BARNACLE-DWELLING EGOTYPES OF THREE BRITISH LITTORINA SPECIES AND THE STATUS OF LITTORINA NEGLECTA BEAN

On the basis of collections from SW Wales and W Ireland it isshown that small (<5 mm) black and/or white Littorina occurringin the barnacle zone on exposed shores, of the L. neglecta shelltype, comprise three species. From their reproductive anatomythese correspond to L. saxatilis, L. arcana and L. nigrolineata,and are interpreted as analogous barnacle-dwelling ecotypesof these species. This adds to the evidence that L. neglectais not a distinct species, but an ecotype of L. saxatilis. (Received 1 June 1992; accepted 23 June 1992)     

A geographically-based study of shell shape in small rough periwinkles

A study using principal component analysis and discriminant analysis was carried out on shell shape variation in 3093 specimens of rough periwinkles, 2500 of which were below 5.5 mm in columella length, from around the North Atlantic. Using a combination of colour plus sculpture, and life history trait, the snails were classified by inspection and examination into Littorina nigrolineata, L. arcana, L. saxatilis and L. neglecta. Principal component analyses indicated that similar aspects of variation were important in the different taxa, but these were sometimes of differing levels of importance between L. saxatilis and L. neglecta. Crossvalidation in a discriminant analysis showed classification of shells larger than 5.5 mm to have at least an 88% accuracy. That of shells below 5.5 mm showed an accuracy of 49% in L. arcana, increasing to 54% in L. saxatilis and 63% in L. neglecta, with 76% accuracy for small L. nigrolineata. This last was a special case as only one site was sampled, therefore comparative data are not available. This geographically-based study reveals that L. neglecta is more homogeneous over its range than recently reported by other workers and shows greater differences from L. saxatilis than the latter does from either L. nigrolineata or L. arcana. Size effects do not account for these differences because L. neglecta is morphometrically distinct from both large and small L. saxatilis. Furthermore, small, mid-shore L. saxatilis classify with large high-shore L. saxatilis in discriminant analysis, not with L. neglecta. These results provide evidence that the taxon L. neglecta is more distinct than has sometimes been suggested.     

THE GENETIC RELATIONSHIPS OF THREE TAXA IN THE LITTORINA SAXATILIS SPECIES COMPLEX (PROSOBRANCHIA: LITTORINIDAE)

Three species in the Littorina saxatilis complex (the ovoviviparousL. saxatilis and the two oviparous species L. arcana and L.nigrolineata) were screened for the products of up to 22 lociusing gel elec-trophoresis. Analysis of allele frequencies andderived genetic distances showed that, with rare exceptionsin L. saxatilis and L. arcana, conspecific populations clusteredtogether. Sympatric pairs of populations showed significantdifferentiation at many polymorphic loci. The results confirmedthe biological reality of the three taxa, and did not suggestthe existence of any previously unrecognized species. The speciesare genetically closely related to each other, with Nei geneticdistances ranging from 0.035 to 0.083. (Received 24 October 1989; accepted 18 April 1990)     

Hybridisation in the Littorina saxatilis species complex (Prosobranchia : Mollusca)

This paper details attempts to breed members of the L. saxatilis species complex in the laboratory and to construct interspecific hybrids between them. Success in reciprocal crosses between animals from the type localities of the species of Littorina saxatilis (Olivi) and Littorina rudis (Maton) indicates that these two taxa are synonymous. Six of the twelve possible reciprocal hybridisation crosses between the four species (L. saxatilis, L. arcana Hannaford Ellis, L. nigrolineata Gray and L. neglecta Bean) have been attempted, with only one proving successful, that between male L. saxatilis and female L. arcana. This hybrid cross produces viable offspring, although at a lower frequency than either of the parental crosses in the laboratory, while the reciprocal cross has, as yet, proved unsuccessful. Limited work on the F1 hybrids shows them to be inter-fertile and also capable of backcrossing with male L. saxatilis, but not with female L. saxatilis or L. arcana. Details are also given of attempts to find natural hybrids in sympatric populations of the species of the complex, using gel electrophoresis.     

