simultaneous polyphenism and cryptic species in an intertidal limpet from new zealand

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Molecular Phylogenetics and Evolution 45 (2007) 470–479

Simultaneous polyphenism and cryptic species in an intertidallimpet from New Zealand

Tomoyuki Nakano a,1, Hamish G. Spencer b,*

a Department of Earth and Planetary Sciences, Nagoya University, Nagoya 464-8602, Japanb Allan Wilson Centre for Molecular Ecology and Evolution, Department of Zoology, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand

Received 11 December 2006; revised 10 July 2007; accepted 19 July 2007Available online 31 July 2007

Abstract

The small intertidal limpets known under the name Notoacmea helmsi occupy a wide variety of habitats in New Zealand and exhibit avariety of shell forms. Phylogenetic analyses of DNA sequences from two genes, mitochondrial COI and nuclear ITS1, reveal that thistaxon comprises at least five morphologically cryptic species, with at least one of these species, N. scapha, consisting of individuals withtwo obviously different shell types. One of these forms is an ecophenotypic response to living on eelgrass (Zostera) fronds. Unlike itsextinct relative, Lottia alveus, N. scapha is not restricted to this substrate, but individuals living elsewhere are larger and have a differentshell shape. Although there is significant overlap in shell form among the different cryptic species, there is some habitat differentiation,with two species predominantly found on exposed shores and three confined to mudflats. One species exhibits distinctive light-avoidingbehaviour, the first known case in which behaviour can be used to separate cryptic species in molluscs.� 2007 Elsevier Inc. All rights reserved.

Keywords: Conservation; Polytypic species; Eelgrass; Behavioural differences; Notoacmea helmsi; Notoacmea scapha; Zostera capricorni

1. Introduction

The ability to correctly identify individuals as belongingto one species or another is a basic requirement of biolog-ical research. The consequences of mistaken assignmentscan be profound, confusing our understanding of basic bio-logical processes as well as misinforming decisions inapplied fields such as conservation and invasive speciescontrol. Even in apparently well-known groups, systematicidentification error can arise from invalid taxonomy due, inturn, to two distinct phenomena—polyphenism and crypticspecies. Polyphenisms can lead to individuals of a singlebiological species being identified as members of two or

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doi:10.1016/j.ympev.2007.07.020

* Corresponding author. Fax: +64 3 479 7584.E-mail address: [email protected] (H.G. Spencer).

1 Present address: Department of Geology and Palaeontology, NationalMuseum of Nature and Science, 3-23-1 Hyakunin-cho, Shinjuku-ku,Tokyo 169-0073, Japan.

more species, whereas the real number of species can beunderestimated if several cryptic species are present.

Perhaps the best known example of polyphenism affectingconservation decisions is the case of the Dusky Seaside Spar-row, Ammodramus nigrescens. Only after significant sums ofmoney were spent on failed preservation efforts, was it real-ized that genetic differences between it and populations ofthe Seaside Sparrow, A. maritimus, from the Atlantic coastwere minimal (Avise and Nelson, 1989). Its status had beenreduced to a subspecies of A. maritimus before its finalextinction in 1987, but even this level of distinction is nowconsidered exaggerated. As Avise and Nelson (1989) put it,‘‘a faulty taxonomy has resulted in well-intentioned but mis-directed efforts in endangered species management.’’

The reverse result—confusing two or more biologicalspecies as one—can arise in the presence of unsuspectedcryptic species and can have equally important implications.The introduction of the invasive Zebra Mussel, Dreissena

polymorpha, to the Great Lakes region of North Americahas led to major economic damage, but the various control

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T. Nakano, H.G. Spencer / Molecular Phylogenetics and Evolution 45 (2007) 470–479 471

programmes were complicated by the unsuspected presenceof a second, cryptic species, D. bugensis (Spidle et al., 1994).In another case, the conservation status of various popula-tions of the New Zealand endemic Tuatara (Sphenodon),the only living genus of the reptilian order Rhynchocepha-lia, has been compromised by neglected taxonomic issues(Daugherty et al., 1990; Hay et al., 2003).

