The Invasive Alien Freshwater Flatworm Girardia Tigrina

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Contributions to Zoology 88 (2019) 236-256 CTOZ brill.com/ctoz

The invasive alien freshwater flatworm Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida) in Western Europe: new insights into its morphology, karyology and reproductive biology

Giacinta Angela Stocchino Dipartimento di Medicina Veterinaria, Università di Sassari, Via Vienna 2, 07100, Sassari, Italy [email protected]

Ronald Sluys Naturalis Biodiversity Center, P.O. Box 9517, 2300, RA Leiden, The Netherlands

Abdel Halim Harrath Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

Lamjed Mansour Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia

Renata Manconi Dipartimento di Medicina Veterinaria, Università di Sassari, Via Vienna 2, 07100, Sassari, Italy

Abstract

Invasions of alien species form one of the major threats to global biodiversity. Among planarian flatworms many species are known to be invasive, in several cases strongly affecting local ecosystems. Therefore, a detailed knowledge on the biology of an invasive species is of utmost importance for understanding the process of invasion, the cause of its success, and the subsequent ecological impact on native species. This paper provides new information on the biology of introduced populations of the freshwater flatworm Girardia tigrina (Girard, 1850) from Europe. This species is a native of the Nearctic Region that was ac- cidentally introduced into Europe in the 1920s. Since then, numerous records across the European continent bear witness of the invasiveness of this species, although only a few studies focused on the biology

© stocchino et al., 2019 | doi:10.1163/18759866-20191406 This is an open access article distributed under the terms of the prevailing cc-by license at the time of publication. Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 237 of the introduced populations. We report on the morphology of sexualized individuals from a fissiparous Italian population, representing the second record of spontaneous sexualization of fissiparous individuals in this species. A detailed morphological account of the reproductive apparatus of these ex-fissiparous animals is presented. Our results increased the number of morphological groups previously recognized for European populations of G. tigrina, thus corroborating the hypothesis on multiple independent introductions to this continent. Karyological results obtained from our fissiparous Italian individuals revealed a constant diploid chromosome complement of sixteen chromosomes. Further, we document the marked intraspecific variation in several morphological features of this species.

Keywords ectopic hyperplasic ovaries – ex-fissiparous Dugesiidae – Italian non-native species – morphotypes – supernumerary copulatory apparatus

1 Introduction et al., 1970; Dahm & Gourbault, 1978; Ball & Reynoldson, 1981; Kawakatsu et al., 1985, 1993; Invasions of alien species are considered to Ribas et al., 1989; Benazzi, 1993; Sluys et al., be one of the major threats to global biodi- 1995, 2005; Vila et al., 2004). versity and to form the second major cause The first European record of this alien of animal extinctions (Gherardi et al., 2007 species was from Germany (Meinken, 1925), and references therein). Among planarian after which it was reported from France flatworms or triclads many species are known (De Beauchamp, 1946; Gourbault, 1969; Vila to be invasive, in several cases strongly affect- et al., 2004), Switzerland (Dahm, 1955), Great- ing local ecosystems (cf. Sluys, 2016). There- Britain (Reynoldson, 1956), The Netherlands fore, detailed knowledge on the biology of (Den Hartog, 1959), Luxembourg (Hoffmann, an invasive species is of utmost importance 1964), Iberian Peninsula and the Balearic for understanding the process of invasion, Islands, Azores (Baguña et al., 1980; Ribas the cause of its success, and the subsequent et al., 1989; Malhão et al., 2007; Vila-Farré et al., ecological impact on native species (Ducey 2011 and references therein), and also from et al., 2005; Boll et al., 2015). To this end, we the Balkan peninsula from countries such as provide in this paper further insights into Romania (An der Lan, 1962; Babalean, 2018) the biology of introduced populations of the and Herzegovina (Knezović et al., 2015). In freshwater flatworm Girardia tigrina (Girard, the Italian peninsula, since the 1960s popula- 1850). tions of G. tigrina were recorded from many Girardia tigrina is a widespread, native spe- localities (Stagni & Grasso, 1965; Stella & Mar- cies of the Nearctic Region that has been ac- garitora, 1966; Benazzi, 1981, 1993; Stocchino cidentally introduced into Europe since the et al., 2013a). A fissiparous population of G. 1920s, very likely through the international tigrina was very recently discovered in a river trade in aquatic plants and the activity of on the island of Sardinia, Italy (Stocchino, aquarists. Subsequently, it has been recorded 2018). also for South America, Israel, Japan, and Aus- In its native Nearctic Region G. tigrina is tralia (Marcus, 1946; Gourbault, 1969; Benazzi represented by sexual, asexual (fissiparous)

