Sonderdruck aus Z. f. zool. Systematik u. Evolutionsforschung

Band 23 (1985), Heft 1, S. 38^9

V E R L A G P A U L P A R E Y • S P I T A L E R S T R A S S E 12 • H A M B U R G 1 Alle Reclitc, audi die dcr Obersetzung, des Nachdrucks, der photomechanischen Wiedergabe und der Speicherung in

Datenverarbcitungsanlagen, vorbchaltcn. (C) 1985 Verlag Paul Parey, Hamburg und Berlin

L a b o r a t o r i i i m v o o r Algemene D i e r k u n d e ( D i r . P r o f . D r . W. N . V e r h e y e n ) , R i j k s i m i v e r s i t a i r C e n t r u m A n t w e r p e n

Estimation of genie similarity within and between Arion hortensis s. 1. and A. intermedins by means of isoelectric

focused esterase patterns in hepatopancreas homogenates (Mollusca, Pulmonata: Arionidae)

By. T. BACKELJAU Received o n 2 0 . J u l y 1 9 8 4

U . S. Copyright Clearance Center Code Statement: 0044-3808/85/2301-0038/$ 02.50/0 Z . zool . Syst. Evolut.-forsch. 23 (1985) 38^t9 © 1985 Verlag Paul Parey, Hamburg und Berlin ISSN 0044-3808/InterCode: Z Z S E A A

Abstract

Over 200 specimens of A r i o n hortensis s. 1. and A . i n t e r m e d i n s were surveyed for general esterase patterns in hepatopancreas homogenates by means of isoelectric focusing. Zymograms were compared with the band counting method. The counts were used to calculate genie similarities. A n A N O V A was employed to test differences among the calculated similarities. Five major conclusions could be drawn from this analysis. 1. A . hortensis s. s.,A. d i s t i n c t u s and A . o w e n i i , three species which until recently were lumped together

as A . hortensis s. 1., constitute three clearly different gene pools. Hence they are reproductively isolated and may be regarded as biological species.

2. In A . hortensis s. s. and A . d i s t i n c t u s there is a gradual decline in genie similarities from intra-population pairs over conspecific pairs from different populations to interspecific comparisons. This fact confirms the genetic isolation between both species.

3. N o comparable gradual decrease of genie similarities could be detected among A . o w e n i i and A . i n t e r m e d i n s , since both species revealed constant and nearly uniform (monomorphic) esterase patterns. Hence both species are assumed to reproduce by self-fertilization or apomictic partheno­genesis, whereas A . hortensis s. s. and A . d i s t i n c t u s are true outcrossing species showing very poly­morphic zymograms. In A . o w e n i i a mixed breeding system is suggested since in this species sperma-tophores and mating behaviour have been observed.

4. N o evidence was found for splitting A . d i s t i n c t u s in its two genitalial forms. Consequently the presence or absence of an eversible portion in the oviduct is not completely reliable for separating A . d i s t i n c t u s from both other species and so only the structure around the outlet of the epiphallus in the atrium can be used to distinguish the three siblings.

5. In view of the high similarity between A . o w e n i i and A . i n t e r m e d i n s and considering chromosome numbers as well as genitalial features, it is concluded that A . i n t e r m e d i n s is more conveniently placed in the same subgenus as A . hortensis s. 1., i . e. K o b e l t i a . Hence, since A . i n t e r m e d i n s is the type species for the subgenus M i c r o a r i o n , the latter may be suppressed or should be redefined on other species.

Introduction

Recently DAVIES ( 1 9 7 7 , 1 9 7 9 ) has suggested that Arion hortensis de Ferussac, 1 8 1 9 might be a complex of three different species, viz. A . hortensis de Ferussac, 1 8 1 9 s. s., A . distinctus Mabille, 1868 and A . owenii Davies, 1979 . These species were primarily defined on differ­ences in genitalial features, mating behaviour and spermatophores. In addition, also slight differences in external morphology have been reported (DAVIES 1979 ; BACKELJAU 1 9 8 1 ; DE WILDE 1983) , but they appear not to be sufficient to separate the three species (BACKEL-

E s t i m a t i o n of genie s i m i l a r i t y w i t h i n a n d between A r i o n hortensis s. I. a n d A . i n t e r m e d i n s 3 9

F i g . 1 . Proximal parts of the genitalia m A . h o r t e n ­sis s. I. A : A . o w e n i i (Buncrana, Ireland); B : A . hortensis s. s. (Wilrijk, Belgium); C : A . d i s t i n c t u s (3) (South Croydon, Great Britain); D : typical A . d i s t i n c t u s (Wilrijk, Belgium). Scale line indi­cates 5 mm. — • = epiphallus, • = eversible por­tion in the oviduct and > = firm portion in the

oviduct

F i g . 2. Outlines of the structure around the out­let of the epiphallus in the atrium. 1 = seen from above; 2 = lateral view. A : A . d i s t i n c t u s ; B : A . hortensis s. s.; C : A . o w e n i i (the structure in this species may be rather variable: the conically ele­vated entrance as depicted here, need not to be apparent in all specimens). Scale line indicates

1 mm

JAU and M A R Q U E T i n press.). Consequently A h o r t e n s i s s. 1. may be regarded as an aggregate of sibling species.