One of the more poignant examples of the consequencesof species identification error is the first known extinction ofa marine invertebrate in historical times, that of the EelgrassLimpet, Lottia alveus (Carlton et al., 1991). This mollusc,which was previously abundant on the blades of the eel-grass, Zostera marina, on the North American coastbetween Labrador and New York, disappeared unnoticedin the early 1930s when eelgrass populations were devas-tated by disease. Its extinction went unrecognized for almost60 years in large part because most taxonomists of the timeconsidered it to be an ecotype of what is now known as Lot-

tia testudinalis, which lives on nearby rocky shores (Carltonet al., 1991; Nakano and Ozawa, 2004, 2007).

Eelgrass beds have drastically declined in several partsof the world on a number of occasions in the 20th Century.Although these populations have usually recovered, thecase of Lottia alveus shows that the complete associatedeelgrass community need not return. Another lottiid lim-pet, Notoacmea scapha, endemic to New Zealand, has alsobeen described as being restricted to living on eelgrass, theAustralasian species, Z. capricorni. This narrow habitatpreference has led to N. scapha being listed as a threatenedspecies by the New Zealand Department of Conservation(Hitchmough, 2002; see also http://www.doc.govt.nz). Asin North America, New Zealand Zostera populations haveoccasionally disappeared over wide parts of the country(Armiger, 1964). And like L. alveus, N. scapha has oftenbeen considered an ecotype of a related species, a congenerfound on hard substrates, N. helmsi (Oliver, 1926; Powell,1979). Here, we examine shell and radular morphology inconjunction with mitochondrial and nuclear gene DNAsequence to elucidate the species status of N. scapha. Toour surprise, we discover that both polyphenism and cryp-tic species are present in the N. scapha–N. helmsi complex:no limpet species in New Zealand is restricted to living onZostera but five previously unrecognized species, which areall but indistinguishable morphologically, occur over awide range of habitats.

2 Materials and methods

2.1 Field observation and collection of samples

We collected N. helmsi and N. scapha from 32 localitiesbetween the Bay of Islands in the North Island and South-land in the South Island, New Zealand, including the typelocalities of N. helmsi (Greymouth) and N. scapha (Dun-edin). We paid careful attention to the habitats in whichwe sampled: specimens on exposed shores were collectedfrom smooth rock surfaces, rock pools, and the shells of

cat’s eye turban shells (Turbo smaragdus) and dentate lim-pets (Cellana denticulata), whereas, on sheltered shores,individuals were sampled from the fronds of eelgrass (Zos-

tera capricorni), the shells of living cockles (Austrovenus

stutchburyi) and mudflat topshells (Diloma subrostrata),dead bivalve shells and rocks. Specimens of another lottiid,Atalacmea fragilis, were also collected for use as an out-group. Living specimens were preserved in 70% ethanoland returned the laboratory where they were stored at4 �C. All specimens used have been deposited in theMuseum of New Zealand, Te Papa Tongarewa. Detailsof the specimens (their voucher reference numbers andhabitats) are presented in Table 1.

2.2 DNA extraction, PCR amplification and DNA

sequencing

DNA was extracted from a single specimen of each mor-phological form from each locality, and two specimensfrom each of the two type localities. If variation in shellmorphology or color was observed, further individualswere analyzed. A 2 mm3 piece of mantle or foot tissuewas dissected from each limpet and rinsed in distilled waterto remove any ethanol. DNA was extracted in a 5% Chelex100 solution (Walsh et al., 1991).

The universal invertebrate COI primers LCO1490 (50-GGTCAACAAATCATAAGAATATTGG-30) and HCO2198(50-TAAACTTCAGGGTGACCAAAAAATCA-30) (Folmeret al., 1994) were used to amplify a 660 bp fragment ofCOI. In some samples, the HCO2198 primer was replacedwith the primer H7005 (50-CCGGATCCACNACRTARTANGTRTCRTG-30) (Hafner et al., 1994) to obtain a longerfragment of �1.1 kb. The cycling parameters for COI prim-ers were an initial denaturation step at 95 �C (2 min), fol-lowed by 36 cycles of 95 �C (45 s), 40–45 �C (60 s) and72 �C (90 s) and a final extension phase at 72 �C for 4 min.

A fragment of 18S, 5.8S and complete ITS1 were ampli-fied using primers, 18S (50-TAACAAGGTTTCCGTATGTGAA-30) and 5.8S (50-GCGTTCTTCATCGATGC-30)(Armbruster et al., 2000). The cycling parameters forITS1 primers were an initial denaturation step at 95 �C(2 min), followed by 36 cycles of 95 �C (45 s), 55 �C (60 s)and 72 �C (90 s) and a final extension phase at 72 �C for4 min. To confirm that amplifications were successful,2 ll aliquots of PCR amplifications were visualized by aga-rose gel electrophoresis.