Downloaded from Brill.com10/10/2021 05:54:17AM via free access 238 Stocchino et al. and seasonally alternating sexual/asexual 2 Materials and methods populations (Kenk, 1937). In contrast, the great majority of the allochthonous popula- 2.1 Collection and culturing tions in other parts of the world is fissiparous Planarians (n = 6) were first collected in De- (cf. Sluys et al., 2005). cember 2009 from a rounded tank (~3 m in Only a few (n = 5) immigrant sexual popula- diameter) localised outside of the greenhouse tions were discovered until now, all occurring of tropical terrestrial plants at the Botanical in Western Europe, viz. two populations from Garden of the University of Genoa (Liguria, Great-Britain (Reynoldson, 1985; Gee et al., Northwestern Italy), harbouring fishes (red- 1998), and single populations from Menor- white carps) and aquatic plants, such as Nim ca Island (Spain) (Ribas et al., 1989), Italy phaea alba (L.), Nuphar lutea (L.) Sm., and (Benazzi, 1993), and France (Vila et al., 2004). Lemna sp. (fig. 1A–B). The Botanical Garden The Italian sexual population was recorded is located within the city’s historical centre. from a lake in the southern part of the penin- More recently (May 2018), in two other tanks sula. Successive studies revealed a high degree planarians were found on the underside of of sterility of these Italian sexual specimens, semi-submerged leaves of N. alba: a) a small despite the great number of cocoons that was square tank (~60 × 60 cm, water depth 2 cm), laid (Benazzi & Giannini Forli, 1996). in which the planarians were associated with Among fissiparous populations of G. tigri gastropods and larvae of insects; and b) a na, the occurrence of so-called “ex-fissiparous” larger rounded tank (~3 m in diameter, water individuals, i.e. planarians that stop multiply- depth 10 cm) in which the associated fauna ing by fission and acquire the sexual state, has were leeches and gastropods. been scarcely reported (cf. Grasso, 1974 and All individuals were exclusively fissipa- references therein; Ribas et al., 1989). More- rous at collection, with signs of fission being over, these very few records did not report de- visible. The collected specimens were trans- tails on the morphology of the reproductive ferred to the laboratory and were reared in apparatus of these “sexualized” animals. glass bowls under semi-dark conditions at 18 Here we report (1) the occurrence of sexu- +/- 2°C; the worms were fed weekly with fresh alized individuals of G. tigrina from a fis- beef liver, while the bowls were cleaned with- siparous Italian population, representing the in 8-12 h after feeding. second record for this species of spontane- It was observed that in this culture, as ous sexualization of fissiparous individuals; well as in a strain of a Sardinian population (2) a detailed morphological description of (M. Pala & G.A. Stocchino, pers. obs.), animals the reproductive apparatus of ex-fissiparous were extremely sticky, and that, for example, specimens of G. tigrina; (3) the chromosome when they were attached firmly to the surface complement of the Italian population; (4) of the containers or to the paint brushes used, the marked intraspecific variation in several it was very difficult to remove the animals morphological features of this species; (5) from these substrates. some peculiarities of the hyperplasic ovaries as well as morphological aberrations of the 2.2 Morphology and karyology reproductive apparatus in the Italian popu- For morphological study, sexualized speci- lation. Possible means of dispersal and sub- mens were fixed for 24 h in Bouin’s fluid, sequent establishment of populations are dehydrated in a graded ethanol series, then discussed. transferred to clove oil, and, subsequently,

Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 239 embedded in synthetic wax or paraffin. Serial muscle; lvd, left vas deferens; m, mouth; ma, sections were made at intervals of 8 μm and male atrium; o, oviduct; og, oogonia; pb, penis stained with Mallory-Cason. Reconstructions bulb; ph, pharynx; php, pharyngeal pocket; of the copulatory complex were obtained pp, penis papilla; rbc, right bursal canal; ro, by using a camera lucida attached to a com- right oviduct; s, sperm; sba, supplementary pound microscope. blind atrium; sd, spermatids; sg, shell glands; Chromosome metaphasic plates were ob- sp, supplementary penis; spv, spermiducal tained by the squashing method on single vesicles; st, spermatogonia; sv, seminal vesi- caudal regenerative blastemas of 5 fissiparous cles; vd, vas deferens. specimens. Blastemas were first treated with a solution of colchicine (0.3%) for 4 h, then transferred onto glass slides and treated with 3 Results a solution of acetic acid (5%) for 5 min. Subse- quently, they were stained with acetic orcein 3.1 Sexualization for 2 h and squashed using a small coverslip. From the animals obtained during the first The chromosome complement was character- collection three specimens sexualized, after ised on the basis of 5-6 metaphasic plates per having been kept in the laboratory for about specimen. Chromosomal nomenclature fol- five years, during which also fissioning oc- lows Levan et al. (1964). curred. These sexualized animals displayed the characteristic features of ex-fissiparous 2.3 Material examined individuals: large body size, development of ZMA V.Pl. 7283.1, freshwater tank at the Bo- the copulatory apparatus, hyperplasic ovaries. tanical Garden of the University of Genoa Four cocoons were also laid, one of which was (44°25’35” N, 8°54’54” E) Genoa, Italy, Decem- found opened, but no juveniles were observed. ber 2009, coll. Paola Ramoino and Renata Manconi, sagittal sections on 4 slides; CGAS 3.2 Morphological description Pla 18.1, ibid., sagittal sections on 3 slides; Body size of preserved, sexualized specimens CGAS Pla 18.2, ibid., sagittal sections on 3 ranged from 4 to 6 mm in length and 1.2-2 mm slides. in width. Head triangular, with bluntly point- The material is deposited in the collections ed anterior tip. Two eyes are present in the of Naturalis Biodiversity Center, Leiden, The middle of the head at the level of auricles, po- Netherlands (ZMA collection code), and in sitioned close together and located in broad the Giacinta A. Stocchino collection (CGAS), pigment-free patches. Unpigmented auricular University of Sassari. grooves are marginally placed just posteriorly to the eyes (fig. 1C). 2.4 Abbreviations used in the figures The dorsal pigmentation pattern is of the a, atrium; b, brain; ba, blind atrium; bc, bursal “striped type” (sensu Hyman, 1939): on the canal; bro, branch of right oviduct; ca, com- yellowish-brown background colour run two mon atrium; cb, copulatory bursa; cg, cement longitudinal stripes, made up of black spots, glands; cm, circular muscles; csv, common separated by a lighter yellowish-brown mid- seminal vesicle; di, diverticulum; dp, diplo- dorsal band; further there are also black tene oocytes; e, eye; ec, ectopic hyperplasic specks haphazardly distributed on the dorsal ovaries; ed, ejaculatory duct; g, gonopore; h, surface, while a clear zone runs along the en- head; ho, hyperplasic ovaries; lm, longitudinal tire body margin (fig. 1C).

