Journal of Molluscan Studies Advance Access published 11 July 2007

LACK OF GENETIC DIFFERENTIATION AMONG FOUR SYMPATRIC SOUTHEAST AFRICAN INTERTIDAL LIMPETS (SIPHONARIIDAE): PHENOTYPIC PLASTICITY IN A SINGLE SPECIES? PETER R. TESKE 1,2 , NIGEL P. BARKER 2 AND CHRISTOPHER D. M C QUAID 1 1 Department of Zoology and Entomology, Rhodes University, 6140 Grahamstown, South Africa; Molecular Ecology and Systematics Group, Botany Department, Rhodes University, 6140 Grahamstown, South Africa

2

(Received 6 October 2006; accepted 29 March 2007)

ABSTRACT Specimens of four sympatric intertidal limpet species (Siphonaria dayi, S. tenuicostulata, S. anneae and S. nigerrima ) were collected from four localities on the east coast of South Africa and southern Mozambique. Their phylogenetic relationships were investigated using sequences of the mitochondrial COI gene and the intron-containing nuclear ATPSb gene. Two closely related lineages were recovered, which grouped specimens on the basis of geography rather than morphology. One lineage was associated with the subtropical coastline of South Africa’s east coast and the other with the tropical coastline of northeastern South Africa and southern Mozambique. This genetic discontinuity coincides with a biogeographic boundary located in the vicinity of Cape St Lucia. Combined genetic diversity of the four species was lower than that of three other southern African congeners, and fell within the range determined for single southern African marine mollusc species. We suggest that the four limpet species are in fact different morphotypes of a single species.

INTRODUCTION

MATERIAL AND METHODS

Siphonariid limpets (Gastropoda: Pulmonata) occur on temperate and tropical intertidal rocky shores throughout the world (Hodgson, 1999). In southern Africa, a total of nine species have been described, but their taxonomic status remains confused (Chambers & McQuaid, 1994). Four small-bodied species with largely sympatric distribution ranges occur on the high shore of South Africa’s subtropical east coast. Morphologically, these species are mainly distinguished on the basis of shell colour and structure. Siphonaria nigerrima Smith, 1903, is small (7 – 12 mm), with a thin, fragile shell that is dark brown-black, and with fused ribs. Siphonaria anneae Tomlin, 1944 and S. tenuicostulata Smith, 1903 are larger (10 – 15 mm) and have paler shells, with 30 –40 distinct ribs in the former and 50 –60 in the latter. Siphonaria dayi Allanson, 1959, is still larger (15 – 20 mm) and pale cream-white with 40– 50 ill-defined ribs (Chambers & McQuaid, 1994). The shells of the four species also differ in their internal shell markings. Despite fairly clear differences in shell morphology, they appear to have similar microhabitat preferences and distribution ranges. Their taxonomic history is chequered; S. nigerrima was considered to be a synonym of S. carbo by Hubendick (1946), and S. tenuicostulata a synonym of S. anneae by Allanson (1959). All four species have direct development, with fully metamorphosed crawling larvae that emerge from gelatinous benthic egg masses (Chambers & McQuaid, 1994). Direct development presumably increases the possibility of local adaptation, and Chambers & McQuaid (1994) concluded, on the basis of shell morphology, that the four species were closely related, but valid species belonging to the subgenus Patellopsis. In the present study, we reexamine the taxonomic status of these species using two neutral genetic markers, one of them a mitochondrial gene, and the other a nuclear gene containing an intron.