The PCR products were separated from excess primersand oligonucleotides using a PCR Purification Kit (Invitro-gen). Purified DNA was quantified using agarose gel elec-trophoresis and sent to the Allan Wilson Centre GenomeSequencing Service for sequencing in an ABI3730 auto-mated sequencer. Sequencing reactions used the originalprimers, diluted to a final concentration of 0.213 lM.

All sequences determined in this study have been depos-ited in DDBJ and GenBank under Accession numbersAB284836–AB284889 (COI) and AB284890–AB284943(ITS1) are shown in Table 1.

http://www.doc.govt.nz

Table 1Specimen collection data

Lot number Species Habitat COI ITS1

M.184194-A* Notoacmea scapha (Suter, 1907) On Zostera AB284836 AB284890M.184194-B* On Zostera AB284837 AB284891M.184195 On living Austrovenus stutchburyi AB284838 AB284892M.184196 On dead bivalve shells AB284839 AB284893M.184197 On topshell Diloma subrostrata AB284840 AB284894M.184198 On Zostera AB284841 AB284895M.184199 On Zostera AB284842 AB284896M.184200 On Zostera AB284843 AB284897M.184201 On dead bivalve shells AB284844 AB284898M.184202 On Zostera AB284845 AB284899M.184203 on topshell Diloma subrostrata AB284846 AB284900M.184204 Notoacmea sp. A On dead bivalve shells AB284847 AB284901M.184205 On dead bivalve shells AB284848 AB284902M.184206-A On dead bivalve shells AB284849 AB284903M.184206-B On dead bivalve shells AB284850 AB284904M.184224-A Notoacmea sp. B On rocks AB284851 AB284905M.184224-B On rocks AB284852 AB284906M.184225 In tide pool AB284853 AB284907M.184226 In tide pool AB284854 AB284908M.184227 On limpet Cellana denticulata AB284855 AB284909M.184228 In tide pool AB284856 AB284910M.184229 In tide pool AB284857 AB284911M.184230 In tide pool AB284858 AB284912M.184231 In tide pool AB284859 AB284913M.184232 In tide pool AB284860 AB284914M.184233 In tide pool AB284861 AB284915M.184234 On rocks AB284862 AB284916M.184235 In tide pool AB284863 AB284917M.184236 Under rocks AB284864 AB284918M.184237 In tide pool AB284865 AB284919M.184238 In tide pool AB284866 AB284920M.184239 On smooth rocks AB284867 AB284921M.184240 On turban shell Lunella smaragdus AB284868 AB284922M.184241 On smooth rocks AB284869 AB284923M.184242 On turban shell Lunella smaragdus AB284870 AB284924M.184243 In tide pool AB284871 AB284925M.184244 On smooth rocks AB284872 AB284926M.184207 Notoacmea sp. C On dead bivalve shells AB284873 AB284927M.184208 On rocks, in mud AB284874 AB284928M.184209 On rocks, in mud AB284875 AB284929M.184210 On rocks, in mud AB284876 AB284930M.184211 On rocks, in mud AB284877 AB284931M.184212 On rocks, in mud AB284878 AB284932M.184213 On dead bivalve shells AB284879 AB284933M.184214 On rocks, in mud AB284880 AB284934M.184215 On dead bivalve shells AB284881 AB284935M.184216 On rocks, in mud AB284882 AB284936M.184217 On rocks, in mud AB284883 AB284937M.184145-A* Notoacmea helmsi (Smith, 1894) On rocks AB284884 AB284938M.184145-B* On rocks AB284885 AB284939M.184141 Atalacmea fragilis (Sowerby, 1823) Under rocks AB284886 AB284940M.184142 Under rocks AB284887 AB284941M.184143 Under rocks AB284888 AB284942M.184144 Under rocks AB284889 AB284943

The lot number is that for the voucher speciemens lodged in the Museum of New Zealand, Te Papa Tongarewa. An asterisk (*) indicates specimenscollected from the type locality. The numbers in the columns headed COI and ITS1 are the GenBank Accession numbers.