Downloaded from Brill.com10/10/2021 05:54:17AM via free access 240 Stocchino et al.

Figure 1 Girardia tigrina. A. Geographic range of allochthonous sexual populations (filled circles) and populations with sexualized animals (triangles and asterisk) in the Western Palaearctic; asterisk: population from Liguria investigated in the present study. B. Aquatic plants as preferential shaded microhabitat in a tank at the Botanical Garden of the University of Genoa, Liguria. C. Habitus of a living ex-fissiparous specimen of the Ligurian population. Scale bar not available. Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 241

The pharynx is unpigmented, with the inner ZMA V.Pl. 7283.1 and CGAS Pla 18.1, whereas and outer pharyngeal musculature bilayered, they are under-developed in specimen CGAS i.e. without an extra, third, outer longitudinal Pla 18.2. In the mature testes spermatogenesis muscle layer. The position of the mouth open- appears to proceed in a regular fashion, in ing is different in the three specimens exam- that no anomalies, such as irregularly shaped ined. In specimen ZMA V.Pl. 7283.1 the mouth spermatids or spermatozoa, were observed is located at the hind end of the pharyngeal (fig. 3D). Vitellaria are located, as usual, be- pocket (fig. 2A), whereas in specimens CGAS tween the intestinal branches. Pla 18.1 and CGAS Pla 18.2 the mouth open- In ZMA V.Pl. 7283.1 two copulatory appa- ing is shifted anteriad, in that it is located at ratuses are present in the post-pharyngeal about 1/6 and 1/4 , respectively, of the distance region: the fully developed system as well as between the posterior end of the pharyngeal another, second one, the latter located at a pouch and the root of the pharynx (fig. 2B, C). short distance behind the pharyngeal pocket. The ovaries are hyperplasic in all three This second copulatory apparatus is not com- specimens examined, with several scattered pletely developed and consists of only a dor- masses of oocytes and oogonia in the body re- so-ventrally oriented penis. gion directly posterior to the brain, occupying A single, left vas deferens can be observed almost the entire dorso-ventral space of the penetrating the muscular penis bulb of the body (fig. 3A). All oocytes show a regular mei- incompletely developed, second copulatory otic maturation process, from prophase up to apparatus. The penis bulb consists of loosely the diplotene phase, when meiosis ends. Dip- interwoven layers of circular and longitudinal lotene oocytes scattered in the ovarian masses muscle fibres. In this not fully developed sys- do not show degenerative nuclear or cytoplas- tem, the sperm duct enlarges to form a semi- mic stages (fig. 3C). Moreover, in specimen nal vesicle, which continues as a very short ZMA V.Pl. 7283.1 some ovarian masses are lo- ejaculatory duct that opens at the tip of the cated ventrally on the right-hand side of the papilla. The ovoid penis papilla is covered by copulatory apparatus, that is, far into the pos- a partially infranucleated epithelium, which terior part of the body (fig. 3B). Also in these is underlain by a subepithelial layer of circu- ovarian masses no anomalies in the oocytes lar muscle, followed by a layer of longitudinal were observed. muscle fibres. The penis papilla is housed in The anterior portion of the oviducts is ex- a genital atrium, which is lined by a cuboi- panded to form a seminal receptacle or am- dal, partially infranucleated epithelium, sur- pulla, which communicates with the ovarian rounded by a subepithelial layer of circular masses at a variable position, depending on muscles, followed by a layer of longitudinal the hyperplasic condition of the ovaries. muscle fibres. The genital atrium is not divi The infranucleated oviducts run ventrally ded into a male and common atrium and does in caudal direction to the level of the copula- not communicate ventrally with a proper tory apparatus and then curve dorsad towards gonopore (figs 4A, 6A). the vaginal area, subsequently opening sym- Posterior to this incompletely developed metrically into the angled hind part of the copulatory apparatus lies the main, well- bursal canal (figs 4A, 5). developed apparatus (figs 4A, 6A). The fol- The testes are situated ventrally and ex- lowing description is based on the fully tend from the level of the most caudal post- developed copulatory apparatus of ZMA V.Pl. cephalic ovarian masses into the posterior end 7283.1 and on those of CGAS Pla 18.1 and CGAS of the body. The follicles are well developed in Pla 18.2. Downloaded from Brill.com10/10/2021 05:54:17AM via free access 242 Stocchino et al.

