Larval development and allozyme variation in the ...

SC!. MAR .. 59( I): 77- 6

SCIENTIA MARINA

1995

Larval development and allozyme variation in the East Atlantic Columbella (Gastropoda: Prosobranchia: Columbellidae)* MARCO OLlVERIO Dipartimento di Biologia Animale e dell" omo. "La Sapienza" Roma University. Viale dell"Universita 32.1-00185 Roma. Italy.

SUMMARY: A survey on the neogastropod Co/ulllbel/a (Mollusca: Gastropoda: Prosobranchia) in the East Atlantic region was carried out. A morphological slUdy of radular and protoconch (embryonic/larval shell) features. was conducted on populations from all over the distribution range. While radular morphology was rather unifollll in all populations scored. prolOconch examinations gave good indications on the variation in the larval ecology of the species studied. The Mediterranean C. rustica (Linne. 1758). with non-planktotrophic development. extends its range along the West African Atlantic coasts as far as Senegal. The Atlantic African populations South of Senegal all have a planktotrophic development. The latter are here considered con specific with the Macaronesian populations. under the name C. adal/sol/i Menke. 1853. Electrophoretic analysis was performed on one population from Canary Islands (with planktotrophic development) and two from the Mediterranean Sea (with non-planktotrophic development). The Macaronesian population proved to be genetically separated at species level from the Mediterranean ones. Further genetic examinations of population from the entire distribution range are needed. to support the taxonomy of' this group. KC\" \\'ords:

Neogastropoda. CO/I/mbella. speciatioil. East-Atlantic. MeditelTanean. allozyme. SEM. radula. proroconch.

RESUMEN: DES.~RROLO LARVARIO Y VARIABllIDAD ELECTROFORETICA E0I COLU.HBELLrI (GASTROPODA: PROSOJ3RAJ,\CHIA: COLUYlBELlIDAE) DEL ATLA'\TtCO ORtE:-JTAL.- Se lIevo a cabo un examcn del neogasteropodo Co/umbel/a (Mollusca: Gastropoda: Prosobranchia) en la region el Allantico oriental. Se efcctuo un estudio morfologico de las caracterfsticas de la nidula y de la protoconcha (conchas embrionaria y lan'aria) en las poblaciones de toda el area de distribucion. Miel1\ras que la morfologfa de la radula fue bastante uniforme en IOdas las poblaciones consideradas. el examen de las protoconchas dio buenos indicios de la variacion en la ecologfa larvaria de las especies estudiadas. La mediteminea C. rusrica (Linne. 1758). de desarrollo no planctotrofico. extiende su distribucion a 10 largo de las costas arlanticas del oeste africano. lIegando hasta Senegal. Las poblaciones del Atlantico africano al sur del Senegal tienen IOdas un desarollo pIancl0trofico. Estas Cdtimas se consideran aquf como coespecfficas con las poblaciones macaronesicas. bajo cl nombre de C. adal/sol/i Menke. 1853. Se Ilevo a cabo un analisis electroforetico de una poblacion de las lslas Canarias (con desanollo planclOtrOfico) y de dos poblaciones medilerraneas (con desarrollo no planctotrofico). La poblacion macaronesica resulto estar geneticamente separada. al nivel especffico. de las meditelTaneas. Se precisan ulteriores analisis geneticos de la poblacion en toda su area de distribucion para fundamentar la taxonomla de este grupo. (Traducido por el Editor). Pa/abras clm'c:

Neogastropoda. Coll/mbel/a. especiacion. Atlantico oriental. Mediterraneo. alozima. MEB. radula. prolO-

concha.

*Received April 18. 199.+. Accepted August 11. 199.+.