In a multivariate analysis of morphological and anatomical features in A . h o r t e n s i s s. 1., BACKELJAU (1981) has shown that in Belgium A . h o r t e n s i s s. s. and A . d i s t i n c t u s constitute two rather well delimited forms. They can always be distinguished by the shape of the struc­ture associated with the outlet of the epiphallus in the atrium (Fig. 2) and by the presence or absence of an eversible portion in the oviduct (Fig. 1). DAVIES ( 1977 , 1979) however, reported that in Great Britain the latter feature cannot be relied on, as it seems that A . d i s t i n c t u s comprises two genitalial types: one which resembles A . h o r t e n s i s s. s. in having a clear eversible portion in the oviduct, and one lacking such portion in its oviduct (Fig. 1). The latter is here further referred to as typical/!, d i s t i n c t u s or A . d i s t i n c t u s (2) ; the former wi l l be denoted as A . d i s t i n c t u s (3).

In view of DAVIES' ( 1977 , 1979) findings, it is clear that the structure associated with the outlet of the epiphallus is the only constant character by which A . h o r t e n s i s s. s. and A . d i s t i n c t u s may be distinguished unambiguously. The value of this structure becomes even more apparent when also taking A . o w e n i i into account. This is a rare species which seems to

40 7*. B a c k c l j a i i

be restricted to Ireland and some scattered localities in Great Britain (DAVIES 1979) . Like A . h o r t e n s i s s. s. and A . d i s t i n c t u s (3) , A . o w e n i i has an eversible portion in its oviduct (Fig. 1). But again, the species can be recognized by the typical structure around the outlet of the epiphallus (Fig. 2 ) .

Because the morphological and anatomical differences between the three presumed species in A . h o r t e n s i s s. 1. are very subtle, it has been much debated whether they may be regarded as biological species (GITTENBERGER in litt.; NORRIS in litt.; EVERSHAM in litt.). Therefore we felt it necessary to start an electrophoretic survey of A . h o r t e n s i s s. I, in order to demonstrate a possible genetic isolation between the supposed species.

The first results of these investigations revealed constant, species specific protein patterns in albumen gland extracts (BACKELJAU in press). These patterns strongly support the specific rank of DAVIES' (1979) species. However, since no attempt was made to quantify the obser­ved electrophoretic differences, a more formal comparison of the involved gene pools was needed. Therefore we present in this paper genie similarities between the three species, calculated from general esterase patterns in hepatopancreas homogenates resolved by means of isoelectric focusing (IEF).

IEF was preferred not only for its high resolution power (FERGUSON 1980) , but also because it is a rather recently developed technique which until now has been used only very scarcely in mollusc investigations (SALADIN et al. 1 9 7 6 ; ROLLINSON and SOUTH GATE 1979) .

We included A . i n t e r m e d i n s Normand, 1852 in our investigations for three major reasons. Firstly it is usually considered as a well defined and easily recognizable species (ADAM 1 9 6 0 ; GITTENBERGER et al. 1970 ; KERNEY and CAMERON 1 9 8 0 ; CAMERON et al. 1983) ; consequently it can act as a reference to which the genie similarities m A , h o r t e n s i s s. 1. may be compared. Secondly, A . i n t e r m e d i n s is closely related to A . h o r t e n s i s s. 1. Therefore, WALDEN (1976) and DAVIES (1979) have suggested that both species may belong to the same subgenus, i . c. K o b e l t i a Seibert, 1873 . This opinion however, contradicts the current view by which A . i n t e r m e d i n s is placed in its own separate subgenus M i c r o a r i o n Hesse, 1926 . A n electrophoretic investigation may provide here valuable arguments for deciding between both opinions. Finally, MCCRACKEN and SELANDER (1980) reported A . i n t e r m e d i n s as a self-fertilizing and A . h o r t e n s i s s. 1. as an outcrossing species. Hence comparisons of general enzyme patterns in both species may give some insight in the reproductive strategies of the three forms in A . h o r t e n s i s s. 1.

Material and methods

In total 228 specimens of A . hortensis s. 1. a n d A . i n t e r m e d i n s , comprizing 40 populations from Belgium, Great Britain and Ireland, were surveyed for general esterase patterns in hepatopancreas homogenates. The proteins were resolved with IEF. The slugs were collected in autumn 1982 and spring 1983. In the following list the number of individuals examined from each locality is indicated in parentheses.

