Hydnibinlosui .165: 1-11. IWS. J. L. Norenhurg & P. Hoe {eels), Fourth international Conference on Nemerteait Biology. ? 1998 Kiutver Academic Publishers. Printed in Belgium. Genetic differentiation of populations of the common intertidal nemerteans Lineus ruber and Lineus viridis (Nemertea, Anopla) Alex D. Rogers1*, John P. Thorpe1, Ray Gibson2 & Jon L. Norenburg3 1 Port Erin Marine Laboratory, University of Liverpool. Port Erin, hie of Man, IM9 6JA. U.K. - Liverpool John Moores University, School of Biological and Earth Sciences, Byrom Street, Liverpool L3 3AF, U.K. ?* Department of Invertebrate Zoology, National Museum of Natural History, The Smithsonian Institution, Washington. DC 20560, U.S.A. * Present Address: Division of Biodiversity and Ecology, University of Southampton. Biomedical Sciences Building, Bassett Crescent East, Southampton, S016 7PK, U.K. Key words: Heteronemertea, genetic differentiation. North Atlantic Abstract Specimens of ihe common intertidal nemerteans Lineus ruber and L. viridis were collected from sites along the west and Southwest coasts of Britain, northern France and North America, Allcle frequencies of up to 13 putative enzyme loci were estimated for all populations of L. ruber and L. viridis. Estimates of genetic variation were low for populations of L. ruber (A/?hS 0.008-0.052) but were higher for populations of L. viridis (//0iii 0.068-0.153). Exact tests for conformity of observed genotype frequencies to those expected under Hardy-Weinberg equilibrium failed to delect significant deviations for L. ruber or L viridis. Z7-statistics were affected by small sample size and low expected values in some populations, but, F$j was significantly different from zero for most loci examined for both Lineus ruber and L. viridis. This indicated a significant degree of population structuring for both species (only a moderate level of genc-fiow). Intraspeeifie comparisons of genetic distance and genetic identity showed little evidence of genetic differentiation between populations separated by large geographic distances (1000s of km). There was no apparent relationship between genetic distance between populations and the geographic distance separating them. Possible explanations for this lack of genetic differentiation between populations of L. ruber and L. viridis arc discussed. These include a lack of variation in the enzyme loci sampled caused by population dynamics, balancing selection in the enzyme loci sampled, large introductions between populations and passive dispersal. Introduction The anoplan nemerteans Lineus ruber (Miiller, 1774) and L viridis (Miiller, 1774) are commonly found in a variety of intertidal habitats on the coasts of north- ern Europe and North America (Gibson, 1982: Nord- hausen, 1988; Thiel & Reise, 1993). L. ruber has also been reported from the Mediterranean, the North Sea, the Pacific coast of North America, Madeira, Green- land, Iceland, the Faroes, Siberia and South Africa (Gibson, 1982). The vertical distribution of these two species appears to vary according to habitat availabil- ity and season (Rogers et ah, 1995). Both species are predators and feed upon a variety of polychaetes, crus- taceans and molluscs (McDermott& Roe, 1985; Nord- hausen, 1988; Thiel & Reise, 1993). Lineus ruber generally reproduces sexually in late winter to late spring. The exact time varies accord- ing to geographic locality. In the Gulf of Maine it is reported to reproduce in March/April (Coe, 1899; Ris- er, 1974), in the Barents sea July-August (Schmidt & Jankovskaia, 1938) and in the English Channel, Dec em ber-January (Ox ner, 1911; Gontcharoff, 1951; Bierne, 1970). Under laboratory conditions the sea- . ' *??;" .;? ??;. ti'- X sonality of reproduction in L. rwier disappears rapidly (Bierne, 1970; Vernet & Bierne, 1993). It is therefore likely that environmental factors influence the timing