Molecular diversity and phylogenetic relationships of the gastropod

http://africanzoology.journals.ac.za

Molecular diversity and phylogenetic relationships of the gastropod genus Melanoides in Lake Malawi Louise Von Gersdorff Sørensen, Aslak Jørgensen* & Thomas K. Kristensen Mandahl-Barth Research Center for Biodiversity and Health, DBL – Institute for Health Research and Development, Jaegersborg Allé 1D, DK-2920 Charlottenlund, Denmark Received 2 August 2004. Accepted 23 November 2004

The freshwater snails belonging to the genus Melanoides Olivier, 1804 are widespread across tropical regions of the world and endemic species have evolved in the African Lakes Malawi, Mweru and Tanganyika. The endemic Melanoides species of Lake Malawi have been investigated several times during the last century, due to their large conchological variation, but no unambiguous answer regarding the number of species has been given. The phylogenetic relationship between morphs or genetic clones of Melanoides in Lake Malawi was inferred by phylogenetic analyses of DNA sequence data from the mitochondrial genes 16S and COI. Additional sequences from GenBank were included to investigate the relationship to other morphs from different parts of the world. For the first time, a putative secondary structure was developed for a partial region of 16S in this genus to identify the variability of the secondary structure in stems and loops. The molecular analyses indicated that several genetic clones exist in Lake Malawi and that M. tuberculata is a paraphyletic taxon. It is not clear from the results whether invasions or dispersals account for the complex situation in Lake Malawi. The basal position of M. admirabilis, endemic to Lake Tanganyika, in the inferred phylogeny indicates that Africa might be the origin of the genus. The results further indicate that three major clades of Melanoides, consisting of several genetic clones, are present in Lake Malawi; one clade consisting of invasive M. tuberculata, another of native M. tuberculata and a third consisting of the M. polymorpha-complex. It appears as if the unique development of morphs within the Melanoides genus in Lake Malawi has evolved primarily by divergence of genetic clones instead of species differentiation. Key words: Melanoides, Lake Malawi, 16S, COI, phylogenetic relationships.

INTRODUCTION The prosobranch genus Melanoides Olivier, 1804 (Thiaridae) includes both cosmopolitan and endemic species, which live in freshwater lakes and streams (Myers et al. 2000). The snails are euviviparous and the populations mostly consist of females with apomictic parthenogenesis as the common method of reproduction, although sexual reproduction also occurs. Samadi et al. (1998) recognized that within each morph the genotype of the offspring was identical to the mother ’s genotype, regardless of whether it was a bisexual population or not, which suggests that each morph represents a genetic clone. Melanoides is the most abundant snail in Lake Malawi and probably the main competitor of the snail Bulinus nyassanus (Smith, 1877), an intermediate host for the schistosome parasite Schistosoma haematobium (Madsen et al. 2001). Several morphologically distinct Melanoides taxa are presently *Author for correspondence: E-mail: [email protected]

believed to have evolved in Lake Malawi (Brown 1994). Even though various scientists have described Melanoides taxa from Lake Malawi since 1877, confusion still exists concerning the number of species. In 1877, Smith suggested that seven species belonging to the genus Melania (Lamarck, 1801) were present in Lake Malawi. In 1889, Bourguignat added 29 species, resulting in a total of 36 species, which later were reduced to 12 by von Martens (1897). Crowley et al. (1964) reduced the number to six, Mandahl-Barth (1972) suggested nine species and Brown (1994) identified eight species existing in the lake. Eldblom & Kristensen (2003) have recently suggested five species based on shell and radula morphology (morphospecies). Currently the Melanoides classification applies only to asexually reproducing genetic clones (morphs) (Facon et al. 2003). The ongoing investigations of Melanoides illustrate the confusing state of species separation within the genus Melanoides,

African Zoology 40(2): 179–191 (October 2005)


180

African Zoology Vol. 40, No. 2, October 2005

Fig. 1. Sampling localities in Lake Malawi. A, An enlargement of the northern tip of the lake marked in B; B, Lake Malawi.

when applying morphological methods in Melanoides taxonomy. Morphospecies identification is problematic due to high ecophenotypical variation or plasticity (van Damme & Pickford 2003) that make adult shell shape and ornamentation unreliable characters (Davis 1994). The same problem applies to radula morphology (Michel 1994), thus there is a need for other methods besides morphological methods to separate the species. Samadi et al. (1999) constructed a morphological identification system that was supported by their molecular investigations. This system was based mainly on shell morphology and colour and was subsequently used by Facon et al. (2003) and Genner et al. (2004). Facon et al. (2003) investigated Melanoides invasions of the New World using molecular methods and suggested that the morphs were paraphyletic and Genner et al. (2004) discovered a camouflaged invasion of M. tuberculata morphs in Lake Malawi that, according to the authors, originated from Southeast Asia. These two studies are very important because they have contributed significantly to the understanding of genetic clones and phylo-

geography within the genus Melanoides. The purpose of the present study was to investigate the molecular diversity and phylogenetic relationships of the morphs within the genus Melanoides in Lake Malawi by analysis of sequence data. Our 16S and COI ribosomal DNA data from Lake Malawi have been analysed together with sequences from Facon et al. (2003) and Genner et al. (2004). MATERIALS & METHODS Sampling In August/September 2002 Melanoides snails were collected at the northern part of Lake Malawi in Tanzania, from the Songwe River to Nkanda (Fig. 1). The collections were done by scooping and dredging. Most snails were found on the western side of the lake on sandy bottom. The samples were sorted and approximately 3000 snails were sent to the Danish Bilharziasis Laboratory (DBL) in 80% alcohol. Snails collected in Malawi by C. Eldblom and T. K. Kristensen in 2001 and H. Madsen in January 2003 in the southern and western parts of the


