A molecular phylogeny of apple snails (Gastropoda, Caenogastropoda, Ampullariidae) with an emphasis on African species A sl a k J 0 r g en sen , T h om as K . K risten sen & H en ry M ad sen Submitted: 28 September 2007 Jorgensen, A., Kristensen, T. K. & Madsen, H. (2008). A molecular phylogeny of apple snails Accepted: 26 November 2007 (Gastropoda, Caenogastropoda, Ampullariidae) with an emphasis on African species. — doi:l0.llll/j.l463-64°9.2007.00322x Zoologica Scripta, 37, 245-252. Ampullariids are widespread in Africa, Asia, South- and Central America, and the Caribbean Islands. Basal phylogenetic relationships of the African genera Afropomus and Saulea have been inferred based on anatomical evidence. Until recently the Viviparidae was regarded as the sister-group of Ampullariidae, but recent molecular data infer a sister-group relationship with Campanilidae. We have used members of both families as outgroups in the present investiga­ tion on ampullariid phylogeny. We have used data from portions of five molecular loci, that is, the nuclear genes l8S rRNA, 28S rRNA and H3, and the mitochondrial genes l6S rRNA and COI. Our data most often infer a basal position of Afropomus. The West African species Saulea is inferred as the basal member of a clade including the South American M arisa and Pomacea. We hypothesize that evolutionary lineages leading to Saulea and the American genera were isolated from each other by vicariance events (Gondwanaland break-up l3 0 -ll0 Mya). Our individual gene analyses inferred two major clades of the African Lanistes. However, in some analyses they were not inferred as sister-groups making Lanistes paraphyletic. The African and Asian genus Pila is most often inferred to be monophyletic (except for the generally unresolved 28S). Our analyses most often inferred a sister-group relationship between Lanistes and Pila. The very low genetic diversity of the endemic radiation of Lanistes in Lake Malawi suggests that the morphological divergence has happened much faster than the molecular divergence as is also evidenced from the cichlid radiations. Corresponding author: Aslak Jergensen, The Mandahl-Barth Research Centre for Biodiversity and Health, DBL — Centre for Health Research and Development, Department o f Veterinary Pathobio- logy, Faculty o f Life Sciences, University o f Copenhagen, Jaegersborg Alle 1D, DK-2920 Charlotten- lund, Denmark. E-mail: [email protected] Thomas K Kristensen and Henry Madsen, The Mandahl-Barth Research Centre for Biodiversity and Health, DBL — Centre for Health Research and Development, Department o f Veterinary Pathobiology, Faculty o f Life Sciences, University o f Copenhagen, Jaegersborg Alle 1D, DK-2920 Charlottenlund, Denmark. E-mails: [email protected], [email protected] Introduction the genus Pila Roding, l798 natively occurs in both Africa The ampullariids or apple snails are widespread in the tropics and Asia, and the genera Asolene (d’Orbigny, l837), Filipponea occurring in Africa, North- and South America, the Carib­ (Dall, l9l9), Marisa Gray, l824, Pomacea (Perry, l8l0) and bean and Asia. Recent human-mediate introduction of South Pom ella (Gray, l847) natively occur in South America and American ampullariids into Africa and Asia have given them the southern parts of North America (Pomacea). Marisa has the status as invasive pests as they can reduce the outcome been introduced into Africa and Pomacea into Asia, and there of agricultural production especially rice (Cowie 2002). is no evidence for prehistoric non-human mediated dispersal Furthermore, ampullariids can serve as intermediate hosts events between the continents. The Gondwana distribution for the rat lungworm Parastrongylus cantonensis that can cause of the ampullariids are probably caused by vicariance events eosinophilic meningitis in man (Hollingsworth & Cowie 2006). in the early Cretaceous (approximately l3 0 -ll0 Mya) when The genera Afropomus Pilsbry & Bequeart, l927, Lanistes South America and India split from the African continent Montfort, l8 l0 and Saulea Gray, l867 natively occur in Africa, (Berthold l99l). © 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters • Zoologica Scripta, 37, 3, May 2008, pp 245-252 245 A molecular phylogeny of apple snails • A. Jergensen et al. Afropomus Saulea dae, traditionally have been regarded as closely related African Saulea Afropomus genera (Ampullarioidea). Furthermore, we included GenBank sequences Lanistes Lanistes from Campanile symbolicum Iredale, 1917 as a recent study of AfrJAsian Pila Pila genus caenogastropod relationships by Colgan et al. (2007) showed Pomacea Pomacea a sister-group relationship between Ampullariidae and Marisa Marisa American Pomacea Campanilidae. Asolene genera Fellpponea Asolene Pomella