DEVELOP'vIEI\T A:\D ELECROPHORESIS OF E. ATLAj\TIC COU;.\1BELLA 77

INTRODUCTIO The gastropod fauna of the orth East Atlantic area (including the Mediterranean Sea) includes many pairs of species that differ in their larval development (planktotrophic vs. non-planktotrophic). This phenomenon has been already pointed out (VERDCL,\. 1977) in reference to taxonomic implications. Adult prosobranch gastropods usually retain (with very few exceptions) the protoconch (i.e. the part of the shell secreted during the embryonic and larval life). Based on the morphology of the protoconch a species can be usually attributed either to one category or the other (JABLONSKI and LUTZ, 1980. 1983). In fact, in planktotrophic larvae a larval shell is present, separate and built differently (BAl'\DEL, 1982). from the embryonic one; furthermore, the boundary between larval shell and adult teleoconch is often delimited in planktotrophic larvae by a characteristic 'sinusigera' outer lip to accommodate the velar lobes of the veliger (see LIMA and LUTz. 1990 for a review). This morphological approach allows inference of the development in the species if the protoconch is retained by the adult. This phenomenon long held chiefly a taxonomic importance, resulting in the recognition of pairs of 'species' based only or mainly on developmental features (i.e. the morphological ones of the protoconch). The evolution of life-histories has a great relevance in studies of evolutionary biology. In this framework, the phenomenon of the .pairs' is presently the object of an extensive analysis by the author aimed at the comprehension of the mechanisms underlying speciation events with loss of planktotrophy as a leading factor (OUVERIO, 1991, I994a, 1994b). One of the targets in such a study is the complex of populations formerly included under CO/l/mbe//a I'lIsrica (LI:\!\E, 1758). The genus CO/l/mbel/a belongs to the family Columbellidae, a group of neogastropod prosobranchs distributed world wide. early all species of this genus live in shallow water environments from low tide level to a few meters depth - with a tendency to sciaphyly resulting in their dwelling in crevices of rocks and corals, under stones. at the base of Cysroseira and Laminal'ia beds. or finally amidst the rhizomes of phanerogams. In the East Atlantic region the genus is present with a complex of populations distributed all over the Mediterranean Sea, the West African coasts and the Macaronesian archipelagos. It has not been recorded on Atlantic European coasts north of Portugal. 78 M. OLIVERIO

MOOLE\BEEK and HOE\SELAr\R ( 1991 ) discussed the taxonomy of the populations of CO/l/mhel/a inhabiting the Macaronesian area. the Atlantic African coast from Senegal and Mauritania northward to Morocco and the Mediterranean Sea. Basing on radular and developmental features (see also THoRsoN, 1950: BANDEL. 1975, 1977) they recognised two distinct species: CO/l/mhella adansoni Menke, 1853 (with planktotrophic development) from the Macaronesia, and C. I'lIsrica (L., 1758) (with intracapsular, non-planktotrophic development) from the Mediterranean, Moroccan and Senegalese/Mauritanian coasts. The genus CO/l/mhel/a is represented by this lineage also along the West African coasts south of the Senegalese region. BERNARD (1984) reported a gastropod under the name C. I'lIsrica whose shells are "the most common ... of the Gabonese coasts." The southern distribution limit of this group along the West African coasts needed to be checked. The present study was aimed at morphologically checking the identity of the West African CO/l/l11hella from Morocco southward to Gabon and at defining if genetic divergence supported the specific distinction between the Macaronesian (Canary Islands) and Mediterranean populations. Preliminary data on the genetic divergence of populations in CO/l/mhella add to a data-set that strongly neglects (with very few exceptions. e.g. GRA"iT. 1988) noegastropods in the North East Atlantic region. Even among all caenogastropos only littorinids have been widely dealt with by such studies (see WARD, 1990). There are very few exceptions of on other caenogastropod groups, like e.g. those by MLi"iKSGAARD (1990) and by OUVERIO (I 994c) both on rissoids.

MATERIALS AND METHODS Specimens (spms = live collected individuals) and shells (shs = shells without soft parts) of CO/lImhella I'lIsrica from all over the distribution range in the MeditelTanean Sea and along the Moroccan Atlantic coasts (more than 400 sampling localities) were examined: a specimen from the following locality is pictured in the present paper: S. Domino Is .. Tremiti Islands (Italy. Adriatic Sea). 42°07'22"N - 015°29'36"E -11 m on Cysroseim bed (65 spms M. Oliverio leg.). Material from 5 localities. representatives of all the archipelagos of Macaronesia have been studied:

Ponta Galera. Sao Miguel (Azores Islands). (50 shs P. Bouchet. V. Heros. B. Metivier leg.): Funchal Lido, Madeira, 2-5 m depth algae (15 spms R.G. Moolenbeek leg.): Salvajes Pequefia (Salvagen Islands) 15 m (5 shs, F.G. Talavera leg.); Pto. Santiago. Tenerife Is. (Spain, Canary Islands), 1-4 m in crevices of rocks and at the base of Lominorio beds (72 spms M. Oliverio leg.): Sal (Cabo Verde Islands). 1 m amidst rocks (16 spms E. Rolan leg.). Samples from 7 localities of the Atlantic West African coasts from Mauritania southward to Angola and the islands of Sao Thome and Principe were examined: Miss. Port Etienne (Mauritania), low tide (5 shs, M. Marchad leg.); 15°37'N-023°22'W 120-150 m (CALYPSO 1959 expedition, stn 65: 25 shs); Dakar (Senegal), low tide level, amidst sea-weed (10 shs and 5 spms, E. Rolan and S. Gofas leg.); Accra (Ghana), tide level (13 spms, E. Rolan leg.); Ilha de Luanda (Angola) 75-80 m (35 shs, S. Gofas leg.); Santo Antonio (Principe Is.), tide level (60 shs and 13 spms, E. Rolan leg.); Praia Mutamba, (Sao Thome Is.), tide level (32 shs and 14 spms, E. Rolan leg.). Quaternary fossil shells were studied from several Italian outcrops dated from Middle to Late Pleistocene, and from the Canary Islands (Tachero, Tenerife Is., dated MellahianjFlandrian). Material from the Pliocene of Altavilla (RUGGIERI, 1992) was examined, confirming the attribution to the present complex, but was eroded and lacking the protoconch, hampering any inference on larval development. A large part of such material consists of subsampIes from collections of the Museum Nationa1e d'Histoire Naturelle (MNHN Paris, France), Museo Geologico "G.G. Gemellaro" (MGP Palermo, Italy), Museo Insular de Ciencias Naturales (MICN Santa Cruz de Tenerife, Spain), Zoological Museum of Amsterdam (The Netherlands), and private collections of E. Rolan (Vigo, Spain), Marc Streitz (Antibes. France) and Marcel Pin (Dakar, Senegal). Other material has been collected by the author during several oceanographic cruises and field trips, and is presently in the malacological collections at the BAU Dept. ("La Sapienza" Rome University, Italy). Radulae were dissected from alcohol fixed and dried specimens (3 to 10 spms per population) under a stereo microscope and mounted on stubs for SEM examination. Protoconchs of juveniles (3 to 15 shs per population) were cleaned with a Lucas-Dawe

40'. ['\'\'j

~

= =

1000

20'

20'

non p/anktotrophic development planktotrophlc development

2000km

0'

20'

FIG. I. - Distribution of the genus Columbella in the East Atlalllic. Different striped areas distinguish populations with planktotrophic and non-planktotrophic development. According to the present study they should correspond to two different species. ''')'J'' evidenciates lack of sufficielll data on the Columbella of that area. Sam'ples for electrophoresis: • Is. Lampedusa: .A. Is. S. Stefano: • Is. Tenerife.

Ultrasonic cleaner for 20-30 seconds and mounted for SEM. Both radulae and protoconchs were coated with gold/palladium, and studied by a Stereoscan 200 (Cambridge Instruments) with 5-15 kV acceleration. The type of development of each studied individual was inferred on the base of the morphology of the protoconch: presence vs. absence of a larval shell. conical vs. bulbous shape of the protoconch, presence vs. absence of a 'sinusigera' outer lip. The intracapsular development of C. rustico at Banyuls-sur-Mer was termed "direct" by BANDEL (1975), but this telm is embryologically eIToneous (FIOROi\I, 1966). and in the present paper the more informative "non-planktotrophic" category name is employed (JABLONSKY and LUTZ, 1983). In a preliminary electrophoretic study three populations were employed to compare the Columbello from the Mediterranean Sea and the Macaronesian area (Fig. I):

DEVELOPMENT AND ELECROPHORESIS OF E. ATLAi\TIC COLL'MBELLA

79

TABLE I. - Electrophoretic techniques employed in the present study. Staining reference

Loci scored

B A B B A G A

RICH.-\RDSO:-J el al.. 1986 TRACEY el al.. 1975 RtCt-IARDSO" el al.. 1986 AYM.-\ el al .. 1974 AYALA el al .. J972 BREWER and SING. 1970 AYAI.-\ el al .. 1972

2.6.11

BI

AYAL.-\

Aco Acph Ada Ao Aph Ca-I. Ca-2. Ca-3 Est-I. Est-2. Est-3. Est-4 Got

1. I. 1.49

El

AYALr\ 1'1 al.. 1974

G6pd

2.7.1.1 1.1.1.42 3.4.11.1 1.1. 1.27 5.3.1.8

C F A A J

AYALA el al.. 1974 BREWER and St;\G. 1970 AY.-\l.A el al.. 1972 SELA:-JDER 1'1 al.. 1971 HARRtS and HOPKINSO:".. 1978