A . hortensis s. s. Belgium: Wilr i jk , garden (10); Berchem, garden (2); Dinant (Fonds de Leffe), road­side (2); Dinant (near Roche Bayard), abandoned stonepit (13); Marche-en-Famenne, roadside (2); Erezee, waste ground (3); Hot ton, roadside (6); Erezee-Fisenne, garden (22); Rochefort, abandoned stonepit (1); Well in , garden (1); Yvoi r , abandoned stonepit (6). Great Britain: South Croydon (Surrey), garden (1); Sanderstead (Surrey), garden (1); Wilmslow (Cheshire), garden (1).

A . d i s t i n c t u s (2). Belgium: Wilri jk, garden (20); Berchem, garden (13); Dinant (Fonds de Leffe), roadside (3); Verrebroek, orchard (6); Sinaai, waste ground (5); Waasmunster, abandoned farm (3); Dochamps, dumpingground (3); Erezee, waste ground (1); Opont, dumpingground (2); Loenhout, waste ground (37); Brasschaat, waste ground (8); Hoogstraten, waste ground (10); Rochefort, abandoned stonepit (1); Yvoi r , abandoned stonepit (1). Great Britain: Sanderstead (Surrey), garden (1); Wilmslow (Cheshire), garden (1).

A . d i s t i n c t u s (3). Great Britain: South Croydon (Surrey), garden (3); London, Highgate Cemetary

A . o w e n i i . Great Britain: London, Highgate Cemetary (4). Ireland: Buncrana, descendants of the original population of the type locality (13).

E s t i m a t i o n of g e n i e s i m i l a r i t y w i t h i n a n d between A r i o n h o r t e n s i s s. I. a n d A , i n t e r m e d i n s 41

A . i n t e r m e d i n s . Belgium: Leopoldsburg, mixed oak-conifer wood (2); Boeckhout, roadside (12); Ronsele, mixed oak-beech wood (3) ; Lomprez, mixed deciduous wood (1 ) ; Koninksem, roadside (1).

The specimens of A . h o r t e n s i s s. s. and A . d i s t i n c t u s were identified according to the criteria outlined by D A V I E S (1979) and B A C K E L J A U (1981) which are summarized in Figs. 1-2. The specimens of A . o w e n i i were supplied and determined by Mrs . S. M . D A V I E S .

Before IEF, live adult slugs were decapitated and immediately dissected under a stereo-microscope (12 X ) in a Petri dish filled with distilled water. The hepatopancreas was removed and the remains of the slugs, such as the body wall and the genitalia, were preserved in 70% ethylalcohol for later examination. O f the hepatopancreas 10 mg tissue was homogenized in 50 til of an aqueous 20% sucrose solution. Afterwards the homogenates were centrifuged at 25 000 g for 40 min by 4°C . The in this way prepared samples were stored at — 70 °C until analyzed by IEF. Usually this was done within two or three weeks after preparation.

IEF was performed in horizontal 1 % agarose gels (24 X 11 X 0.06 cm) in which a 6.25 % Pharmalyte T M ampholines solution was incorporated, creating a 4-6.5 p H gradient. As electrodes we used L K B electro-focusing strips, wetted with respectively a 0.05 M H , S 0 4 solution (anode) and a 1 M N a O H solution (cathode). To obtain a reasonably good resolution, only 5 til per sample was needed (see Figs. 4—7). The IEF was started at 500 V and during the experiments, power was kept constant. After about 90 min the focusing was interrupted at approximately 1500 V . Then the gels were stained for general esterases with the following receipt (quantities per gel): 40 mg Fast Blue R R in a mixture of 25 ml 0.1 M K H , P 0 4 / N a O H buffer (pH 7), 25 ml F L O and 2 ml cv-naphtylacetate solution (1 % a-naphtylacetate in 50% aceton). The receipt is slightly modified after H A R R I S and H O P K I N S O N (1976). Staining took 45 min. Afterwards the stained gels were dried and finally they were stored in plastic bags.

The resulting esterase patterns were studied on a light board under a magnifying lens. Adjacent patterns were compared with the band-counting method ( F E R G U S O N 1980; T E G E L S T R O M et al. 1982, 1983) . For each pairwise comparison a coefficient of genie similarity was calculated according S = C / M , where C is the number of bands of common mobility in both patterns and M the total number of bands in the individual with the highest count of the two ( F E R G U S O N 1980). A n average coefficient of genie similarity (S) was calculated as the arithmetic mean over all similarity coefficients for a given category of comparisons (e. g. for all comparisons between A . O w e n i i and A . i n t e r m e d i n s , A . d i s t i n c t u s and A . h o r t e n s i s s. s., etc.). The mean genie similarities were tested for significant differences with an analysis of variance, A N O V A ( W O N N A C O T T and W O N N A C O T T 1982).

