Dugesia Girard, 1850

Bromley-Schnur, Heather J., 2021, The genus Phagocata Leidy (Platyhelminthes, Tricladida) in Israel, a new species of Phagocata from Lake Kinneret, and an emended description of Dugesia salina, Zootaxa 4969 (2), pp. 293-317 : 307-315

Genus Dugesia Girard, 1850

Material examined. Neotype: HUJ.Oct.1, Sagittal sections on 18 slides.

Other material: Transverse sections on 24 slides (HUJ.Oct.2), sagittal sections on 11 slides (HUJ.Oct.3) and transverse sections on 11 slides (HUJ.Oct.5). Material was collected during the summers and winters of 1971–1974 and ex-fissiparous specimens were obtained in the laboratory. The material is deposited in the Zoological Collections of the Department of Ecology, Systematics and Evolution, Hebrew University, Jerusalem, Israel.

Type locality: En Sheva (Tabgha), N32.873166 E35.548973, a saline spring on the north-western shore of Lake Kinneret , also known as Lake Tiberias .

Diagnosis. Dugesia salina is characterized by occupying a very distinct saline habitat. It has a low triangularshaped head with hardly discernible auricles; it is pale brown or grey in colour dorsally, and very pale or white ventrally. Ex-fissiparous specimens have a large well-developed penis, which is situated to the left and slightly anterior to the larger adenodactyl which has a well-developed lumen in the distal part. The penis papilla is large and cylindrical and blunt at its tip where it bends ventrally. There is a distinct funnel-shaped diaphragm separating the seminal vesicle and the ejaculatory duct; the opening of the ejaculatory duct is terminal. Chromosome portrait: diploid, 2n = 16.

Habitat notes. En Sheva is a complex of several springs of varying salinities, and Dugesia salina was found on the undersides of basalt rocks in a small spring-fed pool about 5 m south of an octagonal building enclosing the largest spring. The salinity was in the region of 1500 mg /l Cl´ and the temperature was 25–27 oC.

Dugesia salina was also found at two other saline locations, namely, En Soda, N32.518836777 E35.534845127, a saline spring several km south of Lake Kinneret (salinity ± 1200 mg /l Cl´, temperature 24–27 oC), and in the River Jordan near its outlet from Lake Kinneret , N32.677439367 E35.570610941 (temperature 15–26 oC). This latter site is where water from several saline springs on the western lake shore (including En Sheva), which prior to 1965 drained directly into the Lake, are now collected in a saltwater channel (the saltwater carrier) and discharged into the River Jordan south of the Lake. No other triclads were found at any of these three sites.

Animals were collected from the field during all seasons of the year from En Sheva and En Soda and all were asexual, measured 1.5–8.5 mm, and most showed evidence of recent fission. Those collected from the River Jordan during winter months at 15 oC were slightly larger (6–8.5 mm) and did not show any signs of recent fission.

The animals from En Sheva and En Soda were difficult to maintain in the laboratory at 18 oC or 23 oC and many died within a few days of collection, while those from the River Jordan reproduced freely by fission at 18 oC, but none were ever observed to mature. However, several specimens from the type locality of En Sheva, which were kept at a fluctuating room temperature of 12–25 oC, did mature over a period of 3–9 months after collection. A constant temperature of 18 oC or 23 oC did not induce maturation. Saline water from the original habitats was not a prerequisite for laboratory survival or maturation as the animals could be kept satisfactorily in tap water .

Description. The planarians from all three populations were similar in external appearance, the only difference being in a greater degree of speckling in the dorsal pigmentation among the animals from the River Jordan. The following description is based mainly on ex-fissiparous animals from En Sheva. The animals have a low triangularshaped head with hardly discernible auricles, which merge gradually into the body region. They are pale brown or grey dorsally and very pale or white ventrally. The animals from River Jordan have a pale or mid-brown mottled colour dorsally and are pale ventrally. After laboratory culture, some animals attained a length of up to 15 mm.