Twenty-four ingroup specimens were analysed morphologically and genetically. Specimens were collected at four localities throughout the ranges of the species and were identified based on shell morphology as described in Chambers & McQuaid (1994). Three of the sampling sites (Umhlanga Rocks, Blythedale Beach and Sodwana Bay) are in South Africa, and the fourth (Inhaca Island) is in southern Mozambique (Fig. 1). The specimens from both Umhlanga and Inhaca were found in adjacent shallow rock pools, whereas those from both Blythedale and Sodwana were collected from single rocks that were surrounded by sand and that became completely exposed during low tide. We also obtained samples of two other southern African congeners, namely Siphonaria capensis (from Cape Agulhas), and S. concinna (from Port Elizabeth). These two species were selected as outgroup taxa because COI sequence data from 15 Siphonaria species from southern Africa, Australasia and Southeast Asia revealed that they were most closely related to the ingroup (Teske, Barker & McQuaid unpubl.). Samples were immediately preserved in 70% ethanol. Prior to DNA extraction, small tissue samples were removed from the foot of each specimen, air dried and then soaked in distilled water for at least 3 h. Genomic DNA was isolated using the Chelex extraction protocol (Walsh, Metzger & Higuchi, 1991). We sequenced a portion of the mitochondrial COI gene using universal primers (Folmer et al., 1994) and an intron-containing region of the nuclear ATPSb gene. For the latter, we designed a genus-specific forward primer (SiphonariaATPSbf: 50 -TGR ATT CCC TGA TGT TTT TGT GAG-30 ), which was used in conjunction with a universal reverse primer (ATPSbr1: 50 -CGG GCA CGG GCR CCD GGN GGT TCG T-30 ; Jarman, Ward & Elliot, 2002). PCR amplification for COI followed protocols in Zardi et al. (2007). PCR reactions of ATPSb included 2.5 ml 10  NH4 standard reaction buffer (Bioline), 3 mM of MgCl2, 0.16 mM of each dNTP (Bioline), 3 pmol/ml of each primer, 0.2 – 0.5 ml of 10 mg/ml Bovine Serum Albumin, 0.1 ml of BIOTAQTM DNA Polymerase (5 units/ml, Bioline) and 1 ml of DNA extracts

Correspondence: P.R. Teske; e-mail: [email protected]

Journal of Molluscan Studies pp. 1 of 6 # The Author 2007. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.

doi:10.1093/mollus/eym012

P.R. TESKE ET AL. obtained from 10,000 bootstrap replications. We also constructed a parsimony bootstrap tree based on 10,000 bootstrap replications using default parameters in PAUP version 4.0b10 (Swofford, 2002). To compare the amount of genetic diversity of the Siphonaria species with that of other southern African marine molluscs, haplotype diversity and nucleotide diversity (based on Tamura-Nei distances) of COI sequences were determined in ARLEQUIN ver. 3.1 (Excoffier, Laval & Schneider, 2005).

RESULTS

Figure 1. Map of the sampling area showing sampling sites in boldface.

in a total of 25 ml. The PCR profile comprised an initial denaturation step (2 min at 948C), 35 cycles of denaturation (30 s at 948C), annealing (45 s at 608C) and extension (45 s at 728C), and a final extension step (10 min at 728C). PCR products were purified with the Wizardw SV Gel and PCR Clean-Up System (Promega), cycle sequenced both in the forward and reverse direction using BigDyew Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and visualized on an ABI 3100 genetic analyser. As the ATPSb sequences of ingroup and outgroup samples were characterized by length differences, sequences were aligned in BALI-PHY (Redeling & Suchard, 2005). This programme estimates alignment and phylogeny simultaneously in a Bayesian framework. We specified the most complex model presently implemented in the programme (the Tamura-Nei model; Tamura & Nei, 1993), including a gamma distribution parameter and an assumed proportion of invariable sites. One thousand iterations were performed, and the final alignment was a consensus based on all alignments recovered excluding a burn-in of 100 iterations. The procedure was repeated five times to ensure consistency of results. Phylogenies of combined sequence data were then reconstructed using Minimum Evolution and Neighbour-Joining (Saitou & Nei, 1987) using default settings in MEGA version 3.1 (Kumar, Tamura & Nei, 2004). The Tamura-Nei model was specified, missing sites were deleted in a pairwise fashion and nodal support was