Figure 2 Girardia tigrina from Liguria. Photomicrographs of the pharynx; sagittal sections (anterior to the left). A. ZMA V.Pl. 7283.1, mouth opening located at the hind end of the pharyngeal pocket; B. CGAS Pla 18.1, mouth opening located at about 1/6 of the distance between the posterior end of the pharyngeal pouch and the root of the pharynx; C. CGAS Pla 18.2, mouth opening located about at 1/4 of the distance between the posterior end of the pharyngeal pouch and the root of the pharynx. Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 243

Figure 3 Girardia tigrina from Liguria. Photomicrographs of hyperplasic ovaries and testes. A. ZMA V.Pl. 7283.1, hyperplasic ovaries located behind the brain; B. ZMA V.Pl. 7283.1, ectopic hyperplasic ovarian masses located at the level of the copulatory apparatus; C. ZMA V.Pl. 7283.1, magnification of hyperplasic ovaries, with oocytes at different stages of maturation; D. ZMA V.Pl. 7283.1, mature testes with sperm. Downloaded from Brill.com10/10/2021 05:54:17AM via free access 244 Stocchino et al.

Figure 4 Girardia tigrina from Liguria. ZMA V.Pl. 7283.1 A. sagittal reconstruction of the two copulatory apparatuses (anterior to the left); B. sagittal reconstruction of the main copulatory apparatus at the level of the right branch of the bursal canal and the blind cavity (anterior to the left).

The small sac-shaped copulatory bursa is “angled” bursal canal), and at this point re- lined by a columnar, glandular epithelium ceives the separate, symmetrical openings of bearing basal nuclei and it is surrounded by a the oviducts, while, subsequently, it commu- network of muscle fibres. The copulatory bur- nicates with the common atrium. The bursal sae of the three specimens examined contain canal is lined by cuboidal, nucleated, and cili- no spermatophores. ated cells and is surrounded by a subepithelial As usual, the bursal canal runs in a cau- layer of circular muscles, followed by a layer dal direction to the left of the copulatory of longitudinal muscle (figs 4–6). apparatus. The posterior portion of the bur- Shell glands, producing a fine-grained, sal canal curves sharply towards the ventral erythrophil secretion, which is very abundant Downloaded from Brill.com10/10/2021 05:54:17AM body surface (thus constituting the so-called in specimens ZMA V.Pl. 7283.1 and CGASvia Pla free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 245

Figure 5 Girardia tigrina from Liguria. CGAS Pla 18.1, sagittal reconstruction of the copulatory apparatus (anterior to the left).

18.1, open into the bursal canal just ectally, moderately developed in CGAS Pla 18.2, con- i.e. ventrally to the separate openings of the sisting of loosely interwoven layers of circular oviducts. and longitudinal muscle fibres (figs 4–6). From the bursal canal of specimen ZMA The sperm ducts form well-developed V.Pl. 7283.1 originates a left branch at a short spermiducal vesicles, packed with sperm, in distance from the postero-dorsal wall of its specimens ZMA V.Pl. 7283.1 and CGAS Pla 18.1. bursa that, subsequently, curves downward to In ZMA V.Pl. 7283.1 and CGAS Pla 18.2, the vasa communicate with a blind sac or cavity locat- deferentia at first curve dorsad before sym- ed on the left side of the body; this cavity may metrically penetrating the anterior wall of the be considered to represent a small, additional penis bulb. In its ascending course, the left vas genital atrium. deferens of ZMA V.Pl. 7283.1 forms a closed This blind cavity is lined by an infranucle- loop (fig. 4A). In CGAS Pla 18.1 the vasa defer- ated epithelium that is underlain by a subepi- entia recurve considerably in caudal direction thelial layer of longitudinal muscle fibres and before separately and symmetrically pen- receives the openings of several cement glands etrating the antero-dorsal wall of the penis (fig. 4B). At the level of the posteriorly located bulb (fig. 5). Once inside the penis bulb, each ectopic ovaries, from the right oviduct origi- vas deferens enlarges to form a wide vesicle nates a dorsal branch, which ascends to the in ZMA V.Pl. 7283.1, whereas the ducts fuse to supernumerary left branch of the bursal canal form a single intrabulbar vesicle in CGAS Pla and then opens into it just at the point where 18.1 and CGAS Pla 18.2. the canal opens into the blind additional atri- In all specimens the seminal vesicle(s) con- um. Shell glands open into this oviduct just be- tinue as a single ejaculatory duct that opens at fore the duct opens into the bursal canal. the tip of the penis papilla. In ZMA V.Pl. 7283.1 The penis bulb is large and globose in ZMA the ejaculatory duct follows a basically ventral Downloaded from Brill.com10/10/2021 05:54:17AM V.Pl. 7283.1 and CGAS Pla 18.1, whereas it is only course through the papilla, whereas it isvia more free access 246 Stocchino et al.

