A molecular phylogeny of apple snails (, , ) with an emphasis on African

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 . 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 (). 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 Scripta, 37, 3, May 2008, pp 245-252 • © 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters A. J 0rgensen et al. • A molecular phylogeny of apple snails

Table 1 Taxon, locality and gene sampling.

Species Locality 18S 28S H3 16S COI

Outgroups Viviparidae Bellamya rubicunda Uganda, Butiaba EU2744535 EU2744517 EU2744502 EU274483 EU2744556 Viviparus contectus Denmark, Hellebaek EU2744536 EU2744518 EU2744503 EU274484 EU2744557 Campanilidae Campanile symbolicum Australia, GenBank DQ916524 AY296888 AF033683 AY010507 AY296828 Ingroup Ampullariidae Afropomus Afropomus balanoidea Sierra Leone, Makemui —— EU2744504 — EU2744558 Afropomus balanoidea Sierra Leone, Nyamayei — EU2744519 EU2744505 EU274485 EU2744559 La nistes Lanistes carinatus Uganda, Lake Bisinia EU2744537 EU2744520 — EU274486 EU2744560 Lanistes eiiipticus , Mwaya EU2744538 EU2744521 EU2744506 EU274487 EU2744561 Malawi, Msaka EU2744539 EU2744522 EU2744507 EU274488 EU2744562 Lanistes sp. Sudan, El Ginaed EU2744540 EU2744523 EU2744508 EU274489 EU2744563 Zambia, Lake Mweru EU2744541 EU2744524 EU2744509 — EU2744564 Lanistes purpureus Zanzibar, Kinyasini EU2744542 EU2744525 — EU274490 EU2744565 Lanistes solidus Malawi, Chipanda EU2744543 EU2744526 — EU274491 EU2744566 Lanistes varicus Burkina Faso, Mogtedo EU2744544 — EU2744510 EU274492 EU2744567 M arisa Marisa cornuarietis Laboratory stock EU2744545 EU2744527 EU2744511 EU274493 EU2744568 Pila Pila ampullacea Vietnam, Hanoi EU2744546 EU2744528 — EU274494 EU2744569 Pila conica Vietnam, Hanoi EU2744547 EU2744529 EU2744512 EU274495 EU2744570 Pila ovata Uganda, Katunguro EU2744548 EU2744530 — EU274496 — Pila ovata Uganda, Mpunge EU2744549 —— EU274497 EU2744571 Pila ovata Uganda, Rutoto EU2744550 — — EU274498 — Pila polita Vietnam, Hanoi EU2744551 EU2744531 EU2744513 EU274499 EU2744572 Pila spedosa Zanzibar, Kinyasini EU2744552 ——— EU2744573 Pom acea Pomacea bridgesii Laboratory stock EU2744553 EU2744532 EU2744514 EU2744500 DQ093524 Pomacea canaliculata Philippines, Samar EU2744554 EU2744533 EU2744515 EU2744501 EU2744574 Saulea Saulea vitrea Sierra Leone, Wangeh — EU2744534 EU2744516 — EU2744575 Saulea vitrea Sierra Leone, Kpetewaima EU2744555 ——— EU2744576

parts of the alignments for manual editing. The genetic I + G) and COI (GTR + I + G), respectively. In the com­ diversity was calculated using mega 3.1 (Kumar et al. 2004). bined data set analyses the estimation of the likelihood parameters Selected Tm ura-N ei distances (using a T-distribution model), was conducted separately for each gene using character which corrects for multiple hits taking into account the partitions. The analysis was run three times which all resulted substitutional rate differences between nucleotides and the in identical topologies. The posterior probability is given as inequality of nucleotide frequencies, are presented in Table 2. the percentage of trees sampled from the posterior distribution Transition/transversion (TS/TV) plots were made for each that had a particular clade. gene based on uncorrected pairwise distances. Phylogenetic analyses using parsimony were conducted Bayesian analyses were conducted using M rBayes 3.1 using paup* 4.0b10 (Swofford 2001). Cladograms were (Huelsenbeck & Ronquist 2001). Bayesian inferences were constructed for each gene and for the combined data set. run with 1 million generations, topologies were sampled every Parsimony inference was performed via a heuristic search 100 generations and a burn-in of 10 000 generations was using 1000 replicates of random sequence entry, tree-bisection- discarded to allow for convergence. The substitution models, reconnection (TBR) branch swapping, and assuming equal which fitted the data best, were calculated using M rM odeltest weight and unordered character states of all characters. Gaps 2.2 (Nylander 2004). The substitution models were 18S were treated both as missing characters and as the fifth character (K80 + G), 28S (GTR + I + G), H3 (GTR + G), 16S (GTR + state. Clade support was evaluated by 100 replicates of