Von Gersdorff Sørensen et al.: Diversity and phylogeny of Melanoides in Lake Malawi

181

Fig. 2. Morphs collected in Lake Malawi.

lake were also used for the analyses (Fig. 1). Snails from Lake Albert, Uganda, Lake Victoria, Kenya, and Eseka, Cameroon, were also used for the analysis. Snail identification The lineages within the genus Melanoides were identified by conchological examination and were separated according to the characters described by Eldblom & Kristensen (2003) and Samadi et al. (1999). The taxa used for analyses are M. tuberculata (Müller, 1774), M. virgulata (Férrusac, 1827), M. nodicincta (Dohrn, 1865), M. simonsi (Smith, 1877), M. woodwardi (Smith, 1893), M. turritispira (Smith, 1877), M. nyassana (Smith, 1877), M. pubiformis (Smith, 1877), M. truncatelliformis (Bourguignat, 1889), M. magnifica (Bourguignat, 1889) and M. polymorpha (Smith, 1877) (Fig. 2, Table 1). The first four morphs mentioned above were considered M. tuberculata-like snails due to morphological resemblance and they were described according to Facon et al. (2003) and Genner et al. (2004) (Table 2). At least one individual per morph was analysed and the identification is based on shape, sculpture and colour of the shell. The morphs were given a three-letter code indicating the locality where they were found. No morph described by Facon et al. (2003) was found among the individuals studied in this paper but several morphs resembled some of those described by Genner et al. (2004). Nine morphs of M. tuberculata are described in this paper. The last seven morphs are considered to

represent the M. polymorpha-complex as defined by Eldblom & Kristensen (2003) and these morphs were also given a three-letter code referring to their former morphospecies names. Melanoides pergracilis (von Martins, 1897) was only present as dry shells in the Mandahl-Barth collection of shells at DBL (Mandahl-Barth 1972), which was used as a reference. Two morphs used in the analyses appeared morphologically as if they were a hybrids of two morphs (Eldblom & Kristensen 2003) and they are thus indicated by both codes. Outgroup 16S rDNA sequences from Tarebia granifera (Lamarck, 1822) and Cleopatra bulimoides (Olivier, 1804) were used as outgroups. COI DNA sequences from Thiara amarula (Linnaeus, 1758) and Cleopatra bulimoides were used as outgroups in the COI analysis. Tarebia granifera and Thiara amarula was chosen as outgroups because Facon et al. (2003) and Genner et al. (2004) used these taxa as outgroups, respectively. The thiarid Cleopatra bulimoides was used as an additional outgroup to increase the taxon sampling. DNA extraction, PCR amplification and phylogenetic analyses Genomic DNA was extracted from Melanoides preserved in 80% ethanol by standard CTAB (phenol/chloroform) extractions. The genomic DNA provided templates for PCR (polymerase chain reactions). Partial sequences of 16S were


182


Table 1. Origin of samples of the specimens sequenced for 16S and COI. The specimens with * were sequenced for both genes. The specimens with # were only used for the COI phylogeny. GenBank numbers are denoted with the accession number for 16S first, and then a possible accession number for COI. ‘Species’

Location

Morph

GenBank No.

Thiara amarula # Tarebia granifera Cleopatra bulimoides* M. tuberculata Kenya* M. tuberculata Uganda M. nyassana/polymorpha* M. nyassana* M. pubiformis* M. nodicincta M. virgulata M. polymorpha* M. tuberculata* M. woodwardi M. woodwardi/truncatelliformis M. polymorpha M. truncatelliformis* M. woodwardi M. polymorpha M. simonsi M. magnifica* M. woodwardi M. tuberculata M. tuberculata* M. tuberculata M. virgulata M. woodwardi M. woodwardi M. turritispira M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. tuberculata M. amabilis M. admirabilis M. tuberculata # M. tuberculata # M. tuberculata # M. tuberculata # M. tuberculata # M. tuberculata # M. tuberculata # M. tuberculata # M. tuberculata #

Mayotte, Comores Archipelago Martinique + Cuba + Tahiti Lake Albert, Uganda Lake Victoria, Kenya Lake Albert, Uganda River Songwe, Tanzania Kiwira, Tanzania Kiwira, Tanzania Kiwira, Tanzania Nsesi, Tanzania Njisi, Tanzania Itungi, Tanzania Itungi, Tanzania Mwaya, Tanzania Mwaya, Tanzania Mwaya, Tanzania Njisi/Itungi, Tanzania Njisi/Itungi, Tanzania Mwaya, Tanzania Mwaya, Tanzania Mwaya, Tanzania Eseka, Cameroon Southern end of Lake Malawi Chembe, Malawi Chembe, Malawi Nkhata Bay, Malawi Nkhota Kota, Malawi Nkhota Kota, Malawi The Philippines Choroni, Venezuela Figuig, Morocco San Jeronimo, Columbia Martinique, French West Indies Martinique, French West Indies Afilayia, Oman Kisumu, Kenya Bouaké, Ivory Coast Seychelles Malawi Malawi Moorea, French Polynesia Huahine, French Polynesia Tumbes, Peru Florida, USA Lombok, Indonesia Martinique, French West Indies Lake Tanganyika, Tanzania Bangkok, Thailand Kambiri point, Malawi Lower Selatar Reservoir, Singapore Nkhata Bay, Malawi Dambulla, Sri Lanka Mkungula, Malawi Mwanza Gulf, Tanzania Upper Selatar Reservoir, Singapore Pandan Reservoir, Singapore