S1 S2 S3 S1 S2

F i g . 3. T w o possible ways in comparing general esterase patterns. A : 'chain wise' comparison yielding mutually dependent similarities; B : ' two-by-two' comparisons yielding statistically independent simila­

rities

Such an analysis requires that the pairwise similarity coefficients are statistically independent obser­vations. This is achieved when each sample is used in only one comparison. Consequently a gel on which at most 24 samples can be studied simultaneously, yields maximally 12 similarity coefficients (Fig. 3 B) . This may seem rather wasteful in comparison with a 'chain-wise' analysis (Fig. 3 A ) , which would yield in our situation 23 similarity coefficients. The latter coefficients however, are not independent. Hence they cannot be compared with statistical methods.

Finally the mean genie similarities were used to construct a dendrogram by means of the un­weighted-pair-group-arithmetic-average ( U P G M A ) method as outlined by F E R G U S O N (1980).

A l l the material used in this study is kept at the Laboratorium voor Algemene Dierkunde, Rijks-universitair Centrum Antwerpen. Later on it wi l l be deposited in the collections of the Koninkli jk Belgisch Instituut voor Natuurwetenschappen ( K B I N ) , Brussel.

Results

A general inspection of Figs. 6 - 7 reveals that both A . o w e n i i and A . i n t e r m e d i n s have very constant and uniform esterase patterns, even over a relatively large geographical area. More­over, from Fig. 7 it appears as if the respective zymograms of the two species are very much alike. In A . h o r t e n s i s s. s. and A . d i s t i n c t u s (Fig. 4—5) on the contrary, no constant patterns

42 T. B a c k e l j t i i t

titiiMie-i^i

Ifi5i-"r2="=S-!U3=l Fig. 4. Variation of esterase patterns in one population of A . distinctus (2) (Loenhout, Belgium)

are apparent. Even among conspecific individuals from the same populations, there seems to exist a large variation (Fig. 4). So at first glance our material can be divided into two groups: one containing A . o w e n i i and A . i n t e r m e d i n s with their monomorphic patterns, the other comprizing A . d i s t i n c t u s and A . h o r t e n s i s s. s. with their extremely polymorphic patterns.

Although within the two afore mentioned groups no clear distinction can be made between the zymograms of the studied species, it nevertheless appears from Table 1 that the S values in each group show a conspicuous decline when interspecific comparisons are made. It is also clear that the mean similarity between species of different groups is remarkably low.

In Table 2 the S values from Table 1 are tested for significant differences with an ANOVA. Therefore we compare the mean similarity within a species with the S values for the three

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Fig. y Esterase patterns of A . hortensis s. s. and of the two genitalial forms in A . distinctus. 1-3: A . distinctus (2), Dochamps; 4: idem, Sinaai; 5-7: A . hortensis s. s., Erezee; 8: A . distinctus (2), Erezee; 9: idem, Waasmunster; 10: A . distinctus (3), South Croydon; 11 : A . distinctus (2), South Croydon; 12—13:

A . distinctus (3), South Croydon

E s t i m a t i o n of genie s i m i l a r i t y w i t h i n a n d between A r i o n h o r t e n s i s s. I. a n d A . i n t e r m e d i n s 43

0

0 1 2 3 4 5 6 7 8 9 10 11 12 13

F i g . 6. Esterase patterns i n A . o w e n i i . 1, 6—13: Buncrana, Ireland; 2-5: London, Great Britain

possible interspecific comparisons comprizing that species. Obviously all observed differ­ences are highly significant, since in every case P ( H 0 ) < 0 . 1 % .

However, such significant differences do not necessarily indicate that different species are involved. It can be expected that even within a species significant different S values may be observed depending on whether intra- or inter-population comparisons are considered. So an intraspecific analysis is needed. To achieve this, we have arranged the intraspecific compa­risons in two groups: one containing all the intra-population comparisons within a species (this group is further referred to as 'wp' , within populations) and the other embracing all comparisons between conspecific individuals from different populations ('bp', between populations).

0 1 2 3 4 5 6 7 8 9 10 11 12 13

F i g . 7. Esterase patterns i n A i n t e r m e d i n s and comparison w i t h A . o w e n i i . 1 : A . i n t e r m e d i n s , Leopolds-burg; 2^1: idem, Ronsele; 5-8: idem, Boeckhout; 9: idem, Leopoldsburg; 10: A . o w e n i i , London; 11 :

A . i n t e r m e d i n s , Boeckhout; 12: A , o w e n i i , Buncrana; 13: A . i n t e r m e d i n s , Ronsele

44 T. B a c k e l j a n

T a b l e I

Mean genie similarities (S) between A . hortensis s. s., A . distinctus, A . owenii and A . intermedius

d h

0.68 0.31 0.13 0.18 (0.18) (0.10) (0.06) (0.11)

0.76 0.14 0.33 (0.15) (0.06) (0.05)

0.98 0.66 (0.02) ( - - )

0.94 (0.05)

Standard deviations are indicated in parentheses. Abbrev. d = typical A . d i s t i n c t u s , h = A . h o r t e n s i s s. s., i = A . i n t e r m e d i n s and o = A . o w e n i i .

