P r o c e e d i n g s of t h e Third I n t e r n a t i o n a l Symposium o n L i t t o r i n i d B i o l o g y , 9-24 (1992)

J. G r a h a m e , P . J . M i l l & D . G. R e i d (Eds)
C o p y r i g h t - The M a l a c o l o g i c a l Society of L o n d o n 1 9 9 2

T H E P H Y L O G E N E T I C RELATIONSHIPS OF T E N A T L A N T I C
LITTORINIDS ASSESSED BY A L L O Z Y M E ELECTROPHORESIS

THIERRY B A C K E L J A U and THIERRY W A R M O E S

1 2

^Koninklijk B e l g i s c h I n s t i t t i u t v o o r N a t u u r w e t e n s c h a p p e n , Afdeling Malacologie, Vautierstraat 29, B-1040

B r u s s e l , B e l g i u m . ^ U n i v e r s i t e i t A n t w e r p e n (RUCA), L a b o r a t o r i u m v o o r Algemene Dierkunde,
Groenenborgerlaan 171, B-2020 Antwerpen, Belgium.

ABSTRACT bias the interpretation of the results, the

Ten Atlantic littorinid species were surveyed
electrophoretically at nine enzyme loci in order to
test some phylogenetic hypotheses. Three measures
of genetic distance were assessed (Nei, Cavalli-
Sforza & Edwards and Prevosti) and two tree-making
methods were compared (UPGMA and distance
Wagner). It is shown that tree topologies depended
on the tree-making method, the genetic distance
measure and the rooting procedure, but not on the
order of data input. Because of the presumed
evolutionary rate heterogeneity among the taxa
investigated, it is suggested that a rate-independent
tree-making method, combined with outgroup
rooting, should give a more reliable phylogeny
estimate. Trees obtained in this way confirm the
clustering of L i t t o r i n a s t r i a t a with L . l i t t o r e a , L .
s a x a t i l i s s . l . and L . o b t u s a t a s . l . They also support
the currently accepted relationships between the last
three species, even though they cluster L . a r c a n a
with L . n i g r o l i n e a t a , which is at variance with the
results of previous electrophoretic analyses. In
opposition to the L i t t o r i n a species, there is a
bifurcation containing N o d i l i t t o r i n a and L i t t o r a r i a .
Finally, the distant relationship of M e l a r h a p h e
n e r i t o i d e s to the L i t t o r i n a species is confirmed.
INTRODUCTION
The systematics of several Atlantic littorinid
species are still much debated. This is for
example the case for the generic position of
L i t t o r i n a n e r i t o i d e s (Linnaeus, 1758), L . s t r i a t a
King & Broderip, 1832, L . p u n c t a t a (Gmelin,
1791) and L . cingulifera Durtker, 1845, as well
as for the subgeneric relationships of L .
s a x a t i l i s (Olivi, 1792) s . l . and L . o b t u s a t a
(Linnaeus, 1758) s . l . A summary of the most
important alternative opinions on these subjects
is given in Table 1. Actually, only one of the
species listed has never provoked systematic
controversy, viz. L . l i t t o r e a (Linnaeus, 1758),
the type species of the genus. In order not to
generic name L i t t o r i n a will be used in its
widest sense throughout this paper.
Until now, the classification and
phylogenetic relationships of the Littorinidae
were inferred from shell characters, radula,
operculum and anatomical features, mainly of
the reproductive system. Such data were used
by Reid (1986, 1989, 1990), who constructed a
most parsimonious cladogram, which served as
basis for the (infra)generic division of the
family. However, a cladogram is an
hypothesis of phylogenetic relationships, which
must be tested by analysing complementary
data sets (Reid, 1989, 1990).
Electrophoretic comparisons of allozymes in
several littorinid species have supported some
of Reid's (1989, 1990) phylogenetic
interpretations. This was the case for the
relationships between L . angulifera and the L .
ziczac species complex (Janson, 1985), as well
as for the relationships between L . n e r i t o i d e s ,
L . s a x a t i l i s s . l . , L . o b t u s a t a s . l . and L . l i t t o r e a
(Warmoes, 1986; Ward, 1990).
In a similar way, i.e. by using
electrophoretic allozyme comparisons, the
present work aims at providing additional data
on the phylogenetic relationships between 10
Atlantic littorinids (Table 1).
MATERIALS AND METHODS
A total of 257 specimens from 22 populations
representing 10 species were analysed by means of
vertical polyacrylamide gel electrophoresis (PAGE).
A sample and species list is presented in Table 2.
Specimens were transported alive to the laboratory
where they were stored at -20°C or at -80°C.
Individual body homogenates were prepared by
thawing frozen animals, crushing their shells and
dissecting the soft parts in distilled water. Usually,
shell fragments, penes, radulae, opercula and
sometimes complete oviducts were separated and put
into 70% ethanol for storage as reference material.
10 T. B A C K E U A U & T. WARMOES

Table 1. Comparison of different viewpoints on the systematics of some Atlantic littorinid

species. References: (1) Bequaert (1943); (2) Fischer (1967); (3) Rosewater (1970); (4)
Sacchi (1974); (5) Bandel (1974); (6) Rosewater (1981); (7) Bandel & Kadolsky (1986); (8)
Reid (1989). *indicates species complex.

Ref. striata punctata neritoides cingulifera saxatilis* obtusata* littorea

1 _ _ Littorina Littorina Littorina Littorina

- - (Melarhaphe) (Littorivaga) (Neritrema) (Littorina)

2 Littorina _ Littorina _

(Melarhaphe) - (Melarhaphe) - - - -

3 Nodilittorina Littorina Littorina Littorina Littorina Littorina Littorina

(Granulilittorina)(Austrolittorina) (Melarhaphe) (Littoraria) (Littorina) (Littorina) (Littorina)

4 Littorina Littorina Littorina _ Littorina Littorina Littorina

(Melarhaphe) (Melarhaphe) (Melarhaphe) - (-) (-) (-)

5 Littorina Littorina Littorina - Littorina Littorina Littorina

(-) (-) (Melarhaphe) - (Neritrema?) (Neritrema?) (-)

6 Nodilittorina Littorina Littorina Littorina Littorina _ _

(Liralittorina) (Austrolittorina) (Melarhaphe) (Littoraria) (Littorina) - -

7 Nodilittorina Nodilittorina Melarhaphe - Littorina Littorina Littorina

(Liralittorina) , (Nodilittorina) (Melarhaphe) - (-) (-) (-)