– – – KLV ULA NYA/POL NYA PUB TAK TAN POL TAI WOO WOO/TRU POL TRU WOO POL TAM MAG WOO CAE MAC 1 MAC 1 MAC 2 WOO WOO TUR ND CHO MOF COL FAL PDC OMW KIS BOU ND ND ND MOO MOO TUM BCI ND – – BAN LMI1 LSS LMN1 SRI LMN3 VIC USR PAN

AY575997 AY283069 AY791935/AY791934 AY791931/AY791913 AY791930 AY791932/AY791925 AY791933/AY791926 AY791920/AY791919 AY791927 AY791906 AY791921/AY791922 AY791910/AY791909 AY791901 AY791900 AY791924 AY791917/AY791916 AY791905 AY791923 AY791918 AY791928/AY791929 AY791904 AY791914 AY791911/AY791912 AY791915 AY791907 AY791903 AY791902 AY791908 AY456618 AY283084 AY283074 AY283078 AY283075 AY283071 AY283073 AY283076 AY283072 AY283077 AY456617 AY456616 AY283083 AY283082 AY283080 AY283079 AY283081 AY283068 AY456615 AY575971 AY575992 AY575989 AY575998 AY575981 AY575991 AY575996 AY575979 AY575975



183

Table 2. Shell trait scores of Melanoides tuberculata-like populations; characters were scored using the categorical scoring system of Facon et al. (2003). Background colour

Ornaments

Columellar band

General shape

Sculptures

Morph

IN

TI

HE

DO

SP

SO

HO

SH

SC

CO

RO

GR

RI

RD

MAC 1 MAC 2 TAK TAI ULA TAN TAM CAE KLE

2 2 1 2 2 2 2 3 2

1 1 1 1 1 3 1 1 1

0 0 0 0 0 0 0 0 0

2 2 1 2 3 2 2 3 2

2 2 3 3 1 2 3 3 3

2 2 1 1 2 2 3 2 1

2 2 1 2 2 1 2 3 1

2 2 2 1 1 2 2 3 1

3 2 2 – – 2 2 3 –

1 1 2 2 2 2 3 2 2

2 2 3 3 3 1 1 3 3

1 1 3 3 2 1 3 1 2

0 0 3 3 3 0 2 0 3

– – 3 2 1 – 3 – 1

– indicates the trait was absent. IN, intensity of shell background colour: (1) very pale, (2) pale, (3) medium, (4) dark. TI, background tint of the shell: (1) yellow to brown, (2) greenish, (3) orange to reddish, (4) white. HE, heterogeneity of the background colour on the shell whorl: (0) homogeneous, (1) distinctly darker band below the suture. DO, overall density of colour ornaments on the whole shell, except the zone just below sutures: (0) no ornaments, (1) medium, (2) dense. SP, ornament type, expressed as proportion of spots to flames: (0) only flames, (1) more flames than spots, (2) more spots than flames, (3) only spots. SO, size of the ornaments: (1) small spots or narrow flames, (2) medium, (3) large spots or wide flames. HO, heterogeneity of ornamentation among different parts of the whorl: (1) homogeneous, (2) slightly different ornaments below suture, (3) ornaments below suture very different from the rest of the shell. SH, presence and sharpness of a dark band on the axial edge of the aperture columellar band: (1) absent, (2) diffuse, (3) sharp. SC, size of the columellar band, when present: (1) narrow, (2) medium, (3) wide. CO, conicity of the shell: (1) acute, (2) medium, (3) blunted cone. RO, roundness of body whorls: (1) flat, (2) slightly rounded, (3) well-rounded. GR, spiral grooves: (0) absent, (1) shallow grooves, (2) intermediate, (3) very deep grooves. RI, density and width of axial ribs: (0) none, (1) a few narrow ribs, (2) a few large ribs, (3) many narrow ribs. RD, elevation of axial ribs when present: (1) shallow, (2) medium, (3) deep.

amplified using primer pair 16Sf, 5’ CGC CTG TTT ATC AAA AAC AT 3’ and 16Sr, 5’ CCG GTC TGA ACT CAG ATC ACG T 3’ (Remigio & Blair 1997). For the partial sequences of COI primer pair LCO1490, 5’ GGT CAA CAA ATC ATA AAG ATA TTG G 3’, and HCO2198, 5’ TAA ACT TCA GGG TGA CCA AAA AAT CA 3’ (Folmer et al. 1994) was used. The PCR contained 20 µl: 2 µl DNA, 1 µl 1 mM dNTP, 2 µl 10 × reaction buffer, 1 µl 25 pmol of each primer, 13 µl deionized water and 0.1 µl (5 units/vl) Taq polymerase (Roche). The PCR settings for 16S and COI included a initial preheat step at 95°C for 5 min followed by 37 cycles consisting of a denaturation step at 95°C for 10 s, an annealing step at 40°C for 30 s, and an extension step at 70°C for 60 s and finally an extension step at 72°C for 10 min. This was run on a Thermal Cycler from Techgene, Techne, Albany, New York. The samples were sequenced from both directions using an ABI Prism Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) and run on an ABI 310 Automated Sequencer.