Hk IdhLap-I. Lap-2 Ldh Mpi

3.4.11 5.3.1.9 2.7.5.1 1.15.1.1 1.2.137

A C D B A

WARD and BEARDMORE. 1977 BREWER and StNG. 1970 BREWER and StNG.. 1970 AYAL1\ ('{ al .. 1972 SELAf\DER el al .. 1971

Pep-I. Pep-2 Pgi Pgm To Xdh

Codes

Enzymes

E.Cn°

Buffer system'"

ACO ACPH ADA AO APH CA EST

Aconitate hydratase Acid phosphatase Adenosine deaminase Aldehyde oxidase Alkaline phosphatase Carbonic anydrase Esterase

-1.2.1.3 3.1.3.2 3.5.4.4. 1.2.3.1 3.1.3.1 4.2.1.1 3.1.1.1

GOT

GlutamlTIic-oxalacetic transam inase GIlIcose-6-Phosphate dehydrogenase Hexokinase Isocitrate dehydrogenase Leucine amino peptidase Lactate dehydrogenase Mannose phosphate Isomerase Peptidase Phosphogl ucose isomerase Phosphogl ucose mutase Tetrazolium oxidase Xanthine dehydrogenase

G6PD HK !OH LAP LDH MPI PEP PGI PGM TO XDH

" as in DE MATTHAEtS

el

al .. 1975

al.. 1983.

C. rustica - Lampedusa Is., on Cystoseira beds at 2-5 m depth, collected during 'Urania 92' cruise (M. Oliverio legit, 2-XII-1992). S. Stefano Is. (Ponziane Islands, LT Italy) collected in crevices at 5 m (M. Oliverio legit, I9-IX-I 992). C. adansoni - Punta del Hidalgo, Tenerife Is. (Canary Islands, Spain), in crevices of rocks, at the base of Laminaria, and on Cystoseira beds at 0.5-3 m depth (M. Oliverio and D. Luchetti legit, VII-1993). The crude homogenates of the entire body added to 1000 microliters of water were centrifuged at 10,000 r/min for 5' at nearly 0 QC: this procedure allowed the storage of aliquots of the supematant to be used in successive analyses or replicates. Electrophoresis was performed on horizontal starch gel (HARRIS and HOPKINSO'\i, 1976). Buffer systems and staining techniques employed are listed in Table 1. Interpopulation genetic distance indices (0: NEI, 1978: R: ROGERS, 1972) were calculated and an UPGMA Cluster Analysis (S'\iEATH and SOKAL, 1973) was performed to draw dendrograms on the base of the distance matrices. Diagnostic alleles were identified sensu AYALA (1983). Chi-squares 80 M.OLlVERIO

el

were computed (WORKMAN and NISWANDER, 1970) to test heterogeneity in the allele composition of the three populations, and of the two Mediterranean ones. The packages BIOSYS-1 (SWOFFORD and SELANDER, 1981) and NTSYS 1.8 (ROHLF, 1993) were employed.

RESULTS After examination of the protoconchs available, the distribution of the developmental types was defined as in Figure 1. All Mediterranean and Moroccan Recent popu1ations studied invariably display the bulbous protoconch characteristic of non-planktotrophic development (Fig. 2). The surface of the protoconch appears smooth, and the particular shape of the initial part of the protoconch, irregular and not perfectly spherical, is due to the presence of nurse-eggs (BA'\iDEL, 1975) that provide additional food supply to the viable embryos, and that deform the shell of the embryos before their calcification. The boundary between the protoconch and the teleoconch is simple with no evidence of a Sll1Uslgera

FIG. 2. - ProlOconch of the non-planktotrophic Iype: Is. Tremiti (Italy. Adriatic Sea). The arrows indicate the boundary between prolOeonch and teleoconch. Scale bar = 100 ~tm

lip. The same pattem is shared by all Quatelllary specimens from the MeditelTanean area (Italian and Iberian Middle Pleistocene to Holocene outcrops: OUVERIO and SABELLI, unpublished observations). A limited amount of material from Mauritania and Senegal was examined: all specimens with intact protoconch from this area indicate a non-planktotrophic development. The Macaronesian populations have a protoconch with an embryonic shell (ca 0.7-0.8 whorls) secreted during the intracapsular life. and a larval shell (of ca 1.5 whorls) produced by the veliger during the planktonic life (Figs. 3. 4). A clear-cut demarcation between the embryonic and the larval shell is not evident in all specimens. Rather. an area of transition from the embryonic to the larval shell is discemible (Fig. 4). The entire protoconch is covered by microtubercles and at the protoconch/teleoconch boundary a sinusigera-like outer lip confinm the development by a planktotrophic veliger. All Quatemary fossil specimens from the Canary Islands display the same planktotrophic development. All specimens from Angola, Ghana and Sao Thome and Principe Islands have identical protoconchs of the planktotrophic type.