Specimens with pale shells (Siphonaria dayi, S. anneae or S. tenuicostulata ) were found at all four localities, whereas S. nigerrima was only found at Blythedale Beach and Sodwana Bay (Fig. 2). Although the shells of most specimens could readily be assigned to one of the four species, there were considerable morphological differences between localities. For example, specimens of S. dayi from Inhaca Island were small and oval, with finely ribbed white ventral ridges, whereas those from Sodwana Bay were larger, had thicker shells, more irregular outlines and broader white ventral ridges with coarser ribs. Specimens of S. anneae from Umhlanga Rocks were flatter and lighter both dorsally and ventrally than those from Blythedale Beach and Sodwana Bay. One specimen from Sodwana Bay (SO6) could not be assigned to any species with confidence, while another (SO5), although tentatively identified as S. anneae, was distinctly darker than other such specimens. Partial COI and ATPSb sequences were 570 and 289 nucleotides in length, respectively. Six unique haplotypes were recovered for COI and four for ATPSb. These have been deposited in GenBank (Accession no. EF418589 – EF418602). The mean and maximum number of base differences among ingroup specimens were 3.26 and 7 (0.6% and 1.2%) for COI, and 1.46 and 4 (0.4% and 1%) for ATPSb. All variable sites in the COI sequences were in third codon positions, and no heterozygotes were identified for ATPSb. Phylogenetic reconstruction using combined COI and ATPSb data recovered two major lineages (Fig. 3). A northern lineage included specimens from Inhaca Island and Sodwana Bay, and a southern lineage comprised specimens from Blythedale Beach and Umhlanga Rocks. The COI sequences of all specimens from the southern group were identical, whereas two ATPSb haplotypes were recovered from this region. Samples collected from the two sites north of Cape St Lucia (Fig. 1) were characterized by two distinct but closely related clusters, each confined to a single sampling locality (Sodwana Bay or Inhaca Island). Samples from Inhaca Island were all identical for both COI and ATPSb, whereas those from Sodwana Bay had four COI haplotypes and one ATPSb haplotype. None of the four species studied were recovered as monophyletic lineages. Combined genetic diversity indices of COI sequences of the four Siphonaria species investigated in this study not only fall within the range for single marine mollusc species, but are even lower than those of three other southern African Siphonaria species (S. capensis, S. concinna and S. serrata; Table 1).

DISCUSSION Discrepancies between shell morphology, protein expression and genetics The question of how useful shell morphology is in resolving phylogenetic relationships among closely related marine gastropods is a matter of debate. Vermeij & Carlson (2000) suggested that while anatomical soft tissue characters are too conserved, shell morphology tends to be sufficiently variable to resolve such relationships. However, like the limpets investigated in

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LACK OF GENETIC DIFFERENTIATION AMONG FOUR LIMPETS

Figure 2. Dorsal and ventral view of 24 limpets from four localities in south-eastern Africa. Tentative species identifications are indicated by means of five symbols. Specimen codes correspond to those used in Figure 3 and represent the following populations: IN ¼ Inhaca Island; SO ¼ Sodwana Bay; BD ¼ Blythedale Beach; UM ¼ Umhlanga Rocks.

the present study, many marine gastropods have simple shells that lack readily identifiable morphological features, and the problem is exacerbated by plasticity in shell morphology (Collin, 2003). While the inclusion of shell morphology characters into data-sets comprising anatomical and/or genetic data may result in better-resolved phylogenetic trees (Schander & Sundberg, 2001; Collin, 2003), the use of shell morphology characters on their own can be highly misleading. Intraspecific phenotypic plasticity of morphological characters is common in many marine molluscs and is generally related to environmental conditions. Examples include the development of larger apertural teeth or increased shell thickness in the presence of shell crushing predators (Appleton & Palmer, 1988; Trussell & Smith, 2000), a larger body size at sites with high wave exposure as compared to sheltered sites (Savini et al., 2004), increase in shell size at lower temperatures (Atkinson, 1994; Trussell, 2000), as well as lighter pigmentation and greater shell height in individuals living higher in the intertidal zone (Branch, 1975; Etter, 1988; Rugh, 1997; Sokolova & Berger, 2000). Because of the sensitivity of molluscan shell morphology to environmental conditions, the number of genetic studies that have synonymized species is likely to be higher than for most other marine invertebrate phyla (Knowlton, 2000). Our analyses based on sequence data did not recover the four Siphonaria species as distinct genetic clusters. This contrasts with