The sequences were edited manually using the Staden Package (Staden 1996) and aligned in CLUSTALW1.8 (Jeanmougin et al. 1998). Additional Melanoides sequences of 16S and COI from GenBank were used in the analyses (Table 1). All sequences obtained in the present study have been submitted to GenBank with the following accession numbers AY791900–AY791935. To investigate the possibility of substitutional saturation of 16S a transition/transversion plot was used to examine the nucleotide substitution patterns. The bivariate plot made pairwise comparisons of the number of transitions and the number of transversions versus genetic distance (P-distance = the proportion (p) of nucleotide sites at which the two sequences compared were different) to investigate if the number of Tv exceeded the number of Ts and if a linear relationship existed. If substitutional saturation is indicated the effect of differential weighting using step matrices should be explored. Transitions, transversions and P-distance were calculated using MEGA2.1 (Kumar et al. 2001).


184


For all the alignments Tajima’s D test (Tajima 1989) was done in DnaSP ver. 3 (Rozas & Rozas 1999) with default settings to investigate if the substitutions deviated significantly from that expected under neutral evolution (P > 0.10). The HKY + G substitution model, which was used in the maximum likelihood analysis, was inferred using Modeltest 4b (Posada & Crandall 1998). Analysis of the 16S and COI data was done in PAUP* version 4.0 (Swofford 2001) by maximum likelihood (ML) and maximum parsimony (MP) with a heuristic search of 100 random replicates. Bootstrap values were calculated as a measure of branch support. The molecular diversity (Pdistances) between the major clades is given in Appendixes 1 and 2. We have many incomplete sequences (unpubl. data) that support our findings. In addition, when more than one sequenced specimen from one location represented the same morphs and the specimens had identical sequences (as in every case), only one sequence is represented in the analyses. All shells are deposited in the MandahlBarth collection at DBL. Recently, sequences of COI from the study of Genner et al. (2004) have become available in GenBank. The present study has used a selection of these sequences to investigate the unique position of the M. polymorpha-complex. Furthermore, the sequences have been used to investigate the dispersal events of native and invasive morphs, as suggested by Genner et al. (2004). The COI data matrix is preliminary but the analysis is justified due to the importance of our 16S findings with regard to the M. polymorpha-complex. A combined matrix (unpublished, available at DBL) has been made only for the specimens collected in Lake Malawi. The computation time for bootstrap analyses including the sequences from GenBank in the dataset was unacceptable. Secondary structure The secondary structure of 16S in Melanoides was made from a molluscan consensus diagram from Lydeard et al. (2000) to investigate the variable regions. The secondary structure was made from a partial sequence from a specimen belonging to Melanoides tuberculata from Cameroon (Fig. 3A). Ambiguous loops were further investigated in the program RNAdraw1.0 (Matzura & Wennborg 1996). A sliding window analysis, designed to reveal patterns of variability along nucleotide sequences by sliding a window of a certain length

(in this case 7 bp) along the sequences, was also made to investigate the position of the variation in the alignment (Fig. 3B). RESULTS 16S rDNA sequences New sequences from 22 Melanoides specimens from Lake Malawi together with single specimens from Cameroon, Uganda and Kenya and C. bulimoides constituted the data matrix together with GenBank sequences from 19 Melanoides specimens and Tarebia granifera. A partial region of 16S with a length of 465 nucleotides was used for the analyses of the present study. Three hundred and twentyfive (69.9%) nucleotide positions were found to be invariant. A total of 90 (19.4%) positions varied and were all parsimony informative. Fifty variable characters (10.8%) were parsimony uninformative. Specimens belonging to the same morph in Lake Malawi displayed the exact same sequences and furthermore several different morphs shared identical sequences. This would be expected of recently derived parthenogenetic genetic clones. Melanoides tuberculata from Cameroon (CAE) differed by three substitutions from the M. tuberculata specimens from Lake Malawi, belonging to the same clade. In clade 5 M. nodicincta (TAK), M. simonsi (TAM) and M. tuberculata (6f) (TAI) had identical sequences. In clade 4 the M. virgulata specimens (MAC 2 and TAN) and the two M. tuberculata specimens from Lake Malawi (2 × MAC 1) had identical sequences. This clearly illustrates that closely related morphs, or morphs with identical sequences, can have different shell morphologies, as both Facon et al. (2003) and Genner et al. (2004) found. The Tv-Ts plot (not shown) did not indicate substitutional saturation and the phylogeny was inferred using unweighted data. Tajima’s D test of neutrality indicated random substitutions (D = –1.26746, P > 0.10). Secondary structure A putative secondary structure model for 16S was made for the first time for Melanoides snails (Fig. 3A). The structure followed the consensus diagram by Lydeard et al. (2000). Where lines are drawn the secondary structure is uncertain and RNAdraw1.0 did not give a useful suggestion. A sliding window analysis (Fig. 3B) further illustrated that the highest rate of variation was in loop



185

Fig. 3. A, The putative secondary structure made from a consensus diagram (Lydeard et al. 2000) of the sequenced partial region of 16S from a specimen belonging to Melanoides tuberculata from Cameroon. It covers approximately 10745–10292 bp of the mitochondrial genome of Katharina tunicate (Lydeard et al. 2000). Uppercase letters illustrate invariable nucleotides and lowercase letters illustrate variation within the Melanoides spp. investigated. The line arcs denote regions where the secondary structure was uncertain. The Roman numerals represent different domains within 16S (Lydeard et al. 2000) and the capital L followed by a number represents different loop regions within 16S (Stothard et al. 2001). The bars between nucleotides indicate that the nucleotides bind strongly. Dots indicate that nucleotides bind weakly. B, A sliding window analysis that reveals variable regions of the partial region of 16S investigated. L7 and L10 are particularly variable. The x-axis represents numbers of bases in the region and the y-axis represents numbers of variations.