The dorsal epithelium is about 15 µm thick and contains many rhabdites, while the ventral one is about 7.5 µm thick with very few rhabdites. The outer muscle sheath of the pharynx consists of two layers only, an outer, subepithelial longitudinal layer 4–5 µm thick, followed by a circular layer, 5–7.5 µm thick. Minute pigment granules are scattered amongst the dorsal body wall muscle layer and its underlying mesenchyme. The inner epithelium of the pharynx is underlain with a thick layer of circular muscle followed by 2–3 layers of longitudinal muscle.

The eyes are placed just anterior to the auricles and are surrounded by a fairly large unpigmented area. They are slightly closer together than their distance from the lateral margins of the head and from the anterior tip of the head. The eyes may attain a size of 180 µm in antero-posterior direction. The intestine is highly branched in large specimens. In smaller animals, there are 5–6 lateral branches on the anterior ramus and about 9 lateral branches on the posterior rami. The anterior intestinal trunk forms an unbranched extension in the midline, reaching between the eyes and sometimes just anterior to the eyes ( Fig. 9 ).

The insertion of the pharynx in an asexual animal just prior to division is almost halfway down the body and its length is ¼ to ⅕ of the total body length. In laboratory cultured ex-fissiparous animals, the pharynx occupies a more forward position and its length is about 1/7 th of the total body length. The mouth opens slightly posterior to the end of the pharyngeal cavity. The mouth is just over halfway down the body and the gonopore occupies a position just over ¾ down the body length.

The two ovaries lie ventrally, just internal to the ventral nerve cords, between the 2 nd and 3 rd or 3 rd and 4 th lateral intestinal branches. They are located just behind the brain, at about one-fifth to one-sixth of the distance between the brain and the root of the pharynx. The ovaries are round to oval in shape and may attain a maximum diameter of 140 µm. The oviducts lead out laterally from the posterior end of the ovaries and open separately, at more or less the same level, into the bursal canal at its junction with the common atrium. The left oviduct enters the base of the bursal canal ventro-laterally, while the right oviduct enters dorso-laterally.

The bursa is large and laterally compressed and takes up much of the dorso-ventral space of the body. It attains a lateral diameter of 150–200 µm and a maximum dorso-ventral diameter of about 300 µm. The bursal canal emerges from the posterior part of the bursa and lies dorsally, to the left of the midline, overlying the penis. The glandular, nucleated epithelium of the bursal canal is surrounded by a thin layer of longitudinal muscle and an outer thicker layer of circular muscle. A few extra longitudinal fibres of ectal reinforcement can be seen where the bursal canal enters the common atrium. The canal remains narrow here with hardly any dilatation. The epithelium is ciliated with basal nuclei. Shell glands open into the bursal canal from both anterior and posterior directions in the region of the oviduct openings.

The testes are principally dorsal and begin at the level of the ovaries. They continue posteriorly to within 550 µm from the posterior tip of the tail. Pre-pharyngeally, there are 3–4 testes across each longitudinal row; in the pharyngeal and copulatory regions there are 1–3 across each row, and posterior to the gonopore, there are single testes across each row. The testes are oval, flattened laterally and reach up to 130 µm in maximum diameter. Spermatogenesis in these laboratory matured animals appeared normal, with the development of normal sperm. The vitelline glands are predominantly ventral in the mesenchyme, but some are also situated dorsally, between the testes.

The copulatory apparatus consists of a large and well-developed penis, which is situated to the left and slightly dorsal and anterior to the larger adenodactyl. The penis bulb is 200–250 µm in diameter and is composed of alternating layers of longitudinal and circular muscle externally. The central tissue, anterior to the seminal vesicle, consists of parenchyma traversed by diagonal muscle fibres. The vasa deferentia expand to form spermiducal vesicles about halfway down the pharynx length. After having decreased in diameter, the vasa deferentia enter the lateral walls of the penis bulb, more or less symmetrically, just anterior to where the penis bulb and the adenodactyl bulb appear to be united. They open into the anterior, lobed part of the seminal vesicle, which has a flattened epithelium. The seminal vesicle may contain eosinophilic material, possibly related to the formation of a spermatophore. There is a large valve-like diaphragm separating the seminal vesicle from the ejaculatory duct. The opening of the ejaculatory duct is terminal but the tip of the penis papilla is bent downwards so that the opening appears ventral. The ejaculatory duct appeared widened distally in two specimens where a spermatophore was present. The penis papilla is large, blunt and cylindrical. It is fairly muscular with outer, subepithelial circular and inner longitudinal muscle layers. Eosinophilic tissue interspersed with longitudinal muscle fibres lies internal to the longitudinal muscle layer ( Figs 10 , 11 ).

