BULLETIN DE L'INSTITUT ROYAL DES SCfENCES NATURELLES DE BELGIQUE, BIOLOGIE, 71-SUPPL.: 43-65, 2001 BULLETI N VAN HET KONINKLIJK BELGISCH INSTITUUT VOOR NATUURWETENSCHAPPEN, BI OLOGIE, 71 -SUPPL.: 43-65 , 2001

Periwinkles (Gastropoda, Littorinidae) as a model for studying patterns and

'' dynamics of marine biodiversity '' by Thierry BACKELJAU, Antonio M. FRIAS MARTINS, Elizabeth GOSLING, John GRAHAME, Peter MILL, Carlos BRITO, Andrea CLARKE, Roberto MEDEIROS, Maureen SMALL, Craig WILDING, Iain WILSON, Birgitta WINNEPENNINCKX, Richard CLARKE, & Hans DE WOLF

Abstract intraspecifi qu e, il s ne représentent pas suffi samment la di versité non-moléc ul aire pertinente du point de vue d'evolution. This paper presents a summary sy nthesis of an international EC­ Mots-clés: Gastro poda, Li ttorinidae, bi odiversité marin e, généti­ fund ed research program in whi ch peri winkles are used as a mode! qu e de populati ons, taxo nomi e, systématique molécul aire. gro up to study pattern s and dynami cs in marine biodiversity. ln the first part of th e paper, a number of techniques and markers to assess bi odi versity in periwin kles are illustrated and evalu ated, while protocols fo r DNA-extrac ti on, Single-S tra nd Confo rmati on Introduction Polymorphi sms (SSCP) and PCR-ampli fica ti on of a number of nu­ clear and mitochondri al DNA markers are pro vid ed. The second Biodi versity can be defin ed as "The vari ability arn ong li ving part of the paper presents a brief overview of the appli cati on of organi srn s from ail sources, including inter a/ia terrestrial, th ese techniques in the analys is of geneti c di versity in two peri win­ m arine and other aquati c ecosystern s and the ecological com­ kl e tax a with co nt rastin g developmental modes, viz. Littorina plexes of whi ch they are part; this includes di versity within striata and th e L. saxatilis complex . Fin all y, th ese results are di s­ species, between species and of ecosysterns" (CONVENTION cussed in the li ght of the implementati on of th e Evo lutionaril y Si g­ OF RI O, 1992), or as MEFFE & CAROLL ( 1994) put it "The nifi ca nt Unit (ES U) and Ma nagement Uni t (MU ) co nce pts in (ma­ vari ety of li vin g organi sms considered at ail levels, frorn ge­ rine) conservati on biology. It is concluded th at, although th ese op­ neti cs through species, to hi gher taxonomie levels, and in­ erational concepts are more appropriate than species level systemat­ ics fo r delineating relevant in tras pecific di versit y, th ey still may not cl uding the vari ety of habitats and ecosystems". M any more adequ ately rep resent non-molec ul ar evolu tionaril y relevant, diver­ de finiti ons of bi odi versity exist, but ail entai! the sarn e basic sity. tenet, viz. that biodi versity is the property of li vin g systems to be distinct and vari abl e (SOLBRI G, 1994). It is precisely Key-words: Gastro poda , Littorini dae , marin e bi odi versity, popul a­ this property th at all ows li vin g systems to evolve and adapt ti on genetics, taxonomy, molec ul ar systematics. in an ever changin g environment, and bi odiversity therefore represents no Jess than future evolutionary potential. In addi­ ti on, bi odi versity offers a wide vari ety of economic, social Résumé and cultural benefits, the value of which is increasingly rec­ Cet article présente une brève synthèse d'un projet in te rn ati onal ogni zed and documented in the literature (e.g. ÜLDF IELD, fin ancié par la CE, dans lequel les littorines so nt choisies comme 1984; BARBIER & A YLWA RD, 1996; LOOM IS & WHITE, 1996; groupe modèle pour !'.étu de de la stru cture et la dy nami que de la SAGOFF, 1996; BENGTSSON et al., 1997; COSTANZA et al., biod iversité marin e. Dans la premi ère parti e de l' article, on présente 1997; EDWA RDS & AB IVARDI , 1998; TEN KATE & LAIR D, une illustrati on et une évalu ati on d'un nombre de techni ques et de 1999). marqueurs molécul aires utili sés dans l'étude de la biodi versité des litto rines. De plus on propose des pro tocoles po ur l' extractio n Hence, the curre nt worldwide concern s with respect to global d' ADN, pour d'analyses cle "Sin gle-Strand Co nformati on change and the concomitant Joss of biodiversity are m ore Polymorphi sms (SSCP)" et pour l'ampli fi cat ion par PCR d'un than justified, since humans are responsible fo r possibly the nombre de marq ueurs d' ADN nucléaires et mit ochondriaux. La fastest and most drastic wave of extincti on in the history of deuxième parti e cl e ! 'art icle donne un bref aperçu cl e l'app li cation de li fe (WILSON, 1988). Hitherto, this problem has been taken ces techni ques cl ans l'analyse cle la diversité généti que cl ans deux seri ously in terrestrial ecosystems, where anthropogenic in­ tax ons cle li ttorin es avec des modes de déve loppement altern atifs: Littorina striata et le complexe de L. saxatilis. Finalement, les ré­ fl uences such as habitat fragmentati on, deforèstati on, species sultats cle ces analyses so nt di scutés dans le cadre cle l' app li ca ti on translocations, urbani sati on, pollution, agricultural practices des conceptes comme les "Evo lut ionaril y Signifi cant Uni ts (ESU )'" and overexploitation of natural habitats, are ha vine: devastat­ et les "' Management Un its (MU )" cl ans la biologie de co nservati on ing effects on the biosphere. Biocli versity in mari71e ecosys­ (marin e). Bien qu e ces "ES Us" el "MUs" soient meill eurs que la tems, on the contrary, is usua ll y beli cved to be much less vul ­ systémati que au ni veau d'es pèces, pour délimiter la di versité nerable to global extincti ons caused by such anthropogenic 44 THTERRY BACKELJAU et al. factors. Yet, recent compelling evidence increasingly indi­ Periwinkles (Mollusca, Gastropoda: Littorinidae: genus cates ( 1) that marine species may be at a far greater risk of Littorina) are such a group of organisms and were therefore extinction than is generâlly assumed (ROBERTS & HAWKINS , chosen as a mode! group. 1998) and (2) that cmTently there is an enormous Joss of local marine biodiversity due to local population declines and The present contribution is a synthesis of the main research, extinctions (CARLTON, 1993). However, losses of marine activities, results and conclusions of the AMBIOS project biodiversity remain largely unmeasured because of the rela­ (see also MILL et al. , 1998). In particular we will discuss (!) tive neglect of marine taxonomy and population genetics a number of different markers and techniques to assess (MCKINNEY, 1998). Moreover, marine conservation has biodiversity, (2) the patterns and dynamics of diversity in much Jess of a theoretical basis than terrestrial conservation planktonic developing (L. striata) vs. direct developing (L. (ALLISON et al., 1998). It is therefore tempting to implement saxatilis) periwinkles, (3) phylogenetic patterns in Littorina, experiences from the latter in the marine realm. However, and (4) the implementation of 'Management Units' (MUs) marine ecosystems differ fundamentally from terrestrial sys­ and 'Evolutionarily Significant Units' (ES Us) sensu MORITZ tems, both in their spatio-temporal scales and in the variabil­ (1994, 1996) as operational concepts in the conservation and ity of processes (ALLISON et al., 1998). It is therefore no sur­ management of marine biodiversity. prise that marine conservation biology lags far behind terres­ trial conservation biology (NOR SE, 1994 ). Furthermore, just as teITestrial conservation requires a sound knowledge of the Periwinkles as a mode) group patterns and processes that govern the dynamics (i .e. origin, maintenance and Joss) of biological variation, so the devel­ Littorinid snails are important grazers, and hence structuring opment of marine conservation strategies will also need such agents, in the rocky intertidal. They occur in huge numbers information. ail along the European coasts, where currently nine species are recognized: Melarhaphe neritoides (LINNAEUS, 1758), Against this background, the European Community launched Nodilittorina (Echi11olittori11a) pu11ctata (GMELIN, 1791 ), within the 'Marine Science and Technology' (MAST-III) Littorina ( Liralittorina) striata KING & BRODERIP, 1832, L. work programme an action plan to support fondamental re­ ( Littori11a) littorea (LINNAEUS , 1758), L. (Neritrema) search aiming at a better understanding of patterns and dy­ obtusata (LINNAEUS , 1758), L. (N.) faba!is (TURTON , 1825), namics in marine biodiversity. One of the supported projects L. (N.) compressa JEFFREYS, 1865, L. (N.) arcana was AMBIOS ('Integrating Environmental and Population HANNAFORD ELLIS, 1978 and L. ( N.) saxatilis (ÜLIVI, 1792). Variation: A Mode] for Biodiversity Studies'), which aimed The first four species have planktonic eggs and larvae, and (!) to evaluate a number of techniques to assess diversity at consequently show substantial gene flow between even very various taxonomie levels and (2) to provide a comprehen­ distant populations, so that genetic differentiation is consid­ sive, integrated picture of how diversity may be generated ered to be low (e.g. JOHANNESSON , 1992; BACKELJAU et al. , and patterned in organisms with contrasting life histories and I 995a). Nevertheless, as we will outline further in this paper, living in a spatio-temporally variable marine environment. this large-scale homogeneity may need to be reconsidered in The rationale behind the AMBIOS project was that under­ three of the four species. L. obtusata, L. fabalis, L. compressa standing marine biodiversity requires the consideration of at and L. arcana, in contrast, are egg-layers (REID, 1996), least five issues: (1) how do we measure diversity, (2) which showing considerable population differentiation even over part of biological variation is genetic and which is relatively short distances. The taxonomie distinction of the ecophenotypic, (3) how is diversity structured in space and fiat periwinkles L. obtusata and L. fabalis is well-established time, ( 4) how are these spatio-temporal patterns generated, (SACCHI & RASTELLI , 1966; REID, 1990), even though recent maintained and lost, and (5) which measures or operational work seems to suggest that L. fabalis might in fact comprise concepts can be implemented to conserve and manage ma­ two sibling taxa (TATARENKOV & JOHANNESSON, 1998, rine biodiversity. Obviously, points (2), (3) and (4) involve 1999). Similarly, the taxonomy of L. compressa and L. the questi on as to h o~ reproducti ve barriers are foITned and arcana is no ·longer contentious (e.g. RAFFAELLI, 1982; hence how independent genetic entities ( often 'species') WARD, 1990; REID, 1996). Both of these species belong to arise under field conditions. This in turn refers to the basic the 'rough' periwinkles, as does L. saxatilis. However, this problem of delimitin g 'species' and the concomitant problem last taxon is an extremely variable one containing live-bear­ ing (ovoviviparous) animais. It comprises a number of of what kind of evolutionarily relevant units of diversity have forms, two of which have, in the past, been accorded species to be considered for conservation and management. These L. taxonomie problems indeed fonned an important component status, i.e. neglecta BEAN, 1844 (a small 'barnacle dwell­ of the AMBIOS project. ing' form with a smooth, glossy and nearly globular shell, usually with an obtuse point and a large last whorl covered The most efficient way to achieve the AMBIOS goals was by with numerous transverse, dark, interrupted bands) (e.g. work.ing on a group of widespread, common organisms, REID , 1993) and, L. tenebrosa (MONTAGU, 1803) (a lagoonal whi ch (l) show considerabl e intra- and interspeci fic m orpho­ form). Although L. te11 ebrosa can apparently li ve in the same logical, behavioural, life-history and population genetic lagoons as L. saxatilis s.s., it differs from the latter both mor­ diversity, (2) whi ch are clearl y in the process of divergence, phologically and behaviourally since it is small er, has a frag­ and (3) which li ve in th e coastal zone as an example of a il e and mooth shell and li ves permanentl y submergecl on spatio-temporally, hi ghl y variable, marine environment. macrophytes (Muus, 1967). Nevertheless, BARNES ( 1993) Periwinkles and marine biodiversity 45 considers that it should be afforded only 'variety' status, i.e. urements and computer-aided image analysis) and thermal L. saxatilis var. lagunae. tolerance experiments. Genotypic variation was assessed by protein electrophoresis and a number of DNA techniques. Three other forms of rough periwinkles are here further re­ The former included starch gel electrophoresis (SGE), poly­ ferred to as L. saxatilis H, L. saxatilis M, and L. saxatilis B. acrylamide gel electrophoresis (PAGE), isoelectric focusing The H form lives high Ôn the shore, has a thin shell, is patu­ (IEF) and two-dimensional electrophoresis (2DE) of lous and produces large embryos. The M form , in contrast, allozymes, esterases (EST), radular myoglobins (Mb) and lives on the mid shore, has a massive, thick shell with a small general proteins (GP). The DNA techniques involved Ran- · aperture and produces smaller embryos (HULL et al. , 1996). dom Amplified Polymorphie DNA (RAPD) fingerprinting, Sometimes intermediates between these two fom1s are found restriction site polymorphisms of nuclear genes identified and such individuals produce two size classes of embryos, from RAPDs and calmodulin intron sequencing, Single­ corresponding to those of fo1ms H and M. Moreover, a high Strand Confo1mation Polymorphisms (SSCP) of mitochon­ proportion of embryos from intermediate specimens abort, drial cytochrome b (Cytb), screening microsatellite DNA suggesting that the intermediates are real hybrids and that variation, nucleotide sequencing of calmodulin intrnns, nu­ there may be a barrier to gene flow between L. saxatilis H clear rDNAs (!SS, 28S, ITS-1) and the mtDNA genes Cytb and M (HULL et al. , 1996). L. saxatilis B is a 'barnacle dwell­ and cytoclu·ome oxidase I (COI). ing' form that lives sympatrically with L. neglecta, from which it is distinguished by its sculptured, non-banded shell with a an ovoid aperture. Generally, form B resembles L. Evaluation of some markers and methods used by saxatilis s.s. but matures at a smaller size. Part of the AMBIOS AMBIOS work focused on the biological interpretation of different fonns within L. saxatilis. INTRODUCTORY REMARKS In addition to this morphological diversity, L. saxatilis shows Sorne of the techniques and markers implemented by a vast geographic di stribution and occupies a very wide AMBIOS to measure bi odiversity were well-established range of habitats from extremely wave-exposed shores to sait (e.g. morphometry and allozyme electrophoresis) and there­ marshes and isolated, brackish lagoons. Ali other European fore did not require an extra assessment of their suitability for littorinids have much more restricted habitat niches and/or the purposes of the project, although refinements were made distributional ranges. As the AMBIOS project mainly dealt to the shell measurements used in the morphometric studies with Littorina species we refer to REID ( 1996) for a compre­ and a computer-aided image analysis system had to be devel­ hensive and up to date background on the biology, taxonomy oped to allow more variables to be measured and to increase and phylogeny of these animais. resolution. In contrast, ail other analytical methods employed by AMBIOS had to be evaluated with respect to their useful­ Materials and Methods ness for the research program. In some cases techniques had to be adapted for particular applications or even had to be developed from scratch. As these technical developments The AMBIOS study concentrated on two of the pl anktonic were one of the aims of AMBIOS, we briefly review in this breeders, L. striata and L. littorea, on two oviparous species, section our methodological survey and evaluation, and dis­ L. arcana and L. compressa, and on the ovoviviparous L. cuss some of the relevant data obtained during the technical saxatilis complex. A number of specific studies involved still survey of the different methods and markers. The further in­ other species. tegration of these data (insofar as it is relevant) is provided in AMBIOS fieldwork was carried out at a number of key sites the sections on L. striata and L. saxatilis. such as Filey Brigg, Robin Hoods Bay, Peak Steel, Ravenscar, Old Peak and Holkham in England, St. Anne's TWO-DIMENSIONAL PROTEIN ELECTROPHORESIS (2DE) Head, Great Castl e Head and the Gann Estuary in Wales, St. Abbs Head in Scotland, Golam Head, Baile na hAbhann and Severa! protocols for nati ve and denaturing 2DE of radular the Aran Islands in Ireland , the Scheldt Estuary in The Neth­ Mb/GP were tested and compared. However, although they erl ands, Ri a Ferrol in Spain , the Canary Islands, Madeira, the ail provided a considerabl y hi gher resolution than one-di­ Cape Verde Islands, and the Azores (the Iast four together are mensional electrophoretic techniques (e.g. SGE, PAGE and referred to as Macaronesia) (Fig. 1) . However, whenever ap­ IEF), 2DE appeared to be a Jess effi cient method to screen propriate, materi al from other regions (in and outside Eu­ biodiversity because it entailed several major drawbacks. rope) was al so included to test specifi c hypotheses, to pro­ Firstl y, 2DE is very time consuming, taking more than fiv e vide phylogeneti c reference tax a (e.g. outgroups) or to serve hours to obtain a sin gle individual profile, and onl y a fe w as comparative test materi al during the development and individual profil es could be generated simultaneously. This evaluati on of an alytical methods. is because each individual profile requires a complete elec­ trophoreti c gel, so that the number of profiles that can be pro­ In order to screen, quantify and analyze bi ologi cal di versity duced at the same time will depend on the number of gels th at in Littorina, a wide array of techniques was implemented by can be run simultaneously. Secondl y, scoring 20-gels was the AMBIOS consortium. Phenotypi c vari ati on was assessed hi ghly problemati c, even when commercial internai markers with morphometric analyses of shell features (calipers meas- were used as reference, and gels were screened by dcnsitom- 46 THIERRY BACKELJAU et al.