Figure 6 Girardia tigrina from Liguria. Photomicrographs of the copulatory apparatus; sagittal sections. A. ZMA V.Pl. 7283.1, supernumerary penis and the main, fully developed copulatory apparatus; B. CGAS Pla 18.1, copulatory bursa with the bursal canal, penis, male atrium, and common atrium with diverticulum; C. CGAS Pla 18.2, copulatory bursa with the bursal canal, penis, and male atrium. Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 247 centrally located in CGAS Pla 18.1 and CGAS 3.3 Karyology Pla 18.2 (figs 4–6). In CGAS Pla 18.2 the ejacu- Metaphasic plates revealed that the fissipa- latory duct is very short. rous animals are characterized by a constant In all specimens examined the intrabul- diploid set of 16 chromosomes with n = 8 as bar seminal vesicles, including that of the haploid number (fig. 7). The karyotype con- supernumerary penis in ZMA V.Pl. 7283.1, are sists of 8 pairs of metacentric chromosomes filled with a longitudinally oriented web-like in descending order, with the first four chro- fibrillate secretion. In CGAS Pla 18.2 many mosomes being metacentric isobrachial, erythrophil granules are also present in the while the other chromosomes are metacentric seminal vesicle (figs 4–6). heterobrachial, with the exception of chro- In ZMA V.Pl. 7283.1 the penis papilla has the mosome 8, which is at the border between shape of an elongated cone; in CGAS Pla 18.1 metacentric and submetacentric. it is a stubby cone, while it is barrel-shaped and more dorso-ventrally oriented in CGAS Pla 18.2 (figs 4–6). The penis papilla is covered 4 Discussion by an infranucleated epithelium in ZMA V.Pl. 7283.1 and CGAS Pla 18.1, but in CGAS Pla 18.2 4.1 Reproductive modes and it is provided with a nucleated epithelium. sexualization process The epithelium of the papilla is underlain Sexualization of individuals from fissiparous with a subepithelial layer of circular muscle strains of freshwater planarians has been fibres, followed by a layer of longitudinal highlighted since the early 1970s for species muscles. of the genus Dugesia (cf. Benazzi, 1974). These In CGAS Pla 18.1 and CGAS Pla 18. 2 the gen- planarians were called “ex-fissiparous” and ital atrium is clearly divided into a common are characterized by (a) an increase in body atrium and a male atrium, which communi- dimensions, (b) development of a complete cate via a pronounced narrowing. In ZMA V.Pl. copulatory apparatus, (c) hyperplasic ovaries, 7283.1 this division into two atria is less obvi- (d) underdeveloped testes, and (e) sterility ous, very likely due to the elongation of the or, at least, low fertility (cf. Stocchino et al., penis papilla, which occupies the entire com- 2012, 2014; Harrath et al., 2013, 2014). mon atrium, with its tip extending into the Only a few cases of sexualized individu- gonopore (figs 4–6). The atria are lined by an als from fissiparous strains of G. tigrina have infranucleated epithelium, which is underlain been documented (Grasso, 1974 and refer- by a subepithelial layer of circular muscle, fol- ences therein; Ribas et al., 1989). Grasso (1974) lowed by a layer of longitudinal muscle fibres. obtained sexualization of many fissiparous The common atrium opens ventrally through individuals from a north-Italian population the gonopore and it receives the coarsely gran- (Lake Maggiore), but only after the worms ular, xanthophil secretion of very abundant had been fed for many weeks with crushed cement glands in ZMA V.Pl. 7283.1 and CGAS tissues of sexually mature specimens of Poly Pla 18.1, while in CGAS Pla 18.2 these glands celis nigra (Müller, 1774). Typical hyperplasic are only moderately developed (figs 4–6). ovaries were reported for these ex-fissiparous In CGAS Pla 18.1 a well-developed posterior animals. It is noteworthy that this worker did diverticulum is present in the hind wall of its not succeed in obtaining sexualized animals common atrium, whereas in ZMA V.Pl. 7283.1 from this population when the flatworms and CGAS Pla 18.2 this diverticulum is much were fed for more than ten years with the less pronounced (figs 4–6). usual live TubifexDownloaded worms from or withBrill.com10/10/2021 minced 05:54:17AMbeef. via free access 248 Stocchino et al.

Figure 7 Girardia tigrina from Liguria. A. Metaphasic plate; B. Karyogram.