and length of the reproductive cycle in this species (Vernet & Bierne, 1993). During reproduction in Lineus ruber, a male and female enclose themselves in a gelatinous mucous cocoon, secreted by the female and firmly attached to a rock (Gontcharoff, 1951). The male releases sperm of a modified type (Franzen, 1983) and these fer- tilise the eggs that are released into the mucus cocoon. Gontcharoff (1951) observed sperm in the gonoducts and ovaries of L ruber which probably act as recep- tacula seminalis (Cantell, 1975). Reproauction in L. ruber may be regarded as a form of pseudocopula- tion (Riser, 1974; Franzen, 1983). The adults leave the cocoon after reproduction and die 2-3 weeks later (Riser, 1974), Lineus ruber has encapsulated larvae (Desor larva) of which only 12-13% hatch within the mucus cocoon (Schmidt, 1946) (approximately 10-15 larvae accord- ing to Gibson [1972]). The remaining larvae/eggs are eaten by the hatchlings which then leave the cocoon as miniature worms (Schmidt, 1946; Gontcharoff, 1951; Gibson, 1972; Bierne, 1983). The juvenile worms feed immediately on leaving the cocoon and initially show a weak positive or more usually indifferent response to light that rapidly develops into a strong negative phototaxis (Gontcharoff, 1951). Lineus viridis shows a similar type of reproduction to L. ruber but larval development in the two species is different. In L viridis the first larvae to hatch do not devour the rest. As a result 400-500 juvenile worms hatch from each mucus egg string. Also in contrast to L rw&er,juvenileL. viridis are strongly photo tactic and do not feed for 2-3 weeks after hatching (Gontcharoff, 1951; Gibson, 1982). Both Lineus ruber and L. viridis are small inver- tebrates with limited powers of locomotory dispersal as adults. Both exhibit a form of pseudocopulation and direct development with a very limited capacity for lar- val dispersal, though L viridis is more fecund than L ruber. It is conceivable that the behaviour of juvenile L viridis after hatching may make it more likely that they will be carried up in to the water column than those of L. ruber. It would be expected that with such a life history geographically separated populations of both species would show marked genetic differentia- tion due to poor gene flow (see Burton, 1983; Wapies, 1987; Ward, 1989; Piertney & Carvaiho, 1994). Gene flow is defined as genetically effective migration, i.e. an exchange between conspecific populations of suc- cessfully fertilising gametes or individuals that survive to reproduce in the population into which they have migrated (Hedgecock, 1986). In this study levels of genetic differentiation of geo- graphically separated populations of Lineus ruber and L. viridis are estimated using starch gel electrophoresis of allozymes. The populations for which differentia- tion is estimated are located on the west coast of the Britain, northern France and the east coast of North America and are separated at spatial scales of tens to thousands of kilometres. Allozyme electrophoresis has been used previously to investigate the systematics of nemerteans (Cantell & Gidholm, 1977; Williams et al., 1983; Sundberg & Janson, 1988; Rogers, 1993; Rogers et al., 1993, 1995) but this study is the first to consider intraspecific population differentiation within the phylum. Materials and methods Samples Various nemerteans were collected between 1989 and 1992 from around the west and Southwest coasts of Britain, northern France and the east coast of North America, The sites are listed in Table 1 along with the numbers of Lineus ruber and L. viridis collected from each site (see also Rogers et al.. 1995 for map). All specimens were collected intertidally beneath stones and rocks lying in silt, muddy sand, sand or fine shelly gravel. Specimens were carefully removed from the substratum and transported back to Port Erin Marine