70°

. ·"'"' 60°

so·

40°

•• AZORES

.,,•MADEIRA

30' CANARY 0

20'

CAPE VERDE ISLANDS

10'

30' 20' o· 10'

Fi g. l. - Map of the stud y area covered by th e AMBIOS progra m.

etry and im age analys is softw are. Finall y, even when ge ls provide new evidence suggestin g that ( J) L. saxatilis and L. were scored app ropriately, it remained difficult if not impos­ arcana are more cl osely related to each other th an either is to sibl e to inte rpret the spot pattern s as ge notypes, so that onl y a L. compressa, (2) L. compressa is the ge neti call y least vari­ pheneti c 'spot counting' analys is was poss ibl e. Ob viously, abl e spec ies of ro ugh periwinkle, and (3) Azorean L. saxatilis fo r a compl ex, time-consuming and qui te cos tl y (e.g. sil ver­ is geneti ca ll y not depauperate or significantl y differenti ated stainin g, Phas tSystem gels) method thi s is a rather poor from British L. saxatilis. The cl oser relati onship between L. amount of info rmati on. Hence, 2DE was not furth er consid­ saxatilis and L. arcana supports previ ous studies (e.g. ered as a ro utine method to survey bi odi versity. wA RD, 1990) (but see BACKELJ AU & wAR MOES , l 992 and the SS CP wo rk here below), while the reduced geneti c va ri ­ Nevertheless, based on a Jaccard 's simil arity analys is of 60- ability of L. compressa confirrns earli er observa ti ons by 105 protein spots in over 100 specimens, the 2DE data did WARD ( 1990). Periwinkles and marine biodiversity 47

RADULAR MYOGLOBINS (Mb) AND GENERAL tian in 142 specimens of L. obtusata and L. fabalis along a PROTEINS (GP) vertical intertidal transect in Galicia (NW Spain) (ÜLABARRIA et al., 1998). Here, the Mb patterns revealed a Prior to AMBIOS, only two studies had used Mb in the popu­ high incidence of rare Mb profiles in L. obtusata from the lation genetics and systematios of periwinkles (WIUM­ Fucus serratus zone compared to specimens from the algal ANDERSEN, 1970; JONES, 1972), and the presence of radular belts higher up in the intertidal. Possibly this heterogeneity Mb was only known in 17 Iittorinid species (MEDEIROS et al., reflected the marginal situation of L. obtusata from the F. ser­ 1998). Because of this Jack of data and because of the prom­ ratus belt. No comparable differentiation was observed in L. ising nature of WIUM-ANDERSEN's (1970) results, we de­ fabalis, for which the F. serratus zone is a 'normal' habitat. cided to screen Iittorinid Mb. Hence, using PAGE and IEF, Interestingly, the Mb data cou Id be interpreted genotypically several aspects of littorinid Mb/GP variation were investi­ in various ways, but none of these was consistent for ail gated. littorinids investigated (including the data from the litera­ ture). Moreover, the 'traditional' picture of a dimeric Mb Intraspecific Mb/GP variation was very low in L. striata. For controlled by a single locus (e.g. READ, 1968; TERWILLIGER example, in 160 specimens there was no detectable Mb/GP & READ, 1969) could not be confirmed. However, assuming variation or differentiation, not even between islands sepa­ that two loci code for a monomeric (or dissociated dimeric) rated by more than 2000 km (Azores vs Cape Verde Islands) Mb a data set was produced for the flat periwinkles in which (DE WOLF et al., l 998a). no significant deviations from Hardy-Weinberg expectations were detected. However, this interpretation could not ac­ In contrast, L. littorea (272 specimens screened) showed a count for ail available Iittorinid Mb data (ÜLABARRIA et al., considerable amount of intraspecific Mb/GP variation, par­ 1998). ticularly in French populations where a higher number of 'unusual' Mb/GP profiles was detected (MEDEIROS et al. , The Mb patterns also provided taxonomie and phylogenetic 1998). This observation is surprising because L. littorea has data, even though the Mb profiles did not distinguish consist­ bee~ reported to show macrogeographic homogeneity over ently between highly similar and closely related species such thousands of km (JOHANNESSON, 1992). However, the Mb/ as l. obtusara and l. fabalis or L. saxarilis, l. compressa and GP data support JOHANNESSON's (1992) suggestion that L. arcana. For this latter group the Mb patterns initially ap­ there may be a cryptic taxon in French L. littorea. Unfortu­ peared to separate l. arcana from bath other species nately, the Mb/GP variation in L. littorea could not be inter­ (MEDEIROS et al., 1998), but a more extensive analysis has preted genotypically and was difficult to quantify. shown that this is not the case and that L. arcana and L. saxarilis are polymorphie for the same Mb variants. Despite Nevertheless, a genotypic interpretation and numerical this Jack of resolution at the level of closely related species, analysis was attempted for microgeographic Mb/GP varia- Mb profiles yielded interesting results at supraspecific lev-