8 Littorina Nodilittorina Melarhaphe Littoraria Littorina Littorina Littorina

(Liralittorina) (Echinolittorina) (-) (Littoraria) (Neritrema) (Neritrema) (Littorina)

All remaining body parts were rinsed in distilled
water, blotted on Kleenex paper, weighed and
transferred into a 20% (w/v) aqueous sucrose
solution (5/tl solution per mg tissue, except for L .
n e r i t o i d e s for which the ratio was 8(tl solution per
mg tissue).
Crude homogenates were centrifuged for 45 min at
27200g (15000 r.p.m.) at 4°C. The supernatant was
stored at -80°C until required for electrophoresis.
PAGE was performed in 80x80x2.7 mm gel slabs
with a gel concentration of 6% (Backeljau, 1989).
To facilitate comparisons between gels, previously
typed specimens were included in each run.
Electrophoreses were conducted using two
continuous buffer systems: Tris/Citric acid (TC) at
pH 8.0 and Tris/Boric acid/EDTA (TBE) at pH 8.9.
TC buffer was employed to resolve acid phosphatase
(ACP, E . C . 3.1.3.2), fumarase ( F U M , E . C .
4.2.1.2), glucose-6-phosphate isomerase (GPI, E . C .
5.3.1.9), glutamic-pyruvate transaminase (GPT,
E.C. 2.6.1.2), malate dehydrogenase (MDH, E . C .
1.1.1.37), malic enzyme (ME, E . C . 1.1.1.40),
peroxidase (PER, E . C . 1.11.1.7) and 6-
phosphogluconate dehydrogenase (PGD, E . C .
1.1.44). T B E buffer was used for superoxide
dismutase (SOD, E.C. 1.15.1.1).
Electrophoresis was started at 25V for the first 15
min, increased to 50V for the following 15 min and
240 min with the TBE buffer. The temperature was
kept below 5 °C.
Enzyme staining recipes were adapted from
Nyman (1970), Shaw & Prasad (1970) and Harris &
Hopkinson (1976). Alleles (electromorphs) were
designated alphabetically according to decreasing
electrophoretic mobilities (A = fastest allele = the
band with the most anodal position). The BIOSYS-1
computer package of Swofford & Selander (1981)
was used to analyse the data. For reasons outlined
later, we pooled all conspecific individuals in single
'populations' (10 gene pools). Allele frequencies
were calculated and used to derive Nei's (1978)
unbiased genetic identities (I) and distances (D),
Cavalli-Sforza & Edwards' (1967) chord distance
and Wright's (1978) Prevosti distance. 1 values and
Prevosti distances were employed to construct
U P G M A dendrograms. Distance Wagner trees
(Farris, 1972) were derived using the chord and
Prevosti metrics.
Wagner trees were constructed using different
orders of data input and rooting methods, because
these factors may affect the branching sequence of
such trees (Swofford, 1981; Emberton, 1988).
For each species we also provide rough estimates
of percentages of polymorphic loci (P), mean
numbers of alleles per locus (A), mean observed
heterozygosities ( H ) and mean Nei's (1978)
0

then 150V for either 180 min with the TC buffer or unbiased expected heterozygosities ( H ) . Wee
 PHYLOGENETIC RELATIONSHIPS OF LITTORINIDS 11

Table 2. Locality data on the species and populations studied. NT = total number of specimens;
N M = number of males; N F = number of females; NJ = number of juveniles; T(°C) =
temperature at which samples were kept.

Species Abbr. Locality Date NT NM . NF NJ T(°C)

littorea li Breskens, the Netherlands 19/03/1989 10 4 5 1 > -80

Dieppe, France 08/04/1989 10 5 5 - -80

saxatilis sa Breskens, the Netherlands 19/03/1989 10 2 8 - -80

Boulogne-sur-Mer, France 31/07/1988 10 3 7 - -20

Trebeurden, France 04/04/1990 1 - 1 - -80

arcana ar Roscoff, France 10/04/1986 10 4 6 - -20

Trebeurden, France 104/04/1990 17 - 17 - -80

nigrolineata i ni Roscoff, France 10/04/1986 10 5 5 - -20

Trebeurden, France 03/04/1990 18 6 12 - -80

obtusata ob Skarberget, Norway 21/07/1988 10 7 3 - -20

Roscoff, France 10/04/1986 10 5 4 1 -20

mariae ma Zeebrugge Belgium 07/10/1989 10 1 9 - -80

Narvik, Norway 19/07/1988 12 3 9 - -20

striata St Santa Cruz, Flores, Azores 10/07/1989 20 5 15 - -80

Anjos, Santa Maria, Azores 14/06/1990 20 9 11 - -80

Formigas, Azores 09/06/1990 11 2 9 - -80

punctata pu Brucoli, Sicily 31/05/1990 25 6 18 1 -80

Goree, Senegal 31/07/1990 11 3 8 - -80

Dakar, Senegal 02/08/1990 4 - 4 - -80

cingulifera ci Diembereng, Senegal 24/07/1990 10 1 9 - -80

neritoides ne San Laurenco, Santa Maria, Azores 25/06/1990 10 1 8 1 -80

Boca de Ribeirinha, Sao Miguel, 06/06/1990 9 7 2 -80

Azores
Brucoli, Sicily 31/05/1990 10 2 7 1 -80

considered a locus as being polymorphic when more eight loci).

than one allele was detected. However, for The protocols and photographs of the
comparative purposes we also mention the P values electrophoretic runs, and the remaining parts of the
with the 0.95 criterion. Because scoring A C P specimens studied, are deposited in the collections of
zymograms was sometimes difficult, all analyses the Koninklijk Belgisch Instituut voor
were conducted for two data sets: one including ACP Natuurwetenschappen (KBIN), Brussels.
(total of nine loci) and one excluding ACP (total of
12 T. B A C K E L J A U & T. WARMOES

Table 3. Overall allele frequencies at 9 enzyme loci in 10 littorinid species. Names are
abbreviated as in Table 2.
Locus St li sa ar ni ma ob pu ci ne

SOD(N) 44 18 20 23 25 4 14 29 10 14
A - 0.500 - - - - - - - -
B - - 0.450 0.239 0.140 - 0.500 - _ -
C
D
-
-
0.472
0.028
-
0.550
-
0.587
-
0.640
1.000
-
0.036
0.464
-
-
-
-
--
E - - - 0.174 0.220 - -- - - -
F - - - - - - - 1.000 -
G
H
1.000
-
-
-
-
- -
- -
-
-
- -
- 1.000
-
-
-
-
1.000
PGD(N) 51 14 12 17 19 21 15 36 10 26
A - - - - - - - -- - 0.346
B - - - - - - - - 0.654
C 0.961 -- - - _ - - - -- _