Feiipponea Gene sampling Pomella DNA was isolated from individual snails using the Nucleon Fig. 1 A, B. The previously inferred phylogenies based on morphological kit (Amersham Biosciences, UK) with approximately 50­ data by Berthold (1991; A) and Bieler (1993; B). The phylogenies 100 mg of tissue from the foot. Genomic DNA was subse­ differ in the position of Afropomus and Saulea, and in Bieler’s (1993) quently quantified using 2 |lL on a 2% agarose gel and a spectro­ reanalysis M arisa is included in a paraphyletic Pomacea. photometer, and diluted with ddH2O in a series of 10, 20, 40, 60 and 80 times. A standard concentration of 50-100 |Ag/|xL Afropomus balanoidea (Gould, 1850) and Saulea vitrea (Born, was used as template for polymerase chain reaction (PCR) 1780) are monotypic, and have restricted West African distri­ amplification. However, very often the standard concentra­ butions. Lanistes has approximately 19 species with Lanistes tion failed and dilutions had to be used. When the undiluted ovum Peters, 1845 being widespread across the African con­ ampullariid DNA template was loaded into the same PCR tinent. A small intralacustrine radiation has been reported reaction as the positive control, as a check for an inhibiting from Lake Malawi constituting L. nasutus Mandahl-Barth, effect, it often caused the positive controls to fail indicating 1972, L. nyassanus Dohrn, 1865 and L. solidus Smith, 1877. Pila the presence of inhibiting substances in the ampullariid DNA has five African species with P ovata (Olivier, 1804) being extractions. widespread across the continent (Brown 1994). Three nuclear genes, ribosomal 18S, ribosomal 28S and Berthold (1991) investigated the phylogeny of Ampullariidae the protein-coding histone H3 and two mitochondrial genes, using morphological data (Fig. 1A) originating from an extensive ribosomal 16S and the protein-coding cytochrome oxidase study of conchological and anatomical characters. Subsequently subunit I (COI) were chosen. They represent slow (18S, 28S Bieler (1993) reanalysed the data reaching a very similar and H3) and fast (16S and COI) evolving genes, nuclear and result (Fig. 1B). These studies inferred the African ampullariid mitochondrial genes, and structural and protein-coding genes. genera as the most basal within the ampullariid phylogeny Partial sequences were obtained after PCR amplification and the American genera as a monophyletic group derived with primer pairs: for 18S, 18SLYMFOR and 18SLYMREV from an African ancestor. A substantial amount of research is (Stothard et al. 2000), for 28S, C1 and 28SR2 (Morgan et al. currently being conducted to resolve the species status 2002), for H3, H3aF and H3aR (Colgan et al. 1998), for 16S, and phylogenetic relationships of the American ampullariids 16Sar-L and 16Sbr-H (Palumbi et al. 1991), and for COI, (Cowie & Thiengo 2003; Rawlings et al. 2007). LCO1490 and HCO2198 (Folmer et al. 1994). The PCR The present study investigates the phylogeny of Ampul- conditions varied in annealing temperature for the five loci; lariidae with an emphasis on the African genera and the basal preheat step at 95 °C for 5 min, 37 cycles of 10 s denaturation relationships of the group using molecular data from nuclear at 95 °C, 30 s annealing at 40-47 °C and 1 min amplification and mitochondrial genes. The aims are to test previous hypotheses at 72 °C, and finally an extension step of 10 min at 72 °C. based on morphology regarding the basal ampullariid taxon PCR products were purified using High Pure PCR Product and the biogeography of the group. Purification Kit (Roche, Germany) or extracted from an agarose gel using QIAEX II (Qiagen, Germany). The ABI Prism Big Materials and methods Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Taxon sampling Biosystems, Brisbane, CA) and an ABI 310 automated The four African ampullariid genera Afropomus, Lanistes, Pila sequencer were used for sequencing. Forward and reverse and Saulea have been sampled together with Asian Pila species sequences were aligned and edited using the Staden Package and the South American genera M arisa and Pomacea. An (Staden 1996). The gene sampling and GenBank accession overview of the taxon sampling is provided in Table 1. numbers are provided in Table 1. Voucher specimens are deposited in our Mandahl-Barth Collection of mainly African freshwater gastropods. As an Phylogenetic inference outgroup in the phylogenetic analyses we have sampled the The sequences were aligned using CLUSTALX (Thompson Viviparidae species Bellamya rubicunda (Martens, 1879) and et al. 1997). The alignment quality was estimated using the Viviparus contectus (Millet, 1813) as Viviparidae and Ampullarii- alignment score curve and this was used to inspect the variable 246 Zoologica