A molecular cytogenetic comparison of planarians from the ’Dugesia gonocephala group’ (Platyhelminthes, Tricladida)

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A molecular cytogenetic comparison of planarians from the ’Dugesia gonocephala group’ (Platyhelminthes, Tricladida) a

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Renata Batistoni , Leonardo Rossi , Alessandra Salvetti & Paolo Deri

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Laboratorio di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia e Biochimica , Università di Pisa , via Carducci 13, Ghezzano, Pisa, I‐56010, Italy Published online: 28 Jan 2009.

To cite this article: Renata Batistoni , Leonardo Rossi , Alessandra Salvetti & Paolo Deri (1999) A molecular cytogenetic comparison of planarians from the ’Dugesia gonocephala group’ (Platyhelminthes, Tricladida), Italian Journal of Zoology, 66:3, 239-244 To link to this article: http://dx.doi.org/10.1080/11250009909356261

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Ital. J. Zool., 66. 239-244 (1999)

A molecular cytogenetic comparison of planarians from the 'Dugesia gonocephala group' (Platyhelminthes, Tricladida)

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RENATA BATISTONI LEONARDO ROSSI ALESSANDRA SALVETTI PAOLO DERI Laboratorio di Biologia Cellulare e dello Sviluppo, Dipartimento di Fisiologia e Biochimica, Università di Pisa, via Carducci 13, I-56010 Ghezzano, Pisa (Italy)

INTRODUCTION The classical cytogenetic studies of Professor Mario Benazzi and his school have produced in the last 50 years a wealth of information that has greatly contributed to the identification of a wide range of karyological phenomena that occur in freshwater planarians, i.e. numerical and structural chromosomal changes, presence of supernumerary chromosomes and also mosaicisms (cf. the list of publications of M. Benazzi reported by Mancino, 1998). Many of these investigations have placed an emphasis on the taxonomic significance of chromosomal variations, suggesting that, in these organisms, speciation may take place through different mechanisms, and in different ways (cf. Benazzi & Benazzi-Lentati, 1976; Benazzi, 1982). The former subgenus Dugesia, recently elevated to the rank of genus by De Vries & Sluys (1990, comprises many related planarian taxa, very similar at a morphological level (cf. Benazzi, 1982; Deri et al, this volume). Moreover, both the haploid chromosome set, usually made up of similar elements, and the presence of polysomic and polyploid forms, especially in asexual organisms, often make the specific identification of the populations of the complex uncertain. We have applied a molecular cytological approach to define differences among species and populations of the D. gonocephala group and attempt to make some inferences about their genomic relationships. The present paper concerns the molecular arrangement and chromosomal location of ribosomal genes (18+28S rDNA) and a further characterization of a highly repeated DNA family, recently isolated in D. etrusca (Del family, Batistoni et al, 1998).

ABSTRACT Planarians belonging to the Dugesia gonocephala group share extensive karyotype similarities when compared using classical cytological techniques. However, after in situ hybridization with rDNA, differences in localization and number of rDNA loci were identifiable among the species. Species-specific features in the rDNA structure have been observed by restriction pattern analysis. A family of long interspersed repeated elements (Del), isolated in Dugesia etrusca, and conserved only in some species of the group, was also informative in establishing specific genomic relationships among the species. We suggest that an understanding of the organization and evolution of repeated DNA sequences can be a useful tool for studying molecular mechanisms of evolutionary significance in planarians. KEY WORDS: Freshwater triclads - Nucleolus-organizing region rDNA - Repeated DNA. ACKNOWLEDGEMENTS We thank Dr. M. Riutort for kindly providing us with D. tigrina 185 rDNA. This work was supported by a MURST grant. Invited paper at the Symposium on "Structure and Evolution of Genome", 59th National Congress of the Unione Zoologica Italiana held in San Benedetto del Tronto in September 1998.