c c H · H T T K K M N T K p u s s

Fig. 2. - IEF profiles of raclular Mb (and general proteins) in a pH gradi ent of 4-6.5 (wit'l1 pH 4.0 at upper gel sicle), stained with Coomassie Brilliant Blue. Abbreviations: C: Cenchritis 111urica111s, H: Nodili//orina ha111aiie11sis, K: L. keenae, M: pH marker, N: Melarhaphe neritoides, P: L. p/ena, S: L. saxatilis, T: L. striata, U: L. sc11111/a1a. 48 THIERRY BACKELJAU et al. els. Using IEF, M. neritoides had a unique pattern shared by Cape Verde Islands) there was no consistent pattern of EST no other littorinids investigated (Fig. 2), yet with PAGE, the di fferentiation, a conclusion that confirms parallel allozyme Mb profile of M. nei'itoides was indistinguishable from that work by DE WOLF et al. (l 998b). of L. striata (MEDEIROS et al., 1998). Furthermore, the Mb pattern (both PAGE and IEF) of this latter species seemed to Finally, the third investigation foc used on EST variation in be conservative as it was shared with, or very similar to, 637 specimens of L. littorea. This work supported the previ­ those of the species from the genera Nodilittorina, Littoraria ous Mb data suggesting that French populations are differen­ and Cenchritis (Fig. 2). Within the genus Littorina, the Mb tiated from other European l. littorea, and thus may repre­ pattern of L. striata also resembled very strongly that of L. sent a cryptic taxon. More surprisingly, however, it appeared keenae (Fig. 2), which is considered as the next basal species that in l. littorea from the Scheldt Estuary (The Netherlands) in the genus. Interestingly, L. scutulata and L. plena, had a numbers of EST bands per individual and interindividual distinct Mb pattern that differentiated both species from L. EST band similarities seem to correlate with geography striata and L. keenae on the one band and ail remaining (some degree of isolation by distance), salinity and/or heavy Littorina species, on the other (Fig. 2). This latter group of metal pollution. Littorina species had a L. saxatilis-like Mb pattern, except for the two flat periwinkles, which had a distinct Mb profile Clearly, these three studies indicate that the IEF analysis of (MEDEIROS et al., 1998). These results are partially at vari­ EST variation is an easy, fast and useful technique in ance with current taxonomie groupings, even though they are biodiversity studies, even though the genetic interpretation in line with a basal position for L. striata and L. keenae, as of EST variation is usually very difficult. We would therefore well as with a possible taxonomie grouping of L. scutulata qualify it as a 'quick and dirty' method that must only be used and L. plena. The data for the 'higher' littorina species may in conjunction with other techniques have been confo unded by ecophysiological and/or phylogeneti c convergence (MEDEIROS et al., 1998). DNA EXTRACTION PROCEDURES Altogether, the Mb/GP results suggest that, even if the ge­ Within the AMBIOS program several DNA techniques have netic background of these markers in littorinids is still con­ been developed and implemented. This required, in the first troversial, they may provide an interesting tool for phenetic place, suitable DNA extraction protocols. In this respect, analyses of variation, as well as fo r ecophysiological and molluscs often pose severe problems because they secrete phylogenetic purposes. Therefore we suggest that littorinid large quantities of mucopolysaccharides (GRENON & Mb variation is certainly worth further investigation. W ALKER, 1980) and polyphenolic proteins (RZEPEC KI et al., 199 1), which copurify with DNA and interfere with the enzymatic processing of nucleic acids (e.g. CHAPMAN & NON-SPECIFIC ESTERASES (EST) BROWN, 1990; SHIY JI et al., 1992; RUMPHO et al., 1994 ). Non-specific EST have been used with variable success in Severa! DNA extraction protocols have been developed to several littorinids of interest to AMBIOS (e.g. VUILLEUM IER overcome these problems (e.g. JIANG et al., 1997; CHASE et & MATTEO, 1972; BERGER et al. , 1975; NEWKIRK & DOYLE, al., 1998; MIKHA ILOYA & JOHANNESSON, 1998), but we rou­ 1979; MILL & GRAHAMME, 1988; DYTHAM et al., 1992; tinely applied the procedures of ASH BU RNER ( 1989) or MILL & GRAHAME, 1992; MAZON et al., 1998). In the WINNEPENNINCKX et al. ( 1993) (adapted so that tissues were present context we assessed the usefulness of EST in three not ground under liquid nitrogen, but instead were minced case studies based on IEF. using a scalpel). Yet, within the context of AMBIOS we also used faster (i.e. si ngle-step), easier and Jess expensive DNA A first study of 142 specimens of flat periwinkles by extracti on protocols, one of which was particularly well­ ÜLABARRIA et al. ( 1998) showed that ( 1) EST profiles con­ suited to handle individual periwinkle embryos (SI MPSON et sistently distinguished between the two flat periwinkles in al., 1999). Galicia, (2) the mean numbers of individual EST bands (as an indirect measure of heterozygosity) were significantly The procedu re of SIMPSON et al. ( 1999) was as fo llows: indi­ higher in L. obtusata than in L. fabalis, and (3) L. f abalis was vidual embryos (0.4-0.7 mm) were placed in a 0.5 ml more heterogeneous than L. obtusata. Neither species microcentrifuge tube with 10 µ! extraction buffer (50 mM showed significant EST differentiati on between specimens KCI , 10 mM Tris HCI at pH 8.3, 2.5 mM MgCl7, 0.01 % gela­ from different intertidal algal levels or sexes. These observa­ tin , 0.9% Tween-20 and 10 mg/ml Proteinase K). Prior to in­ tions largely agree with previous studies, suggesting that L. cubation, minerai oil (20 µI) was added to each tube. Sam pies obtusata tends to be more heterozygous, but Jess heterogene­ were then incubated for 60 min at 65°C, and subsequentl y ous than l. fabalis (e.g. JA NSON, 1987; BACKELJ AU & denaturated fo r 15 min at 94°C. The extracted samples were WAR MOES, 1992; ZASLAYSKAYA et al., 1992; ROLAN­ either used directl y for PCR ampli fi cation or were kept fro­ ALYAREZ et al., 1995; TATARENKOY, 1995). zen at -20°C fo r up to fi ve months. The buffe r can be made in àdvance and stored at -20°C indefi ni tely, although Proteinase In a second study, DE WOLF et al. ( l 998a) found that indi­ K must be added fresh before use. vidual EST vari ation in 7 10 specimens of L. striata was very high, but unrelated to sex, shell morphology, or wave-expo­ For the SSCP analyses, the DNA extraction protocol of sure reg imes. Even al rn acrogeographi c scales (i .e. Azores vs SMALL et al. ( 1998) was used. This protocol is not onl y fas t Periwinkles and marine biodiversity 49

. (it takes 10 min instead of several hours), but can also be In an analogous way, primer RAPD-X amplified a band performed on the bench rather than in a fume hood. Approxi­ which initially appeared to be diagnostic for L. saxatilis M. mately 0.1 g of tissue was in.cubated in 225 µl extraction The band was similar in size to a band shared by both the M buffer [0.1 % Tween-20, 5% Chelex resin (Bio-Rad, Rich­ and H fonn. Therefore this band region was band stabbed in a mond, CA, USA)] for 15 min at 95°C, and ex tracts were agi­ representative of each form and the amplification products

tated after 7 min. After settling, the extracts were stored at - cloned. A range of PCR product sizes was found from each. 1' 20°C until PCR-amplification. Twelve amplification products were cloned from fonn H and 14 from fo1m M. None of the products appeared to be useful rnarkers, except for one which showed a variable Mspl re­ RANDOM AMPLIFIED POLYMORPHIC DNA (RAPD) striction site (clone X80) (see 'Restriction site analysis'). Because of its speed, ease and relatively low cost, RAPD has become a popular DNA technique in population genetics Obviously, these results show that, although RAPD may be (e.g. HADRYS etal., 1992; BOWDITCH et al., 1993). However, complementary to allozyme data, care must be taken when the method has a number of considerable limitations (e.g. the relying on RAPD bands to 'diagnose' taxa. Morover, the fact dominant/recessive nature of genotypic band profiles) and that we have shown that single RAPD bands may contain presents several interpretative and/or analytical caveats (e.g. several very different sequences supports previous concerns BACKELJAU et al., l 995b and references therein). Therefore on the usefulness of RAPDs as phylogenetic (and to a lesser an assessment of its usefulness in the context of AMBIOS extent population genetic) rnarkers (BACKELJ AU et al. , was necessary. This was done in two specific studies. l 995b and references therein). Nevertheless, given the large amount of data that can be generated with RAPD, we con­ DE WOLF et al. ( 1998b) surveyed RAPD variati on in 320 sider this technique as a convenient ' first step' to uncover specimens of L. striata from five populations (Azores, Ca­ hidden variation, but without further analyses as described nary Islands and Cape Verde Islands) using two RAPD prim­ above, it remains a 'quick and dirty' method, which is best ers (5'-ACGCGCATGT-3' and 5'-TCACGTCCAC-3') yield­ used in conjuncti on with other molecular markers. ing six consistently scorable, polymorphie bands (i.e. loci). Since parallel al lozyme studies on fo ur to five enzyme loci in SINGLE-STRAND CONFORMATION POLYMORPHISMS 66 populations of the same species revealed no consistent de­ (SSCP) viations from Hardy-Weinberg equilibrium conditions, it ap­ peared reasonable to assume that the RAPD polymorphisms The methodological rati onale behind SSCP (and related woul d also conform to Hardy-Weinberg expectati ons. techniques) is well-explained by DEAN & MILLIGAN (1998). Hence, using the statisti cal frarnework of LYNCH & Although this relati vely new method (ÜRITA et al., 1989, MILLIGAN ( 1994), the RAPD data were analyzed as classical HAYASHI , 1991 ; FUJ ITA & SILVER, 1994; KIM et al., 1995) dorninant/recessive di-allelic loci. This yielded heterozygos­ has much to offer to molecular systematics and population ity and F,, estimates that were highly congruent with those geneti cs (e.g. HISS et al. , 1994; BLAC K & DUTEA U, 1997; obtai ned from allozyrne data. Moreover, just as with the ÜRTI et al., 1997), it has hitherto only been applied in one all ozyme data, the RAPD analyses suggested that northern rnolecular genetic study of marine gastropods (KYLE & populati ons (Azores) were Jess heterozygous than southern BOULDING, 1998). Hence, by adapting and applying SSCP, ones (Cape Verde Islands). Obviously, this congruence be­ the AMBIOS project made a fondamental contribution to the tween allozymes and RAPD warranted the further use of the wider use of this technique. To this end we developed a sim­ latter. ple, non-radioactive SSCP method using a new nucleic acid stain, SYBR GOLD(R) (Molecular Probes, Eugene, Oregon, In the other study, involving RAPD variati on in L. saxatilis it USA). Moreover, rather than using a DNA sequencing appa­ appeared that primer RAPD-H (5'-GCCGTGGTTA-3') am­ ratus, we performed SSCP separations on a simple protein pli fied a 700 bp band in form M but not in H (GRAHAME et electrophoresis apparatus. al:, 1997). Thi s prompted us to funher characterize this 'di­ agnostic1 RAPD band by band-stabbing and using the band The protocol was as follows. After DNA extracti on using the ? tab as template for a second round PCR. The resulting band Chelex protocol described above (SMALL et al., 1998), DNA was cloned and, after hybridisation analysis, seven different was PCR-amplified fo r Cytb fragments iii (212 bp) and iv sequences were detected in 24 clones examined. Eighteen of (225 bp) with the primers: the clones yielded one of two different sequences. One addi­ iii F 5'-GGGCTTCTTACCAAAACCAACACAC-3', ti onal sequence was found in two clones and the other fo ur iii R 5'-GAGGGTAGCGTTGTCAACTGC-3', sequences were onl y observeq in single clones. One of these iv F 5'-GCAGTTGACAATGCTACCCTC-3' and !aller contained a microsatellite-like region which was stud­ iv R 5'-GGACTAGGGCCGAAAGTATAAATAAAAGG-3'. ied in some detail (see 'Microsatellite DNA'). When specific PCR was perforrned in 20 µl reaction volumes containing J .5 primers were designed to the other sequences identified from µM of each primer, 80 ~tM of each dNTP and 0.5 µI crude RAPD-H, various problems were encountered (e.g. incon­ DNA extract (SMALL et al., 1998). PCR cycles were pre­ sistent amplification, confounding length vari ation, incorrect ceded by a denaturation incubation fo r 3 min at 94°C, then priming, etc.) and hence they were inappropri ate fo r further held at 80°C whil e adding 0.5 U of DNA polymerase. Thirty analysis. cycles foll owecl: 94°C for 30 s, 45°C for 45 s and 72°C for 30 s, with a final 2 min al 72°C. SSCP samples were then prc- 50 THIERRY BACKELJAU et al.