Laboratory, Isle of Man. Specimens were sub- sequently kept in aerated plastic tanks, in which they were starved prior to electrophoresis. Electrophoresis For electrophoresis 5-15 mm of tissue was removed from the posterior end of each specimen. Specimens were alive to prevent possible reduction of enzyme activity that may have resulted from using frozen tissue (Scozzani et al., 1980). The tissue was homogenised, over ice, in 100 (i\ of 0.06M Tris-HCl pH 8.0 using the tip of a glass rod. The homogenate was absorbed on to filter paper wicks (Whatman No. 3) and applied to a starch gel. Horizontal starch gel electrophoresis was per- formed by standard methods (see Harris & Hopkinson, 1978; Murphy et al., 1990) using 12.5% starch gels (Sigma Chemical Co. Ltd, Poole, Dorset). Two buffer Tahia t. Numbers of Linens ruber and L. vtridis sampled at sites in Britain, France and North A murk::) Site Lai/Long Site eode Line us vuher Linens viridis United States of America Mount Desert Island 44?22'N, 68 O ? d do? d d d SiiSiiiiii3iiiiiS?S SII?3IIIIISIIIII I i I i I I S SJII s 5 i i i i & i i t i o\ O 1 1 o> = 1 ? o = 3 ic ^ , g 8 i o I I I I I o I I I I I 1 I c i I id d d I I I I ! d \C m i i o i i i i i i 8 i ? i i i i i S 5 I I ( I I I I I 3 ? i q e> I ! I I i S Si I I s I I ov I I d o I c I I I I i 3 cr> o d ? d d d o 2 5 I ^ I I r IOEOI i i I I ? d 8 8 I rl I ? 1 I d ? I ? d S i i 5 Pi 2 s o o 3 do ? a I i s I ? i I i i I I I ISI i i i i i i S S i S s o> 3 i i i S ? -J-idddd ? c = 9 ? 1 1 1 I z g 2 a i i IOI i i o. 3 i 5 i d d -i ? 2 3 i i i i 3 5 ? 3 rj 5 3 5 c IT. o 3 ? ? d d d ~ i 5 i r S i i i i i i t I C 1 I ?g \D 2 a . S i as c q I I I I 5 is i i ? oo S odd ? ? d d i 5 I I I 3* d a 3 3 i i 5 i i o I i i i o ? i c i i o i i i ? 3 i 8 i S d d ? ? ~ a 3 i ? rj f*. T v"; i ?< rv| ? n ? fir^^fl^r^M-.^isn-.M ? n ui \o ? r] rr, -r v^ = T= "3 3 .^vM4Silrf* < E >? s 5 Tj- s z 1 o " o 1 g o ? ? ? o a ? 1 o c s , c , d 3 i o c o c o o ? 1 t 1 o 1 1-1 c o 8 c o o I I I I I I I I r*1 r- \o m i i r- i ? t i i IOI i i d d d d * i es I I ii i IIO d d d ? c c c r- ^ r^ a n 3C ^ ff> ? ? o ooooo d oo <-, ic is i o\ 5 3 2 ? o I en d d d dddd d d 8? t-1 ? Tf I o o I I r- I <\i odd d d O 2 < d d d d i ft i i s s I I I I I I I 00 I 1-J I I I I I I I I d d Z c I v. d d d d d c d i i i i i i i S i 3 i i i i ? s d d i "i d s I I I I I I I on i ? I l I d d d I f d I I I I i i o I I I I I z S- d rl I d i ?+ i ? i i I I I I I i i S i c I I I I Z CO O i ? ai ffi O ?i <- j i~i t? un ^o 73 between Nei's (1972) D between populations and geo- graphic distance separating populations for L. viridis and L ruber using the programme Minitab (Minitab, 1989). Results Allele frequencies for all enzyme loci that produced well-resolved staining patterns consistent with known subunit structures for all populations of Linens ruber and L. viridis are given in Table 2, Allele frequen- cies are very similar for intraspeciric comparisons but are significantly different between the two species (see Rogers et al., 1993,1995). Observed heterozygosities, which ranged from 0.008-0.052 (Table 3) were gener- ally low for L ruber compared with those recorded for other eukaryotes (see Nevo, 1978; Nevo et al., 1984). Note that the highest of these values came from the French site that had a very low sample size (7 individ- uals). Higher observed heterozygosities were recorded for populations of L. viridis with a range of 0.068- 0.153. This range is comparable to that found in other eukaryotes (Nevo, 1978; Nevo et al., 1984), Fisher's (1935) Exact Test for deviation from Hardy-Weinberg expectations, with significance levels adjusted for multiple tests, showed no significant devi- ations from expected genotype frequencies for popu- lations of Lineus ruber or L viridis. F-statistics for toci variable across all populations for Lineus ruber and L viridis in which ;V > 25, are given in Table 