Many years later, Ribas et al. (1989) reported copulatory apparatus is present, as well as a on some rare, large ex-fissiparous individuals doubling of some structures in the main, com- from Spanish mainland populations, which pletely developed apparatus, together with never laid cocoons. However, these Spanish an unusual course of the left vas deferens. It specimens were of the spotted morphotype is interesting to note that with respect to the (see below, under “Spotted vs. striped mor- genus Girardia occurrence of supernumer- photypes” section) with strongly and coarsely ary copulatory apparatuses was reported for pigmented pharynx, in contrast to the Italian G. dorotocephala (Woodworth, 1897) by Kenk animals that we examined, which belong to (1935). This worker described that 17 out of 71 the striped morphotype with unpigmented fissiparous specimens from a particular local- pharynx. Unfortunately, both Grasso (1974) ity, which were kept at a constant low temper- and Ribas et al. (1989) did not provide any ature and were fed with beef liver, developed details on the reproductive apparatus of their 2-4 genital pores. These animals were on av- sexualized planarians. The present paper erage larger than those with one genital pore, provides the first detailed description of ex- while they also had a comparatively larger fissiparous individuals of G. tigrina. post-pharyngeal region as compared with the It is noteworthy that in one of the our ani- single-gonopore specimens. Three specimens mals examined an extra, albeit incomplete, (with 4, 3, and 2 gonopores, respectively) that Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 249

Kenk (1935) examined histologically showed was not induced by food items consisting of normal testes, hyperplasic ovaries, and pieces of sexual planarians, which may have multiple copulatory apparatuses, developed supplied sex-inducing substances, as has to greater or lesser extent and sometimes occurred in certain experiments (see Grasso connected to each other, being arranged one & Benazzi, 1973; Sakurai, 1981). Thus, it may behind the other along the anterior-posterior be that under laboratory conditions (constant axis of the body. After sexualization also co- temperature and lighting and regular food coons were laid. supply) in the usual ex-fissiparous animals, Supernumerary sexual structures were also at least in the genus Girardia, sometimes an induced by low temperature in a laboratory extra sexual induction occurs with the effect strain of Dugesia gonocephala (presently D. ja that besides development of supernumerary ponica) from Japan (Ogukawa, 1955). Howev- sexual structures also neoblasts not belonging er, differently from the Girardia species these to presumptive germ cells are able to differen- aberrations occurred only in sexual animals tiate into germ cells. with normal ovaries and testes. Hyperplasic ovaries in ex-fissiparous indi- A peculiar condition encountered in the viduals have been reported for a number of present study is that besides hyperplasic ova- Dugesia species (De Vries, 1986; Pala et al., ries, located as usual just behind the brain, in 1995; Stocchino et al., 2002, 2009, 2012, 2013b, the aberrant specimen ectopic ovarian mass- 2014, 2017; Harrath et al., 2013). In contrast, ex- es are localized ventrally at the level of the fissiparous individuals of D. benazzii Lepori, copulatory apparatus, thus very far from the 1951 and D. etrusca Benazzi, 1946 do not devel- usual anterior position. Such a very peculiar op hyperplasic ovaries (Stocchino & Manconi, phenomenon has never been reported before 2013). for freshwater triclads. Such hyperplasic ovaries are characterised It is surprising that in this case neoblasts not by degenerative processes, which lead to a destined to become germ cells were instead blockage of the maturation of the oocytes induced toward this kind of differentiation. (Gremigni & Banchetti, 1972a). It has been In point of fact, it has been demonstrated that demonstrated that a complex process of early the nanos gene is required for postembryonic autophagy, followed by apoptotic processes, development, regeneration, and maintenance occurs during the cell death of oocytes in the of planarian germ cells (cf. Newmark et al., hyperplasic ovaries of D. arabica and that cy- 2008 and references therein). However, also in tokine-like molecules may contribute to this fissiparous animals nanos-positive cells were pathology (Harrath et al., 2014, 2017). detected at positions in which germ cells are As in species of the genus Dugesia occur- first observed post-embryonically in sexual rence of hyperplasic ovaries has been consid- planarians and, thus, they were considered to ered to be the result of a greater proliferation be “presumptive germ cells” that are unable to of neoblasts into oogonia (Gremigni & complete their differentiation (cf. Newmark Banchetti, 1972b; Benazzi, 1974), we may sur- et al., 2008 and references therein). mise that the same process also produced the It is known that environmental factors, hyperplasia in the ectopic ovaries. especially temperature, influence sexual or Another peculiarity, evident in all three asexual reproduction in populations with al- G. tigrina specimens examined, concerns ternating reproductive modes (Kenk, 1937). the fact that, notwithstanding the hyper- Moreover, sexualization of our G. tigrina and plasic condition, oogenesis appears regular the G. dorotocephala studied by Kenk (1935) and the hyperplasicDownloaded ovaries from Brill.com10/10/2021 lack the typical 05:54:17AM via free access 250 Stocchino et al. degenerative stages that are characteristic of mixing up of “….a secretion of some sort ….” such sexualized specimens in several Dugesia (Ball, 1971, p. 15) and sperm that is also present species (Gremigni & Banchetti, 1972a; Stoc- in the seminal vesicle (Chen et al., 2015). chino et al., 2009, 2012, 2013b; Harrath et al., 2014, 2017). 4.2 Karyology Furthermore, in the aberrant individual, Our karyologycal analysis revealed a constant and also in another specimen examined, diploid chromosome complement (2n = 16; n = testes are also well-developed, exhibiting all 8). The basic haploid complement of 8 chromo- phases of sperm maturation. That spermato- somes is in agreement with previous studies (cf. genesis is regular is also highlighted by (1) the Benazzi & Benazzi-Lentati, 1976; Kawakatsu et presence of sperm inside the vasa deferentia, al., 1981; Ribas et al., 1989; Benazzi, 1993). While of which the posterior tracts are enlarged as for sexual populations only diploid karyotypes spermiducal vesicles, and (2) the presence of have been reported, fissiparous populations allosperm in the oviducts, indicating previous show diploid, triploid or mixoploid (diploid mating(s) with another animal(s). and triploid) karyotypes (Dahm, 1958; Benazzi The regular female and male gametogen- & Benazzi-Lentati, 1976; Kawakatsu et al., 1981; esis may be related to the eudiploid condition Ribas et al., 1989; Benazzi, 1993). of the Italian population (see below), in that it That all chromosomes in our specimens allows a more regular meiosis and that, thus, examined are metacentric is in agreement potential sexual reproduction cannot be ruled with previous data on European populations out, although no juveniles were observed to (Ribas et al., 1989 and references therein). In hatch from the opened cocoon of these sexu- contrast, for several non-European popula- alized animals. tions karyotypes were reported that consisted With respect to sexualized freshwater of 7 pairs of metacentric chromosomes and planarians, only one case of well-developed one pair of submetacentric chromosomes testes has been reported, namely for D. bi (the 6th chromosome set): (1) a sexual popula- fida, albeit that in this species the ovaries are tion from Canada (Puccinelli & Deri, 1991); (2) only weakly hyperplasic. Furthermore, in this sexual and asexual populations from South species the chromosome complement is eu- Brazil (Kawakatsu et al., 1981); (3) asexual pop- diploid, while sexualized animals laid fertile ulations from Japan (Kawakatsu et al., 1985, cocoons (Stocchino et al., 2014). In contrast, 1993; Tamura et al., 1985). the majority of the fissiparous species of the genus Dugesia are triploid or mixoploid, with 4.3 Intraspecific morphological the sexualized specimens exhibiting gonadal variability anomalies (Stocchino et al., 2014). Our results highlight the marked intraspecific The presence of a fibrillate secretion in morphological variability of several features the seminal vesicles has never been reported of G. tigrina, such as: (a) pharynx pigmenta- for G. tigrina and may be compared with the tion, being present or absent; (b) position of “web-like tissue” described by Hyman (1956) the mouth opening, which may be located at for D. diabolis (presently G. dorotocephala the hind end of the pharyngeal pocket or may (Woodworth, 1897)) and later also report- be shifted anteriad (located at about 1/6 or 1/4 ed by Ball (1971) and Chen et al. (2015) for of the distance between the posterior end G. dorotocephala and G. sinensis Chen & Wang, of the pharyngeal pouch and the root of the 2015, respectively. Probably it results from the pharynx); (c) length of the ejaculatory duct,