Fig. 3. - SSCP profiles of va ri ati on in two fragments of Cytb in L. compressa (C), l. saxatilis (S) and l. arcana (A). All ele marker (M).

pared by mixing 2 µl PCR product with 22 µl denaturati on ships in 586 specimens of the three rough periwinkles, L. buffer (95 % formamide, 20 mM EDTA, 10 mM NaOH, saxatilis, L. arcana and L. compressa fr orn Ireland and the 0.05 % bromophenol blue, 0.05 % xy lene cyanol). The SSCP UK (Fi g. 3). A total of 38 hapl otypes were detected, several samples were denatured at 94°C for 5 min , then placed in ice ofwhich (including the most comrnon one) were shared by L. cold water, and 6 µI was loaded into a well in a precooled 160 arcana and l. compressa, but were absent or very rare in l. x 220 x 1.5 mm vertical 9% polyac ryl amide gel (3 7.5: 1 saxatilis. In contrast, the most comm on haplotype in L. acrylamide to bi sacrylamide) with 0.75 x TAE (SAMBROOK saxatilis was rare in both other species (Fig. 3). L. compressa et al., 1989) in both the gel and the buffer, and with 200 ml and L. arcana were genetically more similar to each other buffer in the bottom chamber and 400 ml buffer in the top than each of them was to L. saxatilis . At the population ge­ chamber of a Fisons FEC 175 Adjustable Vertical Gel Sys­ neti c level, the three species showed similar geographic tem. Gels were run for 16 hours at 300 V and 7°C. Staining structuring, in that northern English populations were differ­ was done with 0.5 x SYBR GOLD in water for 15 min and entiated from populations from Cornwall and Ireland illuminati on under UV li ght. A 1OO bp ladder was included in (SMA LL & GOSLING , 2000). each run as was an allele standard constructed from the com­ rnon alleles. These all ele stand ards and ail subsequen tl y dis­ The two other SSCP case stud ies dealt with the population covered mobility vari ants were sequenced. geneti cs and species status of L. saxatilis, L. neg lecta and L. tenebrosa. A total of 1537 specimens from Ireland and the During the developmental phase of thi s SSCP protocol it ap­ UK were investigated. Genetie diversity varied among re­ peared that electrophoreti c rn obilities differed profoundly gions, with the populations from western Ireland , northeast­ depending on whether O.Sx or 1x TAE was used. Further­ ern England and southwestern England tendin a to have more, mobilities va ri ed with buffer vo lume, temperature, higher diversity than those from elsewhere. Neither L. voltage, acrylam ide to bisacrylamide rati o and ge l or buffer tenebrosa nor L. neglecta showed unique haplotypes. In ad­ add iti ves such as Mg++" Tween or KCI. Clearly, SSCP analy­ d1t1 on, both analyses revealed hi ah amounts of aene fi ow be- ses do require astringent stand ardi sation of the protocol. The tween the three taxa, and an ana"'l ys is of molec"'ul ar variance conditions described here were best fo r running th e two Cytb (AMOYA : see EXCOFFIER et al., 1992) showed that, particu­ fragme nts on the sarne gel. larl y for L. tenebrosa, geographi c distance explai ned nearl y twice as much of the variance as hab itat type. Hence gene The usefulness of SSCP analyses in the contex t of AMBIOS now was more restricted by di stance than by habitat type. was dern onstrated with three case studies involving Cytb. Simil arl y it appeared that exposure regime had little effect on The first analys is assessed the ge netic structure and relation- gene now at mos t sites. Ali thi s suggests that the thrce taxa Periwinkles and marine biodiversity S 1

are conspecific, and that L. neglecta and L. tenebrosa should Amplification products of four species (M. neritoides, l. be considered as ecotypes of L. saxatilis. littorea, L. aleutica and l. saxatilis) were cloned and sequenced for further characterisation. Three separate These data prove that SSCP analyses are an efficient and introns (CAL-1, CAL-2 and CAL-3) were recognised, the powerful tool to investigate oiodiversity. Particulary, the pos­ sequences of which displayed little apparent homology. This sibility of screening large numbers of specimens for geno­ suggests that they may be introns of three different typic DNA variation and of limiting sequence efforts to only calmodulin genes. Bath CAL-! and CAL-2 occurred in all those variants that appear to be different, makes the techique Littorina species studied, while CAL-3 was only found in l. highly infonnative, since it gives data on bath the genotypes littorea and l. aleutica. M. neritoides had two introns (CAL­ and the nature of the alleles or haplotypes. Moreover, al­ S and CAL-L), but their homology with those of Littorina though not applied in the AMBIOS work, SSCP of nuclear could not be established. genes allow hetero- and homozygous individuals to be dis­ tinguished, thus enabling classical population genetic assess­ Although the sequences of CAL-! and CAL-2 were variable ments. within the rough periwinkles, they showed no fixed differ­ ences that consistently separated four forms of L. saxatilis (H, M, l. tenebrosa and l. neglecta), or that distinguished L. CALMODULIN VARIATION saxatilis from the two other rough periwinkles, l. arcana and Because the analysis of calmodulin introns is considered to l. compressa. However, the fiat periwinkles differed mark­ be a useful tool for surveying neutral DNA polymorphisms edly from the rough periwinkles, suggesting that the (CORTE-REAL et al., 1994; PALUMBI & BAKER, 1994, 1996), calrnodulin introns may provide a phylogenetic signal tore­ it was decided to test these markers in the context of solve relationships among more distantly related species. AMBIOS. The fact that l. littorea and l. aleutica contain a third intron is also suggestive in this context, because these two species The universal calmodulin primers of CORTE-REAL et al. are separated on the consensus tree of REID ( 1996) by a (1994) were used to amplify calmodulin introns in L. littorea, polychotomy of three branches, two of which were repre­ L. saxatilis (forms H and M), L. sitkana, L. natica, L. sented by l. subrotundata and l. sitkana, which are species aleutica, l. subrotundata, l. compressa, l. arcana, L. that do not seem to have the third intron . obtusata, l. fabalis and Melarhaphe neritoides. The PCR re­ action mixture (50 µ!) contained 50 mM KCI , 10 mM Tris The population genetic usefulness of the CAL sequences was HCI pH 9.0, I.5 mM MgCIJ, 0.1 % Triton X-1 OO, 0.01 % (w/ investigated by searching for polymorphie restriction sites. v) gelatin, 200 µM of each cÏNTP, 25 pmol of each primer, 25 This yielded one variable restriction site (for Taql) in CAL-1 ng DNA and 1U DNA polymerase. The PCR conditions (Fig. 4), due to an inde) between positions 362-366. During were: 1 x (5 min 94°C), 35 x (1 min 94°C, 1 min 52°C, 2 min the restriction digest screening of this locus, a third allele was 72°C), 1 x (5 min 72°C). Amplification products were run on found, the exact sequence basis of which remains to be inves­ a 1% agarose gel containing 0.5 nh/µ1 ethidium bromide. ti gated. Another variable restriction site was found in CAL-2

CAL1 CAL2

Fi g. 4. - Exa mpl e gels of res tri cti on fragment va ri ati on in CA L-! (restri cti on site obtain ed with Taql) ancl CAL-2 (restri ction site obtain ed with Ddel) in L. .rnxatilis. 52 THIERRY BACKELJAU et al.

(for Ddel) (Fig. 4). This polymorphie site yielded fo ur were even higher, with up to 36 alleles for locus Lstri 1.1 51 in alleles. The restriction analysis of these two calmodulin Sao Miguel. The five loci showed large heterozygote defi­ intron loci, combined wi~h similar work on two restriction ciencies, and an overall exact test for Hardy-Weinberg equi­

loci derived from apparent] y diagnostic RAPD bands, will be librium, under H 1 = heterozygote deficit, indicated signifi­ discussed un der the heading 'Restriction site analysis'. cant heterozygote deficits at ail loci in the three populations surveyed [Azores: Flores (n = 32) and Sao Miguel (n = 40); 1 1 Finally, CAL-1 also contained a variable microsatellite-like Cape Verde Islands: Sal (n = 12)]. As scoring the gels was region, whose usefulness will be discussed under the heading straightforward, and since allozyme analyses of four to fi ve 'Microsatellite DNA' . loci in 66 populations of L. striata showed no significant het­ erozygote deviations, we suspect that the consistent under­ In view of these results we think that a further characteriza­ scoring of heterozygotes with the microsatellites is due to a tion and survey of littorinid calmodulin intron variation will be particularly useful for resolving supraspecific high prevalence of null alleles (possibly in combination with phylogenetic problems, but is probably Jess relevant for too small sample sizes compared to the large numbers of alleles and possible genotypic combinations). The presumed studying low level taxonomie problems. occurrence of null alleles is supported by the observati on that ail loci revealed different non-amplifying samples, suggest­ MICROSATELLITE DNA ing that lack of amplification was not due to particular indi­ viduals (e.g. because of poor quality of their DNA). Unfortu­ With Jess than 15 published papers worldwide, gastropod nately it was beyond the scope of AMBIOS to perform breed­ mini- and microsatellite analysis is currently still in its in­ ing experiments to test the inheritance of the observed fancy. Although some work has been done on abalone microsatellite variation. Anyway, the alleged high frequency minisatellites (HUANG et al. , 1997; MUCHMORE et al., 1998), of null alleles (i n the Sal population they reached frequencies there is, to our knowledge, no published in fomrntion avail­ of up to 0.48) imposed severe limitations on the usefulness of able on microsatellites of marine gastropods. This is surpris­ these microsatellites. This was even further exacerbated by ing as microsatellites are often highly polymorphie markers, the Jack of cross-species amplification of the L. striata which are increasingly becoming popular in (population) ge­ microsatellites in Nodilittorina punctata, L. obt11sata, L. netic analyses (e.g. JARNE & LAGODA, 1996; GOLDSTEIN & saxatilis and L. arcana. SCHLÔTTERER, 1999). Therefore AMBIOS aimed at bridging this gap by developing microsatellites fo r peri winkles and, as Apart from the deliberate search for microsatellites in L. a consequence, producing the very first published data on striata, we uncovered microsatellites serendipitously in the prosobranch microsatellites (WINNEPENNINCKX & calmodulin intron CAL-1 and in a RAPD-band obtained with BACKELJAU , l 998a, b). primer RAPD-H (5'-GCCGTGGTTA-3') in L. saxatilis M (see also 'Calmodulin variation', 'Random amplified poly­ The protocols for the development and ro utine screening of morphie DNA (RAPD)' and 'Restriction site analys is'). five microsatellite markers in L. striata were descri bed by WI NNEPEN NINCKX & BAC KELJAU ( 1998b). During the de­ The CAL-1 intron in L. saxatilis contained a complex velopmental phase of the work, it became apparent that the microsatellite-l ike region wi th a variety of dinucleotide and quality of the microsatellite profiles strongly depended on trinucleotide repeat moti fs responsible for a presumably al­ the performance of the DNA polymerase during PCR ampli­ lelic length variation. Hitherto, this microsatellite-like se­ fication, the PCR conditions [i .e. the concentrations of bo­ quence was determined in two specimens of L. saxatilis, vine serum albumin (BSA), dimethyl sul foxide (DMSO), yielding respecti vely (GTCT)2(GTTT)z(GT)p A(GT)2 and magnesium sulfate (MgSO)] and on the type of radioac­ GA(GI)181T(G1\(GC)4(G1)101TG1GA(GI)11(GICT)p AG1C(fG)2 tive labeling used (WINNEPENN INCKX & BAC KELJ AU, AGTTAA and (GTTT)lGT)3GA(GT)2GA(GT)18GA(GT)4 l 998a). For example, [ a-35S]dATP labeling during PCR am­ (GC)2 (GT)27(CTGT\ GAGTTAA. The complex structure of plificati on produced considerably more artificial 'stutter' this repeat region suggested that it would be of only limited bands than end label.ing one of the fl anki ng primers with usefulness for microsatellite analysis. [y-32P]dATP. Moreover, with thi s latter type of labeling ail DNA polymerases tested perfonned well . In contrast, some A more useful microsatellite was fo und in a RAPD-H (5'­ DNA polymerases yielded poor amplifications when incor­ GCCGTGGTTA-3') band amplified in L. saxatilis M porating [o.-35S]dATP. In addition, the performance of some (GRAHAME et al. , 1997). This microsatellite involved a DNA polymerases was negati vely affected by adding DMSO GIT repeat motif which was often perfect, eve n though in (but see SHEN & HOHN, 1992), whereas adding BSA or some specimens it showed a vari ety of interruptions attribut­ MgS04 appeared to hav.e no effects and certainly did not im­ able to point mutations and single base pair deleti ons. The prove the quali ty of the microsatelli te patterns, as is usually primers to amplify the repeat were RAPDRPT I (5'­ claimed (e.g. Roux, 1995; KREA DER, 1996). TTTACCTCATCTGTTGCGTCC-3') and RAPDRPT2 (5'­ AATAACATACAGCGACGACGG-3'). Amplifications with Five rni crosatell ite loci were characterized in 16-35 peci­ these f.J rimers produced one or two bands in an approximate mens of L. striata from several localities. The fi ve loci size range of 95-530 bp, with nearl y no alleles in the size showed a hi gh am ou nt of allelic polymorphi sm, with at least range 300-400 bp. Although some alleles >400 bp repre­ 15 to 27 all eles/locus (average 19.2 alleles/locus). A subse­ sented ampli fi cati on products of unclear nature, they were quent populati on survey showed that the numbers of alleles mostl y good GTT repeats. Fortu nately, the vas t majority of Periwinkles and marine biodiversity 53