D 0.039 - - - - - -
E - - - 0.088 - - - 0.014 - -
F - 0.429 0.667 0.176 - - 0.067 - -
G
H
-
-
0.286
0.286
0.333
-
0.735
-
1.000
-
1.000
-
0.933
-
0.944
0.042
0.100
0.900
--
GPI(N) 50 20 18 25 28 22 19 36 10 26
A - 1.000 - - - - - - - -
B - - - - -- - - - 0.050 -
C - - - -- - - 0.900 -
D - - - - - - - 0.050 -
E - - - - - - 0.053 - - -
F
G
-
-
-
-
0.167
0.639
0.040
0.920
-
0.982
-
0.773
0.105
0.632
_

-
_

- -
H
I
0.010
-
-
-
-
0.167
-
0.040
_

0.018
-
0.227
_

0.211
-
_
_ _

J 0.250 - - - - - _ _ - -
K
L
-
0.010
-
-
-
-
-
- -
- - - 1.000
-
_
_
0.019
_

M
N
0.010
0.710
-
-
-
-
-
_ -
-
-
_
_
-- _

-
0.058
-
O - - - - - - - - - 0.404
P - - - - - - - - - 0.077
Q 0.010 - - - - - - - - 0.423
R - - - - - - - -- - 0.019
s - - 0.028 - - - - - -
PER(N) 47 20 12 15 17 21 10 24 10 12
A - - - - - 0.167 0.050 - - -
B - - 0.208 - - 0.833 0.950 - - -
C - 0.425 0.125 0.100 0.088 _ _ _ - -
D 0.500 . 0.575 0.542 0.900 0.912 - _ - -
E - - 0.125 - - - - - - -
F 0.500 - - - - -- - 1.000 1.000 0.167
G - - - - - -- -- - 0.500
H
I
-
-
-
-
-
-
-
- -
-
-
- -
-
-
0.083
0.208
J - - - - - - - - - 0.042
GPT(N) 42 13 9 17 18 4 6 35 10 26
A - - 0.222 - 1.000 - - - -
B 0.095 1.000 0.556 0.206 -- 1.000 1.000 - -
C
D
0.881
0.024
-
-
-
0.111
-
0.147 -
-
-
-- 0.014
0.957
0.100
0.900
-
-
E - - - - - -- - 0.029 - -
F - - - 0.529 - - - - -
G - - 0.111 0.118 - - - - - -
H - - - - - - - - - 1.000
 PHYLOGENETIC RELATIONSHIPS OF LITTORINIDS 13

Table 3. (cont.)
Locus St li sa ar ni ma ob pu ci ne

MDH(N) 51 19 17 17 21 21 14 36 10 14
A 0.010 - - - - - - - - -
B _ _ _ _ _ - 0.036 - - -
C - - - - - - - - 1.000 -
D - - 0.294 - _ - - - - -
E 0.990 _ _ _
- - - -
F - - _ - - - 1.000 - 1.000
G - - 0.706 1.000 1.000 - - - - -
H - - - - _ 1.000 0.964 - - -
I - 0.105 - - - - - - - -
J - 0.895 - - - - - - - -
FUM(N) 44 17 11 18 18 8 7 35 10 26
A - - - - - - - - 0.500 -
B - 1.000 - - - - - - - -
C - - - - - - - - 0.300 -
D - - 0.273 1.000 1.000 - - - - -
E - - 0.364 - - 0.500 0.429 - - -
F 0.068 - - - - - - - - -
G 0.284 - 0.364 - - 0.500 0.571 - - -
H _ - _ - - - - 0.200 -
I 0.602 - - - - - - - - 0.058
J 0.023 - - - - - - - -
K 0.023 - - _ - - - - - 0.500
L - - - - - - - 0.986 - 0.404
M - - - - - - - 0.014 - 0.038
ME(N) 39 18 14 21 19 21 18 35 10 13
A - 0.944 - - - - - - - • -
B - 0.056 - - - - - - - -
C - - - - 0.048 0.306 - - -
D - - - - - 0.048 - - - -
E - - 0.036 0.095 0.053 0.405 0.694 - - -
F - - - - - 0.500 - - - -
G 1.000 - - - - - - - - 1.000
H - - 0.393 0.143 - - - - 0.400 -
I - - 0.571 0.762 0.895 - - - 0.600 -
J - - - - 0.053 - - - - -
K - - - - - - - 0.929 - -
L - - - - - - - 0.071 - -

ACP(N) 40 14 8 19 23 21 18 30 10 13
A - - 0.625 0.053 - 0.810 0.111 - - -
B - - - - 0.109 0.048 - - - -
C 0.100 - - 0.158 0.435 - - - - 0.038
D - - 0.313 0.158 0.457 0.143 0.889 - - -
E 0.375 - 0.579 - - - - - 0.808
F 0.475 - 0.063 0.053 - - - - - 0.154
G 0.050 - - - - - - - - -
H - - - - - - - 0.100 - -
I - 0.536 - - - - - 0.867 - -
J - 0.464 - - - 0.033 - -
K - - - - - - - - 1.000 -

RESULTS The P and He values we observed are

generally larger than previously reported for
The enzymes screened were assumed to be the same species (no comparative data
coded by nine putative loci. Allele frequencies available for L . cingulifera and L . s t r i a t a ) .
at these loci are given in Table 3. The The only exceptions to this observation are L .
measures of genetic variability assessed are p u n c t a t a and partly L . n e r i t o i d e s (see review
presented in Table 4, which shows that these by Ward, 1990). However, the discrepancies
measures did not depend strongly on whether between He and Ho, are not conspicuously
ACP was included or not. large, especially not if one considers the
14 T. B A C K E U A U & T. WARMOES

Table 4. Summary of genetic variability measures in 10 Atlantic

littorinid species. Upper figures refer to the analysis of 9 loci
(including ACP), the lower ones (in parentheses) to the analysis
without ACP. N = mean sample size per locus; A = mean
number of allele^ per locus; P = percentage polymorphic loci using
no criterion; P = percentage polymorphic loci using the 0.95
criterion; Ho = mean observed heterozygosity (direct count); He =
mean unbiased expected heterozygosity under Hardy-Weinberg
conditions.

