The freshwater planarian Dugesia sicula Lepori from Sardinia (Platyhelminthes, Tricladida)

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Hydrobiologia 310: 151-156, 1995.

) 1995 Kluwer Academic Publishers. Printed in Belgium.

The freshwater planarian Dugesia sicula Lepori from Sardinia (Platyhelminthes, Tricladida) Maria Pala, Rosa Alba Vacca, Salvatore Casu & Giacinta Stocchino Istituto di Zoologia dell'Universita'di Sassari, via Muroni 25, 07100 Sassari, Italy Received 4 May; inrevised form 20 September 1994; accepted 4 October 1994 Key words: Platyhelminthes, Tricladida, Dugesia sicula, morphology, karyology, ecology

Abstract Fissiparous strains of freshwater triclads of the Dugesia gonocephala group were collected from 12 localities in Sardinia all situated not more than 5 kilometers from the coast. Some environmental factors and the sexual status of the specimens were noted at the time of collection. During the laboratory rearing 30% of individuals of each strain became sexual (ex-fissiparous individuals). All the examined strains showed common karyological and morphological characteristics suggestive of the species Dugesiasicula Lepori.The chromosome complement, which was a constant 27+2-3 B chromosomes, was classified as aneutriploid, due to its clearly documented characteristics. The fissiparous populations of D. sicula appear to have a high degree of tolerance to variations in environmental factors, especially temperature. Within the D. gonocephalagroup this species has the broadest distribution in the Mediterranean region. Introduction Dugesia sicula Lepori is one of the numerous related species of freshwater triclads in the Dugesia gonocephala group (Benazzi & Benazzi Lentati, 1976). According to De Vries & Sluys (1991), the former subgenus Dugesia (Ball, 1974) has been elevated to the rank of full genus. Dugesia sicula was described on the basis of sexual specimens from a population comprising both sexual and fissiparous individuals from the Sicilian locality of Cardillo near Catania (Lepori, 1948). It was also recorded on the island of Elba by Benazzi (1950), who suggested passive transfer of the species to this island. The same author reported the first karyological datum (nine bivalents in meiotic oocytes), establishing that the chromosome set of D. sicula is 2n= 18; n = 9 (Benazzi, 1955; Benazzi & Benazzi Lentati, 1976). Since then, only one sexual population of D. sicula has been found, and this was by Gourbault (1981) on the island of Mallorca. Through a precise karyological and karyometric analyses she confirmed the chromosome number 2n = 18; n = 9 of the species.

Recently, Ribas et al. (1988) carried out morphological, karyological and biochemical studies on numerous fissiparous populations from the western Mediterranean region, some of which were karyologically known (Gourbault, 1981; Lepori & Pala, 1982). It was shown that all examined fissiparous populations with karyotype 27 + 2-3 B chromosomes belonged to the species D. sicula. These results, together with those of a morphological nature reported by other authors (De Vries, 1988; Benazzi & Deri, 1988) reveal that D. sicula species occurs practically in the whole Mediterranean region. As far as the island of Sardinia is concerned present knowledge indicates that in this region D. sicula is represented solely by fissiparous populations (Ribas et al., 1988; Casu et al., 1988; Vacca et al., 1988). As previous data proved incomplete, this paper describes morfology, ecology and distribution of the fissiparous Sardinian populations collected up to now, and reports in detail on their chromosome portraits.

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Fig. 2. Ex-fissiparous specimen of Dugesia sicula from Rio Triga Abbreviations: ho, hyperplastic ovaries. x40.

Fig. 1. Geographical distribution of Dugesia sicula in Sardinia 1, spring on the Island of Tavolara; 2, river Silis; 3, Rio Torre Argentina; 4, river Temo; 5, Rio Mannu near Scanu Montiferro; 6, Rio Flumentepido; 7, Rio Acqua 'e sa Murta; 8, Rio Triga; 9, Rio 'e sa Canna; 10, Rio Gutturu mannu, 11, Rio Geremeas; 12, river Flumendosa.