alleles had a size <300 bp. A total of 698 specimens of L. of the method. Nevertheless, by way of experiment, a restric­ saxatilis H and M from eight localities in Ireland and the UK tion site analysis of the L. saxatilis complex was performed were surveyed for approx.imate length variation at this on four nuclear loci. The primers microsatellite locus. This suggested that, on average, the am­ CADlF, 5'-TGCACATCATCATGCCAAA-3' and ,, plification products from L. saxatilis H are larger than those CAD 1RB , 5'-TCTTCAGAGCAGGGTTCCATT-3' from L. saxatilis M. Because alleles were not exactly sized, were used to amplify the intron locus CAL-1, and .' ' 1 1 precise genotype frequencies were not available. Neverthe­ CAD2F, 5'-CTGCAGATGGTGACGCAA-3' and less, confo rmance with Hardy-Weinberg expectations was CAD2R, 5'-CTGACGGTGAGTGACAATCG-3' tested on the basis of alleles 'binned' into 5 bp classes. Each were used to amplify CAL-2 (see 'Calmodulin variation'). 'binned' class was then considered as an 'allele'. Genotype frequencies based on these 'alleles' showed significant de­ Two polymorphie loci obtained from cloning and sequencing viations from Hardy-Weinberg conditions in ail populations, apparently diagnosti c RAPD bands (GRAHAME et al., 1997) except for L. saxc1tilis M at one site in SE England (Folke­ were also screened. One of these (X-80) was identified from stone). In ail cases these deviati ons were due to serious het­ sequences generated with the RAPD-X primer (OPY-01 , 5'­ erozygote deficiencies, the cause of which is still unclear GTGGCATCTC-3') and was amplified with the primers (null alleles?). Anyway, despite this possible fl aw, and as­ X80a 5'CATCTCTGTGTTGAAAGAGGGT-3' and suming neutrality of the repeat motif (and no linkage to loci X80b 5'-CAGAACTAAACTGAAGAAACCCG-3'. under selection), the differential size distribution of the alleles in L. saxatilis H and M may suggest some sort of ge­ The other RAPD-locus (referred to as DELETION) was de­ netic divergence between the two forms. rived from RAPD-H band containing the microsatellite re­ peat motif described under the heading 'Microsatellite ln conclusion, despite the current popularity of micro­ DNA'. The flanking sequence of this GIT repeat indeed re­ . satellites, we found the development and applicati on of these vealed a deletion useful fo r a restriction site analysis, 111 · markers in the context of AMBIOS too cumbersome, time­ which the locus was amplified with the primers consuming, costly and unreliable (cf. the high prevalence of RAPDRPT3, 5'-AGTAACGGCAGACGCCAT-3' and null alleles) to merit further pursuit. RAPDRPT4, 5'-TTATTCTTGTCTTCTCTGCC-3'.

Ali PCR amplifications were performed in 25 µl reaction RESTRICTION SITE ANALYSIS mixtures containing 50 mM KCI, 10 mM Tris-HCI pH 9.0,

The application of restriction site analyses is widespread and 1.5 mM MgCl1 , 0.1 % Triton X- 100, 0.01% gelatin, 200 µM very popular in population genetics (e.g. AVISE, 1994), so of each dNTP, -12.5 pmole of each primer, 12.5 ng DNA and that it was fe lt superfluous to provide a technical evaluation 0.5 U DNA polymerase. PCR cycling conditions were: 1 x (5

Littorina saxatilis 97 95@ Littorina compressa @ Littorina obtusata

73 Littorina littorea 80 Littorina striata

Littoraria undulata

Nodilittorina ptmctata A

Nodilittorina ptmctata M

Melarhaphe neritoides

Fig. 5. - Maximum Parsimony (with successive weighting) and Maximum Likelihood tree of littorinid relati onshi ps based on l 8S rDNA sequences. Values at the branchi ng points are bootstrap or support values obtained with fastDN Aml (below), PUZZLE (encircled) and Max imum Parsimony (above). A: Atlanti c; M: Mediterranean. 54 THIERRY BACIŒLJAU et al. min 94°C), 35 x (!min 94°C, 30 s 55°C, I min 72°C) and I x the unstable phylogeny produced by the restnctlon site (5 min 72°C), except for CAL- I in which the annealing tem­ analysis, meant that this method was considered Jess useful perature was 57°C and DELETION in which annealing was for AMBIOS purposes, particularly as the SSCP analyses at 57°C and the extension time 30 S. allowed a much higher resolution.

The allelic variations in CAL-!, CAL-2 (Fig. 4) and X-80 NUCLEAR RIBOSOMAL AND MITOCHONDRIAL DNA ' were screened with the restriction enzymes Taql, Ddel and SEQUENCING Mspl respectively, and the restriction digests were separated on 2% agarose gels. The alleles at the DELETION locus Apart from the application of Cytb sequences in the SSCP were detected by their size difference after electrophoresis of analyses, DNA sequencing was also used on its own, mai ni y the PCR amplification product in a 3% agarose gel. to infer phylogenetic relationships. Evidently, from a techni­ cal point of view DNA sequencing did not require an evalua­ The genolypic profiles obtained were analyzed as all ozyme tion. or microsatellite data. As such, the allele frequency data at the four loci were used to derive phylogenetic relationships Nuclear rDNA sequences were used to investigate the between L. arcana, L. compressa, L. saxatilis, L. neglecta phylogenetic position of L. striata and to test its suitability as and L. tenebrosa (in total 789 specimens from 32 locations in a marker to resolve recent divergences that took place Jess the UK, Ireland and Sweden). The resulting dendrograms than 40 Myr ago (WINNEPENNINCKX et al. , 1998). This was clustered populations more or Jess according to the three tested by comparing complete l 8S rDNA sequences (±1800 well-recognized species L. saxatilis, L. arcana and L. bp) in nine littorinids (Nodilittorina punctata from the Medi­ compressa, with the former two as sister taxa. However, te1i-anean and the Cape Verde Islands, and Melarhaphe never were ail populations placed correctly at the same time neritoides, littoraria undulata, L. striata, L. littorea, L. and no restriction patterns appeared to be diagnosti c for any obtusata, L. compressa and L. saxatilis). Using distance ma­ of the taxa included. trix, parsimony and maximum likelihood methods, these se­ quence data placed L. striata consistently at the base of the After correcting for multiple testing, only three out of 128 Littorina clade, although only with the maximum likelihood tests showed significant deviations of Hardy-Weinberg equi­ methods (fastDNAml and PUZZLE) and the successive librium conditions and there was no sign of linkage in the weighted parsimony analysis did this placement receive data. However, the population genetic analysis of the four bootstrap values >70 % (Fig. 5). The topology within the loci provided little new information that was not already ob­ Littorina clade completely agreed with current ideas on vious from allozyme and SSCP data. This fact, together with Littorina relationships (REID, 1996). Interestingly, Atlantic

Table !: Relative performance of l 2S rDNA (l 2S), l 6S rDNA (165), Cytochrome b (Cytb) and ! SS rDNA sequences in resolving relationships between three closely related littorinid species of the subgenus Neritrema (after WINNEPENNINCKX et al., 1998).

12S 16S Cytb ISS

Number of ali gned si tes 374 444 651 1841

Parsimony informati ve si tes 9 8 52 li

Bootstrap values for: 2.4 1. 8 8.0 0.6

L. compressa+ L. saxatilis

NJ (Jukes and Cantor) 73 NA 100 94

MP NA NA 100 98

ML NA NA 100 96 ' L. compressa + L. saxatilis + L. obtusata

NJ (Jukes and Cantor) 60 100 100 97

MP 56 100 100 93

ML NA 100 99 96 Periwinkles and marine biodiversity 55

Table 2: Heat Coma Temperatures (HCT) and their standard errors (s.e.) in littorinids of the subgenus Neritrema. N: number of specimens examined.