Distribution of the species of rough periwinkle (Littorina) in Great Britain

The status of the four currently recognised species in the Littorina saxatilis species-complex, i.e. Littorina nigrolineata Gray, L. arcana Hannaford Ellis, L. saxatilis (Olivi) and L. neglecta Bean is reviewed briefly, with notes on their characteristic features and location on the shore. Since the taxonomy of these rough periwinkles has only become stable relatively recently much of the previously published information on distribution is of little or no use. In this paper their distribution around the coastline of England and Wales (with some notes on Scotland) is described and discussed. L. saxatilis is found in a wide range of habitats from exposed peninsulas to estuaries on all shores where there is a suitable rocky or stony substrate; also in salt marsh pools. L. arcana has a more restricted distribution and is notably absent along much of the south English coast, central Cardigan Bay and possibly northern Scotland; it is not found in estuaries. L. nigrolineata has an even more restricted distribution, although it occurs both on exposed coasts and in estuaries; it has only been found by us in one locality on the east coast. L. neglecta is probably fairly widely distributed but we have few details so far. The implications of the different patterns of distribution are discussed.     

ESTERASE VARIABILITY IN THE GASTROPODS LITTORINA SAXATILIS (OLIVI) AND L. ARCANA ELLIS

The esterase system has been studied using isoelectric focusingin two closely related rough periwinkles, Littorina saxatilisand L. arcana, in an investigation of possible differences betweenthe two species at this level of discrimination. Collectionsof both species were obtained from Yorkshire, north Cornwalland south Devon; L. saxatilis was also obtained from sites insouth Cornwall (in and near the estuary of the Tamar), Dorsetand Scotland, where L. arcana was not present. There is considerablevariation both within and between sites which tends to maskany overall differences between the two species. However, ingeneral there is a much greater degree of heterogeneity withinsamples of L. saxatilis than within samples of L: arcana whenboth species are found together; this is reflected in the ShannonWiener diversity index for bands. Also the number of esterasebands is greater for L. saxatilis than for L. arcana at eachof the five sites where both occurred. A dendrogram derivedfrom the percentage occurrence of each band present in eachsample produced a reasonable degree of separation of the twospecies (Received 8 January 1988; accepted 24 February 1988)     

A GENETIC ANALYSIS OF SYMPATRIC SUBPOPULATIONS OF THE SIBLING SPECIES LITTORINA SAXATILIS (OLIVI) AND LITTORINA ARCANA HANNAFORD ELLIS

A detailed analysis of shell characteristics (four parameters)and allozyme allele frequencies (eight to nine polymorphic loci)of sympatric Littorina saxatilis and Littorina arcana subpopulationsfrom Filey Brigg on the east coast of England supports theiridentification as closely related but separate species. Oneenzyme system, esterase-2, was nearly species diagnostic. Resultswere generally comparable with an earlier study of the two speciesfrom East Lothian, Scotland. Littorina saxatilis was again foundto be more variable than L. arcana. (Received 14 December 1983;     

A potential species-specific molecular marker suggests interspecific hybridization between sibling species Littorina arcana and L. saxatilis (Mollusca, Caenogastropoda) in natural populations

Three sister species of rough periwinkles, viz. Littorina saxatilis (Olivi 1792), L. arcana (Hannaford Ellis 1978) and L. compressa (Jeffreys 1865) from the Barents Sea (Russia), the White Sea (Russia) and the Norwegian Sea (Norway) were studied. The identification of two sibling species L. saxatilis and L. arcana is often difficult as both species have extremely similar shell morphology and reproductive systems. Only mature females can be unambiguously distinguished, with a jelly gland present in female L. arcana, but which is replaced by a brood pouch containing developing embryos in L. saxatilis. No clear-cut diagnostic features have been found to discriminate between males or juveniles of the two species. The very first diagnostic DNA marker (DNA fragment A2.8, 271 bp length) for L. arcana and L. saxatilis separation was developed. The marker was derived from apparently species-specific L. arcana DNA fragments obtained via Random Amplified Polymorphic DNA (RAPD) analysis. This fragment was cloned and sequenced, whereupon specific primers were designed and the amplification was surveyed in a large number of morphologically well-identified females of both species. Subsequently, the specific DNA marker was used for the identification of male L. arcana and partners in copulating pairs. In this way, we obtained evidence of possible interspecific hybridization between the sibling species L. arcana and L. saxatilis living in sympatry in natural populations: the presence of A2.8 fragment in 12% of morphologically well identified L. saxatilis females and its absence in 14% of morphologically well identified L. arcana females. The A2.8 fragment never amplified in L. saxatilis from sites without L. arcana. The A2.8 fragment did not amplify in L. compressa, not even in microsympatric populations, and we did not observe interspecific copulations between L. arcana and L. compressa.     