T a b l e 2

Results of the analysis of variance on the interspecific comparisons from Table 1

Compared mean Degrees of f reedom Observed F Probability Conclusion similarities freedom value level

d-d/h 1,53 48 P < 0.001 H» rejected h-d/h 1,39 87 P < 0.001 H 0 rejected d-d/o 1,47 45 P < 0.001 H 0 rejected o-d/o 1,10 216 P<0.001 H 0 rejected h-h/o 1,32 37 P < 0.001 H 0 rejected o-h/o 1,9 390 P < 0.001 H 0 rejected d-d/i 1,45 37 P<0.001 H 0 rejected i-d/ i 1,9 1226 P<0.001 H 0 rejected h-h/i 1,32 76 P < 0.001 H 0 rejected

H 0 rejected i-h/ i 1,10 1254 P<0.001 H 0 rejected H 0 rejected

o-o/i 1,6 49 P<0.001 H 0 rejected i-o/ i 1,7 318 P < 0.001 H 0 rejected

P indicates the probability that the observed difference between the compared S values is merely the result of chance; the nulhypothesis (H 0 ) is for each comparison 5, = S,. The same abbrevations are used as in Table 1. Example: in the first comparison, d/d-d/h, the difference between respectively S, = 0.68 and S, = 0.31 is tested.

T a b l e 3

Results of the analysis of variance on the intraspecific comparisons in A . distinctus and A . hortensis s. s.

Compared mean similarities

Degrees of freedom

Observed F value

Probability level

Conelusion

d(wp)-d(bp) 1,41 3.15 P<0.05 H 0 rejected d(bp)-d/h 1,18 18.60 P<0.001 Ho rejected h(wp)-h(bp) 1,27 7.80 P<0.05 H 0 rejected h(bp)-d/h 1,15 15.97 P<0.01 H 0 rejected d(2)-d(2)/d(3) 1,9 2.07 P>0.1 H , rejected

For the S value of d/h see Table 1. The other S values are (standard deviations in parentheses): d(wp) = 0.70 (0.17), d(bp) = 0.58 (0.18), h(wp) = 0.79 (0.12), h(bp) = 0.60 (0.21) and d(2)/d(3) = 0.46 (0.05). Note : the 5 value for d(2) is the same as for d(bp).

E s t i m a t i o n of genie s i m i l a r i t y w i t h i n a n d between A r i o n hortensis s. I. a n d A . i n t e r m e d i n s 45

The results of our intraspecific analysis f o r A . d i s t i n c t u s and A . h o r t e n s i s s. s. are presented in Table 3 . As expected, there is a significant difference between the S value within popula­tions and the similarity among individuals from different but conspecific populations. A comparison of the latter S value in respectively A . d i s t i n c t u s and A . h o r t e n s i s s. s. with the interspecific similarity between both species, reveals again highly significant differences. A n intraspecific analysis in A . i n t e r m e d i n s and A . o w e n i i was not carried out as both these species show apparently monomorphic patterns (Figs. 6 - 7 ) , in which no appreciable differ­ences could be detected.

A final comparison was made between typical A . d i s t i n c t u s and A . d i s t i n c t u s (3) (Fig. 5) . Here we wanted to find out if both these forms belong to the same species or not. From Table 3 it may be concluded that this is a very probable assumption since it is the only com­parison for which P largely exceeds 1 0 % , indicating that the observed difference between the involved S values is probably the result of chance and not of underlying specific differ­ences.

Discussion

For A . d i s t i n c t u s and A . h o r t e n s i s s. s. the results in Table 2 and Table 3 , reveal a gradual decrease of genie similarities from intra-population comparisons, over conspecific inter-population comparisons to at last comparisons between the two species. Since in our study A . d i s t i n c t u s and A . h o r t e n s i s s. s. were found together at 8 localities in Belgium and Great Britain (Wilrijk, Berchem, Dinant, Erezee, Rochefort, Yvoir , Sanderstead and Wilmslow), they cannot be regarded as merely subspecies for these constitute by definition allopatric populations (MAYR 1969) . Moreover, biochemical systematics are probably not the most appropriate tool in elucidating subspecific relationships (AVISE 1975) . Consequently the observed decline in genie similarities can only be interpreted as being the result of a repro­ductive isolation between A . d i s t i n c t u s and A . h o r t e n s i s s. s. Hence, they are regarded as bio­logical species. This opinion is further supported by differences in genitalial features (Figs. 1-2) , spermatophores (QUICK 1946 ; WEBB 1 9 5 0 ; DAVIES 1977 , 1979 ; BACKELJAU and MARQUET in press), mating behaviour (QUICK 1 9 6 0 ; DAVIES 1977 , 1979) and general protein patterns in albumen gland extracts (BACKELJAU in press).