the results of a study using polyacrylamide gel electrophoresis of total soluble proteins (PAGE; Chambers et al., 1996). This previous study identified each species as being monophyletic, except that a single specimen of the planktonic disperser S. capensis was in a derived position within the S. tenuicostulata cluster. Moreover, most of the specimens of the planktonic disperser S. concinna were recovered as the sister taxon of the direct developer S. tenuicostulata. In the present study, both planktonic dispersers were identified as being distinct from the ingroup specimens by both molecular markers, and for that reason were used as outgroup taxa. The discrepancy between the two studies may be explained by the fact that the same genotype may express different phenotypes depending on its biotic or abiotic environment (Agrawal, 2001), and this manifests itself in the synthesis of different proteins as well as the expression of different morphological characters. Species that live in the intertidal zone are strongly affected by extremes in temperature and desiccation stress and exhibit physiological and behavioural adaptations that allow them to deal with the stresses caused by prolonged emersion ( Johannesson, 1989). The distribution of colour morphs is difficult to explain, but this should perhaps not be unexpected. Shell colour can be particularly variable as pigmentation is controlled by different factors in different taxa. For example, the shell pigments of archaeogastropods are thought to be formed as digestive residues

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P.R. TESKE ET AL.

Figure 3. A minimum evolution consensus tree constructed from Tamura-Nei distances of combined COI and ATPSb sequences of 24 specimens of Siphonaria anneae, S. dayi, S. tenuicostulata and S. nigerrima collected at four sampling localities in northeastern South Africa and southern Mozambique, as well as two outgroup species. Bootstrap values from 10,000 pseudoreplications that were greater than 75% are shown for three methods of phylogenetic reconstruction: Minimum Evolution, Neighbour-Joining and Parsimony. Tentative species identifications are indicated by means of symbols, and specimen codes correspond to those used in Figure 2.

Table 1. Genetic diversity indices (+SD) of the COI sequences of various southern African marine molluscs. The four Siphonaria species investigated in this study were treated as a single species (first row). Species

Siphonaria (ingroup

n

24

No.

Haplotype

Nucleotide

haplotypes

diversity

diversity

6

0.74 + 0.06

0.006 + 0.003

spp. combined) S. capensis †

12

8

0.92 + 0.06

0.007 + 0.004

S. concinna †

17

16

0.99 + 0.02

0.025 + 0.013

S. serrata †

16

11

0.93 + 0.05

0.031 + 0.017

Nassarius kraussianus †

80

17

0.72 + 0.04

0.003 + 0.002

Perna perna

82

41

0.87 + 0.02

0.007 + 0.004

58

10

0.49 + 0.08

0.002 + 0.001

140

51

0.87 + 0.02

0.020 + 0.009

13

5

0.54 + 0.16

0.003 + 0.002

(western lineage)‡ P. perna (eastern lineage)‡ P. perna (combined)‡ Octopus vulgaris § †

Teske, Barker & McQuaid unpubl. Zardi et al., 2007. Teske et al., in press..