1 South Wales Northeast England ', Species N Mean HTC (°C) ± s.e. N Mean HTC (°C) ± s.e.

l. saxatilis H 27 31.64 ± 0.34 28 31.96 ± 0.36

l. saxatilis M 30 33.00 ± 0.3 1 29 31.97 ± 0.20

l. QJ:CQ/IQ 91 33.13 ± 0.18 28 32.70 ± 0.44

l. compressa 30 34.16 ± 0.43 - does not occur

l. littorea 21 33.43 ± 0.33 31 31.48 ± 0.30

l. obtusata 21 35.93 ± 0.57 30 32.06 ± 0.68

and Meditenanean Nodilittorina punctata showed the same group and did not provide evidence for a species status for L. degree of sequence divergence as L. saxatilis and L. neglecta and L. tenebrosa. It did, however, consistently dis­ compressa, suggesting that possibly Atlantic and Medite1rn­ tinguish between the rough and fiat periwinkles. In contrast, nean Nodilittorina punctata belong to different taxa. Clearly, combined with SSCP analyses, the Cytb provided useful the 18S data do contain information to resolve more 'rapid' population genetic data (see 'Single strand conformation cladogenetic events. In fact, in comparison with previous polymorphisms'). Whereas the COI sequences were more markers, only Cytb performed better than 18S rDNA in this variable than Cytb, they were insufficient to yield well-sup­ respect (Table 1) (WJNNEPENNINCKX et al.' 1998). ported phylogenies. However, the simultaneous use of both data sets produced a stable phylogeny in general agreement In a subsequent analysis of the higher (generic) relationships with the current, consensus opinion (REID, 1996). A compan­ of the Littorinidae, the performance of l 8S rDNA was further ion restri ction fragment analysis of both genes revealed that, compared with that of partial 28S rDNA sequences (31 taxa although there were RFLP patterns typical of L. saxatilis, L. included, each sequenced for± 1800 bp of l 8S rDNA and a compressa and L. arcana, there were no truly diagnostic pat­ 750-800 bp stretch of28S rDNA). This analysis hitherto sug­ terns (i.e. occurring in all specimens of a given taxon and gests that, at this level, 28S rDNA outperfo1ms l 8S rDNA, never occu1Ting in another taxon). In contrast, there was a even though both data sets contain a substantial amount of degree of haplotype sharing between rough periwinkle spe­ phy logenetic information. cies and forms. It is still unclear whether this is due to incom­ plete lineage sorting of ancestral polymorphisms or to hy­ Severa) attempts to sequence ITS- 1 in several littorinids bridisation events. fai led despite amplification of this gene posing no problems. Although sequencing was unambiguous as long as the actual In order to design the primers for the previous sequencing rDNA subunit stretches were involved (i.e. the priming re­ work, fragments of L. saxatilis mtDNA were cloned and gions), it lapsed into apparently overlain sequences after en­ 8022 bp were sequenced, i.e. about half the complete mtDNA tering the ITS-1 zone. This suggests the presence of different of this species. Since this was not a primary goal of ITS- 1 copies, a problem which deserves further attention, but AMBIOS, we refer to WILDING et al. (1999) for a detailed which made ITS- 1 unsuitable for the AMBIOS goals. account of this mtDNA work.

The relative performance of the mitochondrial Cytb and COI In summary, the 18S and 28S rDNA sequences appear to be genes was also eyaluated. To this end a 537 bp portion of very useful for littorinid phylogeny inference at a variety of Cytb (of whi ch 2.05% is parsimony informative) and a " 400 taxonomie levels. However, the combined mtDNA se­ bp portion of the COI gene (of which 4% is parsimony in­ quences investigated seem to provide a better resolution of formative) were sequenced in a variety of Littorina species, the rough periwinkle relationships. Hence nuclear rDNA and including L. tenebrosa and L. neglecta. It appeared that COI intDNA sequences seem to complement each other nicely. was more variable than Cytb and that COI based phylogenies For population genetic work, DNA sequencing is most rel­ usuall y show better bootstrap support than Cytb phylogenies. evant if combined with restriction analysis or, better still, The Cytb portion here studied did not resolve the phylogeny SSCP. of the rough peri winkles, was nol diagnosti c in this species 56 THIERRY BACKELJAU et al.

ANALYSIS OF THERMAL TOLERANCE polymorphie and two relatively well-defined shell morphotypes can be distinguished: one with a clearly nodu­ Given that littorinids occur in the intertidal zone, where they lose shell, the other with a 'smooth' shell (REID, 1996, DE may be- subject to a wide range of very different thermal WOLF et al., 1997). Both shell types . occur micro­ Ioadings, and considering that temperature moderates the ef­ sympatrically, which mises the question as to the ecological, fects of almost ail environmental and biological factors, it genetic and taxonomie significance of such a polymorphism was decided thatAMBIOS should consider thermal tolerance in a planktonic developing species. as a phenotypic estimate of biodiversity at the physiological level. This could possibly shed light on the functional mean­ In a first analysis, the spatiotemporal distribution of the two ing of other phenotypic traits (e.g. shell morphology, physi­ morphotypes was investigated at Ilheu de Vila Franca do ology, behaviour) that contribute to biological variation, irre­ Campo (further referred to as 'Ilheu'), a drowned volcanic spective of whether these are adaptive or ecophenotypic. crater, about 1000 m off the south coast of Sao M iguel, Azores (DE WOLF et al. , 1997). This key site has been exten­ Thermal tolerance was assessed by estimating mean heat sively described and characterized by FRIAS MARTINS coma temperatures (HCT) as defined by MCMAHON (1976), (1976), MORTON (1990), DE WOLF et al. (1997) and using a method modified from MCMAHON ( 1990). Acute up­ MORTON et al. (1998). The crater consists of two small is­ per lethal thermal limits (LT50 values: estimated temperature lands, surrounding a circular lagoon which is connected with of 50% sample mortality) were also measured as described the ocean by a small channel and six narrow fissures. As a by MCMAHON ( 1990). Generally, thermal tolerance ap­ consequence, the lagoon is extremely sheltered, while the peared to be stable within populations, but highly variable outer margins of the crater are strongly wave-exposed. between populations and species. Ali taxa examined (l. Hence, environmental conditions differ considerably be­ littorea, l. obtusata, l. /abatis, l. compressa, l. arcana, l. tween the lagoon and the outside, providing an excellent set­ saxatilis H, l. saxatilis M, l. tenebrosa, lagoonal l. saxatilis, ting to investigate possible ecological correlates of shell and l. 11 eglecta) had HCT values in the range of 30-36°C polymorphism. Therefore, l. striata was collected annually (Table 2), while acute upper lethal temperatures were about (summers of 1992, l 993 and 1994: these samples were col­ 10°C higher than the HCT values (CLARKE et al., 2000b ). Iected prior to the start of AMBIOS) at eight sites at Ilheu. There was no clear correlation with shore levels, yet Four sites were Iocated in the lagoon, the other four on the eulittoral fringe species tended to possess higher HCT values outside of the crater. Of these latter, two were in the splash than eulittoral species. This is in accord with MCMAHON's zone; the other two in the barnacle belt (i.e. midshore). (1990) work on tropical littorines. HCT values also varied Specimens were separated into nodulose and smooth shelled, seasonally and geographically, but showed no correlation and five shell traits were measured with calipers. In total with individual size (measured by columella length), except 1078 specimens were included. The statistical analysis of in l. littorea, where animais with a columella length >30 mm these data showed that ( 1) nodulose shells are more common showed significantly lower thermal tolerances (CLARKE et in the sheltered lagoon, whereas smooth shells dominate the al. , 2000a). Finally, the du ration of acclimation in the labora­ wave-exposed outside, (2) on average nodulose shells are tory had no effect on HCT values, yet higher acclimation smaller than smooth ones, (3) regardless of morphotype, temperatures increased HCT values (CLARKE et al. , 2000b). shells from the Iagoon have a smaller aperture and are Jess globose than those from the outside, (4) within an exposure In general, thermal tolerance seemed to be a hi ghly plastic regime, smooth specimens have a larger aperture, (5) this trait showing little if any correlation with the genetic markers morphological patterning remains consistent over time (DE investigated by AMBIOS. Moreover, HCT and LT50 values WOLF et al., 1997). do not determine the in situ survival of periwinkles because the tolerance towards a single environmental factor such as The ecological differentiation between nodulose and smooth temperature may well be greater than the tolerance towards a l. striata was further tested with a translocation experiment combinati on of (interacting) environmental factors (SOUTI-1- at Ilheu (DE WOLF et al. , 1997). To this end, 240 specimens WARD, 1958). Hence, although thermal tolerance investi ga­ of comparable size were collected in the lagoon and on the tions may provide clues to explain certain patterns ofbiologi­ outside of the crater. Each shell was measured and specimens cal variation, they seem less suitable and quite uninformative were then divided into four groups: those transplanted from for assessing genetic and taxonomie components of the lagoon to the outside (n=60), those transplanted within biodiversity. the lagoon (n=60), those transplanted from the outside to the lagoon (n=60) and those transplanted on the outside (n=60). Each specimen was marked with a group-specific colour dot, Biological ~ariation in a planktonic developing and animais were recaptured and remeasured every rnonth. littorinid: The case of Littorina striata The experiment was stoppecl after five months. lt showecl that aperture height of specimens transplantecl from the Ja­ L. striata was intensively studied throughout its entire geo­ goon to the outsicle increa ecl significantly, whereas the other graphic range, which comprises the Macaronesian region, three groups showecl no significant rnorphometric cliffer­ including the Azores, Madeira, the Canary Islands and the ences between the start and the end of the experiment (DE Cape Verde archipelago. The species is conchologicall y W OLF et al., 1997). Periwinkles and marine biodiversity 57

45° 15° 0

····.L.C. N.A.C.

1 • • ',' , / , , ,. i ,.· ...... ·- ...... ' GULF STREAM . - - ...... ··~' ,,,,, ~, 45° . --- . . . . . ' . --···- ... , . ' ·. ·-·-- ... ,, •,

ATLANTIC OCEAN

~---- · ·· -·- 15° ...... _ NORTH --- EQUATORIAL .... CURRENT -- -- -·-

Fig. 6. - Proportional gene fl ow patterns of l. stria/a in Macaronesia, projected on a map with the trajeclori es of major surface currents (data fro m private all ele frequencies in fi ve all ozyme loci). Abbrevi ati ons: AZ: Azores; CA: Canary Islands; CV: Cape Verde Islands; LC: Labrador Current; MA: Madeira; NAC: North Atl antic Current.

An unexpected occasion to evaluate shell polymorphism in The final analysis of shell van at1on in L. striata tested L. striata occurred in December 1996, when an exceptionall y whether the shell polymorphism in this species showed the heavy storm hit the Azores (DE WOLF et al., 1999). Immedi ­ latitudinal trend that, as heat stress and predation increase ately after this storm the same sites were sampled at Ilheu as and wave acti on decreases towards the tropics (e.g. VERMEIJ , be fore ( 1992, 1993 and 1994). The expectation was that, af­ 1973, 1978), rocky shore gastropods from lower latitudes ter the stom1 , wave-exposed specimens should have shi fted tend to have more scul ptured (e.g. nodulosity, ... ),more glo­ to a larger aperti.J re size, whereas the sheltered lagoon speci­ bose, shelis wirh smaller apertures and more shell repair mens should have remained largely unaffected. Yet, no clear marks, than those from higher latitudes (e.g. VERMEIJ , 1978). eut morphological response was observed, since different This mode] was tested on L. striata using 1640 mature speci­ statistical techniques (i.e. MANOVA + canonical discrimi­ mens from 4 1 popul ati ons covering the entire range of the nant analysis vs. nested ANOVA) arrived at opposite conclu­ species (from the Azores, Madeira, the Canary Islands and sions. However, the preferred approach, based on a nested the Cape Verde archipelago, between 39°4 1' N and 14 °20' N). ANOVAdesi!!n contrasting the fo ur years (1992, 1993, 1994 Ali shells were measured as before and scored for sculpture and 1996) with tl;e factor 'year' nested within the factor (nodulose vs smooth) and the presence of repair marks. The 'storm', showed no significant storm effect (DE W OLF et al., main results of this work were: (1 ) shell size and weight in­ 1999). Obviously, it remains to be decided whether this Jack creased towards the south, (2) there was no consistent pattern of response indicates that shell form is not directly related to in nodulosity between archipelagos, and (3) nodul ose shells wave-exposure or whether it is simply due to the possibility displayed fewer shell repair marks than smooth shell s, but at that the exposure to increased wave-action during the storm the same time showed more repair marks in northern was too brief. populations than in southern ones. The fact that thesc obser- 58 THIERRY BACKELJAU et al.