186


regions L7 and L10 as found in the pulmonate snail genus Bulinus (Stothard et al. 2001). Phylogeny The maximum parsimony analysis of 16S produced a consensus tree (of 12 trees), where Cleopatra bulimoides was placed as an outgroup in an unresolved node with M. admirabilis (Fig. 4). Melanoides spp. was divided into four clades. The maximum likelihood test with the substitution model HKY + G inferred by Modeltest (Posada & Crandall 1998) only differed from the most parsimonious tree in that Cleopatra was placed basal to M. admirabilis and in that the topology of some of the internal branches differed. Clade 1 placed the M. polymorpha-complex as a closer relative to Tarebia granifera than to the other specimens of Melanoides, which was very surprising. Tarebia granifera differed from the M. polymorpha-complex by 14 substitutions and the clade was supported by a bootstrap value of 100 both with parsimony and maximum likelihood analyses. Clade 2 included all M. tuberculata morphs and M. amabilis and is divided into clade 3 and 4. Clade 4 included six morphs from the Old World and three morphs from the New World placed in two clades. The five new sequences were included in a clade with specimens from Peru and Florida. The other clade in clade 4 included M. tuberculata from Indonesia and M. amabilis from French West Indies. Clade 5 included 13 morphs from the Old World and five invading morphs from the New World and consisted of three clades. The two new sequences from Kenya and Uganda were included in a clade together with specimens from Oman, Venezuela and Morocco. The four new sequences from Lake Malawi were included in a clade together with two other specimens from Malawi, one from Kenya, one from the Ivory Coast and one from the Seychelles. The third clade included four invading genetic clones from the Philippines, Colombia and two from the French West Indies. Clade 6 included two haplotypes from French Polynesia. M. admirabilis was placed together with the outgroup Cleopatra bulimoides in an unresolved node. That is probably an artefact caused by the unrooted tree, but the position as basal to the rest of the Melanoides is determined. The molecular diversity within 16S (Appendix 1) is high compared to species of the gastropod genus Bulinus (Jorgensen 2003), especially taking

into consideration that the Melanoides specimens were sampled within one lake. The diversity between clades 4 and 5, which include M. tuberculata, is 11.2%, between clade 1 (the M. polymorpha-complex) and 5 the diversity is 11.9% and between clades 1 and 4 it is 13.0%. It is interesting that the diversity between Tarebia and the M. polymorpha-complex is only 5.1%. This difference is less than the difference between other clades within Melanoides. The within clade diversity is 0.0% for clade 1, 1.6% for clade 4 and 2.0% for clade 5. COI The phylogeny in Fig. 5 includes three major clades. The first clade includes all the specimens from the M. polymorpha-complex. The second clade includes a specimen sequenced in this paper from the southern part of Lake Malawi (MAC 1) and specimens from Thailand (BAN), Singapore (LSS) and Kambiri Point, Lake Malawi (LMI1). LMI1 belongs to the morphs that Genner et al. (2004) suggested were invasive morphs, therefore MAC 1 probably also belongs to these invasive morphs. The third clade includes two sequences from this paper; a specimen from Itungi, Tanzania (TAI) and one from Lake Victoria, Kenya (KLV) and six specimens from Genner et al. (2004): Nkhata Bay, Lake Malawi (LMN1), Dambulla, Sri Lanka (SRI), Mkungula, Lake Malombe (LMN3), Mwanza Gulf, Lake Victoria (VIC), Upper Selatar Reservoir, Singapore (USR) and Pandan Reservoir, Singapore (PAN), respectively. Since TAI is a part of a clade with native morphs, defined by Genner et al. (2004) it will be considered as a native morph as well. In this analysis the M. polymorpha-complex is more related to the invasive morphs than to the native morphs. The molecular diversity (Appendix 2) shows that the differences regarding COI between clade 2 (native morphs) and 4 (invasive morphs) is 13.4%, which is very similar to the diversity reported by Genner et al. (2004) for COI in Melanoides (13.7%). The diversity between clade 3 (the M. polymorphacomplex) and 4 is 15.7% and it is 15.8% between clades 2 and 3. The within diversity for clade 2 is 2.8%, 0.0% for clade 3 and 2.9% for clade 4. The within diversity regarding invasive (LMI1 and MAC 1) and native morphs (LMN1, LMN3 and TAI) is slightly different from what Genner et al. (2004) found. We found 0.62% and 0.0% and they found 0.36% and 0.26%, respectively. A preliminary phylogeny (unpublished, avail-



187

Fig. 4. The most parsimonious tree based on a mitochondrial fragment (16S) using maximum parsimony. The sampling site is indicated for each morph. Cleopatra bulimoides was used as an outgroup. Bootstrap values are indicated. * indicates that the sequence was found in Genbank. Cl. = clade.

able at DBL) of a combined matrix, only including specimens from Lake Malawi, places specimens from clade 4 (Fig. 4) basal to the M. polymorphacomplex and specimens from clade 5 (Fig. 4) basal to clade 4. These preliminary results confirm both that the M. polymorpha-complex constitutes a clade and that we have invasive and native

morphs of Melanoides. To resolve the relationships between the different clades more investigations are needed. DISCUSSION Samadi et al. (1999) discovered that M. tuberculata was divided into genetic clones and Facon et al.