A karyological analysis was made both in single individuals and in entire specimens. In the first case chromosome metaphasic plates were obtained by the squashing method, and in the second by the air-drying method described in Vacca et al., 1993. Relative length (r.l.), centromeric index (c.i.), mean and standard deviation were calculated on five or six chromosome plates classifying the chromosomes according to Levan et al. (1964).

Results Morphology

Materials and methods Natural strains were collected in 12 Sardinian localities including 2 small nearby islands. Samples were taken from rivers, streams and springs, over a period of ten years, 1979-'89 (Fig. 1). During each sampling the following environmental factors were considered: pH, water temperature, flow and current. At the time of collection all strains were fissiparous and a karyological analysis only was carried out. During the long period of laboratory rearing 30-35% of the individuals in each strain lost their fissioning capacity, developed hyperplastic ovaries and a copulatory apparatus, thus becoming 'ex-fissiparous'. In each strain studied, 5-6 ex-fissiparous specimens were fixed in Bouin's fluid, embedded in paraffin and serially sectioned at 7 am; sagittal and transverse sections were stained with Mayer's haemalum and eosin.

The comparative study of the morphology of the copulatory apparatus was made on large, sexually mature ex-fissiparous individuals, measuring 15-18 mm in which the testes were normal whereas the ovaries were yperplastic (Fig. 2). Meiosis was anomalous and cocoons when laid were always sterile. In all the specimens studied, the anatomical structures were very similar to those described by Lepori (1948) on original type material of Dugesia sicula (Fig. 3). According to his diagnosis, the main characteristics are the following: - The genital atrium is divided into a common one and a male one. - A short, blunt penis papilla occupies the entire male atrium; the ejaculatory duct passes through it, curving sharply downwards and opening ventrally, rendering an asymmetric penis papilla (De Vries, 1988).

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Fig. 3. A and B Dugesia sicula from Rio Triga, sagittal sections through the copulatory apparatus of ex-fissiparous specimens. Abbreviations: ed, ejaculatory duct; mat, masculine atrium; pp, penis papilla. x 85.

- The entrance of the oviducts into the bursal canal is asymmetrical, the left oviduct opening in a more ventral position than the right one. Karyology The karyological analysis was carried out on metaphasic plates obtained from a minimum of 5 fissiparous specimens of each strain studied. The chromosome complement was always 27+2-3 B chromosomes. The chromosomes were arranged in triplets according to their length (Fig. 4). Chromosomes of the same length frequently showed clear differences in the centromere position. The resulting polymorphisms would have made it possible to arrange some chromosomes in quadruplets or pairs. This arrangement could be applied to any of the chromosomic complements studied. The chromosome polymorphism regards all strains examined. The karyometric values were calculated referring to a haploid set obtained from the single triplets by choosing the chromosome showing at least a second homologue. Such values indicate that relative lengths of the

chromosomes in the various populations are essentially similar. The values of the centromeric indices on the contrary, vary greatly. This is true not only for chromosome complements of different populations, but also for those within the same population. The karyometric data indicate that all chromosomes except the last two are metacentric heterobrachial. Chromosome 5 was metacentric, bordering on submetacentric. The karyological and karyometric data shown in Fig. 4 and Table 1 are of two representative populations. The idiogram does not vary (Fig. 5). Ecologicalnotes In Sardinia Dugesia sicula was collected from rivers, streams and in one case from arill fed by a small spring. All the localities sampled are situated not more then 5 kilometers from the coast. All the water-courses sampled, regardless of flow, have in common a torrent-like character. In winter the current is strong and there is a tendency to flooding, whereas in the summer there is very little water and the water-courses are often nearly dry and only small pools

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Fig. 4. Karyotypes of Dugesia sicula from Sardinia. A, Rio Triga; B, Tavolara;C, Rio mannu near Scanu Montiferro; D, Rio Torre Argentina; E (Tavolara) and F (Rio Torre Argentina), chromosome complements arranged in triplets. Note polymorphisms in various triplets and B chromosomes. Scale bars indicate 10 /m.

remain. The seasonal variation of the flow matches a variation of temperature which ranges from 10-11 °C during the winter to 25-26 ° C during the summer. The pH showed values between 7 and 8.