© 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters • Zoologica Scripta, 37, 3, May 2008, pp 245-252 247 A molecular phylogeny of apple snails • A. J 0rgensen et al.

Table 2 Range in the corrected genetic diversity (Tamura-Nei distance using a T-distribution) of selected taxon comparisons.

Taxon/Gene 18S 28S H3 16S COI

Viviparidae—Campanilidae 8.2-9.0 9.7-10.1 18.6-22.5 50.9-62.7 29.5-34.9 Viviparidae-Ampullariidae 6.3-11.3 8.4-12.2 13.1-21.7 35.9-46.2 23.6-33.8 Campanilidae—Ampullariidae 4.6-8.9 8.5-11.3 11.3-21.7 50.4-70.9 27.9-47.5 Afropomus-other ampullariids — 4.8-8.2 8.5-11.6 24.1-35.6 20.9-28.1 Sautea-other ampullariids 1.0-4.7 3.4-7.5 9.2-16.2 — 23.8-31.0 Lanistes-other ampullariids 2.2-4.8 1.4-7.5 7.2-16.2 20.7-29.4 18.7-27.8 Within Lanistes 0-3.6 0-2.7 0-5.5 0.4-25.4 0-27.2 Pla-other ampullariids 2.0-4.8 1.4-6.5 7.0-15.3 21.5-31.8 18.7-28.9 Within P la 0-2.2 0-1.9 0.3 0.2-10.4 7.8-18.6 Marisa-other ampullariids 0.9-5.0 2.2-6.7 2.3-14.8 12.9-24.5 17.1-28.3 Pomacea-other ampullariids 1.0-4.7 2.8-6.4 2.3-17.8 14.4-31.8 17.0-28.6 Within Pomacea 0.7 1.1 2.3 10.1 19.8

nonparametric bootstrapping. Bremer support (Decay index) the sister-group. Lanistes was divided into two major clades was also calculated (Bremer 1988). (L. ovum (L. purpureus (L. nyassanus (L. ellipticus Martens, 1866, L. solidus)))) and (L. va ricu s (Muller, 1774) (L anistes sp., Results L. carinatus (Olivier, 1804))), which were highly supported. The The results from the combined data set and single genes are African and Asian Pila species were sister-groups. The clade presented in Figs 2 and 3 (A-E), respectively. The combined (Lanistes, Pila) was the sister-group to the clade (Saulea (Marisa, data set resulted in an alignment of 2349 bases of which 463 bp Pomacea)). However, this sister-group relationship was not came from 18S, 384 bp from 28S, 376 bp from H3, 500 bp highly supported (91, 84, 7). from 16S and 626 bp from COI, respectively. The alignment of the combined data set had 1574 invariable characters, 601 Individual gene analyses parsimony informative characters and 174 variable but parsi­ A 18S sequence could not be obtained for A. balanoidea. mony uninformative characters. No topological difference Saulea vitrea was basal in an unresolved relationship with the was inferred between the analyses treating gaps as missing or clades (Lanistes, Pila) and (Marisa, Pomacea) (Fig. 3A). Lanistes fifth characters. The genes are expected to show huge varia­ was paraphyletic and the Lake Malawi species constituted a tion in the resolution of the phylogenetic relationships of the clade including L. purpureus. Pila was monophyletic with the Ampullariidae. The two nuclear ribosomal genes 18S and relationship of the African species being unresolved with 28S are very conservative, the two mitochondrial genes 16S regard to the Asian clade. A TS/TV plot indicated that the (ribosomal) and COI (protein-coding) are very variable, and substitutions of the 18S sequence were unsaturated with up the nuclear protein-coding gene H3 show intermediate sequence to 5.0% sequence variation within Ampullariidae (Table 2). variation (Table 2). The expected variation in ‘resolution The phylogenetic relationships inferred by 28S were mostly power’ at this taxonomic level was clearly observed in the unresolved (Fig. 3B). Saulea vitrea was inferred to be part of phylogenetic trees (Fig. 3). All analyses inferred the Ampul­ the American clade (Marisa, Pomacea). A TS/TV plot indicated lariidae to be monophyletic and C. sym bolicum to be its that the substitutions of the 28S sequence were unsaturated sister-group. with up to 8.2% sequence variation within Ampullariidae (Table 2). Combined data set analyses The Bayesian analysis of H3 inferred A fropomus as the Afropomus balanoidea was inferred to be the basal taxon by the sister-group to the clade (Lanistes, Pila) (Fig. 3C). However, this combined data set (Fig. 2). Saulea vitrea was inferred to be relationship was not supported by the maximum parsimony sister-group to the American clade (Marisa, Pomacea). This analysis. Saulea was inferred to be the sister-group to the relationship was highly supported by the posterior probability clade (Marisa, Pomacea). Lanistes was monophyletic and the and Bremer support (100 and 14, respectively), but not by the sister-group to Pila. A TS/TV plot indicated that the substi­ bootstrap value (81) of the parsimony analysis. Both Lanistes tutions of the H3 sequence were unsaturated with up to 17.8% and Pila were monophyletic and sister-groups. These clades sequence variation within Ampullariidae (Table 2). were highly supported by posterior probabilities, bootstrap The analysis of 16S inferred Afropomus as the basal taxon support and Bremer support. The Lake Malawi Lanistes (Fig. 3D). A 16S sequence could not be obtained for S. vitrea. species formed a clade with L. purpureus (Jonas, 1839) as L anistes was inferred to be paraphyletic with a clade of

