . . (Meier et al., ). Objective clus- Identification of P. scutata and congeners from tering was performed using the SpeciesIdentifier (TaxonDNA South-east Asia ..; Meier et al., ) to determine molecular operational taxonomic units (mOTUs) at a range of thresholds (–%), Based on examination of Pila spp. collected for this study to test stability of the results. In objective clustering sequences (with the exception of Pila from Java that was loaned are grouped according to uncorrected pairwise distances from ZMB, and collected in ) from the known range (Meier et al., , ; Srivathsan & Meier, ); i.e. mem- of P. scutata, we identified P. scutata from Java, Indonesia bers of a set of putative conspecific sequences have at least one (ZMB), and from Neuva Ecija, Philippines (ZRC. match to a sequence in the set that falls within a given threshold MOL.; Plate ). From the states of Kelantan, distance. Previous studies have shown the intraspecific genetic Terengganu, Perak, Selangor and Perlis in Peninsular distance for the majority of gastropods is , % for COI (Meier Malaysia three other congeners were identified (Plate ): et al., ;Laytonetal.,). Sequences were checked against P. ampullacea, Pila angelica and P. gracilis. Ampullariids the GenBank database (Benson et al., ) under default collected from Viet Nam were identified as Pila erythrochila, parameters in MegaBLAST (OMICtools, Le-Petit-Quevilly, P. gracilis, and Pila virescens (Plate ). France). Further analysis was not carried out because of the COI and S from  P. scutata individuals were suc- low variation within and among the populations (see cessfully sequenced. From Singapore, these included  Results). All sequences were deposited in GenBank each from Kranji, Tampines, Sarimbun and Pandan, six (KY–KY,KY)andBOLD(SEAAM– from Seletar, and one from Jurong; from Peninsular ). Malaysia,  from Ipoh; from the Philippines,  from Neuva Ecija; from Indonesia, one from Java. Among the  Results other species, both COI and S were sequenced from P. ampullacea and P. gracilis from Peninsular Malaysia, whereas  Historical distribution of P. scutata in the Malay peninsula only COI was sequenced from P. angelica, and only S was sequenced from P. gracilis and P. virescens from Two Pila species are known from Singapore, P. scutata and Viet Nam (all one individual each). Pila ampullacea, with the latter species recorded as a single Sequence clustering of COI using objective clustering population that is now considered extirpated (Ng et al., (Meier et al., ) produced five mOTUs that were mostly

FIG. 1. Historical and current distribution of Pila scutata and other ampullariids in the Malay Peninsula (Peninsular Malaysia and Singapore). Sites with extant P. scutata populations: , Ipoh; , Sarimbun; , Kranji; , Seletar; , Tampines; , Pandan; , Jurong. Records within a  km radius in Peninsular Malaysia, and within a  km radius in Singapore were treated as single localities.

congruent with the morphologically-identified species over pairwise distance). Pairwise distances were .% be- –% thresholds (Table ). Across these thresholds, the num- tween the Malay peninsula population and that of the bers of clusters remained stable, with no specimens involved Philippines, –.% between the Philippines population in mOTU reassignment between thresholds. Over the same and individuals from Hawaii (Rawlings et al., ; threshold range P. scutata sequences separated into two Tran et al., ), and .–.% between the Javan individual mOTUs: a Javan specimen on its own, and the other  se- and all other P. scutata. For S, P. scutata from Singapore, quences as a single mOTU (Table ). At % threshold, the Peninsular Malaysia, the Philippines, and Hawaii shared a Javan P. scutata sequence merged with the larger P. scutata single S haplotype (% uncorrected pairwise distances), cluster. Results for S were similar to those for COI; the whereas the pairwise distances between the Javan individual Javan specimen remained a separate mOTU up to the % and all other P. scutata were .–.%. threshold. The mOTUs for COI for other Pila spp. remained stable across –% thresholds, corresponding to the morphologically-identified species. Discussion All specimens that were identified as P. scutata based on morphology were matched to GenBank COI sequences of Historical and current distribution of P. scutata in the P. scutata collected from Hawaii (as Pila conica in Malay peninsula Rawlings et al., ; Tran et al., ) with % identity, except for the Javan P. scutata, which was a % match The first record of an ampullariid in Singapore (as (Supplementary Table ). Other than P. scutata, only P. gra- Ampullaria sp.) by Traill () lacks figures or specimens, cilis (Peninsular Malaysia and Viet Nam) and P. virescens preventing identification of the species. We found that mu- (Viet Nam) had high matches (–% identity) to avail- seum and literature records corroborate historical accounts able sequences on GenBank (Supplementary Table ). of P. scutata being formerly widespread and common in Singapore (Johnson, ) and Peninsular Malaysia (Berry, ). Yet in our exhaustive surveys we discovered only six Mitochondrial DNA diversity of P. scutata populations populations in Singapore, and only one population in Peninsular Malaysia. Five of these populations overlapped All  P. scutata individuals from Singapore and Peninsular with previous records (Supplementary Table ), with only Malaysia shared a single COI haplotype (% uncorrected Sarimbun and Pandan being newly reported localities. All