Under these environmental conditions, D. sicula was the only species of planarian that was found, with the sole exception of a D. Benazzii polyploid biotype found in the river Silis (Vacca et al., 1988). In fast

155 Table 1. Means and standard deviations of the relative lengths (r.i.) and centromeric indices (c.i.) of five chromosome sets. A, population from Rio Torre Argentina; B, from Tavolara.

Chromosome

A r.l.

I 2 3 4 5 6 7 8 9

14.83 ± 13.00 ± 12.40 ± 11.45 i 10.74 i 10.35 i 9.75 i 9.17 i 8.31 i

0.51 034 0.43 0.51 0.43 0.55 0.35 0.68 0.44

43.88 43.04 41.05 42.58 40.24 42.97 41.48 49.67 48.63

20

eo

15

10 11 1

2

3

4

5

6

7

B r.l.

c.i.

8

9

chromosomic no. Fig. 5. Idiogram of fissiparous populations of Dugesiasicula from Sardinia.

flowing water-courses D. sicula was normally found near the river mouth, while other species of the same genus were collected along the course of the river.

Discussion A survey of recent literature, has revealed the presence of D. sicula throughout the Mediterranean region (Ribas et al., 1988; Casu et al., 1988; De Vries, 1988; Benazzi & Deri, 1988; Vacca et al., 1988). However, its distribution is disjunct, which seems to suggest an ancient phylogenetic origin (De Vries, 1988). As we can see, at present, all known Mediterranean populations of D. sicula are fissiparous except the Mallorcan one, due to the fact that the Catania population is now extinct (our unpublished data). All the strains

± 2.95 ± 2.29 ± 1.96 4- 3.11 ± 2.16 ± 2.70 ± 4.20 ± 0.56 ± 1.61

14.34 13.73 12.40 11.77 11.08 10.17 9.43 9.01 8.07

c.i. ± ± ± ± ± ± ± ± ±

0.67 0.84 0.27 0.20 0.52 0.33 0.47 0.49 0.14

44.18 41.85 40.27 41.68 40.81 43.27 42.52 48.15 47.30

± ± ± ± ± ± ± ± ±

0.68 4.42 5.22 4.17 4.37 3.13 3.87 0.61 0.94

that were studied karyologically showed a constant chromosome complement of 27 + 2-3 B chromosomes (Ribas et. al., 1988; Casu et al., 1988). The present study confirms that all the Sardinian fissiparous populations with 27 + 2-3 B chromosomes belong to the species D. sicula. They are morphologically, karyologically and ecologically very similar. A comparison of karyological and karyometric data indicates that the karyotype is unique and numerically constant. The values of the chromosome relative lengths correspond and they are similar to those reported by Gourbault for the Mallorca population. However, there are differences in the values of the centromeric indices which were also observed in the Sardinian populations we studied. These differences are the most important characteristic of their chromosome complements. Moreover, frequent chromosomic polymorphisms caused by chromosome rearrangements can be observed both within an individual population and between populations. Due to the distinct irregularities in the morphology of the chromosomes and the presence of B chromosomes, the chromosome complement of the fissiparous populations of D. sicula may be aneutriploid not eutriploid. Chromosome banding could be used to study the nature of the anomalies and to identify the chromosomes (Canovai et al., 1994). The fissiparous populations studied showed a strong resistence to variations in environmental factors, especially temperature. Research being carried out presently in our laboratory has shown that fissiparous strains of D. sicula have a greater tolerance to temperature variations than the sexual diploid ones of

156 the high degree of tolerance of the fissiparous populations of D. sicula is related to their mode of reproduction. Fissioning, as reproductive strategy, permits a rapid population increase by avoiding gametogenesis and the development of cocoons, processes which require more favorable environmental conditions.

Acknowledgments We are grateful to Prof. N. G. Lepori for his suggestions and criticisms of the text. Work supported by grants from Italian Ministero dell'Universita e della Ricerca Scientifica e Tecnologica (40% and 60%) and from E.E.C. 'Interreg program'.

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