MATERIALS AND METHODS Animals Most of the specimens examined in this study were collected from natural populations of planarians from the D. gonocephala group. The species, collected in the areas where their presence had already been described, are indicated after the sampling site. The precise localities were as follows: northern Italy: Revigliasco (Torino), Saliceto (Cuneo), Colli Berici (Vicenza), S. Vittoria di Liguria (Genova), Taggia (Imperia), Monte Moro (Savona), Carcare (Savona), Carrodano (La Spezia), Sestri Levante (La Spezia); central Italy: Rio di Calci (Pisa), Rio Grande (Pisa), Donoratico (Livorno) CD. etrusca race monoadenodactyla, Deri et al., this volume), Parrana (Livorno) CD. etrusca race labronica, Lepori, 1950), Marciana (Elba island, Livorno) CD. ilvana, Lepori, 1948), Stoppe DArca (Arezzo), Torrente Fiumicello (Arezzo), Fiume Farma (Grosseto), Montieri (Grosseto) CD. etrusca race monoadenodactyla, Lepori, 1947), Torniella (Grosseto), Rocca Albegna (Grosseto), Rapolano Terme (Siena) CD. etrusca race biadenodactyla, Benazzi, 1946), S. Vittore del Lazio (Frosinone); southern Italy: Amaseno (Frosinone), Guardavalle (Catanzaro), Munita (Reggio Calabria), Bova Marina (Reggio Calabria); Sicily: Alcantara (Catania) CD. sicula, Benazzi & Deri, 1988); Sardinia: Rio Gutturu Mannu (Cagliari) (D. sicula, Pala et al, 1995); Albania: Pogradec. Specimens from Roccapina (Corsica) CD. benazzii, Benazzi, 1968), Tineghir (Morocco) and Dhamar (Yemen) belong to laboratory stocks.

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Fig. 1 - Metaphase chromosomes of Dugesia etrusca (Parrana, Livorno) after in situ hybridization (FISH) with 18S rDNA. Note the subterminal position of the NORs, visualized by fluorescein. Scale bar, 8 |xm.

Dugesia gonocephala s.s. specimens were provided by Dr. C. Vreys (Diepenbeek, Belgium). Methods Restriction analysis was performed on genomic DNA extracted from pools of animals for each population; Southern blot hybridizations were performed as described (Batistoni et al, 1998). Chromosomal preparations and fluorescence in situ hybridization (FISH) experiments were conducted using a previously described method (Filippi et al, 1998). Planarian 18S ribosomal DNA (rDNA) (kindly supplied by Dr. M. Riutort, Barcelona, Spain) was used as a probe. After FISH, preparations were studied with a Zeiss photomicroscope equipped with epifluorescence filters. The images were processed using Adobe Photoshop software. The pXcr7 clone, containing a complete genetic unit of Xenopus laevis rDNA (see De Lucchini et al, 1993), was employed as a probe to visualize the rDNA restriction enzyme patterns in the filter hybridizations. The 1.4 kb Hind III fragment of Del repeated DNA of D. etrusca (p20 clone, cf., Batistoni et al, 1998) was used as a probe for analysing the genomic conservation of Del elements.

RESULTS

Chromosomal location and restriction enzyme analysis of rDNA In situ hybridization (FISH) experiments allowed us to localize the nucleolus-organizing region(s) (NOR) on the mitotic chromosomes of some species of the D. gonocephala group. As found in differently related species of planarians, such as Polycelis tennis, Dendrocoelum lacteum (Joffe et al, 1998), Dugesia tigrina and Cura pinguis (Batistoni, unpubl. data), 18S + 28S ribosomal genes preferentially colonize telomeres of a limited number of chromosomes in planarians of the D. gonocephala group. Up to eight rDNA loci, located at both telomeres of a triplet of similar chromosomes and near one telomere of two slightly smaller chromosomes, have been counted in D. sicula specimens, which possess a chromosome set of 27 + 2-3 B chromosomes (Filippi et al, 1998). Moreover, a major cluster of ribosomal

Fig. 2 - Metaphase chromosomes of a specimen of the Dugesia gonocephala population collected near Pogradec (Albania). Selected NOR-bearing chromosomes, visualized after in situ hybridization with 18S rDNA, are shown (inset). Scale bar, 8 (im.