Downloaded from Brill.com10/10/2021 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 251 which may be long or very short; (d) number duct, both conditions having been reported in of the seminal vesicles, which may be single the present paper and also in earlier studies or double; (e) course of the vasa deferentia, (e.g. Ball, 1971, figs 5, 7; Kenk, 1972; Kawakatsu which, before penetrating the antero-dorsal & Mitchell, 1981). wall of the penis bulb, in some cases simply Another character showing marked differ- curve dorsad, while in other cases they recurve ences among populations concerns the ar- considerably in caudal direction. These fea- rangement of muscles around the bursal canal. tures will be discussed below. Sluys et al. (2005) suggested that the Nearctic Although a pigmented pharynx was con- and Neotropical forms of G. tigrina concern sidered a diagnostic character for G. tigrina sibling species, in view of the fact that South (cf. Kenk, 1972; Ball & Reynoldson, 1981), fur- American populations have a bursal canal ther studies have also reported on specimens musculature consisting of a well developed with unpigmented pharynx (Ribas et al., coat of intermingled circular and longitudi- 1989; Benazzi, 1993). Nevertheless, Sluys (2001) nal muscle fibres, whereas North American considered the pigmented pharynx to be an forms are characterised by a simple coat of apomorphic character for the genus Girar muscle around the bursal canal made up by dia, in spite of the fact that a few species are a thin, subepithelial layer of circular muscle, polymorphic, in that individuals may have followed by an equally thin layer of longitu- either pigmented or unpigmented pharynges, dinal muscle. The latter condition was found and very few have an unpigmented pharynx. also in introduced populations, such as those Our finding of specimens with unpigmented from Spain, France, southern Italy (Ribas pharynx thus confirms the variability of this et al., 1989; Vila et al., 2004; Sluys et al., 2005), character. and our population from Liguria, northern The position of the mouth opening was Italy. This may be an indication that all of the considered a feature that warranted further above-mentioned introduced European pop- consideration, since the mouth was found to ulations originate from the Nearctic Region. be at different positions in the pharyngeal pocket of animals from different geographic 4.4 Spotted vs. striped morphotypes localities (Sluys et al., 2005). However, this With respect to its external features, G. tigrina variability appears to be much less due to a is a polymorphic species that varies from geographic gradient than to intraspecific vari- spotted to striped, both types of individuals ation, as in our three animals from Italy the sometimes occurring in the same population location of the mouth turned out to be highly in its native area (Kenk, 1972). Several of these variable, even within a single population. conditions are expressed also in European Presence of two seminal vesicles in one of populations: 1) all sexual populations are of our individuals and a single intrabulbar vesicle the striped type (Ribas et al., 1989; Benazzi, in the other two individuals confirms previ- 1993; Gee et al., 1998; Vila et al., 2004); 2) the ous reports on the variability of this character majority of fissiparous populations are spot- (cf. Kenk, 1972; Ribas et al., 1989). According to ted without exhibiting sexualization process- Kenk (1972), a single seminal vesicle would be es (i.e. producing ex- fissiparous animals) (cf. a transitory condition, but we interpret it as Ribas et al., 1989 and references therein); 3) a being the result of intraspecific variation. single case of a fissiparous striped population The seminal vesicle(s) may continue either and with development of ex-fissiparous speci- as a long ejaculatory duct or as a very short mens (present paper); 4) some fissiparous