In an analogous way we can interpret our data for A . i n t e r m e d i n s and A . o w e n i i . These species differ not only clearly in their external appearance and anatomy, but also the genie similarity between both species decreases sufficiently to support their specific rank (Tables 1-2) .

The S values within and among A . o w e n i i and A . i n t e r m e d i n s are appreciably higher than the corresponding values in A . d i s t i n c t u s and A . h o r t e n s i s s. s. They may be the result of fundamental different reproductive strategies, whereby the latter two species are clearly out-crossers. The monomorphic patterns in A . i n t e r m e d i n s and A . o w e n i i however, may be explained by assuming regular self-fertilization. In A . i n t e r m e d i n s this has already been reported by MCCRACKEN and SELANDER (1980) , who also found nothing but mono­morphic enzyme patterns in this species. However, with their data it is not possible to decide whether actually self-fertilization or parthenogenesis is involved. Therefore breeding experiments with known heterozygote individuals should be carried out.

In this context it is noteworthy that recently NICKLAS and HOFFMANN (1981) found that the pulmonate land slug D e r o c e r a s l a e v e (Miiller 1774) usually reproduces by apomictic parthenogenesis, although reciprocal outcrossing may occur occasionally. Important is that both hermaphrodite and female individuals (the sexual status of these slugs is controlled by environmental factors) reproduce parthenogenetically. Despite this mixed breeding system, there is only very little heterozygosity in natural populations (NICKLAS and HOFFMANN 1981) . This may indicate that apparently monomorphic enzyme patterns, even if they show a low degree of heterozygosity, are not sufficient to decide which breeding system is involved.

46 T. B a c k e l j a u

Nevertheless for A . i n t e r m e d i u s reproduction by cross fertilization seems highly unlikely. Three arguments support this thesis: (1) spermatophores of A . i n t e r m e d i u s are unknown (OKLAND 1922; QUICK 1960; WIKTOR 1973; DAVIES 1979), (2) the congress has never been described (OKLAND 1922; QUICK 1960) and (3) individuals reared in isolation produce viable eggs (CHICHESTER and GETZ 1973; DAVIES 1977).

In A . o w e n i i on the contrary, spermatophores and mating behaviour have been reported (DAVIES 1977, 1979). So in view of its monomorphic esterase patterns, we assume that A . o w e n i i has a mixed breeding system comprizing as well cross- as self-fertilization. Such facultative self-fertilization has also been reported in the land snail R u m i n a d e c o l l a t a (L . , 1758) (SELANDER and KAUFMAN 1973). However again, the available data in R u m i n a decol­l a t a and A . o w e n i i do not enable us to distinguish self-fertilization from parthenogenesis.

fig. 6". Dendrogram showing the rela­tionships among A . hortensis s. 1. and A . intermedins based on genie similari­ties calculated from general esterase pat­terns in hepatopancreas homogenates re­solved with isoelectric focusing. The dendrogram was constructed according

to the U P G M A method

From the foregoing discussion it is clear t h a t A o w e n i i , A . i n t e r m e d i u s , A . d i s t i n c t u s and A . hortensis s. s. constitute four well defined biological species. In Fig. 8 we present a dendrogram showing the interspecific relationships. Again the two initially recognized species groupings appear. Important is however, that/I. o w e n i i seems more closely related t o A . i n t e r m e d i u s than it is to both other species, with which it until now has been confused. It is also clear that A . d i s t i n c t u s and A . hortensis s. s. are electrophoretically much more dissimilar than A . o w e n i i a n d A . i n t e r m e d i u s . The superficial resemblance among the esterase patterns of the latter two species is indeed remarkable (Fig. 7). However, a comparable result has been reported by UKOLI (1973) who stated "that though on purely morphological grounds populations of B i o m p h a l a r i a pfeifferi, B . a l e x a n d r i a n a and B . s u d a n i c a were recog­nizable as distinct species, their esterase patterns indicated only minor variations". The same author also pointed out that "though esterase patterns can be relied on to demonstrate popu­lation differences within a species, they are not necessarily equally reliable in evaluating rela­tionships between different species". Nevertheless other authors have successfully used general esterases in resolving systematical problems in B i o m p h a l a r i a and B u l i n u s (WIUM-ANDERSEN 1973; JELNES 1979). Our results here indicate that also in A r i o n esterase patterns may be useful to elucidate systematical problems.