472 T. Nakano, H.G. Spencer / Molecular Phylogenetics and Evolution 45 (2007) 470–479

2.3 Phylogenetic analyses

COI sequences were manually aligned using MacClade4.03 (Maddison and Maddison, 2002), with reference tothe translated amino acid sequence. The fragments of 18S

and 5.8S were deleted since they were byproducts ofobtaining the ITS1 sequences. The ITS1 sequences werealigned using ClustalX alignment program, run using thedefault parameters (Thompson et al., 1997). Further man-ual adjustments to improve ITS1 alignments were made by


eye. The models of nucleotide substitution for the Neigh-bor-Joining (NJ), Maximum-Likelihood (ML) and Bayes-ian analyses were selected using Modeltest (Posada andCrandall, 1998), giving GTR+I+G for COI andTVM+I+G for ITS1, and these models were also used tocalculate pairwise molecular distances among individuals.The partition-homogeneity test (Swofford, 2002; the ILDtest Farris et al., 1995) was performed to test whether theCOI and ITS1 sequences contained the same phylogeneticsignal and could thus be analysed as a single dataset.

Phylogenetic analyses were performed with PAUP* ver-sion 4b10 (Swofford, 2002) for NJ (Saito and Nei, 1987),equally weighted maximum parsimony (MP) and ML, aswell as their associated bootstrap values (Felsenstein,1985, 1988). MrBayes v.3.1.2 (Huelsenbeck and Ronquist,2001; Ronquist and Huelsenbeck, 2003) was used to esti-mate Bayesian posterior probabilities.

The NJ bootstrap analysis consisted of 10,000 repli-cates, whereas the MP bootstrap analysis comprised1000 replicates of a heuristic search (with 10 randomaddition sequence replicates and TBR branch-swapping),and the likelihood bootstrap analysis was performed with100 replicates using a heuristic search. MrBayes was runwith the following settings for the two partitions (i.e.,genes); the maximum-likelihood model employed sixsubstitution types (nst = 6); rate variation across siteswas modeled using a gamma distribution, with a propor-tion of the sites being invariant (rate = invgamma); theshape, proportion of invariable sites, state frequency,and substitution rate parameters were estimated for eachpartition separately. The Markov-chain Monte–Carlosearch was run with four chains for 3,000,000 generations,with trees being sampled every 100 generations and the

Fig. 1. Bayesian phylograms, showing posterior probabilities and ML

first 5000 trees (i.e. 500,000 generations) were discardedas burnin.

2.4 Examination of radular morphology

The radula was dissected and placed in 20% KOH atroom temperature overnight, and rinsed in distilled waterbefore examination by scanning electron microscopy.

3. Results

3.1 Molecular data

PCR amplification of COI gave a product of approxi-mately 660 bp, and subsequent sequencing of this productroutinely yielded approximately 621 bp of readablesequence. The ITS1 product was usually 550–600 bp long,and sequencing routinely gave a 530–580 bp read, which,after alignment, gave a total of 636 bp. The partition-homogeneity test confirmed that there was no significantdifference in the phylogenetic signal between the COI andITS1 gene sequences (1000 replicates, P = 0.11), and thusthe two genes were subsequently concatenated and ana-lyzed as a single dataset. The combined aligned dataset of1257 bp characters (621 bp for COI and 636 bp for ITS1),including the outgroup taxon (Atalacmea fragilis), had523 variable and 492 parsimony-informative characters.

3.2 Molecular phylogeny

All the phylogenetic trees, whether constructed usingNJ, equally weighted MP, ML or Bayesian analyses, gavefive well-supported clades (Figs. 1 and 2): bootstrap sup-

bootstrap values, generated from COI and ITS1 data, separately.

Fig. 2. Bayesian phylogram generated from the 1257 bp combined COI and ITS1 data. The dataset was partitioned to allow model parameters to beestimated for each gene separately. Numbers above the branches are Bayesian posterior probabilities/ML bootstrap values calculated from 100 replicatesusing a heuristic search.


port for these clades ranged from 99 to 100% for the NJ,MP and ML analyses, and the Bayesian analysis gave pos-terior probabilities of between 0.97 and 1.00. We desig-nated these clades N. helmsi, N. scapha, N. sp. A, N. sp.B and N. sp. C. The topologies of the ML and Bayesiantrees estimated from the COI and ITS1 data were identical(Fig. 1), as were those for NJ and MP (not shown). Thetrees (ML and Bayesian versus NJ and MP) differed onlyin the placement of N. sp. B, which the latter two methodsgrouped as sister to N. helmsi, albeit with no bootstrap sup-port. Consequently, we analyzed our sequence as a singledataset, as described above.