4, For L. ruber two out of nine loci showed highly significant Fis values. In both of these loci there was one common allele and 1-3 rare alleles. This may have led to small expected values in tests of significance for Fis, especially for Pgi'but may also indicate a degree of inbreeding within populations. Mean Fis for L ruber was not significant. FST values for populations of L. ruber were highly significant for five out of nine variable loci and signif- icant overall. The number of migrants per generation between populations (7Vem) indicated a moderate level of gene flow between populations. However, as with FJS, significance of FST was likely to have been influ- enced by low expected frequencies due to low genetic variation and low sample sizes in some populations. The only locus that showed substantial variation in L, ruber was Odh and it is notable that FST was highest for this enzyme locus. It is therefore likely that the degree of population structuring indicated by FST is underestimated. Although the genetic variability (mean heterozy- gosity) of the enzyme loci scored for L. viridis was higher than in L. ruber (especially for Odh), most enzyme loci still consisted of a single common allele with several rare alleles. For populations of L. viridis Fis values were significant for Got-I, Mdh-l andPgm but the overall Fts value was not significant. Most FST values for L viridis were highly significant, as was the overall FST value. Though the significance of FST values was probably influenced by low expected fre- quencies for rare alleles and low sample sizes at some sites, this will have been offset to some degree by the low variability of the loci studied (leading to an overestimate of gene flow). These results therefore do indicate a degree of population structuring in L. viridis. The number of migrants per generation between pop- ulations was lower in L. viridis than in L. ruber, this is probably the result of the difference in variability of the loci studied between the two species. Nei's (1972) genetic identity between populations of Lineus ruber is very high with a range of 1.000 to 0.996 (see Table 5). This lack of genetic differentiation is at least partially due to the low variability of the loci studied. Genetic identity and distance estimates for L. viridis suggest more structured populations for this species, but there is still very little genetic divergence across the geographic range sampled (/ = 0.957-0.995, D = 0.003-0.044; see Table 6). The correlation coef- ficient of genetic distance between populations and geographic distance separating populations indicated little relationship between these parameters (L ruber r - -0.366; forL viridis r = 0.009). Discussion Fisher's (1935) Exact Test on populations of L. ruber and L viridis failed to detect any significant deviations from genotype frequencies expected under Hardy- Weinberg equilibrium. Although this test takes into account small sample size, it does so by pooling geno- type frequencies and may therefore underestimate the significance of deviation from Hardy-We in berg expec- tations (Lessios, 1992). Fis values for Lineus ruber were affected by small sample size and low variability in the enzyme loci studied. Overall F,s values for L. viridis were not sig- nificant. FST values for Lineus ruber gave highly signifi- cant results for over half the loci examined. The mean FsT value was higher than that found over similar geo- graphic distances in several molluscan species with :%##; Table J. Mean observed Laid expected heterozygosity (under Hardy-Weinberg expectations) for popu- lations of Lineita ruber and L viridis. Mean sample size per locus and percentage of polymorphic loci (at 0.95 and 0.99 levels) are also given Population Mean sample Percentage Percentage Mean Mean size per polymorphic polymorphic observed expected locus loci (95%) loci (99%) heterozygosity