248 Zoologica Scripta, 37, 3, May 2008, pp 245-252 • © 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters A. Jurgensen et al. • A molecular phylogeny of apple snails

- Viviparus contectus • Bellamys rubicunda - Campanile symbolicum

66 ■ Pila speciosa 63°r Pila ovata Rutoto lr Pila ovata Mpunge

100 79I Pila ovata Katunguro 100 12 Pila polita 100 Pila ampullacea 90H r 10 0 L 100' Pila conica 100 100 100 13 Lanistes ovum 100 14 99 100 46 - Lanistes purpureus

98 Lanistes nyassanus 91 _ r 84 10000 b Lanistes i ellipticus 7 100 10000 I 94 331!3l00l ool Lanistes] solidus 7 100 6— Lanistes various

100I r Lanistes sp. 1001— 11 100L 100 Lanistes carinatus 26 Saulea vitrei 100 100 Marisa cornuarietis— 18 100 Fig. 2 The phylogeny inferred by Bayesian 81 —, 14 100oq-I-| I------' Pomacea bridgesii analysis of the complete data set. Note the 944 M position of Saulea basal to the American clade 5 100I____ Pomacea canaliculata 97 (Marisa, Pomacea). The numbers at each node 15 Afropomus balanoidea Makemui is the posterior probability (Bayesian inference, 100I BI), bootstrap support (Maximum parsimony, 100 Afropomus balanoidea Nyamayei MP) and Bremer support (MP), respectively. 0.1 16

Lanistes species being sister-group to Pila. The other Lanistes of transitions and transversions indicating that substitutions clade had L. purpureus as the sister-group to the Lake Malawi of the COI sequences were saturated within Ampullariidae clade. The American clade (Marisa, Pomacea) was the sister-group (Fig. 4B). Corrected (Tamura-Nei distance using a T-distri- to the clade (Lanistes (Lanistes, Pila)). A TS/TV plot showed bution) genetic diversity showed up to 31.0% sequence an overlap of transitions and transversions indicating that the variation (Table 2). 16S sequences were saturated within Ampullariidae (Fig. 4A). Corrected (Tamura-Nei distance using a T-distribution) genetic Discussion diversity showed up to 35.6% sequence variation (Table 2). The analyses of the individual genes and the combined data The basal phylogenetic relationships inferred by COI are set strongly supported the sister-group relationship of incongruent compared to the other genes (Fig. 3E). Afropomus Ampullariidae and Campanilidae. These results further empha­ was the sister-group to the part of Lanistes genus and Saulea size that the Ampullarioidea J. E. Gray, 1824 (Ampullariidae was the sister-group to the rest of the African taxa including and Viviparidae) is not a monophyletic group. This conclusion the South American genus Marisa. Pila was monophyletic is in accordance with the molecular-based studies first by with an Asian clade and Pila speciosa (Philippi, 1849) as the Harasewych et al. (1998) and recently by Colgan et al. (2007). basal member. The American genera were polyphyletic with In the analyses of the combined data set, a monophyletic Pomacea canaliculata inferred as the basal ampullariid followed Ampullariidae was highly supported. However, Ampullariidae by P. bridgesi. The clade support was very low or missing for was not equally well supported by the individual genes most of the inferred clades. A TS/TV plot showed an overlap (except for 16S).