Genetic distances between the five clades ranged from5.68 to 36.5% (for COI), 0.51 to 34.4% (for ITS1), whereasdistances within species from 0.00 to 3.47% (for COI), 0.00to 3.08% (for ITS1) (Table 2). Only N. sp. C had significant

within-species variation (0.16–3.47%: COI, 0.00–3.08%:ITS1). Indeed, this degree of ITS1 differentiation is greaterthan the distances between N. scapha and N. sp. A, and it ispossible that significant geographical structure exists withinthis species.

3.3 Ecology

Notoacmea scapha lives on various substrates, from theleaves of Zostera, to living Austrovenus stutchburyi andDiloma subrostrata individuals and the dead shells of sev-eral bivalve species (Austrovenus stutchburyi, Macoma lili-

ana and Cyclomactra ovata). It is apparently restricted tomudflats. N. sp. B is also found on a variety of hard sur-faces, from other limpets (Cellana denticulata) and cat’seye turban shells (Turbo smaragdus), to smooth rock sur-

Table 2Genetic distances: intraspecific (in bold on diagonals) and interspecific pairwise comparisons

1 2 3 4 5 6

COI

1. N. scapha 0.00–0.09

2. N. sp. A 5.68–6.21 0.00–0.48

3. N. sp. B 27.3–29.7 27.3–27.6 0.00–0.16

4. N. sp. C 27.3–28.2 27.2–28.1 23.8–24.7 0.16–3.47

5. N. helmsi 35.8–36.0 36.2–36.5 36.0–36.5 34.8–35.2 0.16

6. A. fragilis 44.6–44.9 47.4–48.0 41.7–43.5 42.3–43.2 38.6–39.1 0.00–0.16

ITS1

1. N. scapha 0.00–0.34

2. N. sp. A 0.51–0.68 0.00–0.51

3. N. sp. B 8.21–9.62 7.99–8.62 0.00–2.41

4. N. sp. C 9.48–11.4 9.22–10.3 5.41–6.65 0.00–3.08

5. N. helmsi 32.6–34.4 31.8–32.4 27.7–29.8 27.6–30.0 0.68

6. A. fragilis 27.7–30.4 27.5–29.8 24.2–26.2 23.6–25.8 29.4–30.9 0.00–0.89

Figures are the sequence difference calculated using the models selected by Modeltest: GTR+I+G for COI and TVM+I+G for ITS1.


faces in tide pools. Compared to N. scapha, however, thisspecies prefers more exposed shores. N. scapha and N. sp.B are widely distributed in New Zealand, but they do notoccur in the same localities (Fig. 3; the two apparent sitesof overlap in this figure are due to the maps’ coarse scale:the localities for these two taxa are geographically closebut quite different in degree of wave exposure, e.g., RaglanHarbour and Whale Bay, Raglan). N. sp. C is commonlyfound on rocks in sheltered inlets, but like N. scapha andN. sp. A can also attach to dead bivalve shells, usuallythe inside of valves and away from the light. When suchbivalve shells were turned over, N. sp. A moved quicklyto avoid direct light, a behaviour not seen in any of theother species. (It is possible this movement was a responseto the limpets being turned upside down, rather than beingphototaxic. Nevertheless, the bivalves were held horizon-tally, so the limpets were not detecting any vertical dis-placement.) N. helmsi lives only on smooth rock surfaceson highly exposed shores.

3.4 Morphology

Examination of the radular and conchological morphol-ogy of the five taxa discriminated by molecular analyses

Fig. 3. The geographical distributio

revealed both inter- and intra-specific differences. N. sca-

pha, in particular, is remarkably variable in shell morphol-ogy (Fig. 4). Although the individuals of this species thatare attached to Zostera invariably have long, narrow andlaterally depressed shell (Fig. 4A), those found on livingcockles or dead shells are larger and have rounder patelli-form shells (Fig. 4B). N. sp. A and N. sp. C have very sim-ilar, circular shells (Fig. 4C, G), but N. sp. A is smaller andtends to have lighter coloured shells. N. sp. B collectedfrom rocks, cat’s eye shells and limpets is most often small,with a narrow shell outline (Fig. 4D), but individuals foundin tide pools have two other variations, namely black orwhite coloured depressed patelliform shells (Fig. 4E andF), which look like two other New Zealand congeners, N.

badia and N. parviconoidea, respectively, although theyare genetically distant from these species (data not shown).N. helmsi has a broadly ovate, elevated shell, ornamentedwith greenish-brown radial lines (Fig. 4H).