Species N A P P* H 0
H e

striata 45.3 2.9 77.8 55.5 0.211 0.271

(46.0) (2.8) (75.0) (50.0) (0.232) (0.227)
littorea 17.0 1.9 66.7 66.7 0.253 0.282
(17.4) (1.9) (62.5) (62.5) (0.275) (0.253)
saxatilis 13.4 3.0 100.0 100.0 0.448 0.559
(14.1) (3.0) (100.0) (100.0) (0.488) (0.562)
arcana 19.1 2.8 77.8 77.8 0.156 0.338
(19.1) (2.5) (75.0) (75.0) (0.176) (0.302)
nigrolineata 20.9 1.9 55.6 44.4 0.064 0.171
(20.6) (1.8) (50.0) (37.5) (0.062) (0.117)
mariae 15.9 1.9 55.6 55.6 0.183 0.234
(15.3) (1.8) (50.0) (50.0) (0.206) (0.222)
obtusata 13.4 2.2 88.9 77.8 0.315 0.286
(12.9) (2.3) (87.5) (75.0) (0.340) (0.297)
punctata 32.9 1.9 55.6 33.3 0.035 0.066
(33.3) (1.8) (50.0) (25.0) (0.039) (0.044)
cingulifera 10.0 1.8 55.6 55.6 0.111 0.192
(10.0) (1.9) (62.5) (62.5) (0.125) (0.216)
neritoides 18.9 2.7 55.6 55.6 0.224 0.306
(19.6) (2.6) (50.0) (50.0) (0.243) (0.302)

pooling procedure we employed.
Tables 5-7 present different measures of
genetic 'relatedness' between the species
investigated. Apparently also these data are
only slightly influenced by whether or not ACP
was considered. The interspecific genetic
distances we obtained are comparatively large
for littorinids. For example, the mean genetic
distance between species of the L . s a x a t i l i s
complex is D=0.275 (+ACP) or D=0.223 (-
ACP) (Table 7), while the largest (single) D
value Ward (1990) reported in this species
aggregate was D=0.237 (L. s a x a t i l i s l L .
n i g r o l i n e a t a ) . The largest mean distance Ward
(1990) observed among species in L . s a x a t i l i s
s.l. was between L . a r c a n a and L . n i g r o l i n e a t a
with D=0.140 (range: 0.052-0.227). Yet, in
our study, these two species revealed the
smallest genetic distance (D=0.149) (Table 5).
The genetic distances between L . o b t u s a t a
and L . m a r i a e , v i z . D=0.231 (+ACP) and
D=0.156 (-ACP) (Table 5), are smaller than
the value of Moyse et a l . (1982) (D=0.501),
but larger than the value of Morris (1979)
(D=0.087).
All other genetic distances between L .
l i t t o r e a , L . s a x a t i l i s s . l . and L . o b t u s a t a s . l .
(Tables 5 and 7) are larger than the D values
mentioned by Ward (1990), while they are of
the same magnitude as the genetic distances
between L . angulifera and L . ziczac s . l .
(Janson, 1985).
As there exist no comparative data with
respect to interspecific genetic distances
 PHYLOGENETIC RELATIONSHIPS OF LITTORINIDS 15

Table 5. Nei's (1978) unbiased genetic identities (above diagonal) and distances (below
diagonal) between 10 Atlantic littorinid species. Upper figures are based on 9 loci (including
ACP), the lower ones (in parentheses) were obtained after excluding ACP (8 loci). Species
names are abbreviated as in Table 2. * = infinity.

Species St li sa ar ni ma ob pu ci ne

striata - 0.059 0.090 0.117 0.071 0.035 0.040 0.207 0.088 0.236
(0.063) (0.092) (0.080) (0.069) (0.038) (0.044) (0.223) (0.098) (0.193)

littorea 2.834 - 0.260 0.172 0.125 0.263 0.206 0.103 0.042 0.000
(2.766) (0.287) (0.185) (0.133) (0.288) (0.228) (0.042) (0.047) (0.000)

saxatilis 2.407 1.347 - 0.740 0.688 0.482 0.551 0.073 0.118 0.002
(2.383) (1.247) (0.793) (0.723) (0.422) (0.548) (0.081) (0.135) (0.000)

arcana 2.146 1.760 0.301 - 0.861 0.275 0.340 0.118 0.109 0.079
(2.520) (1.689) (0.231) (0.892) (0.288) (0.349) (0.128) (0.122) (0.000)

nigrolin- 2.640 2.083 0.375 0.149 - 0.259 0.342 0.119 0.086 0.002
eata (2.674) (2.017) (0.325) (0.114) (0.269) (0.311) (0.128) (0.096) (0.000)

mariae 3.344 1.334 0.729 1.290 1.353 - 0.794 0.124 0.014 0.000
(3.264) (1.244) (0.863) (1.244) (1.313) (0.856) (0.137) (0.016) (0.000)

obtusata 3.227 1.582 0.596 1.079 1.073 0.231 - 0.120 0.014 0.000
(3.131) (1.476) (0.602) (1.053) (1.168) (0.156) (0.134) (0.016) (0.000)

punctata 1.576 2.270 2.618 2.135 2.126 2.087 2.120 - 0.255 0.219
(1.500) (3.178) (2.509) (2.056) (2.052) (1.989) (2.007) (0.288) (0.242)

cingulifera 2.427 3.178 2.137 2.213 2.448 4.260 4.293 1.366 - 0.025
(2.324) (3.065) (2.002) (2.106) (2.347) (4.135) (4.153) (1.244) (0.028)

neritoides 1.442 + 6.249 2.539 6.012 * * 1.520 3.700 -

(1.645) (*) (*) (•) (*) (*) (*) (1.417) (3.570)

involving L . s t r i a t a , L . p u n c t a t a , L . cingulifera subclusters: one involving L . p u n c t a t a and L .

and L . n e r i t o i d e s , we can only note that they cingulifera and one composed of L . s t r i a t a and
yield very large D values both in comparisons L . neritoides.
with true L i t t o r i n a species, as in comparisons The Wagner trees we constructed are
amongst each other (Table 5). presented in Figs 2-4. The data from which
The UPGMA dendrograms inferred from they were inferred are given in Table 6. The
Nei's (1978) unbiased genetic identity and topologies obtained did not depend on the in-
Wright's Prevosti distance are presented in or exclusion of ACP. Fig. 2 shows that the
Fig. 1. Both measures yielded the same the branching sequence of Wagner trees is affected
branching order, while the in- or exclusion of by the choice of the genetic distance measures.
ACP did not affect the tree topology. It is also clear that the two Wagner trees in
The UPGMA dendrograms reveal three Fig. 2 are at variance with the UPGMA trees
notable features (Fig. 1): in Fig. 1.
1) L . l i t t o r e a , L . s a x a t i l i s s . l . and L . o b t u s a t a The effect of four different data input
s . l . form a single cluster in which three combinations upon the topology of a Wagner
subclusters can be distinguished, each tree was tested. Yet, each data input order
coinciding with a species or species complex. yielded the same tree topology (Fig. 3).
2) Within the L . s a x a t i l i s complex, L . a r c a n a is In Figs 2 and 4, finally, it is shown that both
clustered with L . n i g r o l i n e a t a , while L . the rooting method and the choice of outgroup
s a x a t i l i s s.s. appears to be somewhat less affect the branching sequences of Wagner
closely related. trees.
3) In opposition to the cluster containing L . By using midpoint rooting (no outgroup), we
l i t t o r e a , L . s a x a t i l i s s . l . and L . o b t u s a t a s . l . , obtained a tree, whose topology largely
there is a cluster consisting of two bifurcated corresponds to the branching pattern obtained
16 T. B A C K E L J A U & T. WARMOES