Microsatellite cross-species amplification in the genus Littorina and detection of null alleles in Littorina saxatilis

Microsatellite DNA is widely used as population genetic marker,but the cost of using microsatellites is high, as they usuallyneed to be developed and optimized for each species separately.Cross-species amplification of microsatellites is thereforecommonly applied to bring down the cost, but it can also involvegenotyping errors. We studied cross-species amplification ofmicrosatellites in four species of the Atlantic group of Littorina(Neritrema): L. saxatilis (Olivi, 1792), L. obtusata (Linnaeus,1758), L. fabalis (Turton, 1825) and L. arcana Hannaford Ellis,1978 to investigate whether markers originally developed fora more distantly related Pacific species [L. subrotundata (Carpenter,1864)] suffered from more amplification problems than markersdeveloped for one of the species in the Atlantic group (L. saxatilis).We also compared variation in amplification success among thespecies and among different regions in the NE Atlantic. Approximatelyhalf of the 12 primers developed for L. subrotundata and theseven primers developed for L. saxatilis were successfully amplifiedin other species of the subgenus. The success was dependenton phylogenetic distance among species within the subgenus.On the other hand, the variation in performance of the locibetween geographically remote populations of the same specieswas as high as variation among the species. In earlier studiesstatistical analyses indicated that several loci showed a heterozygotedeficiency due to null alleles. The presence of null alleleswas confirmed by a segregation analysis of the microsatelliteloci in eight half-sib families of L. saxatilis. (Received 2 April 2007; accepted 19 November 2007)     

Detecting shape differences in species of the Littorina saxatilis complex by morphometric analysis

We investigated variation in shell size and shape of marinesnail species of the Littorina saxatilis complex (L. saxatilis,L. compressa and L. arcana) using geometric morphometric methods.These morphologically similar periwinkle species that are commonin the European intertidal have presented many problems fordiagnosis based on morphology alone. A discriminant analysisdemonstrated that geometric morphometrics is very efficientfor diagnosing individuals to species among sympatric populations.We successfully diagnosed an average of 96% of the specimens(with 85.7–100% correct diagnosis for specific comparisons).The diagnosis capability of this method is absolute at the populationlevel. This makes the technique potentially useful for the designof manipulative field or laboratory experiments. Moreover, ageometric-morphometric analysis was also accomplished in twosnail ecotypes (H and M) of L. saxatilis from rocky shores ofNE England which are apparently adapted to different degreesof wave exposure. We found that the H (exposed) ecotype hasa relatively rounded shell shape with a bigger aperture, whereasthe M (protected) ecotype has a smaller aperture. (Received 23 March 2006; accepted 25 January 2007)     

Resource allocation,demography and the radiation of life histories in rough periwinkles (Gastopoda)