The radulae of these limpets are, as expected, docoglos-sate, consisting of three pairs of lateral teeth and lackingcentral and marginal teeth (Fig. 5). Although N. scapha

individuals collected from Zostera have a straight cuttingedges on their radular teeth (Fig. 5A), those found livingon cockle shells have rounded teeth (Fig. 5B), very like

n of the five Notoacmea species.

Fig. 4. The shell morphology of the limpets used in the present study. (A) N. scapha living on the stems of Zostera. (B) N. scapha living on the cockleshells. (C) N. sp. A. (D) N. sp. B living on rock surfaces. (E,F) N. sp. B living in the tide pool. (G) N. sp. C. (H) N. helmsi. Bars under each specimenindicate 1 cm.


those of N. helmsi (Fig. 5F). The radula of N. sp. A is sim-ilar to that of N. scapha from cockle shells and N. helmsi,but the first lateral teeth are trapezoid with rounded outeredges and the second lateral teeth are slightly longer(Fig. 5C) compared to the wider and more rounded teethof the other two species. The radulae of N. sp. B and N.sp. C are similar to each other, but easily distinguishablefrom those of the other species (Fig. 5D and E): their firstand second lateral teeth are very long and pointed, and thethird lateral tooth is reduced and rounded.

4. Discussion

Our molecular analyses unequivocally show that at leastfive biological species are concealed under the nameNotoacmea helmsi, used by recent molluscan taxonomists(e.g., Powell, 1979; Spencer et al., 2006) for small, concho-logically variable, intertidal limpets from New Zealand.Intriguingly, the most morphologically distinct form

(Fig. 4A), with a small narrow, straight-sided shell thatlives only on the leaves of the eelgrass, Zostera capricorni,

is part of a polyphenic species, N. scapha. Limpets foundon nearby hard substrates on mudflats are, by genetic cri-teria, also N. scapha, although their shells are larger anda more typically limpet ovate shape (Fig. 4B), very likethose of two further genetically distinct species from similarhabitats, N. sp. A and N. sp. C (Fig. 4C and G). Thus, thisgroup of limpets harbours both cryptic and polyphenicspecies.

Our resolution of the status of N. scapha clarifies a long-standing debate on the subject and means that this namecan be removed from the New Zealand Department ofConservation’s list of threatened species. The holotype ofN. scapha is of the straight-sided form that lives on Zostera

(Suter, 1907), and, in all the literature to date, this namehas been restricted to individuals with this distinctive shellshape. Oliver (1926), however, considered that N. scapha

differed from N. helmsi only in shape and, given his concept

Fig. 5. The radular morphology of the limpets. Scale bar = 50 lm (A), 100 lm (B–F). (A) N. scapha living on the stems of Zostera. (B) N. scapha living onthe cockle shells. (C) N. sp. A. (D) N. sp. B. (E) N. sp. C. (F) N. helmsi.


of this latter species, which included N. scapha withrounded shells (as well as N. sp. A and N. sp. C), ourresults show he was at least partially correct. Powell(1979) noted that there was little evidence either way aboutthe distinctiveness of the two, but essentially followed Oli-ver (1926) in considering the difference to be ecologicallydriven. Previously, however, Powell (1961) had given sca-

pha subspecific rank, a treatment also used by Mortonand Miller (1973).

It would be particularly interesting to investigate theplasticity exhibited by N. scapha to see if the ability torespond to substrate is retained throughout life, or if thephenotype is determined once and for all soon after settle-ment. In the intertidal snail, Echinolittorina australis, forinstance, Yeap et al. (2001) used transplantation experi-ments to show that individuals could switch between twomorphologies long after settlement. Similar experimentaltranslocations caused changes in substrate-induced ecophe-notypes of the northeastern Pacific limpets Lottia asmi andL. digitalis (Lindberg and Pearse, 1990). L. pelta also exhib-its distinctive ecophenotypes depending on the substrate towhich individuals are attached (Sorensen and Lindberg,1991). These differences are extreme enough that the vari-ous forms were often considered by 19th Century workersto be separate species or subspecies. Nevertheless, individ-ual limpets can move from one substrate to another, andtheir subsequent dimorphic shell form reflects this habitatchange, thereby raising their predation risk (Sorensen andLindberg, 1991).