188


Fig. 5. The most parsimonious tree based on a mitochondrial fragment (COI) using maximum parsimony. The sampling site is indicated for each morph. Cleopatra bulimoides and Thiara amarula were used as outgroups. Bootstrap values are indicated. * indicates that the sequence was found in Genbank. Cl. = clade.

(2003) surveyed invasions in the New World of Melanoides and suggested that the genetic clones were paraphyletic. The results inferred from analyses of 16S in the present study support these views. As in Facon et al. (2003) we have a clade with representatives from Africa and the Middle East (clade 5, Fig. 4) and a clade from the Pacific (clade 6, Fig. 4) and one from Asia, Africa and America (clade 4, Fig. 4). The exception from Facon et al. (2003) is the presence of a well-supported fourth clade (clade 1, Fig. 4) consisting of the M. polymorpha-complex and Tarebia granifera. This clade, which is highly supported, could be a result of homoplasy or convergence in the 16S sequences. The phylogeny inferred from the analyses of the COI sequences (Fig. 5) indicates that the M. polymorpha-complex constitutes a clade, but unfortunately the study by Genner et al. (2004) has not included any morphs belonging to the M. polymorpha-complex, thus comparison is not possible. It is difficult to conclude anything about the invasion history of Melanoides in Lake Malawi from these trees, but Africa appears to be the origin of this genus, since M. admirabilis and clade 1 is basal to the rest of the Melanoides. This is in contrast to the findings by Facon et al. (2003) and Genner

et al. (2004). They both found specimens from Southeast Asia to be basal to the African specimens, but they did not include any morphs from the M. polymorpha-complex, thus it is likely that the genus originated from Africa. To disclose the true origin of Melanoides further investigations are needed. Our results also contrast the findings of Eldblom & Kristensen (2003), who based their investigation on morphology and discovered five morphospecies. We only discovered three major clades, each consisting of several genetic clones, using molecular methods. This illustrates that molecular methods are needed for these kinds of studies, which Stothard et al. (1997) also illustrated by separating two species of Bulinus (B. globosus and B. nasutus) on Zanzibar, which was conchologically confused. Figure 5 placed the M. polymorpha-complex as a sistergroup to clade 4, which included the invasive M. tuberculata, whereas Figure 4 placed the M. polymorpha-complex basal to clade 4, 5 and 6. Perhaps, according to Fig. 5, the M. polymorpha-complex and the invasive morphs have had a more recent common ancestor than the M. polymorha-complex and the native morphs. The incongruence of the results from the 16S and COI genes indicates that further studies are necessary to solve the phylo-



genetic and zoogeographical problems, but it can be concluded that the M. polymorpha-complex constitutes a clade not formerly recognized. There are many unresolved relationships in the terminal clades in Fig. 4 and this is because the 16S gene is not variable enough to resolve the complete evolutionary history of this genus. The preliminary results for COI (Fig. 5) also include unresolved relationships, but the sample size is too limited to comment on this. The present results indicate that the morphological characters are poor determinants of Melanoides taxa, especially within the M. polymorpha-complex, where many shapes are represented and the sequences are identical. Following the argument by Facon et al. (2003) we recognize one invasion event if a specimen from both the Old World and the New World are represented in the same clade. In the present study we find six invasion events, which correspond to the number reported by Facon et al. (2003). The paraphyly of M. tuberculata arose due to Tarebia granifera and M. amabilis being part of clades 1 and 4 (Fig. 4), respectively. It would be reasonable to assume that M. amabilis simply is a morph of M. tuberculata. However, the surprising position of T. granifera is not easily explained but might be the result of homoplasy in the sequences. It is also possible that Tarebia granifera and the M. polymorpha-complex have gone through a convergent evolution in the 16S gene. It is very likely that M. simonsi (TAM), M. nodicincta (TAK) and M. virgulata (TAN and MAC 2) are morphs of M. tuberculata, due to both sequence and morphological similarity. Melanoides tuberculata morphs from Lake Malawi were distributed in clade 4 and clade 5 (Fig. 4), which indicate the presence of two different clonal lineages of M. tuberculata in the lake. According to Fig. 5 and Genner et al. (2004) these two clonal lineages probably correspond to invasive and native lineages, respectively. The limited specimen and gene sampling, the unresolved clades and the problems with Tarebia as a member of the ingroup keeps us from discussing the phylogenetic relationships within the different clades, likewise the complex scenario of dispersal events awaits a better geographical coverage of the African continent and the inclusion of further genes. The molecular diversity or sequence difference was very high within M. tuberculata, but was similar in 16S (11.2–13.0%) and COI (13.4–15.8%) between the three major clades (Fig. 4, clades 1, 4