‡ §

(Cole, 1975), so that colour and patterning can be determined by the nature of the primary food source (Underwood & Creese, 1976). In contrast, shell colour in the meso- and neogastropods has a strong genetic basis (Reimchen, 1979; Atkinson & Warwick, 1983) and morph-dependent selection can be driven by factors such as thermal effects and predation (McQuaid, 1996; Parsonage & Hughes, 2002). In fact, the land snail Cepaea has been used as an example of the complexity of the control of shell colour in the Pulmonata by Jones et al. (1977), who concluded that at least eight evolutionary forces act on shell colour and that one factor alone rarely explains the frequencies of colour morphs fully. We found no obvious spatial separation between light and dark shells at localities where both types were found (Sodwana Bay and Blythedale Beach). Moreover, shells were found that could be classified as being intermediate between the light and dark species in terms of their shell coloration (Fig. 2, specimens SO5 and SO6). At both of these sites, specimens were more exposed to desiccation stress than at the sites where only light shells were found in rock pools (Inhaca Island and Umhlanga Rocks), suggesting that the hypothesis that shells with lighter pigmentation occur higher on the shore (where desiccation stress is greater; e.g. Branch, 1975; Etter, 1988) does not hold in this case. Although some specimens were difficult to allocate to species, we are left with the problem that individuals that were apparently very clear examples of different species occurring within the same habitat were grouped together in the genetic analysis.

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LACK OF GENETIC DIFFERENTIATION AMONG FOUR LIMPETS For example, most of the limpets from Umhlanga Rocks and Blythedale Beach were genetically indistinguishable, but included typical examples of both S. anneae and S. nigerrima. Such specimens differed strongly not only in shell colour, but also in aspects of shell shape such as apex height, presence/ absence of ribbing and robustness. Closely related molluscs can show rapid intergenerational convergence of shell form when grown under laboratory conditions (Wullschleger & Jokela, 2002), but this situation is the reverse. The problem becomes one of explaining why animals that share the same habitat on the same shore differ so dramatically in shell morphology when we cannot invoke a genotypic explanation.

Biogeographic patterns Explaining the genetic structure of these populations proves easier than explaining their morphologies. Recent phylogeographic studies of southern African coastal invertebrates identified a number of species that are subdivided into genetically distinct geographic units. In most cases, these are associated with different marine biogeographic provinces (e.g. Teske et al., 2006; Zardi et al., 2007), with the boundary between the cool-temperate west coast and the warm-temperate south coast, as well as the boundary between the south coast and the subtropical east coast, being of particular importance. Bolton et al. (2004) identified a discontinuity in the composition of benthic shallow-water macroalgae in the vicinity of St Lucia (Fig. 1), a region that may be considered the south-western boundary of the tropical Indian Ocean flora. The genetic discontinuity between the two main lineages of the Siphonaria species studied coincides with this boundary, and this result represents the first indication that the St Lucia biogeographic boundary could be important at what appears to be an intraspecific level. However, as the differentiation between lineages was small and there were large sampling gaps between populations, additional samples from the region between Blythedale Beach and Sodwana Bay are required to assess the importance of the St Lucia biogeographic boundary to the phylogeography of the Siphonaria species.

Conclusion In this study on four southeast African intertidal limpets, a northern and a southern genetic lineage of what is apparently a single species were recovered. Each lineage contains at least two morphs that are almost identical genetically, but different in terms of shell morphology. Apparent conflict between genetic and morphological data is itself is not problematic, but the fact that morphs are shared between the lineages is difficult to interpret. One possible explanation is that these morphs arose prior to genetic divergence of the two genetic lineages. This apparent morphological stasis and the fact that different morphs were found adjacent to each other in the same habitat suggests that there is no directional selection on shell morphology. However, this seems highly unlikely in terms of the stresses of desiccation and temperature that are characteristic of the species’ habitat.

ACKNOWLEDGEMENTS This is a contribution from the African Coelacanth Ecosystem Programme. We are grateful to Nerosha Govender (Greater St Lucia Wetland Park Authority) for the personal and efficient way in which our permit application was handled. Research funding for this study was provided to NPB by the National Research Foundation (GUN 2069119).