Table 6. Prevosti distance (above diagonal) and Cavalli-Sforza & Edwards' chord distance
(below diagonal) between 10 Atlantic littorinid species. Upper figures are based on 9 loci
(including ACP), the lower ones (in parentheses) were obtained after excluding A C P . Species
names are abbreviated as in Table 2.

Species St li sa ar ni ma ob pu ci ne

striata - 0.934 0.893 0.873 0.933 0.958 0.958 0.829 0.931 0.796
(0.926) (0.887) (0.923) (0.938) (0.953) (0.953) (0.808) (0.922) (0.842)

littorea 0.857 - 0.782 0.848 0.891 0.805 0.843 0.900 0.957 1.000
(0.851) (0.754) (0.829) (0.878) (0.780) (0.823) (0.959) (0.952) (1.000)

saxatilis 0.833 0.765 - 0.436 0.508 0.618 0.537 0.951 0.869 0.993
(0.835) (0.746) (0.398) (0.485) (0.667) (0.532) (0.944) (0.853) (1.000)

arcana 0.815 0.780 0.442 - 0.256 0.773 0.697 0.900 0.890 0.926
(0.850) (0.764) (0.406) (0.203) (0.769) (0.685) (0.888) (0.876) (1.000)

nigrolin- 0.855 0.817 0.516 0.397 - 0.774 0.700 0.895 0.922 0.996
eata (0.861) (0.806) (0.486) (0.360) (0.770) (0.720) (0.882) (0.913) (1.000)

mariae 0.865 0.781 0.634 0.747 0.771 - 0.301 0.895 0.989 1.000
(0.861) (0.765) (0.667) (0.750) (0.774) (0.245) (0.882) (0.988) (1.000)

obtusata 0.864 0.798 0.546 0.679 0.707 0.387 - 0.896 0.989 1.000
(0.859) (0.784) (0.560) (0.680) (0.725) (0.365) (0.883) (0.988) (1.000)

punctata 0.796 0.825 0.855 0.836 0.850 0:850 0.852 - 0.772 0.822
(0.782) (0.864) (0.849) (0.827) (0.844) (0.844) (0.846) (0.743) (0.799)

cingulifera 0.841 0.866 0.823 0.819 0.846 0.884 0.885 0.767 - 0.981
(0.833) (0.861) (0.813) (0.808) (0.839) (0.882) (0.883) (0.749) (0.979)

neritoides 0.762 0.900 0.895 0.857 0.894 0.900 0.900 0.783 0.880 -
(0.801) (0.900) (0.900) (0.900) (0.900) (0.900) (0.900) (0.767) (0.877)

Table 7. Mean genetic distances (Nei, 1978)

between L i t t o r i n a species as inferred from Table 5.
Species names are abbreviated as in Table 2.

Species comparison D(+ACP) D(-ACP)

among sa-ar-ni 0.275 0.223

among ob-ma 0.231 0.156

between l i and sa-ar-ni 1.730 1.651

between l i and ob-ma 1.458 1.360

between sa-ar-ni and ob-ma 1.020 1.041

with the U P G M A procedure. Only the (Fig. 2A), whereas in the U P G M A trees they
positions of L . striata and L . neritoides differ form a single cluster (Fig. 1).
in the two tree topologies, for in the Wagner The outgroup method revealed a different
tree these species are separated by two nodes picture. Using L . punctata+L. cingulifera as
 PHYLOGENETIC RELATIONSHIPS OF LITTORINIDS 17

0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0 90 100

-. , 1 1 , 1 r i 1 t r n 1 1

si
ne
pu

li
sa
ar
ni
ma
ob

1.00 0.90 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10

si
ne
pu
ci
li
sa
ar
ni
ma
ob

Figure 1. U P G M A trees for 10 Atlantic littorinid species. A : using Nei's (1978) unbiased genetic identity
and including A C P ; B: using Wright's (1978) Prevosti distance (ACP included). Species names are
abbreviated as in Table 2.

outgroup we obtained a Wagner tree in which
the clustering of L . l i t t o r e a , L . s a x a t i l i s s . l . and
L . o b t u s a t a s . l . is confirmed. This cluster is
joined with L . s t r i a t a , while the resulting
subtree is in turn connected with L . n e r i t o i d e s .
Hence, in this case L . s t r i a t a and L . n e r i t o i d e s
are separated by a single node (Fig. 4A). The
same topology is obtained when L . p u n c t a t a
and L . cingulifera are used separately as
outgroups. In either case, the remaining
species of the couple is placed near the root.
However, if L . n e r i t o i d e s is the outgroup, this
species is separated from L . s t r i a t a by two
nodes, for now the L . p u n c t a t a + L . cingulifera
cluster becomes inserted between L . n e r i t o i d e s
and L . s t r i a t a (Fig. 4B). Thus, this tree
topology contains two clusters: one
encompassing L . p u n c t a t a and L . c i n g u l i f e r a ,
the other one involving L . s t r i a t a , L . l i t t o r e a ,
L . s a x a t i l i s s . l . and L . o b t u s a t a s . l .
Note that in all trees the configuration of L .
s a x a t i l i s s . l . and L . o b t u s a t a s . l . did not
change.
DISCUSSION
Methodology ofphylogeny inference
The merits and drawbacks of various methods
for phylogenetic tree construction from
molecular data have been discussed and
compared excessively in the literature (e.g.
Swofford, 1981; Tateno et a l , 1982; Nei et
a t . , 1983; Tateno & Tajima, 1986; Patton &
Avise, 1983; Fitch & Atchley, 1987; Nei,
1987; Hartl et a l . , 1990; Sundberg et a l . ,
1990; Swofford & Olsen, 1990). Yet, the
conclusions of these studies are sometimes
contradictory with respect to the reliability of
the different algorithms or procedures.
One of the issues provoking much debate is
whether a tree should be constructed under the
assumption of (nearly) equal evolutionary rates
in all branches (e.g. UPGMA) or whether it
should allow for a non-constant evolutionary
clock (e.g. distance Wagner algorithm,
maximum likelihood methods). Although
18 T. B A C K E L J A U & T. WARMOES