heterozygosity L. ruber US 65.1 0.0 23.1 0.008 0.008 CAN 3.0 7.7 7.7 0.019 0.019 OBN 80.0 7.7 385 0029 0.032 PTE 13.0 7.7 15.4 0.018 0028 CST 380 15.4 30.8 0.031 0.038 LDN 45.2 9 1 45.5 0.036 O.04O ANG 70.7 0.0 15.4 0.009 0.009 FRA 7.0 30.7 30.7 0.052 0.075 L, viridis US 22.4 45.5 54.5 0.153 0.165 CAN 98 27.3 27.3 0.118 0.109 OBN 46.0 30.8 38.5 0.074 0.087 WTN 33 I 18.2 27.3 0.068 0.088 BRW 13.0 9 1 18.2 0.084 0.072 BNC 28.8 27.3 36.4 0.119 0.130 LDN 53.2 23.1 38.5 0.105 0.107 ANG 25.2 23.1 46.1 0.100 0.113 STA 107 1 182 45.5 0.123 0 102 PLY 18.5 36 4 36.4 0.086 0.095 WMY 18.3 27.3 45.5 0.118 0.133 FRA 7.0 27.3 27.3 0.09! 0.103 long-lived planktonic larvae (Levington & Suchanek, 1978; Johnson & Black, 1984; Benzie & Williams, 1992). This suggests that, as expected, L. ruber, a species with direct development, shows a lower gene flow between populations than in some species with planktonic larval phases (e.g., Burton, 1983; Hedge- cock, 1986; Waples, 1987; Hunt & Ayre, 1989; Ward, 1989). FsT values obtained for L ruber though, were not as high as those obtained for species with plank- tonic larvae that do show a degree of population dif- ferentiation (Smith & Potts, 1987; Macaranas et ah, 1992). FST values for Lineus viridis were significant at almost all loci and were higher than those found in L. ruber. It is initially surprising that gene flow is lower than that observed in L. ruber. L. viridis is expected to have a greater potential for dispersal than L. ruber, it is more fecund and its larvae are positively photo lactic and therefore more likely to become suspended in the water column (see Hagerman & Rieger, 1981; Dobbs & Vozarik, 1983) . However the differences in FST between L. ruber and L. viridis are not large and are probably confounded by the low variation of allozyme loci sampled for L. ruber (see below). While populations of Lineus viridis and L. ruber show some degree of differentiation, estimates of Net's (1972) genetic distance and identity between popula- tions show genetic cohesiveness over very large (trans- Atlantic) geographic distances. Other groups of inter- tidal marine invertebrates that have pelagic larvae have shown marked genetic differentiation between popula- tions located on the east and west sides of the North Atlantic ocean. In the cimpede, Semibalanus bal- anoides, allele frequency differences as the Pgi and Mpi loci indicated the presence of two populations in the North Atlantic (Flowerdew, 1983), one extending along the coasts of Europe from Spain in the south to Spitsbergen north of Norway, the other along the east coast of the USA through to Newfoundland and Ice- land. Studies on Mytilus edulis have shown differences Table 4. Summary of F-statistics at all loci for populations of Lineus ruber and L. viridis in which N > 25. " P < 0.01 Locus fis % FsT %e Lineus ruber Ap -0.028 -0.008 0.020" Got-1 -0 019 -0.007 0.012 Icd-l -0.025 -0.010 0.015" Icd-2 -0.011 -0.002 0.009 Mdh-l -0.048 -0.013 0.033** Mdh-l -0 010 -0.004 0.006 Odh 0.182" 0.236 0.065" Psi 0.416" 0.434 0.030" Pgm -0.017 -0.010 0.007 Mean 0.116 0.155 0.044" 543 Lintus viridis Got-1 -0.151" -0.035 0.100" Mdh-l 0.170** 0.301 0.158** Mdh-2 -0.051 -0.011 0.038 Odh 0.012 0.142 0.131" Pgd -0.035 -0.006 0.028" Pgi -0.036 -0.010 0.025** Pgm 0.166" 0.240 0.089" Mean 0.055 0.168 0.119" 1.85 in allele frequencies for leucine aminopeptidase (Lap), Ap, Pgi and Pgm between populations located on the east and west sides of the North Atlantic (Gosling, 1992). The population dynamics of Linens ruber and L. viridis may explain the low genetic distances between geographically distant populations. L ruber and L. viridis are short lived species with populations that are often small and numerically variable, over a period of years. Both these species are found generally under stones and other epibenthic materials (Gibson, 1982; Thiel & Reise, 1993) that