REFERENCES AGRAWAL, A.A. 2001. Phenotypic plasticity in the interaction and evolution of species. Science, 12: 321–326. ALLANSON, B.R. 1959. On the systematics and distribution of the molluscan genus Siphonaria in South Africa. Hydrobiologia, 12:149–180. APPLETON, R.D. & PALMER, A.R. 1988. Water-borne stimuli released by predatory crabs and damaged prey induce more predator-resistant shells in a marine gastropod. Proceedings of the National Academy of Sciences of the USA, 85: 4387– 4391. ATKINSON, D. 1994. Temperature and organism size – a biological law for ectotherms? Advances in Ecological Research, 25: 1–58. ATKINSON, W.D. & WARWICK, T. 1983. The role of selection in the colour polymorphism of Littorina rudis Maton and Littorina arcana Hannaford-Ellis (Prosobranchia: Littorinidae). Biological Journal of the Linnean Society, 20: 137–151. BOLTON, J.J., LELIAERT, F., DE CLERCK, O., ANDERSON, R.J., STEGENGA, H., ENGLEDOW, H.E. & COPPEJANS, E. 2004. Where is the western limit of the tropical Indian Ocean seaweed flora? An analysis of intertidal seaweed biogeography on the east coast of South Africa. Marine Biology, 144: 51– 59. BRANCH, G.M. 1975. Ecology of Patella species from the Cape Peninsula, South Africa. IV. Desiccation. Marine Biology, 32: 179–b88. CHAMBERS, R.J. & MC QUAID, C.D. 1994. Notes on the taxonomy, spawn and larval development of South African species of the intertidal pulmonate limpet genus Siphonaria (Gastropoda: Pulmonata). Journal of Molluscan Studies, 60: 263– 275. CHAMBERS, R.J., MC Quaid, C.D. & Kirby, R. 1996. Determination of genetic diversity of South African intertidal limpets (Gastropoda: Siphonaria ) with different reproductive modes using polyacrylamide gel electrophoresis of total cellular proteins. Journal of Experimental Marine Biology and Ecology, 201: 1–11. COLE, T.J. 1975. Inheritance of juvenile shell colour of the oyster drill Urosalpinx cinerea. Nature, 257: 794– 795. COLLIN, R. 2003. The utility of morphological characters in gastropod phylogenteics: an example from the Calyptraeidae. Biological Journal of the Linnean Society, 78: 541–593. ETTER, R.J. 1988. Physiological stress and colour polymorphism in the intertidal snail Nucella lapillus. Evolution, 42: 660–680. EXCOFFIER, L., LAVAL, G. & SCHNEIDER, S. 2005. Arlequin ver. 3.0: an integrated software package for population genetics data analysis. Evolutionary Bioinformatics Online, 1: 47–50. FOLMER, O., BLACK, M., HOEH, W., LUTZ, R. & VRIJENHOEK, R. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3: 294–299. HODGSON, A.N. 1999. The biology of the Siphonariid limpets (Gastropoda: Pulmonata). Oceanography and Marine Biology: An Annual Review, 37: 245– 314. HUBENDICK, B. 1946. Systematic monograph of the Patelliformia. Kungliga Svenska Vetenskapsakademiens Handlingar, 23: 1– 93. JARMAN, S.N., WARD, R.D. & ELLIOTT, N.G. 2002. Oligonucleotide primers for PCR amplification of coelomate introns. Marine Biotechnology, 4: 347– 355. JOHANNESSON, K. 1989. The bare zone of Swedish rocky shores: why is it there? Oikos, 54: 77–86. JONES, J.S., LEITH, B.H. & RAWLINGS, P. 1977. Polymorphism in Cepaea: a problem with too many solutions. Annual Review of Ecology and Systematics, 8: 109–143. KNOWLTON, N. 2000. Molecular genetic analyses of species boundaries in the sea. Hydrobiologia, 420:73–90. KUMAR, S., TAMURA, K. & NEI, M. 2004. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Bioinformatics, 5: 150–163. MC QUAID, C.D. 1996. Biology of the gastropod family Littorinidae. I. Evolutionary aspects. Oceanography and Marine Biology: An Annual Review, 34: 233–262.