0 0.07 0.13 0.20 0.27 0.34 0.40 0.47 0.54 0.60 0.67

i f—i 1 — "i 1 1 1 1 1 r
pu

ob

0.06 0.11 0.17 0.23 0.28 0.34 0.40 0.45 0.51 0.57
I 1 1 1 1 1 1 1 1 1 i 1 1 1 1 1 1 1 1

pu

ob

Figure 2. Distance Wagner trees for 10 Atlantic littorinid species. A : using Wright's (1978) Prevosti
distance; B: using Cavalli-Sforza & Edwards' (1967) chord distance. Both trees were rooted by midpoint
rooting. ACP was included. Data input order: pu-ci-li-sa-ar-ni-ma-ob-st-ne. Species names are abbreviated
as in Table 2.

phylogenetic relationships inferred from
allozyme data are usually summarized in
UPGMA dendrograms, Sundberg et a l . (1990)
concluded that at least for populations of L .
s a x a t i l i s s.s. and L . a r c a n a the most reliable
trees are obtained by using algorithms which
do not rely on a constant evolutionary clock.
The reason for the poor performance of
UPGMA in the study of Sundberg et a l . (1990)
may be twofold: (1) the assumption of rate
homogeneity may be unrealistic, even at the
level of closely related species such as L .
s a x a t i l i s and L . a r c a n a , and (2) the number of
loci surveyed (16) may have been too small.
This latter constraint is expected to produce a
tree which is subject to a large stochastic error
(Nei et a l . , 1983; Nei, 1987). In general it is
recommended that for phylogenetic inferences
a minimum of 30 loci should be surveyed in
order to have a reasonable chance of
recovering a correct tree (Nei et a l . , 1983;
Nei, 1987). This is particularly true when
genetic distances are small, as is the case in the
study of Sundberg et a l . (1990). Nevertheless,
Nei (1987) also ascertained that when distance
estimates are subject to large stochastic errors,
UPGMA is often superior to other tree making
methods based on distance matrices. However,
if rates vary greatly along branches, the
distance Wagner algorithm (or other rate-
independent procedures) should perform better
(Nei, 1987).
Whether the assumption of rate homogeneity
is applicable to the study of Sundberg et a l .
1990) is difficult to decide. Fuerst & Ferrell
1983) suggested that interspecific
differentiation of electromorphs is not
equivalent to intraspecific change. Similarly,
Eldredge (1985) and Crother (1990) claimed
that evolutionary change operates on different
time scales, which are evidenced by two
patterns: a within-species pattern and an
 PHYLOGENETIC RELATIONSHIPS OF LITTORINIDS 19

0 0.08 0.16 0.24 0.33 0.41 0.49 0.57 0.65 0.73 0.81

n 1 1 1 r -i 1 i 1
i r—i r st

ob
pu

Figure 3. Distance Wagner tree obtained with different data input combinations of 10 Atlantic littorinid
species, using Cavalli- Sforza & Edwards' (1967) chord distance. The tree was rooted with L . n e r i t o i d e s as
outgroup. A C P was included. The data input orders were: 1) st-ne-li-sa-ar-ni-ma-ob-pu-ci, 2) st-li-sa-ar-ni-
ma-ob-pu-ci-ne, 3) pu-ci-li-sa-ar-ni-ma-ob-st-ne and 4) li-sa-ar-ni-ma-ob-st-pu-ci-ne. Species names are
abbreviated as in Table 2.

0 0.08 0.16 0.24 0.32 0.40 0.49 0.57 0.65 0.73 0.81
l 1 1 r i 1 r 1 1 1 1 1 r ~ i i

ob
. st
ne
pu

0.09 0.18 0.26 0.35 0.44 0.53 0.62 0.70 0.79 0.88
i 1 1 1 1 1 1 1
-i n — i 1 r li