genes has been identified at one of the ends of two medium-sized chromosomes in the diploid complement (2n = 16) of D. etrusca (Fig. 1). Two similar chromosomes are labelled in the diploid set of D. gonocephala specimens collected near Sestri Levante (data not shown). The planarians collected near Pogradec are asexual and have a constant chromosome number of 24 + 1 B chromosome (cf. Deri et al, this volume). In this population the major clusters of rDNA are placed at one end of two medium-sized chromosomes. In addition, a third site, located in intercalary position of an element tentatively indicated as chromosome 3, was constantly found in the metaphases of the five analysed individuals (Fig. 2). Accordingly, two or three intense label signals were recognized in interphase nuclei (data not shown). The structural organization of rDNA was examined in planarians of the group, collected in different localities (see Materials and Methods). This analysis, performed with Bgl II and Eco RI restriction enzymes, revealed the presence of species-specific patterns of hybridization. In fact, Bgl II digestion of rDNA produced a single fragment of about 11-12 kb, suggesting the presence of a single recognition site per rDNA unit in D. gonocephala s.S., whereas two heavy hybridization bands, which differ in length, could be observed in D. etrusca (2.7 kb and 8 kb), in D. sicula (4.5 kb and 7.9 kb) and in D. benazzii (3-9 kb and 4.5 kb). Fainter bands at about 11-12 kb also occurred in these species, probably due to the presence of sequence polymorphisms at the Bgl II sites of rDNA repeats (Fig. 3). An informative pattern of hybridization was obtained by Eco RI (Fig. 4). Four bands (2.6 kb, 2.9 kb, 4.5 kb, 5.5 kb) were visible in D. sicula: a very similar pattern

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1.6 Fig. 3 - Southern blot hybridizations of genomic DNA obtained from planarians of the D. gonocephala group, digested with Bgl II and probed with pXcr7. a) D. gonocephala s.S.; b) D. etrusca; c) D. sicula; d) D. benazzii. Molecular sizes are expressed in kilobases (kb).

has been found in Eco Rl-digested rDNA of specimens collected in the Alcantara river (Sicily) and in Rio Gutturu Mannu (Sardinia). D. ilvana and D. etrusca also show species-specific rDNA fragments, because two

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Fig. 4 - Southern blot hybridization of genomic DNA obtained from planarians of the D. gonocephala group, digested with Eco RI and probed with pXcr7. a) D. sicula (Alcantara, Sicily); b) D. sicula (Rio Gutturu Mannu, Sardinia); c) D. ilvana, d) D. etrusca; e) D. gonocephala s.S. Molecular sizes are expressed in kilobases (kb).

Fig. 5 - Southern blot hybridizations of genomic DNA obtained from planarians of the D. gonocephala group and probed with pXcr7. Bgl II-digested DNA of D. sicula (a) and Pogradec population (b). Eco Rl-digested DNA of Yemen population (c) and D. sicula (d). Molecular sizes are expressed in kilobases (kb).

(2.9 kb and 4.9 kb) and three (2.9 kb, 4.5 kb, and 4.6 kb) hybridization bands were detected in Southern hybridizations, respectively. A more complex rDNA restriction pattern occurred in D. gonocephala s.S.: five fragments, of about 1.6 kb, 1.8 kb, 2.9 kb, 4.5 kb and 5 kb in length, were found after hybridization of Eco Rldigested DNA. Patterns of hybridization comparable with those described above were found in the other D. gonocephala planarians analysed. In fact, planarians coming from populations in southern Italy as well as Moroccan specimens showed a D. sicula-Yike restriction pattern of rDNA. Similarly, a D. etrusca-like rDNA pattern was shared by all the analysed populations from central and northern Italy. Peculiar Bgl II and Eco RI restriction fragments are shown by both Pogradec and Yemen populations. As an example, Figure 5 illustrates the rDNA bands obtained with Bgl II and Eco RI in specimens from Pogradec (Albania) and Dhamar (Yemen) populations, respectively. Bgl II and Eco RI D. sicula-like rDNA patterns are shown for comparison. Conservation of Del repeated elements within D. gonocephala genomes Previous studies have characterized a family of long interspersed repeated DNA sequences, originally isolât-