189

and 5). As expected the protein coding COI gene shows the highest molecular diversity due to the limited selection on mutations in the third position base. The very high diversity is probably caused by the parthenogenetic isolation of different genetic clones. For the 16S gene it is very interesting that the sequence diversity between Tarebia and the M. polymorpha-complex only is 5.1%, which is less than half of the diversity between the Melanoides clades. A simple explanation could be similarity due to convergence in the 16S gene, but unfortunately the COI sequence for Tarebia is not available in GenBank for comparison with the 16S diversity. This study is unique since it investigated morph speciation in Lake Malawi. The studies by Berthold (1990) and Michel (2004) investigated the speciation of Lanistes in Lake Malawi and Lavigeria and other species in Lake Tanganyika, respectively, but are very different because the snails are sexually reproducing In conclusion, it appears as if Melanoides in Lake Malawi is divided into three major clades, the M. polymorpha-complex and invasive and native M. tuberculata morphs. Melanoides tuberculata might constitute a taxon comprising several more or less distinct genetic clones, which harbour a very high degree of sequence diversity. Several of the currently recognized African morphs of Melanoides might be part of this taxon, but the results of the present study indicate that at least M. admirabilis, endemic to Lake Tanganyika, is not part of the M. tuberculata taxon. Further progress in our understanding of the African Melanoides taxa is dependent on further specimen and gene sampling and a better geographical coverage of the specimens. ACKNOWLEDGEMENTS We are most grateful to the Danish Council of Development Research for funding L.v.G.S.’s and A.J.’s trips to Africa and the Danish Bilharziasis Laboratory for supplying us with working facilities, and for laboratory assistance to Benedikte Løhr Wilken and Ivan Baehr. A. Bwathondi, Tanzanian Fisheries Research Institute (TAFIRI) in Dar es Salaam and John Mwambungu (TAFIRI) in Kyela are thanked for their logistic support in Tanzania. The Villum Kann Rasmussen Foundation, which financed the ABI 310 Automated Sequencer that we used for sequencing. J. Gow, University of Aberdeen, Scotland, is thanked for the M. tuberculata specimens from Cameroon.


190


REFERENCES BERTHOLD, T. 1990. Phylogenetic relationships, adaptations and biogeographic origin of the Ampullariidae (Mollusca, Gastropoda) endemic to Lake Malawi, Africa. Verhandlungen des Naturwissenschaft lichen Vereins in Hamburg, NF 31/32: 47–84. BOURGUIGNAT, J.R. 1889. Mélanidées du lac Nyassa suivies d’un apercu comparatif sur la faune malacologique de ce lac avec celle du grand lac Tanganyika. Bulletins de la Société Malacologique de France 6: 1–66. BROWN, D. 1994. Freshwater Snails of Africa and their Medical Importance (2nd edn). Taylor & Francis, London CROWLEY, T.E., PAIN, T. & WOODWARD, F.R. 1964. A monographic review of the Mollusca of Lake Nyasa. Annales Musée Royal de l’Afrique Centrale, 8. Zoologiques 131: 1–58. DAVIS, G.M. 1994. Molecular genetics and taxonomic discrimination. The Nautilus, Suppl. 2: 3–23. ELDBLOM, C. & KRISTENSEN, T.K. 2003. A revision of the genus Melanoides (Gastropoda: Thiaridae) in Lake Malawi. African Zoology 38: 357–369. FACON, B., POINTIER, J.P., GLAUBRECHT, M., POUX, C., JARNE, P. AND DAVID, P. 2003. A molecular phylogeography approach to biological invasions of the New World by parthenogenetic thiarid snails. Molecular Ecology 12: 3027–3039. FOLMER, O., BLACK, M., HOEH, W., LUTZ, R.A. & VRIJENHOEK, R.C. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3:294–299. GENNER, M.J., MICHEL, E., ERPENBECK, D., DE VOOGD, N., WITTE, F. & POINTIER, J-P. 2004. Camouflaged inversion of Lake Malawi by an Oriental gastropod. Molecular Ecology 13: 2135–2142 JEANMOUGIN, F., THOMPSON, J.D., GOUY, M., HIGGINS, D.G. & GIBSON, T.J. 1998. Multiple sequence alignment with Clustal X. Trends in Biochemical Science 23: 403–5. JORGENSEN, A. 2003. Diversity and phylogeny of African freshwater gastropods with special emphasis upon the molecular phylogeny of ‘Ancyloplanorbidae’ and Biomphalaria and snail biodiversity within the Great East African Lakes. Ph.D. thesis, Faculty of Science, University of Copenhagen. KUMAR, S., TAMURA, K., JACOBSEN, I.B. & NEI, M. 2001. MEGA2: Molecular Evolutionary Genetics Analysis software. Bioinformatics 17: 1244–124. LYDEARD, C., HOLZNAGEL, W.E., SCHNARE, M.N. & GUTELL, R.R. 2000. Phylogenetic analysis of molluscan mitochondrial LSU rDNA sequences and secondary structures. Molecular Phylogenetics and Evolution 15: 83–102. MADSEN, H., BLOCH, P., PHIRI, H., KRISTENSEN, T.K. & FURU, P. 2001. Bulinus nyassanus is an intermediate host for Schistosoma haematobium in Lake Malawi. Annals of Tropical Medicine and Parasitology 95: 353– 360.mm MANDAHL-BARTH, G. 1972. The freshwater Mollusca of Lake Malawi. Revue de Zoologie et de Botanique Africaines 86: 257–273. MATZURA, O. & WENNBORG, A. 1996. RNAdraw: an integrated program for RNA secondary structure