ob

pu

B
Figure 4. Effect of different rooting procedures upon distance Wagner trees for 10 Atlantic littorinid species,
using Wright's (1978) Prevosti distance. A C P was included. A : outgroup = pu-ci; B: outgroup = ne.
Order of data input: li-sa-ar-ni-ma-ob-st-pu-ci-ne. Species names are abbreviated as in Table 2. For the
effect of midpoint rooting, see Fig. 2A.
20 T. B A C K E L J A U & T. WARMOES
among-species pattern. Thus intra- and
interspecific genetic distances would not
measure the same process. If this is correct,
then it is evident that in trees in which different
species are compared at the population level
there must be an a p r i o r i rate heterogeneity.
This was probably the case in the study of
Sundberg et a l . (1990) and as such it may
partially explain why U P G M A performed so
poorly.
The two constraints mentioned above are
also expected to hold for our study. We
screened even fewer loci than Sundberg et a l .
(1990), while our trees consisted of both
closely and distantly related species. Thus, in
this study it seems unrealistic to assume a
constant evolutionary clock, even if we pooled
conspecific populations to avoid problems of
rate heterogeneity due to simultaneously
assessing intra- and interspecific relationships.
Our analyses confirm the observation of
Sundberg et a l . (1990) that for a given data set
rate-dependent (UPGMA) and rate-independent
(Wagner procedure) distance matrix algorithms
may produce different trees (compare Figs 1
and 2). Because of the considerations outlined
above, we tentatively follow Sundberg et a l .
(1990) in preferring rate-independent
algorithms (in this case the Wagner procedure)
when inferring littorinid phylogenies. This
premise is also supported by the work of
Knight et a l . (1987), who applied the Wagner
algorithm to L . s a x a t i l i s s.s. and L . a r c a n a
and obtained in this way a much better tree
than with U P G M A . The Wagner procedure
has furthermore been advocated by Swofford
(1981), Buth (1984), Emberton (1988) and
Swofford & Olsen (1990).
As our work indicates that the topology of a
Wagner tree depends on the distance measure
and rooting method used (Figs 2 and 4), we
must evaluate these factors too.
We used three genetic distance measures, of
which only two were employed to construct
Wagner trees. Indeed, because Nei's (1978)
unbiased estimate is non-metric, it is not
appropriate for the Wagner procedure
(Swofford & Selander, 1981; Buth, 1984).
Nevertheless, we included Nei's (1978)
measure in Table 5, for it allows a direct
comparison with the literature on littorinids, in
which this is the only genetic distance measure
hitherto used.
Cavalli-Sforza & Edwards' (1967) chord
distance and Wright's (1978) Prevosti distance
are much less widespread throughout the
literature, even though these metrics are highly
appropriate for phylogenetic inferences
(Swofford & Selander, 1981; Felsenstein,
1984; Emberton, 1988; Swofford & Olsen,
1990). In the present work, we prefer the
Prevosti distance over the chord distance, since
the former recovers the generally accepted
relationships between L . l i t t o r e a , L . s a x a t i l i s
s . l . and L . o b t u s a t a s . l . , whereas the chord
distance does not (Fig. 2). Swofford &
Berlocher (1987) also argued for the
superiority of the Prevosti distance.
Since we preferred a rate-independent tree-
making algorithm, we also preferred an
outgroup method rather than midpoint rooting,
for this latter assumes a constant rate of
evolutionary change (Nei, 1987).
Two alternative tree-making procedures
appeared inappropriate in the present context.
The maximum likelihood method of Felsenstein
(1981) was not employed since its underlying
assumptions (Brownian motion model) are
unrealistic (Nei, 1987). Probably the
algorithm is only applicable for problems
involving intraspecific populations or closely
related species (Swofford & Berlocher, 1987).
A Hennigian analysis of electrophoretic data
(e.g. Patton & Avise, 1983; Buth, 1984;
Richardson et a l . , 1986; Emberton, 1988), was
not performed in view of the drawbacks
pointed out by Swofford & Berlocher (1987)
and Sundberg et a l . (1990). Particularly the
small number of loci we studied and our
relatively small sample sizes make a Hennigian
approach suspect.
Electrophoretic techniques
Most electrophoretic studies on littorinids have
involved starch-gel electrophoresis, while only
six relied on PAGE (Wium-Andersen, 1970;
Vuilleumier & Matteo, 1972; Berger, 1973,
1977; Caugant, 1979; Warmoes, 1986). Yet,
the detectability of protein differences does not
only depend on characteristics of the proteins
themselves, but also on the electrophoretic
conditions employed (gels, buffers, pH, etc.)
(e.g. Coyne et a l . , 1979; Ramshaw et a l . ,
1979; Singh, 1979; Beaumont & Beveridge,
1983; Nei, 1987; Backeljau, 1989; Backeljau
& DeBruyn, 1990; Murphy et a l . , 1990).
Thus both the choice of proteins under study
(e.g. slow versus fast evolving loci), and the
running conditions, may influence the genetic
data obtained. Heterozygosity estimates, for
example, may vary considerably between
different data sets for a given taxon (e.g.
inclusion or exclusion of esterases) (Simon &
Archie, 1985).
Three of the nine enzymes surveyed have not
been investigated previously in littorinids
(PER, GPT, FUM). At the six remaining loci,
our data confirm the numbers of alleles
reported in the literature and where they
deviate from previous studies, they usually
reveal higher numbers of alleles. At GPI in L .
s a x a t i l i s , L . a r c a n a and L . m a r i a e , for
example, we observed the same numbers of
alleles as Snyder & Gooch (1973), Moyse et
a l . (1982), Ward & Janson (1985), Knight et
 PHYLOGENETIC RELATIONSHIPS OF LITTORINIDS
a l . (1987) and Sundberg et a l . (1990) to name
a few. At the same locus in L . n e r i t o i d e s , L .
n i g r o l i n e a t a and L . o b t u s a t a , on the contrary,
we found more alleles than respectively Nevo
& Lavie (1989), Beardmore & Morris (1978)
and Moyse et a l . (1982). For L . l i t t o r e a and
L . p u n c t a t a we observed fewer alleles than
reported in the literature. Similar observations
can be made for the other loci studied.
However, electrophoresis can only
demonstrate allozyme differences (Allendorf,
1977; Ferguson, 1980; Richardson et a l . ,
1986). Hence, when we found certain loci to
be polymorphic, while the same loci were
monomorphic in starch-gel studies, it is
possible that this is the result of the inability of
the starch-gel technique to detect differences
revealed by PAGE (and v i c e v e r s a ) .
These observations could account for the
relatively larger P, H and D values (or lower I
values) we generally detected. Note that the
genetic identities we observed among sibling
species of L . s a x a t i l i s s . l . (1=0.763) and L .
o b t u s a t a s . l . (1=0.794) (Table 5) correspond
well to the data provided by Thorpe (1979,
1982, 1983). This author suggested that in
about 97% of interspecific comparisons Nei's
mean genetic identity between "good" animal
species is lower than 1=0.850, whereas in 98%
of intraspecific comparisons the mean identity
is higher than 1=0.850. Moreover, the genetic
distances we obtained between species
currently assigned to different subgenera or
genera, are of the same magnitude as the
genetic distances Janson (1985) reported
between L . angulifera and the L . ziczac
complex, species which are now placed in
different genera (Reid, 1989).
Thus, starch-gel electrophoresis and PAGE
may produce divergent population genetic and
phylogenetic data. Even among studies
involving only starch gel electrophoresis, there
may be large differences with respect to the H ,
P and D values reported. This is illustrated by
the data of Noy et a l . (1987) and Ward (1990)
for L . n e r i t o i d e s or by the genetic distances
Morris (1979) and Moyse et a l . (1982)