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Fig. 6 - Geographical distribution of planarian populations from the D. gonocephala group analysed for the presence of Del DNA repeats. The collection localities are indicated in Materials and Methods. Open circles (o) indicate samples possessing Del elements in the genome. Filled circles (•) refer to populations lacking Del repeats.

ed from the genome of D. etrusca (Del family) and conserved only in some D. gonocephala species (Batistoni et al, 1998). Conservation of Del elements was further investigated within the group by Southern blot hybridization. Figure 6 illustrates the geographical distribution of planarians analysed so far. The results indicate that sequences homologous to Del occur in the genomes of D. etrusca, D. ilvana, D. gonocephala s.s. as well as in all the analysed populations of northern and central Italy and Pogradec. In contrast, the probe does not hybridize to the genomes of D. sicula, D. benazzi and of populations from southern Italy, Morocco and Yemen (Figs 6, 7). In accordance with what has been found in previous studies, no evident differences in copy number and structural organization of Del sequences have been observed in the genome of planarians that possess these elements (cf. Batistoni et al, 1998). In fact, blot hybridizations of Hind Ill-digested genomic DNAs produced, upon total cleavage, only the expected 1.4 kb fragment. Sometimes a darker smear in the upper part of the gel, due to partially digested molecules, could be observed (cf. Batistoni et al, 1998) (Fig. 7). An exception to this pattern is shown by planarians collected near Pogradec. Although the intensity of the signal did not appear to change significantly, suggesting that no difference in the abundance of Del elements occurred, only the darker smear was present in Hind Ill-digested DNA (Fig. 7). The lack of 1.4 kb Hind m hybridization fragments suggests that a certain sequence heterogeneity occurred in Del elements of these planarians.

DISCUSSION The data presented reveal the presence of a number of genomic differences within the species of the D. gonocephala group. At the chromosome level, the analysed species differed in the number and distribution of the NOR loci. In fact, eight sites were observed in five chromosomes of D. sicula (Filippi et al, 1998), whereas only two chromosomes were found to carry 18S + 28S rDNA clusters in D. etrusca. The different ploidy level of the examined individuals could be only partially account for the differences observed between the two species. In fact, D. sicula metaphases showed, besides having a larger number of NOR-bearing chromosomes, a peculiar distribution of rDNA loci at both telomeres on three similar chromosomes. As a common feature, shared by other planarian taxa so far analysed Qoffe et al, 1998), 18S + 28S rRNA genes appeared preferentially located on telomeric regions of medium-sized chromosomes. The chromosomal location of NOR sites observed in the Pogradec population is, up to now, the only exception to this rule. These planarians have two telomeric NOR loci, as well as a ribosomal site located in an intercalary position on the long arm of one of the largest chromosomes. The intercalary position of this NOR may reflect a structural chromosome rearrangement, such as a paracentric inversion, suggesting that, in accordance with Benazzi's view, "minor mutations have accompanied the speciation of D. gonocephala forms" (Benazzi, 1982). Genes encoding the processed 18S + 28S rRNA are

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Fig. 7 - Southern blot hybridization of Hind Ill-digested genomic DNA probed with p20 clone, containing the 1.4 kb HindlW fragment of Del repeated DNA. a) D. etrusca; b) D. sicula; c) D. benazzii; d) Pogradec population. The 1.4 kb autoradiographic band is indicated.