In the analysis above we only considered typical A . d i s t i n c t u s (Fig. 1). Concerning A . d i s t i n c t u s with an eversible portion in the oviduct, we think our results show that this form should be considered conspecific with typical A . d i s t i n c t u s . However, in view of the limited number specimens used in this study, we suggest that a further electrophoretic analysis should be undertaken in order to clarify the relationships between both genitalial forms.

In view of the close relationship between A . i n t e r m e d i u s and A . o w e n i i and considering the relatively large distance between A . d i s t i n c t u s and A . hortensis s. s. (Fig. 8), we think there is no ground to exclude A . i n t e r m e d i u s from the subgenus K o h e l t i a . This thesis is supported by the work of BEESON (1960). He compared chromosome numbers in different A r i o n species. For both A . i n t e r m e d i u s and A . hortensis he found a haploid number of n = 28. The corresponding figures in other species are: A ( C a r i n a r i o n ) c i r c u m s c r i p t u s n = 29, A . ( M e s a r i o n ) subfuscus n = 25 and A . ( A r i o n ) r u f u s l a t e r n = 26. So if A . i n t e r m e d i u s and A . hortensis are placed into one subgenus, i . e. K o b e l t i a , the chromosome numbers exactly match the subgeneric division. DAVIES (1977, 1979) pointed out that there are also morpho-

A. intermedius

A.owenii

A. hortensis s.s.

A. distinctus

E s t i m a t i o n of genie s i m i l a r i t y w i t h i n a n d between A r i o n hortensis s. I . a n d A . i n t e r m e d i n s 4 7

logical and anatomical arguments for regarding A i n t e r m e d i n s as a K o b e l t i a . That spermato­phores of this species are still unknown, is no objection, since the strongly serrated sperma­tophores of A o w e n i i and A h o r t e n s i s s. s. also do not correspond with the typical K o b e l t i a spermatophore, which lacks any appreciable serration (HESSE 1926) . Concluding, we think there is enough evidence to place A i n t e r m e d i u s in K o b e l t i a . This may imply that the subgenus M i c r o a r i o n , for which A i n t e r m e d i u s is the type-species, can be suppressed or should be redefined on other species such as A v e j d o v s k y i Babor & Kostal, 1893 or A obesoductus Reischiitz, 1973 .

Acknowledgements

We are most indebted to Mrs. S. M . D A V I E S (Great Britain), Mrs . S. D E R I D D E R (Wilrijk, Belgium) and D r . J . F . G E Y S , University of Antwerp, for providing us valuable material. Lie. J . V A N R O M P A Y , Univer­sity of Antwerp, and T. IR . M . S E L E N S , University of Antwerp, kindly helped with the electrophoretic analysis. D r . V . V A N D O R E N , University of Antwerp, and D r . L . L E M M E N S , University of Antwerp, are greatfully acknowledged for their comments on the statistical treatment. We thank Prof. Dr. W. N . V E R -H E Y E N , University of Antwerp, for providing us the necessary working facilities. Special thanks are due to Prof. D r . I. H . H U L S E L M A N S , University of Antwerp, for linguistic help. The author is grant holder of the Instituut tot Aanmoediging van het Wetenschappelijk Ondcrzoek in Nijverheid en Landbouw, I . W . O . N . L .

Resume

E s t i m a t i o n des s i m i l a r i t e s genetiques entre A r i o n hortensis s. I. et A . i n t e r m e d i n s p a r f o c a l i s a t i o n i s o ­e l e c t r i c des esterases des e x t r a i t s de l a g l a n d e digestive ( M o l l u s c a , P u l m o n a t a : A r i o n i d a e )

Extraits de la glande digestive de plus que 200 individus de A r i o n hortensis s. 1. et de A . i n t e r m e d i u s out ete soumis a une focalisation iso-electric. L'activite des esterases non-specifiques a ete recherchee et les differentes zymogrammes ont ete companies avec la methode de la numeration des bandes. Avec ces donnees la, nous avons calcule des similarites genetiques. Differences entre ces dernieres ont ete verifiees avec une analyse de variance ( A N O V A ) . Cinq conclusions importantes sont formulees. 1. A . hortensis s. s., A . d i s t i n c t u s et A o w e n i i , trois especes qui jusqu'a present ont ete considerees en­

semble comme A hortensis s. 1., constituent clairement trois entites genetiques isolees par rapport a leur reproduction. Elles doivent done etre considerees comme de especes biologiques.

2. Les similarites genetiques parmi A hortensis s. s. et A d i s t i n c t u s montrent une decroissance graduelle en comparant respectivement d'abord des individus de memes populations, puis des individus ap-partenant a des differentes populations du meme espece et finallement des individus des differentes especes. Ce fait confirme l'isolation genetique entre les deux especes.