FIG. 3. - Protoconch of the planktotrophic type: Tenerife Is. (Canary Islands. Spain). The arro\\"S indicate the boundary between prolOconch and teleoconch. Scale bar = I 00 ~m

FtG. -+. - Protoconch of the planklOtrophic type (top \ iew): Sal (Cabo Verde Islands). Black arrows indicate the protoconch-teleoconch boundary: \\ hite arrows indicate the transition area between embryonic and larval sheik Scale bar = I00 ~m

All dissected specimens sampled from the Mediterranean. West African coasts (Morocco to Ghana). Suo Thome and Principe Islands. and the Macaronesian Islands (excluding the Azores).

DEVELOPMEI\'T Ai\D ELECROPHORESIS OF E. ATLAi\TIC COLL·.v/BELLI 81

display a radula of the same type (Fig. 5, 6). The central tooth is vestigial, and reduced to a slightly arched plate (Fig. 5). A single pair of massive lateral teeth is present, with three cusps: the distal one is narrow, the central one is slightly enlarged at the base, and the proximal one is quadrangular and api-

cally curved. On the outer side of the teeth there is a basal cusp, whichis narrow and with a triangular base (Fig. 6). The zymograms for 19 enzymes displayed 27 activity zones interpreted as the results of as many presumptive encoding loci. Allele frequencies and

TABLE 2. - Allele frequencies in the assayed populalions. An asterisk marks a diagnostic locus. All populations share the same allele at the following 15 monomorphic loci: Ada. Ca-I. Ca-2. Ca-3. Est-I. Est-2. Est-3. ESI-4. GOI. Hk. Idh. Ldh. Pep-I. Pep-2. Xdh. Population: Lam = Lampedusa Is.: Stc = S. Stefano Is.: Ten = Tenerife Is. mSliea-X' = Chi square of heterogeneity among Lam and Ste populations: total-X' = Chi square of hetcrogcneity among all three populations. Probability in parentheses: ,',,' = probability> 0.05.

e. adallsolli

e. I"IIsliea LOCUS Allele

Lam

Ste

ACO A B C

1.000 0.000 0.000 ,

.,

0.000 1.000 0.000 17

0.917 OJJ83 12

0.889 0.111 9

1.000 0.000 12

1.000 0.000 17

0.000 1.000 33

0.000 1.000 12

0.000 1.000 17

1.000 0.000 33

0.000 1.000 12

0.000 1.000 17

1.000

1.000 0.000 12

1.000 0.000 17

0.000 1.000 33

0.000 1.000 12

0.000 1.000 17

1.000 0.000

0.000 1.000 12

0.000 1.000 17

1.000 0.000 33

0.000 0.833 0.167 12

0.000 0.833 0.167 12

0.000 1.000 12

0.000 1.000 17

0.083 0.542 0.125 0.250 12

0.056 0.361 0.194 0.389 18

1.000 0.000 12

1.000 0.000 17

(N)

ACPH A B (N)

"'AO-I A B (N)

"AO-2 A B (N)

*APH A B (N)

*G6PDH A B (N)

*LAP-I A B (N)

"LAP-2 A B (N)

MPI A B C (N)

"PGI A B (N)

PGM A B C D (N)

*TO A B (N)

82 M. OUVERIO

ms/iea-x'

40.000 (0.000)

0.092"" (0.761)

Ten

0.263 0.684 0.053 19 0.893 0.107 28

total-x' 35.511 (0.000)

0.124"" (0.940) 124.000 (0.000) 124.000 (0.000) 124.000 (0.000) 0.000

33 124.000 (0.000) 124.000 (0.000)

r

- -.)