Applicability of life-history theory to higher levels of comparison (from populations, through ecotypes to sibling species) was investigated in rough periwinkles, whose life histories have diversified since colonization of the North Atlantic by an oviparous ancestor in the upper Pliocene. Comparisons were made among populations of the ovoviviparous Littorina saxatilis, between L. saxatilis and its ecotype, L. neglecta (with an annual life history) and between the sibling species L. saxatilis and L. arcana, the latter of which retains the ancestral oviparity. Resource-allocation priority, reproductive effort and related trade offs were compared between the ecotypes and the sibling species by measuring changes in flesh mass and reproductive output in snails subjected to different degrees of food deprivation, and by measuring mortality rate of snails stressed by desiccation, high temperature and low salinity. Body size had a marked effect on all parameters, but after statistically removing this effect there remained no significant differences in allocation among ecotypes or species. Published demographical data were reviewed for correlations between habitat, mortality regime and life-history characteristics. Populations of L. saxatilis varied principally in size at birth and in adult size. Theoretical premises based on density-dependent versus density-independent mortality regimes could not explain these trends. Instead, size at birth may have reflected the mechanical, physiological or biological nature of mortality risk rather than its density dependence or independence. Adult size reflected the available sizes of crevices used for shelter and perhaps also the quality of feeding conditions. Radiation of life histories within the rough periwinkles is interpreted as a series of adaptations to a progressively wider range of habitats. The transition from oviparity to ovoviviparity allows colonization of estuaries, saltmarshes and pebble beaches too hazardous for naked egg masses. The transition from a perennial to an annual life history in barnacle ecotypes follows from allometric re-scaling of morphological and physiological parameters, enabling reproduction and brooding to occur at the small body size necessary for life within empty barnacle tests. This suite of adaptations allows exploitation of a relatively benign microhabitat that occurs almost ubiquitously on exposed rocky shores of the temperate North Atlantic. The persistence of oviparous forms, presumably in the face of competition from sympatric ovoviviparous forms, remains unexplained.     

Different growth responses of C3 and C4 grasses to seasonal water and nitrogen regimes and competition in a pot experiment

Understanding temporal niche separation between C₃ and C₄ species(e.g. C₃ species flourishing in a cool spring and autumn whileC₄ species being more active in a hot summer) is essential forexploring the mechanism for their co-existence. Two parallelpot experiments were conducted, with one focusing on water andthe other on nitrogen (N), to examine growth responses to wateror nitrogen (N) seasonality and competition of two co-existingspecies Leymus chinensis (C₃ grass) and Chloris virgata (C₄grass) in a grassland. The two species were planted in eithermonoculture (two individuals of one species per pot) or a mixture(two individuals including one L. chinensis and one C. virgataper pot) under three different water or N seasonality regimes,i.e. the average model (AM) with water or N evenly distributedover the growing season, the one-peak model (OPM) with morewater or N in the summer than in the spring and autumn, andthe two-peak model (TPM) with more water or N in the springand autumn than in the summer. Seasonal water regimes significantlyaffected biomass in L. chinensis but not in C. virgata, whileN seasonality impacted biomass and relative growth rate of bothspecies over the growing season. L. chinensis accumulated morebiomass under the AM and TPM than OPM water or N treatments.Final biomass of C. virgata was less impacted by water and Nseasonality than that of L. chinensis. Interspecific competitionsignificantly decreased final biomass in L. chinensis but notin C. virgata, suggesting an asymmetric competition betweenthe two species. The magnitude of interspecific competitionvaried with water and N seasonality. Changes in productivityand competition balance of L. chinensis and C. virgata undershifting seasonal water and N availabilities suggest a contributionof seasonal variability in precipitation and N to the temporalniche separation between C₃ and C₄ species. Key words: Chloris virgata, competition, growth, Leymus chinensis, nitrogen seasonality, water seasonality Received 19 November 2007; Revised 29 January 2008 Accepted 4 February 2008     

Micro-spatial distribution of two sibling periwinkle species across the intertidal indicates hybrdization