PLATE 1 Pila scutata from South-east Asia: (a) Bogor, Java, Indonesia (ZMB), (b) Neuva Ecija, Philippines (ZRC. MOL.), (c) Ipoh, Malaysia (ZRC.MOL.), (d) Pandan, Singapore (ZRC.MOL.). Scale bars =  mm. Photographs by T.H. Ng.

seven sites were at former village areas, which included plantations and ponds (Ponniah, ; Yeap, ; National Archives of Singapore, ). The decline of P. scutata in Singapore has been attributed to habitat loss and canalization of open country streams (Ng PLATE 2 Other ampullariids recorded from Peninsular Malaysia  et al., ; Chan, ). Many previously known P. scutata and Viet Nam: (a) Pila ampullacea, Malaysia (ZRC.MOL. ), (b) Pila angelica, Malaysia (ZRC.MOL.), (c) Pila gracilis, localities no longer exist (Ng et al., ). A similar situation Malaysia (ZRC.MOL.), (d) P. gracilis, Viet Nam (ZRC. was encountered in Peninsular Malaysia, where many MOL.), (e) Pila erythrochila, Viet Nam (ZRC.MOL.), former sites have been modified or have disappeared al- (f) Pila virescens, Viet Nam (ZRC.MOL.). Scale bars =  mm. together. Lack of habitat alone, however, does not complete- Photographs by T.H. Ng. ly explain the decline of P. scutata, as this resilient species was known to be common even in degraded habitats such with the introduction and spread of Pomacea spp. since as stocked fishponds and septic ponds (Ponniah, ; the late s (Ng et al., ). Pomacea spp. were first col- Johnson, ). Furthermore, some concrete canals around lected from a single reservoir in , but are now found in Singapore with banks that had recently been naturalized or most reservoirs and many canals and ponds (Ng et al., ). softened for aesthetic and recreational reasons (Lee et al., The possible displacement of native apple snail species has ) now resemble freshwater habitats of the past that also been reported in other regions invaded by Pomacea spp. were ideal for snails (Johnson, ), and paddy fields (e.g. Halwart, ; Thaewnon-ngiw et al., ; Marwoto where the species thrived are still present in rural areas of et al., ). Efforts by collaborators from the Philippines Peninsular Malaysia. and Indonesia to find comparative material of Pila for this These remaining suitable areas and newly-created habi- study were largely unsuccessful as they encountered an over- tats, however, are being rapidly colonized by South whelming presence of Pomacea instead (Roberto Pagulayan American Pomacea spp. Throughout the Malay peninsula, & Ristiyanti M. Marwoto, pers. comms). Pomacea was also Pomacea spp. appear to have replaced P. scutata in areas established at the same locality in the Philippines where where the latter were once common (Fig. ). Thus, a plau- P. scutata was obtained for this study. sible alternative hypothesis for the decline of P. scutata It is uncertain if the extant populations of P. scutata in may be competition from the confamilial Pomacea spp. the Malay peninsula will survive in the long-term. Two of (but see Ng et al., ; Chan, ; Tan et al., ). The de- the localities with no Pomacea present, Pandan cline in P. scutata in Singapore appears to have coincided (Singapore) and Ipoh (Malaysia) have experienced recent

% habitat modification (THN, pers. obs.), and the third, in  Jurong (Singapore) is connected by a canal to Tengeh P. am- and (SGP,  Reservoir (Daniel J.J. Ng, pers. comm.), where Pomacea is P. virescens P. ampul- (MYS)

een established. The faster growth, higher reproductive capacity, and voracious appetite for aquatic macrophytes of Pomacea P. angelica