vations partly disagree with current ideas on rnorphological Azores (none elsewhere), (2) the mean number of alleles per north-south patterns rnay be due to the unexpected (and indi­ locus was highest in the Cape Verde Islands, (3) the number rectly inferred) increased wave action in the Cape Verde is­ of loci revealing between archipelago heterogeneity was lands cornpared to the three other, more northerly, island highest for comparisons involving the Cape Ver9e Islands, groups. This atypical situation rnay confound the interpreta­ and (4) both a correspondence analysis and a UPGMA tion of rnorphological patterns in L. striata. More generally, dendrogram separated the Cape Verde Archipelago from the it seerns that generalizations about rnacrogeographic shell . other island groups. In addition, the macrogeographic rnorphology patterns based on interspecific cornparisons are morphometric study showed that the morphological varia­ not directly applicable to intraspecific patterns, and may de­ tion in the Cape Verde Islands was larger than elsewhere (DE pend strongly on local conditions, which rnake adequate WOLF et al. , 1998d), white the RAPD and EST data also sug­ sampling and data treatment very difficult (DE WOLF et al. , gested a higher diversity in this archipelago than elsewhere 1998d). (DE WOLF et al., 1998a, b). However, there was no signifi­ cant relationship between geographic and genetic distances. The results of the morphometric studies were interpreted as The macrogeographic pattern of variation was tentatively in­ showing that the nonrandom distribution of both shell terpreted as being the result of ancient and cu1Tent oceanic morphs reflects a functional response to thermal stress and circulations (Fig. 6). L. striata probably originated in the wave exposure. The more globose fom1, Jack of sculpture Cape Verde Islands since: ( 1) the oldest known fossils of L. and larger aperture size (i .e. stronger holdfast on the striata date from Tertiary deposits in this archip_elago substrate) protect the smooth specimens better against drag (SOWERBY, 1844), (2) these islands are the oldest in and dislodgement in wave-exposed conditions, where ther­ Macaronesia (together with Gomera and Fuerteventura in the mal stress is reduced, while the smaller aperture and in­ Canary archipelago) (MITCHELL-THOME, 1976), and (3) both creased reflectance/convection surface protect the nodulose genetic and morphological diversity is highest in the Cape specimens against the higher the1mal stress prevalent in shel­ Verde Islands (see above). Given that in the past the Atlantic tered areas (BRITTON , 1995; DE WOLF et al. , 1997, 1998d). Ocean was Jess wide (ADAMS, 1981), while surface currents The actual underlying mechanism ofthis functional response across the Atlantic rnay have been stronger than now remains unknown, even though the translocation experiment (SCHELTEMA, 1995, REID, 1996), we suggest that, during the at Ilheu tentatively suggests that at least aperture size would Tertiary, larvae originating from the Cape Verde Islands may be an ecophenotypically plastic trait caused by differential have reached the emerging Azores by the westward clock­ growth rates (DE WOLF et al., 1997). wise circulation, i.e. via the North Equatorial Current from the Cape Verde Islands westward to the Caraibes and the east In order to assess whether the patterns of shell variation in L. coast of North America and from there back eastward via the striata reflect genetic barriers or population structuring, a Gulf Stream to the Azores (see GOFAS , 1990). However, in parallel population genetic survey was performed using subsequent periods the currents nearer the surface slowed allozyme electrophoresis (PAGE), IEF of Mb and EST, down and the Atlantic widened. Thus larval westward trans­ RAPDs and microsatellite DNA. A first PAGE analysis of port from south to north decreased and ultimately became four allozyme loci (Gpi, Pgd, Mpi and Mdh) in the 1078 impossible, so that currently the Cape Verde islands may act specimens used for the microgeographic shell morphometric as a sort of 'cul de sac' for gene fl ow coming from the north. survey at Ilheu over the years 1992, 1993 and 1994 (see This tentative scenario could account for the accumulation of above; DE WOLF et al., 1997) showed that ( 1) there were no higher diversity in the Cape Verde Islands, without spreading significant deviations from Hardy-Weinberg equilibrium this diversity to the other archipelagos. conditions, (2) both shell forms share a common gene pool, (3) gene flow between populations was high, (4) the popula­ tion genetic structure remained stable over a sampling period Biological variation in a non-planktonic developing of three years, and (5) genetic and morphological distances littorinid: The case of the Littorina saxatilis complex between populations were not correlated. Hence, the two shell morphotypes of L. striata may be safely considered as The investigations on the L. saxatilis complex focused par­ bein g conspecific, implyin g th at their nonrandom spatial ti cularly on (1) the biological interpretation of the forms H, patterning persists in the presence of intense gene flow (DE M , L. ten.ebrosa and L. neg lecta (see Materi al and Methods), WOLF et al. , 1998c). A similar popul ation genetic survey of (2) rnacrogeographic variation in L. saxatilis, (3) characteri­ five enzyme loci (Gpi, Pgd, Mpi, Mdh and Hbdh) in 41 sation of Azorean L. saxatilis, and (4) the relationships be­ populations (1640 specimens) derived from the four tween L. saxatilis, L. arcana and L. compressa. Macaronesian archipelagos, was performed to complement the macrogeographic morphometric analysis. These For both L. tenebrosa and L. neg lecta, most analyses pointed all ozyme data suggested that, although only very limited to the same outcome: neither of these forrns seem to repre­ amounts of population differentiation were detected, there sent an independent gene pool or show consistent rnolecular were indications of a rnacrogeographic patterning of genetic differentiation from L. saxatilis. For example, the re­ all ozyme variation (Fig. 6), such that at least th e populations striction analysis of CAL-1 , CAL-2, DELETION and X-80, from the Cape Verde Islands appeared different from those the SCCP analysis of Cytb (SMALL & GOSLING , 2000) and from the other archipelagos because: (1) there were eight the restriction analysis of COI provided no evidence to sup­ unique all eles in the Cape Verde archipelago and two in the port a taxonomie separati on of L. neglecta and L. tenebrosa Periwinkles and marine biodiversity 59

at the species level, even though the Cytb RFLP analysis sug­ Azorean L. saxatilis (n = 54) was compared with L. saxatilis gested differentiation between L. saxatilis and L. neglecta on from Robin Hoods Bay (England), Vigo (Spain) and Venice the East coast of England. Furthermore, L. tenebrosa and L. (Italy) (n = 80), L. arcana from Robin Hoods Bay (England) neglecta do not differ from L. saxatilis in heat coma charac­ (n = 20) and L. striata from the Azores (n = 20) by means of teristics (CLARKE et al., 2000b). Nevertheless, two popula­ PAGE of eight polymorphie enzyme loci, 2DE of GP and IEF tion genetic analyses based on SGE of five and 12 allozyme of EST. The allozyme data showed that the Azorean popula­ loci respectively, suggested that gene flow between L. tion was as variable as L. saxatilis from elsewhere and that its saxatilis and L. tenebrosa is somehow reduced compared to allele frequencies did not differ significantly from those of gene flow within L. saxatilis (GOSLING et al. , 1998; WILSON other l. saxatilis populations. These observations were fully et al. , 1999). However, there was no consistent pattern of dif­ supported by the EST and 2DE data. Hence it seems that, in ferentiation between the taxa, and cluster analyses failed to terms of genetic variability and differentiation, Azorean L. separate L. tenebrosa from L. saxatilis as two homogeneous saxatilis does not show the expected pattern of an intro­ groups. Earlier allozyme work by JANSON & WARD (1985) duced, peripheral and highly isolated population. This is also suggested that L. tenebrosa is a distinct form of L. somewhat surprising in a live-bearing species with Iimited saxatilis rather than a separate species. Similarly, allozyme (active) dispersal capacity. However, all allozyme studies analyses performed in the context of AMBIOS suggested perfo1med in the context of AMBIOS did confirm the con­ that gene flow between L. saxatilis and L. 11.eglecta may be spicuous shore Ievel allele frequency differentiation at the reduced. However, once again there is no consistent pattern AAT locus in L. saxatilis (WILSON & GOSLING, 1998) which of differentiation or clustering as homogeneous groups. has been attributed to strong selection (JOH ANNESSON & Moreover, RAPD analyses of L. neglecta were unable to JOHANNESSON, 1989; JOHANNESSON et al. , 1995). separate this form from L. saxatilis (CROSSLAND et al. , 1996; WILDING et al., 1998). In contrast, a survey of shell shape in Finall y, the relationships of the three well-recogni zed rough six Iagoonal and three coastal populations does clearl y sepa­ periwinkles (L. saxatilis s.s., L. arcana and L. compressa) rate out L. tenebrosa from Golam Head, Ireland. were investigated with allozymes, 2DE, nuclear restriction site polymorphisms, SSCP's of Cytb, RFLP's of COI, and The situation for the H and M forms of L. saxatilis is com­ sequences of Cytb, COI and nuclear rDNAs. The cmTent plex. These animais occupy different shore levels, have dif­ allozyme and 2DE data suggest that L. compressais the sister ferent morphologies and show developmental di fferences taxon of the L. saxatilis - L. arcana clade, and thus confirm (HULL et al., 1996). At local scales, both forms can be differ­ most earlier allozyme studies based on SGE (e.g. WARD, entiated by a few RAPD bands, even though th is does not 1990, KNIGHT & WARD, 1991). A similar result was ob­ allow a consistent clustering (WILDING et al., 1998). One 'di­ tained from the restriction site analysis of fo ur nuclear genes agnostic' RAPD band contained a microsatellite showing a (CAL-1 , CAL-2, X-80 and DELETION), even though the differential size distribution between the two fonm. This topology of rough periwinkle relati onships received little may suggest some degree of reproducti ve isolation. How­ bootstrap support and was only evident when using NEI's ever, neither the restricti on site analysis of the other 'diag­ ( 1978) genetic distances, but not when using WRIGHT's nosti c' RAPD band (X-80), nor the all ozyme, calmodulin ( 1978) Prevosti distances. In contrast, the RFLP and se­ and mtDNA data allow a clear eut separation of these two quence analysis of Cytb and COI did not consistently sepa­ forms. Similarly, the two fo rms show no consistent heat rate the three rough periwinkles, made each of them coma tolerance differences (CLARKE et al., 2000b). On the paraphyletic and yielded mainly unresolved and poorly sup­ other hand , there is good evidence that they mate ported tree topologies. Furthermore, the SSCP analysis of assortatively by form (HULL, 1998; PICKLES & GRAHAME, another Cytb fragment suggested a closer similari ty between 1999). It therefore seems that L. saxatilis H and M may be in L. arcana and L. compressa, thus placing L. saxatilis outside the process of speciation wi thout however having reached this group. Although this result agrees with the allozyme data complete reproducti ve isolati on and geneti c differentiation. of BACKELJAU & wA RMOES ( 1992), it should be stressed that the SSCP data were interpreted by ordinati on via non­ In the course of the AMBIOS program still , another special metric multidimensional scaling, which is not a phylogeny ecotype of L. saxatilis was considered, viz. a 'barnacle dwell­ inference technique allowing reconstruction of 'common de­ ing' morphotype, referred to as B (CALEY et al. , 1995), dif­ scent' . Moreover, this approach does not provide support fering from L. neglecta and L. saxatilis. Yet, neither the re­ values, and hence the stability of this result cannot be evalu­ striction fragment analysis of CAL- 1, CAL-2, X-80 and DE­ ated. Finally, the l 8S and 28S rDNA sequences yielded vari­ LETION, nor the RFLP and sequence analysis of Cytb and ous, contradictory, but nevertheless bootstrap supported, COI, could consistently differentiate the B ecotype from L. topologies, depending on the taxa included and the gene frag­ neglecta or typical l. saxatilis, even though at some sites ments considered. Hence REID's ( l 996) morphology-based there was evidence for reduced gene fl ow between this latter proposai to main tain an unresol ved trichotorn y for the rough and the B form. However, on the basis of allele frequencies, periwinkles still seems to be the best refl ection of the current L. saxatilis B seems always to corn e out indistinguishable knowledge on the relationships of these pecies. from L. neglecta, but the two morphs can be different from 'large' L. saxatilis. On the other hand, there is evidence that L. neglecta is an annual species, whereas l. saxatilis B is longer li ved (L. JOH NSON, pers. comm.). 60 THIERRY BACKELJAU et al.