The adenodactyl is of the hollow type with a distinct lumen in the distal part of the papilla. The bulb may be over 250 µm in diameter and is muscular only at the most anterior part, and the muscle fibres mingle with those of the adjacent penis bulb. A central core of darkly staining cyanophilic tissue, interspersed with longitudinal muscle fibres, extends from the centre of the bulb throughout the length of the papilla, surrounding the duct in the distal part. The papilla may reach a length of 600 µm. Beneath the outer epithelium of the anterior part of the papilla there is a thick layer of circular muscle. This is underlain by a thinner layer of longitudinal/diagonal muscle which surrounds the cyanophilous glandular core. In the central part of the papilla, anterior to the lumen, the longitudinal/diagonal muscle layer becomes thinner and is absent from the muscle coat in the region of the lumen. In the posterior part of the papilla, there is a thin outer layer of circular muscle under which lies the glandular core and in the centre of this is the adenodactyl lumen. The lumen is surrounded by a cuboidal epithelium with insunk nuclei, surrounded by a thin layer of circular muscle. The lumen of the duct is filled with a secretion that is usually cyanophilic but may be eosinophilic or a mixture of both.

The male and common atria are usually distinct, although sometimes the tip of the adenodactyl papilla extends into the entrance of the bursal canal. The male atrium is lined by a flattened epithelium and a short gonoduct leads from it to the gonopore.

Karyology. A total of 43 metaphase plates of D. salina were examined from two specimens each from En Sheva and En Soda and revealed a mitotic chromosome number of 16 ( Table 6 , Fig. 12 ). In addition, 34 metaphase plates from 6 specimens from the River Jordan also revealed a mitotic chromosome number of 16. All chromosomes were metacentric, and of these, a few were metacentric heterobrachial bordering on metacentric isobrachial. The chromosomes decreased gradually in size. Pair number 8 was in all cases a little less than half the size of pair number 1 .

Discussion. Most species of Dugesia which have been examined karyologically have a haploid complement of n = 8, but six are known with n = 9 and three are known with n = 7. Other populations may be markedly aneuploid (e.g. Pala et al. 1982). The species of Dugesia from Israel that have been examined karyologically are the widespread, fissioning D. sicula (formerly known in Israel as D. biblica ), which is normally triploid, 3n = 27, + 1-5 supernumeraries, and another ecotype of D. sicula , a normally sexually reproducing diploid form, 2n = 18, which is distributed in the central hilly region of Israel. Another sexually reproducing species, D. golanica , known only from the cold spring-fed waters of Dan and Banias in the very north of Israel, and previously thought to be D. cretica (Meixner, 1928) ( Bromley & Benazzi 1991) was also examined karyologically, 2n = 16 ( Bromley 1974). Thus, regarding karyotypes, D. salina is similar to most other species of Dugesia , including D. golanica , in having a haploid number of n = 8.