P. scutata spp. (Cowie, ; Carlsson et al., ; Morrison & Hay,

P. gracilis ; Chaichana & Sumpan, ) are invasive traits that (MYS),

(MYS, VNM), threaten the continued survival of P. scutata in the remain- 4 mOTUs: MYS, PHL, IDN), lacea (MYS), ing four localities where it co-occurs with Pomacea (i.e. (SGP, MYS, PHL, IDN), Kranji, Sarimbun, Tampines, Seletar). P. gracilis P. scutata

(MYS), Anthropogenic influences and taxonomic confusion

Another factor that may have contributed to the decline of 4 mOTUs: pullacea (VNM) P. scutata in the Malay peninsula is the unexpected lack of (MYS), mitochondrial variation we found across all extant populations. Although the species is considered native to the Malay peninsula, the current low genetic diversity and lack of geo- P. angelica graphical structure may have stemmed from anthropogenic

(MYS, introduction, and a resulting founder effect. Although non- P. scutata (MYS), native species may overcome the limitations of genetic bot- tlenecks experienced during introduction (Roman & 

P. gracilis Darling, ), low genetic diversity is known to affect the fitness of populations (Kinziger et al., ).

P. ampullacea The low level of variation in the Malay peninsula P. scu- (MYS), tata mitochondrial genes mirrors that of the introduced (SGP, MYS, PHL),

(VNM) 

(IDN), population in Hawaii (as P. conica in Tran et al., ). Clustering analysis showed the Hawaiian populations to be closely linked to the Philippines population, strongly in- P. scutata

P. scutata dicating the Philippines as the origin of P. scutata in Hawaii P. ampullacea P. virescens (as P. conica in Cowie, ; Tran et al., ), with the Philippines population showing some slight variation 5 mOTUs: (IDN), VNM), (.–.% uncorrected pairwise distances). The extremely low mitochondrial diversity among Malay peninsula populations of P. scutata is thus not likely to be natural. By com- (SGP, MYS, PHL), parison, preliminary analysis of COI genes of P. gracilis from the Malay peninsula (four sites – km apart) showed that individuals from each population had distinct P. graci- P. scutata P. scuta- P. gracilis (MYS)

S genes. haplotypes. P. virescens P. ampulla-  P. scutata Comparisons of genetic diversity between the native (MYS), (MYS), (SGP, MYS, range and introduced localities, or among non-native re- No. of mOTUs at clustering threshold 1%5 mOTUs: 2% 3% 4% 5% 6% (VNM), (VNM) 6 mOTUs: ta PHL), (IDN), cea lis P. gracilis gions, can uncover introduction pathways (Hayes et al., ; Wong et al., ). Attempts to obtain comparative specimens from the type locality of P. scutata (i.e. Java) and other countries for this study were largely unsuccessful.

species identified based on morphology compared to number of molecular operational taxonomic units (mOTU) at objective clustering thresholds betw It is thus impossible to draw definitive conclusions regard- (SGP, (SGP, ing the true native distribution of P. scutata, nor is it certain Pila P. am- P. am- (MYS)

P. gracilis that the lack of genetic diversity in Malay peninsula P. scu- P. angelica P. virescens tata is the result of a historical genetic bottleneck experi- P. scutata P. scutata enced by a native population (rather than of a founder (MYS), (MYS), P. gracilis effect caused by anthropogenic introduction). To address 1. Number of such biogeographical questions it would be necessary first ABLE T SGP, Singapore; MYS, Malaysia; PHL, Philippines; IDN, Indonesia; VNM, Viet Nam. (MYS), (uncorrected pairwise distances) for COI and No. of morphospecies (by gene) COI 4 species: MYS, PHL, IDN), pullacea 16S 4 species: MYS, PHL, IDN), pullacea (MYS, VNM), (VNM) to conduct a comprehensive revision of the genus in