© 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters • Zoologica Scripta, 37, 3, May 2008, pp 245-252 249 A molecular phylogeny of apple snails • A. Jorgensen et al.

18S B 28S — Viviparus contectus — Viviparus contectus — Bellamya rubicunda Bellamya rubicunda Campanile symbolicum — Campanile symbolicum r - Saulea vitrea 1gSp Lanistes various Pila ampullacea —' Lanistes carinatus Pila conica Lanistes sp. Pila polita 188 Lanistes ovum - Pila ovata Katunguro 14 ■ Pila speciosa — Afropomus balanoidea Nyamayel Pila ovata Katunguro - Lanistes carinatus Pila ovata Rutoto Pila ovata Mpunge Lanistes sp. p Pila pollta i! f oo~|f PUa ampullacea Lanistes ovum Pila conica Lanistes ellipticus Lanistes ellipticus Lanistes solidus Lanistes nyassanus **it "I Lanistes nyassanus ~ M - Lanistes solidus '6|_ Lanistes purpureus ■— Marisa cornuarietis [Satz/ea vitrea \ "->macea canaliculata > Pomacea bridgesii 3 % l r Pomacea bridgesii I (D ip- pt Marisa cornuarietis Pomacea canaliculata 6? H3 16S — Viviparus contectus - Viviparus contectus ■ Bellamya rubicunda ■ Bellamya rubicunda ■ Campanile symbolicum - Campanile symbolicum Afropomus balanoidea Nyamayei rAfropomus balanoidea Makemui Marisa cornuarietis |> ^Afropomus balanoidea Nyamayei Pomacea bridgesii 13 Pomacea canaliculataiT* r Lanistes ellipticus Lanistes various | Lanistes nyassanus Lanistes carinatus I Lanistes ovum Lanistes sp. Pila pollta |> Lanistes sp. Pila ampullaceal" ^I-Lanistes various Pila conica 13 Pila conical^ Pila ovata Rutoto Pila ovata Kinyara Pila pollta § Pila ovata Lwampanga | Saulea vitrea Lanistes purpureus H p Marisa cornuarietis 3 Lanistes nyassanus 3 Lanistes ellipticus ^ “ 921- Pomacea bridgesii S 921 O Lanistes solidus $ Pomacea canaliculata o>□ COI Viviparus contectus ■ Bellamya rubicunda • Campanile symbolicum ■ Pomacea canaliculata — PomaceaE bridgesii ■ Marisa cornuarietis 63|----- ■pm Pila speciosa 88 —H Pila ovata Mpunge 4 M r — Pila pollta |> 2 V - g V | j - Pila ampullaceal!£. - Pila conica |“ — Lanistes various Lanistes carinatus .anistes sp. Lanistes nyassanus Lanistes ellipticus Lanistes solidus Lanistes purpureus Lanistes ovum Afropomus balanoidea Makemui Afropomus balanoidea Nyamayei Saulea vitrea Wangeh River ISaulea vitrea Kpetewaima

Fig. 3 A-E. The inferred phylogenies by Bayesian analyses from 18S, 28S, H3, 16S and COI. The numbers at each node is the posterior probability (BI), bootstrap support (MP) and Bremer support (MP), respectively.