among the most evolutionary conserved genomic sequences, whereas the spacer regions evolve very rapidly, showing high levels of sequence variation (Long & David, 1980). These characteristics of the ribosomal units provide powerful tools for the analysis and comparison of the rDNA molecular organization in differently related organisms. In this work, the entire ribosomal unit from Xenopus laevis (pXcr7) was used as a probe for analysing the molecular structure of rDNA in different D. gonocephala planarians. Blot hybridization experiments, performed on Bgl II- and Eco RI-digested DNAs, revealed the presence of an inter-specific variability in the length and number of rDNA fragments. The species-specific rDNA patterns allowed us to identify precise genome characteristics in different planarian populations of the group. This analysis gave some indication of the possible geographical distribution of the species, representing a useful tool to assess previously uncertain situations, for example in fissiparous (usually with heterogeneous chromosome sets) populations. The data showed a D. etrusca-like rDNA pattern in the genomes of planarians collected from various localities of central and northern Italy (see Materials and Methods), supporting the possibility of a wider distribution of this species, previously described only from some Tuscan areas and from Molara, an island near Sardinia (Benazzi, 1944, 1946; Lepori, 1947, 1950; Pala et al, 1979). Because the sibling species of the D. gonocepha-

la group are considered largely or entirely allopatric (Benazzi, 1982), it is possible that the distributional area of D. etrusca is limited by the Alps in the north. The specific characteristics found in the rDNA structure of D. gonocephala s.s. from Belgium would support precise differences between the two genomes. In this context, the analysis of rDNA in D. iberica, a D. gonocephala species from the Iberian peninsula (Gourbault & Benazzi, 1979), is of particular interest. It is possible that Latium represents the limit of distribution of D. etrusca in the south. In fact, populations from southern Italy, Sicily, Sardinia, and Morocco showed a D. sicula-like rDNA pattern. In the past, the presence of D. sicula has also been described from an area of the island of Elba and in Mallorca (Benazzi, 1961; Gourbault, 1981). On the basis of the wide and disjunct distribution in the Mediterranean basin, an ancient phylogenetic origin of this species was hypothesized by De Vries (1985; 1988). The analysis of the geographical distribution of Del repeats, long interspersed DNA sequences present only in some species of D. gonocephala (Batistoni et al, 1998), confirms the ancient origin of D. sicula (and D. benazzii). Because the loss of all Del repeats seems an improbable event after amplification and dispersal has occurred in the genome, the observation that D. sicula and D. benazzii are devoid of repeated Del elements suggests that these species became separated from a D. gonocephala ancestral stock before amplification of the Del family had taken place. Planarians from the Pogradec population deserve further investigation. Although more detailed studies are needed before conclusions can be drawn, the peculiar Del Hind III pattern observed probably reflects sequence differences that accumulated because this species diverged before D. etrusca or D. ilvana that, at present, share with D. gonocephala s.s. a similar structural organization of Del repeats in the genome. REFERENCES Batistoni R., Filippi C., Salvetti A., Cardelli M., Deri P., 1998 - Repeated DNA elements in planarians of the Dugesia gonocephala group (Platyhelminthes, Tricladida). Hydrobiologia, 383: 139-146. Benazzi M., 1944 - Nuova specie di planaria trovata nel Senese. Atti Accad. Fisiocrit. Siena, Sez. med.-fis., ser. II (12), 19-20. Benazzi M., 1946 - Sopra una nuova planaria di acqua dolce. Arch. zool. ital., 31: 93-102. Benazzi M., 1961 - Les planaires des îles Tyrrhéniennes et leur différenciation raciale, cytologique et génétique. Le peuplement des îles mediterranéennes et le problème de l'insularité. CNRS, Paris, pp. 103-112. Benazzi M., 1968 - Popolazioni di Dugesia benazzii della Sardegna e della Corsica di probabile origine ibrida. Atti Ass. genet, ital., 13: 117-124. Benazzi M., 1982 - Speciation events evidenced in Turbellaria. In: C. Barigozzi (ed.), Mechanisms of speciation. Alan R. Liss, New York, pp. 301-304. Benazzi M., Benazzi-Lentati G., 1976 - Platyhelminthes. In: B. John (ed.), Animal cytogenetics. Gebrüder Borntraeger, Berlin, pp. 1-182. Benazzi M., Deri P., 1988 - Taxonomic perspectives concerning

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