South-east Asia with a wider range of samples from Many of the historical records could not be verified because throughout the region, using multiple molecular markers, of the lack of clear photographs and voucher specimens. The including nuclear genes (Meier, ). example here of poorly resolved taxonomy affecting conser- All extant populations of P. scutata identified in our vation is not unique to ampullariids, it is symptomatic of a study, however, were found in human-disturbed areas. wider issue across understudied invertebrate groups in gen- The pre-modified natural inland waters of Singapore were eral (Collen et al., ), and requires more attention. probably too soft and acidic to sustain a high diversity of molluscs (Johnson, ; Yeo & Lim, ). The historical absence from natural habitats, and invariably close Conclusions association of P. scutata with human-modified habitats, Our findings strongly suggest that present-day populations of supported by the lack of genetic diversity across the popula- P. scutata in the Malay peninsula were probably introduced tions in this study, therefore strongly indicate that present- by people. The questionable origins of the South-east Asian day populations of P. scutata in the Malay peninsula have apple snail P. scutata in its presumed native range necessitates been introduced. a reassessment of the species’ current IUCN Red List status. Further complicating matters, our results also suggest These extant populations pose a dilemma, and the lack of that the species thus far identified as P. scutata may be a spe- genetic diversity is of concern in light of Pila decline through- cies complex. Pila scutata of Java was separated from P. scu- out South-east Asia. Conservation management of P. scutata tata of the Malay peninsula and the Philippines up to a and its other South-east Asian congeners must therefore be .   threshold of % in both COI and S. Intraspecific pair- better informed by greater taxonomic resolution, and more ,  wise difference for the majority of gastropods is % comprehensive investigations of their ecology, both in native   (Meier et al., ; Layton et al., ), although there and introduced ranges. are exceptions: e.g. a terrestrial snail, Cepaea nemoralis, has . % intraspecific divergence (Thomaz et al., ). Interspecific relationships for some Pila species have been Acknowledgements We thank Robert Cowie and an anonymous investigated previously (Hayes et al., b), but intra- reviewer for their critiques, Y.M. Yong, T.S. Ng, N.L. Zulkipeli, and interspecific differences for the genus, especially in R. Meier, W.H. Wong, A. Srivathsan and all staff and students of the South-east Asia, are poorly known. We are unable to deter- Freshwater and Invasion Biology Laboratory (National University of Singapore), for field and laboratory assistance, S.-Y. Chan, J.K. Foon, mine conclusively whether the Javan snails on the one hand B.L. Lim, R.M. Marwoto, R.C. Pagulayan, S. Ambu, M. Reid, and the Malay peninsula and Philippine snails on the other P. Callomon, A. Lawless, J. Gerber, N. Badruddin, J. Ablett, hand represent two distinct species or a single species with B. Tripathy, T. Lee, A.N. van der Bijl, J. Goud, B. Hausdorf, high intraspecific variation. Apple snails are notoriously dif- R. Janssen, T. von Rintelen and C. Zorn for information or material, and access to photographs of museum material, and the National ficult to distinguish by conchological characters (Hayes ’  Parks Board, Singapore, PUB Singapore s National Water Agency (for- et al., ), and possible hybridization sometimes con- merly Public Utilities Board), Ministry of Defence (Singapore), and  founds genetic analysis (Matsukura et al., ). Thus, a Economic Development Board (Malaysia) for research permits and ac- taxonomic revision of South-east Asian ampullariids is cess to sampling sites. We acknowledge financial support from the overdue (Hayes et al., a, ). Department of Biological Sciences, National University of Singapore, the National Research Foundation and the Economic Development Board (SPORE, COY-15-EWI-RCFSA/N197-1), Wildlife Reserves Consequences for conservation Singapore Ah Meng Memorial Conservation Fund (NUS grant R-154-000-617-720), Malacological Society of Australasia Mollusc Outreach campaigns in Peninsular Malaysia over the past  Research Grant 2013, and the Conchological Society of Great Britain years successfully raised awareness of the threat of Pomacea and Ireland Research Grant 2013. among farmers, who actively remove apple snails from paddy fields but are unable to tell the genera apart (Joshi et al., ). Older literature, including medically-related Author contributions Study conception and design: THN, SKT, HHT and DCJY; data collection and analyses: THN, SKT, AA, VTD, studies, identified P. scutata as the most common apple RCJ and WYW; reagents, materials and analysis tools: AA, HHT and  snail on the Malay peninsula (e.g. Berry, ; Lim et al., DCJY; writing: THN, SKT, AA, VTD, RCJ, WYW, HHT and DCJY. ). However, we found only one population of P. scutata, in Ipoh, compared to multiple populations of P. gracilis, along both the east (Kelantan, Terengganu) and west Conflicts of interest None. (Perak, Selangor) coasts of Peninsular Malaysia. Given the morphological similarity between different Pila spp., espe-  Ethical standards All animal handling procedures were in accor- cially juveniles (Keawjam, ), it is possible that some of dance with the ethical standards of the Institutional Animal Care the historical records of P. scutata in the Malay peninsula and Use Committee of the National University of Singapore and com- were of P. gracilis or juveniles of P. ampullacea (Plate ). ply with the journal’s Code of Conduct.

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