calculation and analysis under 32-bit Microsoft Windows. Computer Applications in the Biosciences (CABIOS) 12: 247–249. MICHEL, E. 1994. Why snails radiate: a review of gastropod evolution in long-lived lakes, both recent and fossil. Archiv für Hydrobiologie Beiheft Ergebnisse der Limnologie 44: 285–317. MICHEL, E. 2004. Venundu, a new genus of gastropod (Cerithioidea; ‘Thiaridae’) with two species from Lake Tanganyika, East Africa, and its molecular phylogenetic relationships. Journal of Molluscan Studies 70: 1–19. MYERS, M.J., MEYER, C.P. & RESH, V.H. 2000. Neritid and thiarid gastropods from French Polynesian streams: how reproduction (sexual, parthenogenetic) and dispersal (active, passive) affect population structure. Freshwater Biology 44: 535–545. POSADA, D. & CRANDALL, K.A. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14: 817–818. REMIGIO, E.A. & BLAIR, D. 1997. Molecular systematics of the freshwater snail family Lymnaeidae (Pulmonata, Basommatophora) utilizing mitochondrial ribosomal DNA sequences. Journal of Molluscan Studies 63: 173–185. ROZAS, J. & ROZAS, R. 1999. DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15: 174–175. SAMADI, S., ARTIGUEBIELLE, E., ESTOUP, A., POINTIER, J.P., SILVAIN, J.F., HELLER, J., CARIOU, M.L. & JARNE, P. 1998. Density and variability of dinucleotide microsatellites in the parthenogenetic polyploid snail Melanoides tuberculata. Molecular Ecology 7: 1233–1236. SAMADI, S., MAVAREZ, J., POINTIER, J.P., DELAY, B. & JARNE P. 1999. Microsatellite and morphological analysis of population structure in the parthenogenetic freshwater snail Melanoides tuberculata: insights into the creation of clonal variability. Molecular Ecology 8: 1141–1153. SMITH, E.A. 1877. On the shells of Lake Nyasa, and on a few marine species from Mozambique. Proceeding of the Zoological Society of London 1877: 712–722. STADEN, R. 1996. The Staden Sequence Analysis Package. Molecular Biotechnology 5: 233–241. STOTHARD, J.R., MGENI, A.F., ALAWI, K.S., SAVIOLI, L. & ROLLINSON, D. (1997). Observations on shell morphology, enzymes and random amplified polymorphic DNA (RAPD) in Bulinus africanus group snails (Gastropoda; Planorbidae) in Zanzibar. Journal of Molluscan Studies 63: 489–503. STOTHARD, J.R., BRÉMOND, P., ANDRIAMARO, L., SELLIN, B., SELLIN, E. & ROLLINSON, D. 2001. Bulinus species on Madagascar: molecular evolution, genetic markers and compatibility with Schistosoma haematobium. Parasitology 123: 261–275. SWOFFORD, D.L. 2001. PAUP* Phylogenetic Analysis Using Parsimony (*and Other Methods) version 4.0b8. Sinauer Associates, Sunderland, MA. TAJIMA, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123: 585–595. VAN DAMME, D. & PICKFORD, M. 2003. The late Ceno-



191

zoic Thiaridae (Mollusca, Gastropoda, Cerithioidea) of the Albertine Rift Valley (Uganda–Congo) and their bearing on the origin and evolution of the Tanganyikan thalassoid malacofauna. Hydrobiologia

498: 1–83. VON MARTENS, E. 1897. Beschahlte Weichtiere DeutschOst-Afrikas, pp. 192–202. Dietrich Reimer (Ernst Vohsen), Berlin.

Appendix 1 The molecular diversity (P-distances) between the major clades in Fig. 4. In this table clade 1 does not include Tarebia granifera.

Appendix 2 The molecular diversity (P-distances) between the major clades in Fig. 5.

Clades Cleopatra vs clade 5 Cleopatra vs clade 4 Cleopatra vs clade 6 Cleopatra vs clade 1 Cleopatra vs Tarebia Cleopatra vs M. admirabilis Tarebia vs clade 5 Tarebia vs clade 4 Tarebia vs clade 6 Tarebia vs clade 1 Tarebia vs M. admirabilis M. admirabilis vs clade 5 M. admirabilis vs clade 4 M. admirabilis vs clade 6 M. admirabilis vs clade 1 Clade 5 vs clade 6 Clade 5 vs clade 4 Clade 5 vs clade 1 Clade 4 vs clade 6 Clade 4 vs clade 1 Clade 1 vs clade 6 Variation within clade 5 Variation within clade 4 Variation within clade 1

Mean (S.D.) 0.208 (0.005) 0.213 (0.002) 0.206 0.217 0.217 0.186 0.106 (0.004) 0.109 (0.007) 0.150 0.051 0.099 0.111 (0.006) 0.127 (0.004) 0.127 0.103 0.098 (0.008) 0.112 (0.008) 0.119 (0.005) 0.130 (0.005) 0.130 (0.005) 0.150 0.020 (0.012) 0.016 (0.016) 0

Clades Cleopatra vs clade 3 Cleopatra vs clade 4 Cleopatra vs clade 2 Cleopatra vs Thiara Thiara vs clade 3 Thiara vs clade 4 Thiara vs clade 2 Clade 3 vs clade 4 Clade 4 vs clade 2 Clade 3 vs clade 2 Variation within clade 3 Variation within clade 4 Variation within clade 2 LMI1 vs MAC 1 LMN1 vs LMN3 and TAI

Mean (S.D.) 0.201 0.210 (0.004) 0.203 (0.005) 0.201 0.142 0.174 (0.003) 0.133 (0.003) 0.157 (0.003) 0.134 (0.010) 0.158 (0.007) 0 0.029 (0.028) 0.028 (0.016) 0.0062 0