The two forms of N. scapha are particularly interestingin that their radula teeth are shaped differently (Fig. 5Aand B). Molluscan workers have traditionally emphasizedthe importance of radular characters in the taxonomyand phylogenetic reconstruction of patellogastropods (seelist in Lindberg, 1998; Nakano and Ozawa, 2005, 2007).

Nevertheless, intraspecific radula variation is known insome lottiids, for example, Notoacmea fascicularis (Simisonand Lindberg, 1999), and Patelloida pygmaea and P. ryuky-

uensis (Nakano and Ozawa, 2005), and other gastropodgroups (Padilla, 1998; Reid and Mak, 1999). Moreover,Andrade and Solferini (2006) recently demonstrated thatthe shape of the radula of the Brazilian littorinid snail, Litt-

oraria flava, changed within 40 days of individuals beingtransferred between rocky and mangrove habitats. Radulardifferences may have functional implications related to diet(Steneck and Watling, 1982; Reid and Mak, 1999). Wood-eating limpets have saw-like teeth (Marshall, 1985; Lind-berg, 1990), kelp feeders have teeth with a broad, straightcutting edge (Lindberg, 1979, 1981), and limpets that feedon coralline algae have blunt teeth (Lindberg, 1988; Sasakiand Okutani, 1993a,b). The rounded radula teeth of N. sca-

pha inhabiting on cockles or dead bivalves may be causedtheir hard substrata.

In the ecological literature (e.g., Morton and Miller,1973), the name N. helmsi has been applied most often tovarious of the three mudflat species, N. scapha, N. sp. Aand N. sp. C, all of which have undistinguished brownshells. Our research, ironically, shows that the name isapplicable to none of those species, but instead pertainsto populations from highly exposed rocky shores, withshells strongly marked by greenish-brown radial lines.

This study is one of the number in which molecular tech-niques have revealed cryptic species within molluscangroups (e.g., Echinolittorina, Williams and Reid, 2004;Patelloida, Kirkendale and Meyer, 2004; Nakano andOzawa, 2005; Monodonta, Donald et al., 2005; Tricolia,Williams and Ozawa, 2006; Amborhytida Spencer et al.,2006). Our findings are unique, however, in the degree ofboth habitat and geographic overlap among the geneticallydistinguishable species. For instance, we collected both N.


scapha and N. sp. C from within 1 m of each other at onelocality; specimens of N. sp. A and N. sp. C were alsofound close to one another at two further sites.

Polyphenic molluscan species are well known through-out the world, but rather few cases have been revealed bygenetic studies. One interesting example is that of the Dal-matian freshwater snail, Adriohydrobia gagatinella, whichwas previously considered to belong to three sympatric spe-cies that differed in shell-size characters (Wilke and Fal-niowski, 2001).

Perhaps the closest example to ours is that of the turbi-nid snail Astralium rhodostomum, which consists of twosympatric clades, whose only obvious morphological differ-ence is in the colour of the mantle (Meyer et al., 2005).These clades exhibit significant conchological overlap, butare specifically distinct. Within each clade a number ofreciprocally monophyletic but allopatric clades exist; these,too could be considered separate species. In Notoacmea,however, the potential number of species at any one siteis greater than the maximum of two seen in Astralium

(see above), and the phenotypic differences with N. scapha

are much more obvious than any within Astralium.Also noteworthy is the rapid light-avoiding response of

N. sp. A. Many marine invertebrates that live under rocksexhibit such behaviour (e.g., the chiton Ischnochiton maori-

anus, Powell, 1979), but so far as we are aware, our accountis the first to report such a difference as a distinguishingcharacter for morphologically cryptic molluscan species.

Acknowledgments

We thank James Irwin, Ceridwen Fraser, Andrew Jeffsand Andrea Alfaro who helped us to collect samples andBruce Marshall (Museum of New Zealand) for additionalfield consumables. Martyn Kennedy advised us on phylo-genetic analyses. The manuscript was greatly improvedby comments from Martyn Kennedy, Bruce Marshall,Graham Wallis, Jon Waters, Richard Willan and twoanonymous reviewers. This study was supported by aGrant-in-Aid for Scientific Research project no. 177770to T.N. from Japan Society for Promotion of Science, theDepartment of Zoology at the University of Otago andthe Allan Wilson Centre for Molecular Ecology andEvolution.

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simultaneous polyphenism and cryptic species in an intertidal limpet from new zealand

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