250 Zoologica Scripta, 37, 3, May 2008, pp 245-252 • © 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters A. J 0rgensen et al. • A molecular phylogeny of apple snails

Lanistes ovum and L. ellipticus are present in swamps adja­ cent to Lake Malawi and either could be the most recent common ancestor of the intralacustrine radiation. Our results show that they both could be the most recent common ancestor. Our results show that L. ovum or L. purpureus is the likely most recent common ancestor and that L. ellipticus is part of the Lake Malawi clade. Berthold (1990) found an unresolved group of species including L. ellipticus, L. ovum and L. purpureus to be possible ancestors of the Lake Malawi clade. The very low COI sequence variation of the Lake Malawi clade (0%-0.7%) suggests that the morphological divergence has happened much faster than the molecular divergence. The genetic diversity of the currently recognized species of Lake Malawi falls within the intraspecific variation threshold of 2%-3% established by DNA barcoding (Hebert et al. 2003). The much higher diversity in Lake Malawi between major clades of the predominantly parthenogenetic thiarid M elanoides (13.4%-15.8%; Serensen et al. 2005) and between the planorbids Bulinus nyassanus (Smith, 1877) and B. succinoides (Smith, 1877) (5.2%; Jergensen et al. 2007) sug­ gest different evolutionary histories of these taxa. Further gene and taxon sampling of Lanistes species would probably resolve the status of the most recent common ancestor. Further investigation is also needed to resolve the question Fig. 4 A, B. Transition/transversion plots indicating substitutional regarding the relationships within Lanistes. Two major clades saturation of 16S (A) and COI (B). The plots are based on the uncorrected pairwise distances and substitution number between the of Lanistes are inferred by all analyses; however, these two species. lineages only form a monophyletic group in the analyses of the combined and H3 data matrices. L anistes are not monophyletic in the analyses of 18S and 28S (unresolved), and The African species A. balanoidea was inferred to be the 16S and COI (the non-Malawi clade is sister-group to Pila). basal taxon of Ampullariidae. This relationship is in accord­ The phylogenetic relationship between the African and ance with the morphologically based inferences by Berthold Asian Pila could not be unambiguously resolved. The Bayesian (1991; Fig. 1A) and does not support that of Bieler (1993; analyses of the combined data set inferred the African P. speciosa Fig. 1B). The basal phylogenetic position suggests that the to be the sister-group to the African P. ovata (with weak support; most recent common ancestor was African and that the Fig. 2). COI (Fig. 3E) inferred a basal relationship of P. speciosa, evolution of the recent ampullariids initially took place at but this was not supported by the parsimony analyses. The the African part of Gondwanaland. basal relationship was unresolved for 18S, which is too con­ The position of the West African species S. vitrea basal to servative to provide resolution within the ampullariid genera the American clade (Marisa, Pomacea) has not previously been (Fig. 3A). The preferred hypothesis by Berthold (1991) sug­ inferred by other studies. The genetic distance between Saulea gests that the ancestor of Pila was living in the African part of and the American clade is lower for 28S and H3 than the Gondwanaland (120 Mya) and dispersed into Asia through distance to Afropomus. However, Saulea and Afropomus are Madagascar, where it speciated. Further taxon sampling more similar with regard to the COI sequence. The average of the African Pila is needed to resolve the African-Asian genetic distance between Saulea and Marisa, Pomacea are relationship and to test the hypothesis by Berthold (1991). 4.0% for 28S, 10.0% for H3 and 27.7% for COI. The cor­ The results presented in the current study point to several responding values for Saulea and Afropomus are 8.2%, 11.6% lines of future investigations relating to the African ampullariids. and 24.9%, respectively. In a biogeographical context it is not However, improvement of the taxon sampling is required to surprising that the ancestor of the American genera is to be further increase our understanding of the evolution of found in West Africa. It is hypothesized that evolutionary Lanistes and Pila. Although, obvious, this is a main obstacle as lineages leading to Saulea and the American genera were the geographical distribution (with specific localities) often is isolated from each other by vicariance events (Gondwanaland poorly known and the species can be very rare and show break-up 130-110 Mya; Brown & Gibson 1983). highly patchy distributions.

© 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters • Zoologica Scripta, 37, 3, May 2008, pp 245-252 251 A molecular phylogeny of apple snails • A.Jorgensen et al.

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252 Zoologica Scripta, 37, 3, May 2008, pp 245-252 • © 2008 The Authors. Journal compilation © 2008 The Norwegian Academy of Science and Letters