Populations of periwinkles Littorina saxatilis (Olivi 1792) and L. arcana Hannaford Ellis, 1978 are well suited for microevolutionary studies, being at the same time closely related and intraspecifically diverse. The divergence between these two sibling species, sympatric over large parts of their distribution areas, is small, the only morphological difference being the pallial gland complex structure in females. Molecular identification is possible with the use of a RAPD nuclear marker (cloned A2.8 DNA fragment) typical for L. arcana. However, in some individuals from sympatric populations molecular and morphological criteria suggest conflicting species affiliation, which may be explained either by hybridization or by shared ancestral polymorphism. We tested the hybridization hypotheses examining the micro-spatial distribution of these two species across the intertidal zone in two distant sites at the Barents Sea. We found that (a) the frequency of putative hybrids in sympatric populations was proportional to the frequency of L. arcana; (b) L. saxatilis bearing A2.8 DNA fragment were almost absent in the lower part of the intertidal zone, where L. arcana was absent too; (c) there was a close positive correlation between the distribution of potential parent molluscs and putative hybrids. Moreover, logistic regression models showed a good agreement between the distribution of putative hybrid frequencies and that of parental species frequencies. All our observations taken together support the hypothesis of hybridization between L. saxatilis and L. arcana. Elucidating the mechanisms that support the species status of these sympatric populations is necessary.     

POPULATION GENETICS OF A SNAIL SPECIES COMPLEX IN THE BRITISH ISLES: LITTORINA SAXATILIS (OLIVI), L. NEGLECTA BEAN AND L. TENEBROSA (MONTAGU), USING SSCP ANALYSIS OF CYTOCHROME-B GENE FRAGMENTS

The intertidal snail Littorina saxatilis displays a range ofshell morphologies associated with a variety of habitats. Sincemorphology has an environmental and genetic basis, shell-basedtaxonomy may not accurately reflect genetic relationships. Weexplored genetic structure among adjacent populations of L.saxatilis (the robust open-shore type), L. neglecta (the tinybarnacle-dwelling type) and L. tenebrosa (the fragile brackish-watertype), at nine sites in Britain. Using single-strand conformationalpolymorphism analysis of a 375bp fragment of cytochrome-b wefound no evidence of species distinction. In AMOVA tests significantvariation was contained among populations (68%) and among individuals(32%, both P < 0.001), and insignificant variation was foundamong ecotypes. Genetic patterns suggested gene flow among ecotypesover small scales and a strong random input over larger scales. ^* To whom correspondence should be addressed. (Received 27 March 2000; accepted 10 July 2000)     

Breeding biology of Tenagomysis tasmaniae Fenton,Anisomysis mixta australis (Zimmer) and Paramesopodopsis rufa Fenton from south-eastern Tasmania (Crustacea: Mysidacea)

The number, size and developmental stage of young in the brood pouch of female Tenagomysis tasmaniae, Anisomysis mixta australis and Paramesopodopsis rufa was recorded throughout the year. Breeding was intensive from spring till the end of autumn for the three species. Calculation of the egg ratio for each species showed that their major reproductive peaks occurred during spring and summer. A winter depression in the breeding cycle was observed for T. tasmaniae and P. rufa, but A. mixta australis ceased breeding during winter. Seasonal variation in the length of gravid females and number of young carried was evident for these three species. Females were longer in spring and summer and carried more young than in autumn and winter. A linear relationship between female length and brood size was demonstrated for each species; annual and seasonal equations were calculated for females carrying each developmental stage. The seasonal equations showed that for a female of given length fecundity was greater during spring than any other season. Natality was estimated to be highest during late spring, summer and early autumn for the three species. No seasonal variation in the size of eggs was evident for the three species. The reproduction pattern of T. tasmaniae, A. mixta australis and P. rufa appears to be very similar to that reported for the majority of iteroparous coastal temperate mysids throughout the world.     

Linking ecology,morphology, and metabolism: Niche differentiation in sympatric populations of closely related species of the genus Littorina (Neritrema)