Downloaded from Brill.com10/10/2021 05:54:17AM via free access 252 Stocchino et al. spotted populations exhibiting sexualization chroa (Ball & Reynoldson, 1981), and S. medi processes (Grasso, 1972, 1974; Ribas et al., 1989). terranea (M. Pala & G.A. Stocchino, pers. obs.) Ribas et al. (1989) related the external pig- as this has been observed under laboratory mentation pattern of several Spanish popu- conditions, and thus may imply strong com- lations with the pharynx pigmentation, thus petition with the native planarian fauna. distinguishing three morphological classes: Another possible feature facilitating its A, fissiparous “spotted” with pigmented spread over the world may be related to our pharynx; B, fissiparous “spotted” with unpig- observation that the animals are extremely mented pharynx; C, sexual, “striped” with sticky, which greatly enhances their acciden- unpigmented pharynx. Class A and the group tal passive dispersal on, for example, aquatic made by classes B and C together were con- plants. This may explain their occurrence in sidered as natural groups or races, also on the the botanical garden in Genoa. basis of biochemical data. To these classes we Adhesiveness in flatworms is known to be can now add a fourth class, comprising the facilitated by the secretion of adhesive glands. Ligurian fissiparous, “striped” population with It is not known whether the observed sticki- unpigmented pharynx. ness of G. tigrina is due to an overproduction The presence in Europe of several morpho- of mucus or to particular characteristics of logical groups may be explained as the result the mucus released by the adhesive glands of of multiple independent introductions of G. this species. To our knowledge there are no tigrina to this continent, very likely from the comparative studies on this subject, which Nearctic Region (see above), as already sug- deserves further investigation. gested by Ribas et al. (1989) for the Spanish populations. Future molecular analyses might form appropriate tests of this hypothesis. Acknowledgements

4.5 Dispersal and establishment of This research was supported by the following populations grants to G.A. Stocchino: a grant in memory Many underlying causes have been suggested of Prof. N.G. Lepori (Università di Sassari), a for the remarkable dispersal and establish- Temminck Fellowship from Naturalis Bio- ment capability of G. tigrina, such as (a) fis- diversity Center (Leiden, The Netherlands), siparous reproduction, enabling it to rapidly and by a grant from Prof. R. Pronzato (Diparti- colonize new water bodies (Wright, 1987), mento di Scienze della Terra, dell’Ambiente e (b) extreme tolerance to suboptimal and de- della Vita, Università di Genova). We acknowl- manding environmental conditions, allowing edge financial support from Fondazione di it to spread along water bodies which are de- Sardegna and Regione Autonoma Sardegna nied to native species (Wright, 1987), (c) abil- (RAS2012-LR7/2007-CRP-60215” Conservazi- ity to exploit a wide variety of food resources one e valorizzazione delle grotte sarde: biodi- with overlap in the diets of G. tigrina and versità e ruolo socio-economico-culturale”). native triclads, thus suggesting a potentially Prof. M. Pala is thanked for her kind sup- strong inter-specific competition for food (oli- port. We are grateful to Dr. P. Ramoino and gochaetes, isopods, chironomids, snails, cad- Prof. G. Barberis (Dipartimento di Scienze disflies, mayflies, amphipods, and cladocer- della Terra, dell’Ambiente e della Vita, Uni- ans) (Pickavance, 1971; Gee & Yang, 1993), and versità di Genova) and Dr. E. Mora (Botanical (d) perhaps also cannibalism on other species Garden of Genoa) for their kind help in col- of planarians such as Polycelis spp. and S. poly lecting planarians andDownloaded identification from Brill.com10/10/2021 of plants. 05:54:17AM via free access EX-FISSIPAROUS ITALIAN FLATWORM GIRARDIA TIGRINA 253

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