Identification ofPomacea species using molecular techniques Keiichiro Matsukura1 and Takashi Wada2 1NARO Kyushu Okinawa Agricultural Research Center, Koshi, Kumamoto 861-1192, Japan. Email: [email protected] 2Suya 1635-107, Koshi, Kumamoto 861-1102, Japan. Email: [email protected] Abstract Recent development of molecular approaches has prompted new taxonomic analyses and identification of species in the genusPomacea . Following the sequencing of the mitochondrial cytochrome c oxidase subunit I (COI) gene of many Pomacea species, this marker was used by several research groups to reveal the diversity and distributions of certain key species in both native and invaded areas. Nuclear DNA sequences such as elongation factor 1α and the H3 histone subunit together with mitochondrial sequences permitted assessment of relationships among closely related species. These molecular techniques contributed to improved taxonomic understanding of this genus, and clarification of the distribution and invasion pathways of some important species. Nonetheless, there remains some risk of misidentification when relying on molecular techniques. Additional keywords: Ampullariidae, Asia, DNA sequencing, invasive species, Mollusca, Pomacea canaliculata, Pomacea maculata 33 Introduction Taxonomy and identification of organisms have traditionally been based on morphological features. Identification by diagnostic morphology is usually easy and reliable once the diagnostic traits of the target species are clarified. The remarkable development of molecular biology in recent decades, however, has brought a novel approach to taxonomy. A molecular approach allows us to distinguish organisms that could not be distinguished morphologically, to clarify phylogenetic distances among related species and to examine the possibility of genetic exchange between closely related species. Nowadays, DNA barcoding, sequencing a short genetic marker specific to individual species, is widely used to distinguish a large number of species in almost all groups of organisms (e.g. CBOL Plant Working Group, 2009; Teletchea, 2009; Schlick- Steiner et al., 2010). The taxonomy of the genus Pomacea has historically been extremely confused because of intraspecific variation and environmental influences on the variation of morphological traits (Cazzaniga, 2002; Estebenet & Martín, 2003). The number of nominal Pomacea species was thought by Cazzaniga (2002) to be approximately 50, with fewer than this actually thought to be valid because of unrecognized synonyms. However, rigorous nomenclatural compilations recognized almost 100 valid species (Cowie & Thiengo, 2003; Cowie, 2015; Hayes et al., 2015). Although recent morphological and molecular analysis concluded that P. insularum, P. gigas and a number of other species were synonyms of either P. maculata or of P. canaliculata (Hayes et al., 2012), other as yet un-named species have yet to be described (R.H. Cowie, pers. comm.). Nonetheless, Hayes et al. (2015) still estimated the real number of valid species of Pomacea to be around 50. The taxonomy of Pomacea will gradually become better understood through further morphological and molecular biological analyses. Following the initial clarification of the identities of the non-native species of Pomacea in North America, using mitochondrial DNA sequences (Rawlings et al., 2007), molecular approaches addressing the taxonomy, identification, interspecific relationships and geographic origins of Pomacea species have developed remarkably (e.g. Hayes et al., 2008, 2009; Tran et al., 2008). Here, we first review recent works on the taxonomy, diversity and distribution of Pomacea species based on DNA sequences. We then provide brief technical guidance for DNA analysis of Pomacea, commenting on both the advantages and disadvantages. 34 BIOLOGY AND MANAGEMENT OF INVASIVE APPLE SNAILS Identification ofPomacea in Asia Establishment of techniques for analysis of mitochondrial cytochrome c oxidase subunit I (COI) DNA sequences in Pomacea species contributed greatly to understanding the current status and process of expansion of the invasive species in Asia. Identification of invasive species is one of the most fundamental needs when attempting to manage them. However, even 25 years after the first invasion ofPomacea in East and Southeast Asian countries, rigorous identification of the invasive snails was not possible because identification ofPomacea species based on shell morphology, particularly among closely related species, was difficult and in some cases impossible (Cowie et al., 2006). Analysis of COI sequences of Pomacea species from throughout the region revealed that two species, P. canaliculata and P. maculata, are widely distributed from Malaysia to Japan (Hayes et al., 2008; Matsukura