Regarding the copulatory apparatus, there are only a few species of Dugesia which have a large adenodactyl. Adenodactyls sensu stricto, which have no known function, may vary in number, size and position in relation to the penis papilla, but are defined as strongly muscularized, glandular-parenchymatic folds of the copulatory apparatus and provided with a lumen ( Stocchino et al. 2017). The large, well-developed adenodactyl with a lumen, seen in ex-fissiparous specimens of D. salina , is most similar to that of D. cretica , which also has a single large hollow adenodactyl. However, the adenodactyl of D. cretica described from Crete by De Vries (1984) arises ventrally from the base of the penis papilla, and is positioned ventrally to the penis, whereas the large adenodactyl of D. salina is situated to the right of the penis. In contrast to this, Kenk (1930) described the large adenodactyl of D. cretica (from Crete) as being situated ventrally and to the right of the penis papilla. De Vries (1988) studied material attributed to D. cf. cretica from Cyprus, which had a dorsally situated adenodactyl, while material from the Greek island of Sérifos had an adenodactyl arising laterally to the right of the penis papilla, in a position similar to that of D. salina . However, De Vries (1988) noted that the position of the adenodactyl of D. cretica apparently varies between the geographically isolated populations in the eastern Mediterranean. True adenodactyls sensu stricto, strongly musculo-glandular organs of the type seen in D. salina and D. cretica , are not common among members of the genus Dugesia . They have also been recorded in Dugesia bactriana De Beauchamp, 1959 , which occurs in Afghanistan and Pakistan, and in Dugesia transcaucasica (Livanov, 1951) from the Caucasus ( Stocchino et al. 2017). D. bactriana has three small to medium-sized hollow adenodactyls all near the gonopore, two of which are posterior to the penis and one is ventral to it. D. transcaucasica has three medium-sized adenodactyls with a small lumen, one dorsal to the left, one dorsal to the right and one ventral to the right of the penis.

Other differences between D. salina and D. cretica lie in the head shape ( D. cretica has much more pronounced auricles than D. salina ) and penis shape, which is conical in D. cretica , but is large, cylindrical and somewhat blunt-ended in D. salina , and curves ventrally at its tip. De Vries (1988) noted that earlier records of D. cretica from Turkey, Iran and Israel are invalidated and that this species is known with certainty only from the Mediterranean islands of Crete, Tinos, Sérifos and Cyprus, and also from Romania.

Although ex-fissiparous specimens of D. salina were obtained only from En Sheva, and on which the morphological description is based, the populations from En Sheva and En Soda appear to be identical. However, the population from the River Jordan showed slight differences with regard to pigmentation, karyotype and larger size. The lack of fission of the River Jordan animals during the winter may indicate that this population is a distinct ecotype recently derived from the En Sheva population and dispersed via the salt-water carrier. De Vries (1985) stated that generally, freshwater triclads have a very low resistance to salinity. The animals from these three sites obviously show an adaptation to living in water with a high salinity and it is notable that no other species of planarian was ever found in these habitats. It was suggested by Solà et al. (2015) that Dugesia specimens collected by them from another saline spring site in Israel, Einot Huga, which is close to En Soda, with a salinity of about 2000 mg /l Cl´, may perhaps also be assigned to D. salina on the basis of molecular studies.

Stocchino et al. (2005) referring to the high level of endemicity of planarians of the genus Dugesia in the Mediterranean region, note that a process of allopatric speciation is strongly supported by their fragmented habitat, the present high endemicity values and the morphological divergence in copulatory traits. The unusual and highly saline habitats where Dugesia salina was found, as well as the differences in its copulatory apparatus from its morphologically closest congener ( D. cretica ) support the provisional observations of Whitehouse (1914) that ‘ Planaria salina ’ is a good species and apparently endemic to saline waters where other Dugesia species cannot thrive.

Figures

FIGURE 9. Dugesia salina from En Sheva. A. Dorsal view of mature specimen (from whole mount). B. Dorsal view of live asexual animal. C. Dorsal view of copulatory apparatus, from whole mount and sections. Scale bar represents 1 mm in A and B, and 200 µm in C.

FIGURE 10. Dugesia salina from En Sheva. A. Sagittal reconstruction through adenodactyl. B. Sagittal reconstruction through penis. C. Transverse reconstruction through adenodactyl and penis papilla, as seen from posterior end. A and B based on Neotype, HUJ.Oct.1; C is based on HUJ.Oct.5. Scale bar represents 200 µm.

FIGURE 11. Dugesia salina. Photomicrographs of the copulatory apparatus. A. Sagittal section through adenodactyl. B. Sagittal section through penis. C. Transverse section through adenodactyl and penis papilla as seen from anterior end. A and B are from Neotype, HUJ.Oct.1; C is from HUJ.Oct.5. Scale bar represents 200 µm in A and B, and 300 µm in C.

FIGURE 12. Karyograms and mitotic metaphase plates of Dugesia salina. The metaphase plates A and B have their extracted karyograms beside them. Karyograms A, B & C are from En Soda; D, E & F are from En Sheva.