5 of 6

P.R. TESKE ET AL. PARSONAGE, S. & HUGHES, J. 2002. Natural selection and the distribution of shell colour morphs in three species of Littoraria (Gastropoda: Littorinidae) in Moreton Bay, Queensland. Biological Journal of the Linnean Society, 75: 219–232. REDELING, B.D. & SUCHARD, M.A. 2005. Joint Bayesian estimation of alignment and phylogeny. Systematic Biology, 54: 401–418. REIMCHEN, T.E. 1979. Substratum heterogeneity, crypsis and colour polymorphism in an intertidal snail (Littorina mariae ). Canadian Journal of Zoology, 57: 1070–1085. RUGH, N.S. 1997. Differences in shell morphology between the sibling species Littorina scutulata and Littorina plena (Gastropoda: Prosobranchia). Veliger, 40: 350–357. SAITOU, N. & NEI, M. 1987. The neighbour-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4: 406 –425. SAVINI, D., CASTELLAZZI, M., FAVRUZZO, M. & OCCHIPINTI-AMBROGI, A. 2004. The alien mollusc Rapana venosa (Valenciennes, 1846; Gastropoda, Muricidae) in the Northern Adriatic Sea: Population structure and shell morphology. Chemistry and Ecology, 20: 411–424. SCHANDER, C. & SUNDBERG, P. 2001. Useful characters in gastropod phylogeny: soft information or hard facts? SystematicBiology, 50: 136–141. SOKOLOVA, I.M. & BERGER, V.J. 2000. Physiological variation related to shell colour polymorphism in White Sea Littorina saxatilis. Journal of Experimental Marine Biology and Ecology, 245: 1–23. SWOFFORD, D.L. 2002. PAUP  – phylogenetic analysis using parsimony (  and other methods), version 4.0b10. Sinauer Associates, Sunderland, MA. TAMURA, K. & NEI, M. 1993. Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular Biology and Evolution, 10: 512–526.

TESKE, P.R., MC QUAID, C.D., FRONEMAN, P.W. & BARKER, N.P. 2006. Impacts of marine biogeographic boundaries on phylogeographic patterns of three South African estuarine crustaceans. Marine Ecology Progress Series, 314: 283–293. TESKE, P.R., OOSTHUIZEN, A., PAPADOPOULOS, I. & BARKER, N.P. in press. Phylogeographic structure of Octopus vulgaris in South Africa revisited: identification of a second lineage near Durban harbour. Marine Biology. TRUSSELL, G.C. 2000. Phenotypic clines, plasticity, and morphological trade-offs in an intertidal snail. Evolution, 54: 151–166. TRUSSELL, G.C. & SMITH, L.D. 2000. Induced defenses in response to an invading crab predator: an explanation of historical and geographic phenotypic change. Proceedings of the National Academy of Sciences of the USA, 97: 2123–2127. UNDERWOOD, A.J. & CREESE, R.G. 1976. Observations on the biology of the trochid gastropod Austrocochlea constricta (Lamarck) (Prosobranchia). II. The effects of available food on shell-banding pattern. Journal of Experimental Marine Biology and Ecology, 23: 229–240. VERMEIJ, G.J. & CARLSON, S.J. 2000. The muricid gastropod subfamily Rapaninae: phylogeny and ecological history. Paleobiology, 26: 19– 46. WALSH, P.S., METZGER, D.A. & HIGUCHI, R. 1991. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques, 10: 506–513. WULLSCHLEGER, E.G. & JOKELA, J. 2002. Morphological plasticity and divergence in life-history traits between two closely related freshwater snails, Lymnaea ovata and Lymnaea peregra. Journal of Molluscan Studies, 68: 1–5. ZARDI, G.I., MC QUAID, C.D., TESKE, P.R. & BARKER, N.P. 2007. Unexpected genetic structure of mussel populations in South Africa: indigenous Perna perna and invasive Mytilus galloprovincialis. Marine Ecology Progress Series, 337: 135–144.

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