et al., 2008), and that P. scalaris occurred in Taiwan (Hayes et al., 2008). This result resolved the presumption in a number of papers, based on morphology, that there was more than one species of Pomacea introduced widely in Asia (e.g. Mochida, 1991; Keawjam & Upatham, 1986), belonging to the “canaliculata group”, which includes P. canaliculata and closely related species (Cazzaniga, 2002; Hayes et al., 2009). For example, Pomacea introduced to Japan had been thought to be a single species (P. canaliculata) until 2008 but analysis of COI sequences showed that both P. maculata and P. canaliculata were present (Matsukura et al., 2008). In addition, the molecular analysis revealed that P. canaliculata is dominant and widespread in most parts of southern and central Japan, whereas P. maculata was observed in only three of ten prefectures in the southern region. Revealing the current distributions of Pomacea spp. in Japan has prompted establishment of appropriate management strategies in prefectures in the infested regions. Analysis of COI sequences of P. canaliculata and P. maculata in East and Southeast Asia also revealed that these snails were introduced probably on multiple occasions from their native regions because the COI sequences were significantly divergent and contained several haplotypes found in native populations in Brazil and Argentina (Hayes et al., 2008). Clarification of the invasive process from native to invaded areas is important in permitting attempts to limit further invasion and expansion of the invasive snails. Further molecular analysis (e.g. SNP and microsatellite analysis) of both native and invasive populations could reveal additional details of the invasion process and help to predict the risk of further spread in the invaded areas. IDENTIFICATION OF POMACEA SPECIES USING MOLECULAR TECHNIQUES 35 Clarifying the inter-specific relationship betweenP. canaliculata and P. maculata Analysis of nuclear DNA sequences can be an effective approach for assessing relationships between closely related species, whereas analysis of mitochondrial DNA sequences, often in combination with nuclear DNA, is more often used to examine phylogenetic relationships among species within a genus, family or other taxonomic group of interest. Unlike mitochondrial DNA, nuclear DNA is derived from both parents, and therefore, analysis of nuclear DNA can detect genetic exchange and its frequency between species. The confused taxonomy of Pomacea has been improved remarkably since the advent of molecular approaches using mitochondrial DNA; however, some relationships are still unclear, particularly among closely related species. Matsukura et al. (2013) examined the nuclear elongation factor 1α (EF1α) sequences of P. canaliculata and P. maculata collected from East and Southeast Asia. Phylogenetic analysis of these sequences demonstrated significant divergence of the sequences between the two species, as Hayes et al. (2009) had also reported. However, some snails had both P. canaliculata and P. maculata EF1α sequences. This result indicates that there is genetic exchange between P. canaliculata and P. maculata. The hybrid snails were widely distributed in the region (Matsukura et al., 2013). Similar genetic exchange between the two species also seems to occur in their native South America (Hayes et al., 2012; Matsukura et al., 2013). Knowledge of the origin of the hybrid snails and their adaptive significance would add to our understanding of diversity and distribution in the genus. Similar clarification of relationships among other closely related Pomacea species would be valuable. Technical guidance for DNA analysis of Pomacea Analysis protocols (DNA extraction, amplification by PCR and analysis) for Pomacea, which basically follow general protocols used in animals, differ (but not considerably) among research groups (e.g. Thaewnon-ngiw et al., 2004; Rawlings et al., 2007; Matsukura et al., 2008; Dong et al., 2011; Cooke et al., 2012). In this section, technical guidance for Pomacea DNA analysis is provided. For more details see Matsukura et al. (2008) and Matsukura et al. (2013). 36 BIOLOGY AND MANAGEMENT OF INVASIVE APPLE SNAILS DNA extraction Foot muscle of Pomacea is usually used for DNA extraction (Thaewnon-ngiw et al., 2004; Rawlings et al., 2007; Matsukura et al., 2008) to prevent contamination of DNA by symbiotic microorganisms and gut contents (DNA of prey can be detected in the early stages of digestion). The phenol-chloroform-protease K method (Thaewnon-ngiw et al., 2004; Rawlings et al., 2007), Qiagen’s Dneasy extraction kit (Rawlings et al., 2007; Dong et al., 2011; Cooke et al., 2012) and GE Healthcare’s Nucleon PhytoPure DNA extraction kit (Matsukura et al., 2008) have all been used for DNA extraction,