3. Une decroissance comparable n'a pas ete observee parmi A . o w e n i i et A . i n t e r m e d i u s , puisque ces deux especes montrent des zymogrammes a peu pies monomorphes. Par consequence elles sont supposees de se rcproduire par autofecondation ou par parthenogenese. Pourtant, pour A . o w e n i i nous suggerons an systeme reproductif mixt, car dans cette espece des spermatophores ainsi que des copulations ont deja ete observees. A u contraire, les zymogrammes polymorphes de A . hortensis s. s. et A d i s t i n c t u s montrent que ce sont des vraies fecondateurs reciproces.

4. Nous n'avons aucune evidence pour separer electrophoretiquement les deux formes genitaux de A . d i s t i n c t u s . Done la presence ou absence d'une portion evertile dans l'oviduct, n'est pas suffisante pour distinguer les siblings de A . hortensis s. 1. Seulement la structure autour l'orifice de l'cpiphallus dans l'atrium, pent donner une identification exacte.

5. En considerant la grande similarite entre A . o w e n i i e t A . i n t e r m e d i u s , et en vue des nombres chromo-somales ainsi que des caracteres genitaux, nous pensons que A . i n t e r m e d i n s est mieu place dans le sous-genre K o b e l t i a (espece type: A . hortensis s. 1.). Done le sous genre M i c r o a r i o n , dont A . i n t e r ­medius est l'espece type, peut etre supprime ou doit etre defini de nouveau.

Zusammenfassung

Schatzung der genetischen A h n l i c h k e i t i n n e r h a l b u n d zwischen A r i o n hortensis s. I . u n d A . i n t e r m e d i u s d u r c h i s o - e l e k t r i s c b e F o k u s s i e r u n g von Esterasen i n Hepatopankreas-Homogenaten

( M o l l u s c a , P u l m o n a t a : A r i o n i d a e )

Von iiber 200 Individuen der Arten A r i o n hortensis s. 1. und A . i n t e r m e d i u s wurden die Esterase-Muster durch iso-elektrische Fokussierung von Hepatopankreas-Homogenaten untersucht. Aus der Zahlung der Banden in den Zymogrammen und deren Vergleich werden die genetischen Ahnlichkeiten berechnet und durch Varianzanalysen gepruft. Daraus ergeben sich folgende Schliisse:

48 T. B a c k e l j a u

1. Die bislang zu der Ar t A . h o r t e n s i s s. 1. zusammengefafken Taxa A . h o r t e n s i s s. s., A . d i s t i n c t u s und A . o w e n i i , bilden offensichtlich drei deutlich getrennte Genpools, die reproduktiv isoliert sine). Sie sind daher als drei gute Arten im Sinne des Biospezieskonzeptes aufzufassen.

2. E i n Vergleich der genetischen Ahnlichkeit von A . h o r t e n s i s und A . d i s t i n c t u s ergibt, dafi diese bei Individuen derselben Population am grofken 1st und graduell abnimmt, wenn man Individuen der jeweils gleichen Ar t aus verschiedenen Populationen oder Individuen der beiden Arten vergleicht. Dadurch wird die genetische Isolation der beiden Arten bestatigt.

3. A . o w e n i i und A . i n t e r m e d i u s zeigen dagegen konstante und nahezu gleichformige Esterase-Muster ohne eine graduelle Veranderung beim Vergleich verschiedener Populationen. Daraus wird ge-schlossen, dafi bei beiden Arten (im Gegensatz zu A . h o r t e n s i s und A . d i s t i n c t u s ) Selbstbefruchtung oder apomiktische Parthenogenese vorliegt. Bei A . o w e n i i sind Kopulationen und Spermatophoren beobachtet.

4. Die beiden auf Grund ihrer Genitalstrukturen unterscheidbaren Formen von A . d i s t i n c t u s waren durch Iso-elektrische Fokussierung nicht zu trennen. Demnach ist die A n - oder Abwesenheit eines ausstiilpbaren Teiles am Ovidukt kein zuverlassiges Merkmal zur Unterscheidung von A . d i s t i n c t u s von den beiden anderen Arten. Lediglich die Epiphallusmiindung in das Atr ium erlaubt eine sichere Unterscheidung der drei Arten.

5. Wegen der grofSen Ahnlichkeit zwischen A . o w e n i i und A . i n t e r m e d i u s und unter Beriicksichtigung der Chromosomenzahlen und der Genitalstrukturen sollte/1. i n t e r m e d i u s zusammen r a i t A , h o r t e n ­sis s. 1. in dasselbe Subgenus K o b e l t i a gestellt werden. D a A . i n t e r m e d i u s die Typus-Art fur das Subgenus M i c r o a r i o n ist, muE diese Untergattung ent-weder aufgegeben oder neu definiert werden.

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A u t h o r ' s address: Drs T H I E R R Y B A C K E L J A U , Laboratorium voor Algemene Dierkunde, Rijksuniver-sitair Centrum Antwerpen, Groenenborgerlaan 1 7 1 , B - 2 0 2 0 Antwerpen/Belgium