0.000*" (1.000)

0.103 0.793 0.103 29 1.000 0.000 33

2.500'"'' (0.475)

0.000 0.147 0.135 0.118 34 0.000 1.000 .).1

124.000 (0.000) 5.813"" (0.213)

124.000 (0.000) 46.030 (0.000)

124.000 (0.000)

FIG. 5. - Radula of East Atlantic ColulI/bella.· Accra (Ghana). Scale bar = 100 ~m

FIG. 6. - Radula of East Atlantic Colul1Ibella: Dakar (Senegal).

genetic variability are reported in Tables 2 and 3. Levels of allozyme polymorphism (heterozygosity, nO of alleles and percentage of polymorphic loci: Table 3) seem to be comparable in all populations. It must be stressed that the small sample size, especially at the polymorphic loci, does not allow to elaborate reliable statistics for genetic variation analysis: thus, at present, genetic variability will not be discussed in more detai I here. Diagnostic alleles between the two species have been found at 8 loci that are marked with an asterisk in Table 2. Especially at the To and Pgi loci the mobility difference of the two altemative alleles was very high: they can thus probably be used as routine taxonomic markers in further studies. Chi-squares at pOlymorphic loci highlighted significant divergence between the three populations at all but two (Acph and Mpi)

loci. Comparing the two MeditelTanean populations. they were significantly heterogeneous in the allele composition only at the locus Aco (with fixed, alternative alleles); at the other four polymorphic loci they did not present significant differences. For the taxonomic aims of the present study, the computation of genetic distance indices and the identification of diagnostic alleles at species level yielded important results. Values of genetic distance were relatively low between the two Mediterranean populations (D = 0.039 and R = 0.044). Comparing each Mediterranean population with the Macaronesian one. the values were much higher (D = 0.388-0.416 and R = 0.328-0.346). The resulting pattem is shown in Figure 7 where an UPGMA tree displays the separation of the c. adansoni population from the c. rustica ones.

Scale bar = 50

~m

TABLE 3. - Genetic variability at 27 loci in the assayed populations. - ") A locus is considered polymorphic if the frequency of the most comIllon allele does not exceed 0.99 - Ho = observed heterozygosity - H = expected (Unbiased estimate: see NE!. 1978). Standard error in parentheses.

Population

Mean nO of alleles per locus

Percentage of polymorphic'" loci

Mean Heterozygosity Ho

H

C. I'lIsrica Lampedusa Is.

1.2 (0.1 )

I 1.1

0.040 (0.025)

0.0-\1 (0.026)

C. I'lIsrica S. Stefano Is.

1.2 (0.1 )

I 1.1

0.037 (0.02-\)

0.0-\-\ (0.028)

C. adansoni Tenerife Is.

1.3 (0.1 )

1-\.8

0.05-\

0.05-\ (0.027)

(0.026)

DEVELOPMEt\T AND ELECROPHORESIS OF E. ATLA1\TIC COLLl1BELLA 83

Lampedusa

rust/ca

S.Stefano adansonl

Tenerife

.D NEI.19781

i

0.42

0.30

0.18

0.06

0

FIG. 7. - UPGMA tree portraying genetic relationships among Collll11hella populations and species. based on the D (NEt. 1978) genetic disIance matrix.

DISCUSSION All Moroccan. Senegalese and Mauritanian Atlantic specimens have the same paucispiral and bulbous protoconch as the Mediterranean ones. However. all specimens with intact protoconchs examined from the populations south of Senegal have planktotrophic development according to their larval shells. Their general shell morphology also closely resembles that of the Macaronesian specimens. The lack of data on live collected specimens from the Azores hampers a definitive decision on their specific status (the serrated radula found by MOOLENBEEK and HOE:\SELAAR (1991) in an unspecified 'Macaronesian' population has not been scored in the present study: there is the possibility that it could be found in the Azarian Colul1lbella, for which the name a:orica OROUET, 1958 is eventually available). All other populations (those from the remaining Macaronesian archipelagos and the West African coasts south of Senegal) are here considered conspecific. The morphological data set presently available seems to include the existence of at least two species in the East Atlantic area (see Fig. I): Columbel/a mstica (L. 1758). from the Meditena-nean and the Moroccan. Mauritanian and Senega-lese Atlantic coasts (with non-planktotrophic development): Columbel/a adansoni MD!KE, 1853 from the Macaronesian islands (excluding the Azores?) and the West African coasts south of Senegal (with planktotrophic development). The altemative hypothesis of a single specific entity with variation in the larval development should also be considered. A polymorphism for larval development (poecilogony) was recently contested in marine invertebrates (HOAGLAND and ROBERTSOl\, 1988: BOUCHET, 1989). Nevertheless. evidence does exist that some species can develop following altemative heterochSot M.OUVERIO