may be disturbed frequently, especially during winter storms, possibly causing mor- tality of individual nemerteans and, from time to time, entire local populations (see Osman, 1977; Sousa, 1979, 1980). Other factors such as environmental tem- perature, predation and parasitisation may contribute to mortality in nemertean populations (Roe, 1976). While both species investigated apparently have a large geographic range, possibly consisting of a very large number of individuals, they are divided into many smaller populations over this range; as indicated by estimates of FST in this study. These smaller popula- tions may be affected by many extinction and recolo- nization events due to the nature of preferred habitat type of these species and by other factors. In such a case, extinction and recolonization will reduce the effective size and variation of the total species pop- ulation and reduce the divergence of subpopulations (Wright, 1940; Maruyama & Kimura. 1980; Slatkin, 1985, 1987). This would explain the genetic cohesion of populations of these two species. What is usually interpreted as a moderate level of gene flow may in this case be a suppression of genetic divergence by extinction of local populations. The only apparent difference in the biology of Linens ruber and L. viridis is that the former is less fecund than the latter. It would be expected that with all other things being equal this alone would increase the extinction rate and increase the time for popula- tion recovery in L ruber compared with L. viridis. L ruber would have a smaller effective (and probably actual) population size than L. viridis and would there- fore be expected to have a lower heterozygosity (see Kimura, 1983; Nei, 1987; Sole-Cava& Thorpe, 1991) which fits observations made in this investigation. It is interesting that the cryptic species found in very low numbers with L ruber and L viridis (Rogers et al., 1995) also has a very low heterozygosity. There are several other possible factors that may contribute to genetic homogeneity between popula- tions of Lineus ruber and Linens viridis. There is evi- dence that the inference of genetic structure of natural populations from the geographic distribution of alle- les derived from allozyme data is not as simple as has previously been suggested by the neutral gene theo- ry. Studies of several species of marine animals have shown that population structure can be resolved at dif- ferent levels by allozyme electrophoresis and by mole- cular techniques (see Gonzalez-Villasenor & Powers 1990; Burton & Lee, 1994). Furthermore, in at least one study, apparent geographic unity in allele frequen- cies has been contrasted with pronounced population subdivision indicated by restriction analysis of mito- chondrial DNA sequences (Karl & Avise, 1992). The most likely explanation for the lack of geographic vari- ation in allozyme frequencies in this case was thought to be balancing selection on protein coding loci coun- teracting genetic drift in a species with a subdivided population structure. The apparent lack of genetic differentiation in Lineus populations located on either side of the Atlantic could also be explained by the introduction of large numbers of individuals <'t these species from one side of the ocean to the other The lack of genetic differen- -.v?iV ?:,.'. >tg Table 5. Matrix of Nei's (1972) genetic identity (/) (above diagonal) and genetic distance (D) (below diagonal) for all populations of Lineus ruber Population US CAN OBN PTE CST LDN ANG FRA US - 0.999 0.999 0.999 0.998 0998 1.000 0.997 CAN 0.001 - 1.000 0.998 0.999 U.999 0.999 0.997 OBN 0.001 0.000 - 0,997 0.999 0.999 0.998 0.997 PTE 0.001 0.002 0.003 - 0.998 0.996 0.999 0.997 CST 0.002 0.001 0.001 0.002 - 0.999 0.998 0.996 LDN 0.002 0.001 0.001 0.004 0.001 - 0.997 0.996 ANG 0.000 0.00! 