Divergence of ecological niches in phylogenetically closely related species indicates the importance of ecology in speciation, especially for sympatric species are considered. Such ecological diversification provides an advantage of alleviating interspecies competition and promotes more efficient exploitation of environmental resources, thus being a basis for ecological speciation. We analyzed a group of closely related species from the subgenus Neritrema (genus Littorina, Caenogastropoda) from the gravel‐bouldery shores. In two distant sites at the Barents and Norwegian Sea, we examined the patterns of snail distribution during low tide (quantitative sampling stratified by intertidal level, presence of macrophytes, macrophyte species, and position on them), shell shape and its variability (geometric morphometrics), and metabolic characteristics (metabolomic profiling). The studied species diversified microbiotopes, which imply an important role of ecological specification in the recent evolution of this group. The only exception to this trend was the species pair L. arcana / L. saxatilis, which is specifically discussed. The ecological divergence was accompanied by differences in shell shape and metabolomic characteristics. Significant differences were found between L. obtusata versus L. fabalis and L. saxatilis / L. arcana versus L. compressa both in shell morphology and in metabolomes. L. saxatilis demonstrated a clear variability depending on intertidal level which corresponds to a shift in conditions within the occupied microhabitat. Interestingly, the differences between L. arcana (inhabiting the upper intertidal level) and L. compressa (inhabiting the lower one) were analogous to those between the upper and lower fractions of L. saxatilis. No significant level‐dependent changes were found between the upper and lower fractions of L. obtusata, most probably due to habitat amelioration by fucoid macroalgae. All these results are discussed in the contexts of the role of ecology in speciation, ecological niche dynamics and conservatism, and evolutionary history of the Neritrema species.     

THE LIFE CYCLE, POPULATION DYNAMICS, GROWTH AND SECONDARY PRODUCTION OF THE SNAIL BRADYBAENA FRUTICUM (MULLER, 1774) (GASTROPODA PULMONATA) IN NORTHERN GREECE

The life cycle, population dynamics, growth and secondary productionof the pulmonate land snail Bradybaena fruticum were studiedin northern Greece. The demographic analysis of the populationsof B. fruticum revealed that a) two cohorts exist in the fieldthroughout the year b) the reproductive period started in thebeginning of summer and c) growth was increased during springand autumn. According to von Bertallanffy' method B. fruticum needs 5 yearsto attain its maximum size in the field of 25.40 mm. Mortalityrate increases and life expectancy decreases with increasingage. Net reproductive rate (Ro) was equal to 3. 15 and per capitarate of increase (r^c) was equal to 1. Annual secondary production, calculated by Hynes' size frequencymethod, revealed a mean annual density of 5.9 individuals/m²,a mean standing crop (B) of 8.09 g/m²/year and an annual production(P) of 1.92 ± 0.11g/m²/year. Annual turnover ratio (P/B)was equal to 2.37. (Received 25 May 1989; accepted 17 March 1989)     

MORPHOLOGICAL DIVERGENCE AND EVIDENCE FOR REPRODUCTIVE ISOLATION IN LITTORINA SAXATILIS(OLIVI) IN NORTHEAST ENGLAND

Current work at Old Peak (Ravenscar) on the northeast coastof England indicates that boulder-dwelling Lutonna saxatilis(Olvi) comprise at least two distinct forms, inhabiting high-and mid-shore boulders Using multivariate morphometrics we showthat these forms are significantly different in shape. Rareintermediates between these two distinct forms were occasionallyfound, usually with the low shore animals The two forms showdifferent reproductive strategies. High shore females (L. saxatilisH) mature at a smaller size and produce a smaller number oflarge eggs giving rise to large crawlaways The mid-shore form(L. saxatilis M) matures at a larger size and produces a largenumber of small eggs which give rise to small crawlaways. Anovel observation is that intermediate females (L. saxatilisI) contain eggs of both sizes, giving rise to a bimodal distributionof egg sizes within the brood pouch. The usual incidence ofabortion (observed in H and M forms) is Very low (<1%) However,in the intermediates the large eggs showed a high incidenceof abortion (about 60%) and the incidence of abortion in thesmall eggs was also increased (about 30%) Thus there appearsto be a partial reproductive barrier between the two forms,presumably resulting in reduced fitness due in the intermediatesto reproductive dysfunction. These findings extend to othershores in the region, over total distance of about 66 km. Thefacts are suggestive of a hitherto unrecognised reproductivebarrier with L. saxatilis, the nature and strength of whichis under investigation ^*Present address: University College Scarborough. Filey Road.Scarborough Yo11 3AZ. (Received 12 June 1995; accepted 15 September 1995)