ronic pathways (LEVli\ et al .. 1991: PARKS et al.. 1988: STRATHMA:\:\ et al.. 1992). Genetic distance values here scored indicate a good separation between the population of Tenerife Island and those from the Mediterranean Sea. The observed values (0=0.4) are perfectly within the range referable to allopatric non-conspecific populations (THORPE, 1983). Comparable levels of genetic divergence were scored between sibling species of the Lifforina :ic:ac (GMELI)J) complex (see WARD, 1990). Alternative alleles are fixed in the two species at 8 loci among the 27 scored. Thus the present preliminary electrophoretic study supports the specific distinction between the Mediterranean (non-planktotrophic) populations of C. mstica and the Canarian (planktotrophic) ones. It seems evident that a more detailed study is needed especially on the populations from, and immediately south of, Senegal. This region may represent a transition area between the two species. Due to the origin from Senegal of the taxon C. striata OUCLOS taxonomic problems could be involved. An electrophoretic survey would allow definition of whether a clear-cut separation is present or a hybridisation/introgression area works as a barrier. At present, from the taxonomic point of view. the name adansoni is here maintained for the planktotrophic species from Macaronesia and West Africa. The presence of a locus showing altemative alleles between the two Mediterranean populations can be explained either by the reduced level of gene flow among non-planktotrophic populations (and) or by some effect of selection upon such a locus. Moreover, some effect of the sample size of the Mediterranean populations can not be excluded. Chi-square tests of heterogeneity at the other three polymorphic loci (Acph, Mpi and Pgm) showed non-significant values between the two c. J'lIstica populations. A model of speciation in the Caenogastropoda. involving loss of planktotrophy as a leading factor. has been recently claimed (OUVERIO 1991: SAFRIEL and HADFlELD, 1992) to explain the presence of sibling species differing only or mainly in their larval development and works well under an 'Altemative Adaptation' developmental perspective (WEST-EBERHARD, 1986). Non-planktotrophic populations referable to C. rustica have been scored in Quaternary Mediterranean outcrops. Nearly contemporaneous records from Canary Islands are of the plank(otrophic type. The origin of the Quaternary non-

planktotrophic C. mstica form a Pliocene ancestral species (RUGGTERI. 1992) with planktotrophic development fits well with this pattern: it agrees also with the consideration that the presence of a planktotrophic larval phase is normally the primitive conditions in the Caenogastropoda lineages. It has been suggested that palaeoclimatic changes (and especially cooling phases) operated to produce speciation opportunities in the Mediterranean Sea (OLIVERTO. 1991. 1994a, 1994b). According to the values of genetic distance between C. adansoni and C. rl/sticct (0=0.4), the time of divergence of the two species can be dated approximately to 2 M. Y. It is noteworthy that this time can be correlated to the onset of glaciation. and especially with their extension to southern regions. The present distribution of the planktotrophic C. adansoni along the West African coasts could be explained by its shifting southwards during a cold phase. This could have followed the analogous shifting of boreal species (TAVIA'JI et al., 1991) from northern regions, and the range extension of the nonplanktotrophic species along the Moroccan Atlantic coasts could have contributed to produce the present distribution pattern. The well-known isolation of the benthic fauna of the Canary Islands from the continental West African coasts. due to upwellings and general circulation patterns. contributed to hamper colonization of Moroccan coasts by C. adansoni.

ACKNOWLEDGMENTS S. Gofas (MNHN. Paris). R.G. Moolenbeek (ZMA, Amsterdam), M. Pin (Dakar. Senegal). E. Rolan (Vigo. Spain). M. Streitz (Antibes. France). and EG. Talavera (MIC . S.Cruz de Tenerife). kindly made available material for this study. Profs. B. Baccetti (CNR, Siena) and M. Cobolli ("La Sapienza" Rome University), allowed the author's participation in the oceanographic cruises onboard R/Y Bannock and R/V Urania. E. De Matthaeis ("La Sapienza" Rome University) constantly supported and encouraged the author during this and other studies. Two anonymous referees significantly improved this paper. This work was partly supported with funds by MURST (40C/c). CNR (Comitato Ambiente). and Scuola di Dottorato in Biologia Evoluzionistica ("La Sapienza" Rome University).

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