0.002 0.001 0.002 0.003 - 0997 FRA 0.003 0.003 0.003 0.003 0.004 0.003 0,004 - Table 6. Matrix of Nei's (1972) genetic identity (f) (above diagonal) and genetic distance (D) (below diagonal) for all populations of Liitcus viridh Population US CAN OBN WTN BRW BNC LDN ANG STA PLY WMY FRA US - 0.971 0.965 0.985 0.975 0.974 0.961 0.984 0989 0.970 0.992 0.973 CAN 0.030 - 0.984 0.991 0,991 0.972 0.978 0.988 0.981 0.985 0.983 0.990 OBN 0.036 0.016 - 0.985 0.977 0.957 0.987 0.981 0.987 0.968 0.976 0,987 WTN 0.015 0.009 0.015 - 0.997 0.982 0.981 0.990 0.992 0.988 0.995 0.988 BRW 0.025 0.009 0.023 0.003 - 0.987 0.979 0.985 0.981 0.994 0.987 0.983 BNC 0.026 0.028 0.044 0.018 0.013 - 0.971 0.974 0.971 0984 0.981 0.966 LDN 0.040 0.022 0.013 0.019 0.021 0030 - 0.976 0.972 0.966 0.970 0.977 ANG 0.016 0.012 0.019 0.010 0.015 0.027 0.025 - 0.988 0977 0.990 0.986 STA 0 011 0.019 0.013 0.008 0.019 0.030 0.028 0.012 - 0.973 0995 0986 PLY 0030 0.015 0.032 0.012 0.006 0.016 0035 0.023 0.027 - 0.979 0.977 WMY 0.008 0.017 0.024 0005 0.013 0.019 0.031 0.010 0.005 0,021 - 0.981 FRA 0.027 0.010 0.013 0012 0018 0.035 0.023 0.014 0.014 0.023 0.019 - tiation between populations of the cimpede Elminius modestus in Australia, New Zealand and Europe is thought to be due to such introductions (Dando, 1987). Large introductions of L ruber and L. viridis might have taken place by transport with other organisms, such as cultivated bivalves (see Christiansen & Thorn- sen, 1981), or as a component of fouling communities of ships {see Bertelsen & Ussing, 1936; Carlton, 1985). Passive dispersal by rafting may also maintain a mod- erate level of gene flow between geographically distant populations (see Highsmith, 1985; Edgar, 1987; Jok- iel 1989; DeVantier, 1992). The low level of gene flow maintained by such means may be all that is required to prevent genetic divergence between populations of L ruber&ndl. viridis (see Wright, 1940; Kimura, 1955; Slatkin, 1985; Maynard Smith, 1989). To summarise, the analysis of population structure in Lineus ruber and L viridis has detected a moder- ate level of genetic differentiation between populations at different spatial scales. This genetic differentiation appears to be unrelated to geographic separation of the populations studied. Many studies of genetic differen- tiation, in populations of marine invertebrates, have shown that the dispersal potential of larvae is often not realised (e.g., Todd et al? 1988). Few studies have shown genetic homogeneity over large distances where little dispersal is expected from examination of the life history of the species under investigation (e.g., Sole- Cava et al., 1985). The latter seems to be the case in the present study but genetic differentiation has prob- ably been underestimated due to the low variation of the loci detected or due to an inherent lack of reso- lution in alleles as a consequence of non-neutrality. In fact, since it is evident that the larval forms of L viridis and L. ruber are derived from the pelagic form of heteronemertean larvae, it is possible to argue thai L viridis and L. ruber demonstrate sequential grades of modification in reproductive biology that serve to minimise dispersal. 10 Acknowledgements We gratefully acknowledge the support of the Nat- ural Environment Research Council with grant number GR3/7I68. We would also like to thank Prof. Nathan P. Riser, Northeastern University's Marine Science Institute, Nahant. and Prof. Guy Verne t, Umversite de Reims Champagne Ardenne, for providing specimens from the U.S.A. and France respectively. Thanks are also due to Tim Hoiden and Eric McEvoy for assistance in the collection of specimens in the U.K. References Benzie, J. A. H. & S. T. Williams, 1992. No genetic differentiation of giant clam (Tridaaia s'Sas) populations in the Great Barrier Reef. Australia. Mar. Bio! 113: 373-377 Bertelson, E. & H. Ussing, 1936. 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