Discussion of known DNA fragments and the possibility of sequencing new allelic/haplotypic variants. Hence, SSCP analyses com­ METHODOLOGIC:AL ISSUES bine the advantages of allozymes, RAPDs, RFLPs, microsatellites and DNA sequencing! From a methodological point of view the AMBIOS program clearly demonstrated that not ail techniques and molecular CONCEPTUAL ISSUES markers employed are equally suitable for measuring effi­ ciently genetic diversity and inferring phylogenetic relation­ The investigations here presented illustrate convincingly that ships. For example, despite its higher resolving power than studying and conserving marine biodiversity requires a conventional protein electrophoresis, 2DE was found to be a multidisciplinary approach that must be applied at different Jess efficient technique if large numbers of specimens are to spatial scales. Bath L. striata and L. saxatilis show consider­ be surveyed. Furthermore, the interpretation and statistical able morphological and genetic variation, but the structuring analysis of 2DE profiles in population genetic terms is still and interpretation of the patterns of this variation may differ somewhat problematic. fundamentally. In L. striata, morphological differentiation is apparent at microgeographic scales and is maintained in the Similarly, the genetic background of littorinid Mb variation presence of intense gene flow, even over distances of up to still remains speculative (e.g. ÜLABARRIA et al., 1998) and 3000 km. In L. saxatilis, on the contrary, patterns of morpho­ Mb therefore seems Jess appropriate for population genetic logical variation may covary with different degrees of ge­ analysis. However, littorinid Mbs do appear useful for quali­ netic exchange that are already noticeable at distances of a tative (eco)phenotypic analyses of variation, and for helping few meters (e.g. the H and M forms). These patterns of mor­ to answer taxonomie questions and problems ofphylogenetic phological variation and gene flow largely reflect the devel­ relationships (e.g. MEDEIROS et al., 1998), particularly if opmental differences between these periwinkles, and at the analysed via IEF. This latter technique does indeed uncover sarne Lime point to the possible raie of selection and/or cryptic variation that may remain undetected by PAGE or phenotypic plasticity in generating and maintaining marine SGE (e.g. MEDE!ROS et al., 1998). biodiversity.

More surprising was the relative failure of microsatellites as Generally, L. striata reveals a temporally stable, spatial a population genetic markers. The pervasive presence and structuring of shell variation, most probably in relation to high frequencies of tentative null alleles, the Jack of cross­ wave-exposure and/or thermal stress, but shows little differ­ species amplification and the considerable laboratory efforts entiation according to shore Ievel or predation pressures, required to develop them (W!NNEPENN!NCKX & BACKELJAU, even though thermal stress and shore level effects may de­ l 998b), mean that littorinid microsatellites appear to be Jess pend on the type of substrate (e.g. black vs light coloured sui table for population genetic work than allozyme or RAPD rocks; cf. BRITTON, 1995). L. saxatilis, in contrast, shows data. This is not to say that, in general, the analysis of strong effects of shore level and wave-exposure, but does not microsatellites is not worthwhile pursuing, but rather that, in reveal a consistent pattern in thermal tolerance features. this particular case, considerable extra effort would be Given that these shore level differences may be accompanied needed to produce useful microsatellite data. Similarly, the by various degrees of molecular differentiation, it is obvious calmodulin intron data do not seem to be very useful for that classical taxonomie species boundaries only describe population genetic work in L. saxatilis, but appeared to be biodiversity at a relatively coarse level. Hence restricting the informative for phylogenetic analyses of taxa above the spe­ management of marine biodiversity to the species level will cies level. almost certainly result in important losses of evolutionary relevant variation present at the intraspecific level; this In contrast, conventional allozyme electrophoresis (SGE and would have a serious and profound effect on evolutionary PAGE) and RAPD analyses appear to be highl y cost-effec­ potential. Therefore it seems more appropri ate to delimit tive techniques for performing large scale surveys of genetic 'Evolutionarily Significant Units' (ESU: historically isolated variation. However, the informati on yield per locus is hi gher sets of popul ations that together encompass the evolutionary with allozymes than with RAPD bands, because these latter diversity of a taxon) and 'Management Units' (MU: sets of behave as dominant/recessive markers so that heterozygote populations that are currently demographically independent) genotypes cannot be directl y inferred from gel banding pat­ (MORITZ, 1994, 1996). In practice, these operational con­ terns (e .g. BOWD!TCH et al. , 1993; LYNCH & M rLLIGAN, cepts are recogni zed on the basis of molecular markers such 1994; BACKELJAU et a/., 1995). as mtDNA and allozymes, such that ESUs should be recipro­ cally monophyleti c for mtDNA alleles and also di ffe r sie:nifi­ Finall y, the most efficient technique to screen geneti c cantly fo r all ele frequencies at nuclear loci, whereas ~MUs polymorphisms appears to be the combination of SSCP and only imply significant differences in allele frequencies, with­ DNA sequencing, even though the potential of this approach out considering a phylogenetic component. Hence an ESU is is still severely underscored. The virtues of SSCP reside in · more inclusive than an MU. the relati ve simplicity of the experimental procedures, the possibility fo r inferring indi vid ual genotypes, the high re­ Although the application of the ESU and MU concepts repre­ solving power that all ows detection of point mutations, the sents a fun damental recognition of the importance of possibility of screening large numbers of individuals, the use in traspcci fic variation, and at the sa me ti me prevents man- Periwinkles and marine biodiversity 61

agement decisions being restricted to 'classically named' BACKELJAU, T. & WARMOES , T. , 1992. The phylogenetic relation­ species, ·there are .still a number of issues which should be ships of ten Atlantic littorinids assessed by allozyme electrophore­ considered if management policies were to be limited to the sis. Pp. 9-24 in GRAHAME, J. , MILL, P.J. & REID, D.G. (eds.), strict recognition of ES Us and MUs. The implementation of Proceedings of the Third International Symposium on Littorinid Bi­ ology. The Malacological Society of London, London. these concepts indeed relies solely on population inferences derived from 'neutral' molecular markers. This situation en­ BACKELJAU, T., BREUGELM ANS, K., BRITO, C., DE BRUYN, L. & tails the danger that loci under selection may not receive the TIMMERMANS, J.-M., l 995a. Macrogeographic genetic homogene­ attention they merit as evolutionarily 'active' genetic compo­ ity in lit1orina striata from the Azores (Mollusca, Prosobranchia). nents. In a similar way, the strict implementation of the ESU Açoreana, Supplement 1995: 159-1 7 1. and MU concepts may not deal adequately with non-molecu­ BACKELJAU, T., DE BRUYN, L., DE WOLF, H., JORDAENS, K. , V AN lar diversity. This is well-illustrated in the present research DONGEN, S., VERHAGEN , R. & WI NNEPEN NINCKX, B., 1995b. Ran­ program, where the observed morphological diversity could dom amplified polymorphie DNA (RAPD) and parsimony meth­ not be consistently interpreted in terms of ESUs or MUs. For ods. Cladistics, 11: 119-130. example, the peculiar lagoonal form of L. saxatilis, viz. L. BARBIER, E.B. & AYLWARD , B.A., 1996. Capturing the pharmaceu­ tenebrosa, shows a unique and consistent, apparently non­ tical value of biodi versity in a developing country. E11 viro111nental plastic, different shell morphology (e.g. JANSON & WARD, and Resource Eco 11 0111ics, 8: 157- 181. 1985), even though molecular techniques have so far failed BARNES, R.S.K., 1993. On the nature of coastal lagoon winkles to distinguish this form from other L. saxatilis (JANSON & attributed to lit1ori11a tenebrosa and lillorina saxatilis. Cahiers de WARD, 1985; GOSLING et al., 1998; WILSON et al., 1999). Biologie Marine, 34: 447-495. We therefore propose that the ESU and, even more so, the MU concepts be extended to al so include significant differ­ BENGTSSON, J., JONES, H. & SETÂLÂ, H. , 1997. The value of biodi ­ versity. Tre11ds i11 Ecology and Evo/111io11 , 12: 334-336. ences for other than purely molecular traits. In this way, con­ servation managers may, for example, more appropriately BERGER, V.Y. , PAKHOMOV,A.N. & MUKHLENOV,A.G., 1975 . Study consider ecologically relevant variation, which according to of isozyme composition of esterases and lactate dehydrogenase dur­ recent studies seems increasingly important in the formation ing adaptation of lit1ori11a lit1orea (L.) (Gastropoda) to the changes of species. Indeed it is being argued that ecological separa­ in water salinity. Zlwmal Obshchei Biologii, 36: 579-584. [i n Rus­ sian]. tion inay be as, or even more, important than geographical separation in the evolution of species (e.g. ÜRR & SMITH , BLACK, W.C. & DUTEAU, N.M., 1997. RAPD-PCR and SSCP 1998; MORELL, 1999). However, the logical and statistical analysis fo r insect population genetic studies. Pp. 361-373 in framework for extending the ESU and MU concepts still CRAMPTON, J.M., BEARD, C.B. & LOUIS, C. (eds.), Molecular biol­ needs to be established and entails a new difficulty, viz. ogy of insect disease vectors: a methods manual. Chapman & Hall, whether or not, and how, to distinguish between genetic and London. plastic phenotypic vari ation. This issue will undoubtedly be­ BOWDITCH, B.M., ALBRIGHT, D.G., WILLIAMS, J.G. K. & BRAU N, come more important as soon as attention is focused more on M.J ., 1993. Use of randomly amplified polymorphie DNA markers quantitative geneti cs, a topic which until recently has been in comparati ve genome studies. Methods in Enzymology, 224: 294- neglected far too much in (marine) conservation biology 309. (STORFER, 1996). BRITTON, J.C., 1995. The relati onshi p between position on shore and shell ornamentati on in two size-dependent morphotypes of li11ori11a stria ra, with an estimate of evaporati ve water Joss in these ACKNOWLEDGEMENTS morphotypes and in Melarhaphe neriroides. Hydrobiologia, 309: 129- 142. We are indebted to J.C. BRITTON (Texas Christi an Univer­ sity) for reviewing the manuscript, to Cl. M ASS IN (RBINS) CALEY, K.J ., GRAH AM E, J. & MILL, P.J ., 1995. A geographically­ based study of shell shape in small rough periwinkles. fo r correcting the French résumé and to H. V AN PAESSCHEN Hydrobiologia, 309: 181-193. (RBINS) for the artwork. 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