reported between L . o b t u s a t a and L . m a r i a e
(reviewed by Ward, 1990).
The value of protein electrophoresis in
assessing phylogenetic relationships is limited
by the common observation that allozyme data
are only reliable at intra- and interspecific
levels. Usually, such data should no longer be
relied on for taxa beyond the genus level
(except in birds), because of the high incidence
of allele similarities caused by chance
convergences (Ferguson, 1980; Richardson et
a l . , 1986; Murphy et a l . , 1990).
Despite these limitations we expected that
allozyme comparisons of littorinid genera and
subgenera would yield reliable phylogenetic
estimates, because the genetic distances among
21
L . s a x a t i l i s s . l . , L . o b t u s a t a s . l . and L . l i t t o r e a ,
as hitherto reported, are so small (Ward, 1990)
and because Janson (1985) noted realistic
"intergeneric" genetic distances among L .
angulifera and L . ziczac s . l . In retrospect, it
seems as if our hypothesis was only partially
warranted, for the inclusion of L . n e r i t o i d e s
provoked infinite genetic distances (Table 5),
indicating that the phylogenetic gap between
this species and the remaining ones may be too
large to be evaluated by protein
electrophoresis.
Finally, the use of allele frequency data in
phylogenetic reconstructions has been
questioned by Crother (1990), who argued that
allele frequencies are temporally unstable, as
they are easily modified by random genetic
drift and/or selection (Mickevich & Johnson,
1976). Allele frequencies may even vary over
the span of a few years and therefore they
cannot by definition be synapomorphic
(Crother, 1990).
Although this issue is far from resolved, it
must be emphasized that allele frequencies are
only a way to weigh the presence or absence of
a particular allele (Swofford & Olsen, 1990).
We have tried to alleviate possible temporal
(and geographical) instabilities of allele
frequencies by pooling all conspecific
individuals in single gene pools. Frequency
differences at population levels are thus
averaged, so that local and temporal
fluctuations are expected to have little effect on
the overall frequency. In addition, we avoided
as much as possible the use of loci which are
suspected to be under selective control (e.g.
esterases and aspartate aminotransferase)
(Newkirk & Doyle, 1979; Johannesson &
Johannesson, 1989), although we did include
GPI (Nevo & Lavie, 1989).
Systematic and p h y l o g e n e t i c considerations
Our results are largely in agreement with
recent revisions of littorinid classification and
relationships (Warmoes, 1986; Reid, 1986,
1989, 1990; Ward, 1990). Only the position of
L . a r c a n a and the (uncertain) branching of
N o d i l i t t o r i n a and L i t t o r a r i a are points of minor
disagreement.
L . a r c a n a is, according to Ward (1990),
most closely related to L . s a x a t i l i s , whereas L .
n i g r o l i n e a t a would be a somewhat more
distantly related species. However, the
ovoviviparous reproduction of L . s a x a t i l i s
appears to be the only cladistically relevant
'morphological' character from which the
relationships between the three species have
been inferred. Yet, since ovoviviparity is an
autapomorphy, it is consistent with any
topology. As a consequence, Reid (1990)
arrived at an incompletely resolved trichotomy
for the L . s a x a t i l i s group. This uncertainty
22 T. B A C K E L J A U & T. WARMOES
shows that the relationships within this species
group need further investigation.
The branching sequence of L . l i t t o r e a , L .
s a x a t i l i s s . l . and L , o b t u s a t a s . l . in Wagner
trees based on Prevosti distances (Fig. 4)
corroborate the trees of Reid (1990) and Ward
(1990), who used respectively morphological
and allozyme data. Hence, Reid's (1989)
infrageneric classification of these species
seems to be a robust one.
The clustering of L . p u n c t a t a and L .
cingulifera is inconsistent with the cladogram
of Reid (1989), but consistent with the tree of
Reid (1986). However, the latter tree was
explicitly refuted by (Reid, 1989) because of
the limited data set on which it was based and
because of the apparent high incidence of
parallel evolution in the genera N o d i l i t t o r i n a
and L i t t o r a r i a (Bandel & Kadolsky, 1982;
Reid, 1989). Both these points may have
provoked the clustering of L . p u n c t a t a and L .
cingulifera in the present work too.
The U P G M A dendrograms and the Wagner
trees rooted at the mid point of the greatest
patristic distance, support Fischer's (1967)
opinion, according to which L . s t r i a t a would
be a M e l a r h a p h e . The Wagner trees rooted by
the outgroup method, on the contrary, cluster
L . s t r i a t a with the L i t t o r i n a group and thus
confirm the branching sequence proposed by
Reid (1989, 1990). For reasons outlined in the
first section we favour these latter results.
Note that the tree topologies remained
essentially the same irrespective of whether L .
n e r i t o i d e s or L . p u n c t a t a + L . cingulifera were
used as outgroups.
Our outgroup choice was based on the
cladograms of Reid (1986, 1989, 1990). In the
present context L . n e r i t o i d e s seems more
appropriate as outgroup than the L .
c i n g u l i f e r a + L . p u n c t a t a cluster (or each of
these species separately), for M e l a r h a p h e is the
sistergroup of the clade containing the
remaining Littorininae, including L i t t o r a r i a ,
N o d i l i t t o r i n a and L i t t o r i n a (Reid, 1989).
Indeed, whatever outgroup was used, L . s t r i a t a
was always clustered with L i t t o r i n a rather than
with M e l a r h a p h e . In addition, in none of the
trees was L . s t r i a t a clustered with
N o d i l i t t o r i n a . Thus we are fairly confident that
the inclusion of this species in N o d i l i t t o r i n a
cannot be maintained. Hence, our data support
the transfer of L . s t r i a t a from M e l a r h a p h e or
N o d i l i t t o r i n a (Table 1) to L i t t o r i n a (Reid,
1989, 1990). Nevertheless, L . s t r i a t a appears
to be rather distantly related to the other
members of this genus, and is therefore
appropriately placed in a monotypic subgenus
L i r a l i t t o r i n a (Reid, 1989, 1990). This is
supported by Wium-Andersen (1970), who
noted that the electrophoretic profiles of
haemoglobin in L . l i t t o r e a and L . s a x a t i l i s s . l .
are identical, while those of L . s t r i a t a are
different.
Note finally that our results also confirm the
distant relationships between M e l a r h a p h e and
L i t t o r i n a (Warmoes, 1986; Reid, 1989; Ward,
1990), as can be inferred from the extremely
large genetic distances involved (Table 5).
ACKNOWLEDGEMENTS
We are much indebted to E . Dumoulin
(Knokke), J . M . Azevedo, C. Brito, R.T.
Cunha, A . M . Frias Martins, (all of the
Universidade dos Azores), G. Spada (Bologna)
and A . Waren (Naturhistoriska Riksmuseet,
Stockholm) for collecting parts of the material.
We are furthermore grateful to W . N .
Verheyen and J . L J . Hulselmans (both
University of Antwerp) who provided the
necessary working facilities. J. Van Goethem
(KBIN, Brussels) and an anonymous referee
are sincerely thanked for their comments on
the manuscript. H . Van Paesschen (KBIN,
Brussels) kindly prepared the figures. Travel
grants and financial support were provided by
the Belgian National Fund for Scientific
Research and the "Koninklijk Belgisch Instituut
voor Natuurwetenschappen, Brussel". The
Italian populations were sampled during the
"Mission Sicile, 1990" organized by P.
Bouchet (Museum National d'Histoire
Naturelle, Paris). The Azorean material was
collected during the missions "Flores 89" and
"Santa Maria e Formigas, 1990" organized by
A.M. Frias Martins (Universidade dos
Azores).
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