Madagascar, the fourth largest island in the world, is one of the priority global hotspots for biodiversity conservation (Mittermeier et al. 2004, Vieites et al. 2009) with 5% of the total species in the world and a very high percentage of endemism (70%). Unfortunately, this outstanding species richness is greatly endangered, due to rapidly increasing deforestation, soil erosion, and habitat destruction in large areas of the island (Harper et al. 2007, Andreone et al. 2008). This is particularly true for freshwater environments and their communities of benthic invertebrates.

It has been shown that Madagascar represents a global biodiversity hotspot for land planarians (Sluys 1998, 1999) but, unfortunately, freshwater triclads have received much less attention. With respect to freshwater planarians only representatives of the genus Dugesia Girard, 1850 of the family Dugesiidae Ball, 1974 have been reported from Madagascar. This genus is present with three species, two of which are endemic, viz. Dugesia debeauchampi De Vries, 1988, Dugesia myopa De Vries, 1988, and Dugesia milloti De Beauchamp, 1952 (cf. De Beauchamp 1952, De Vries 1988, Stocchino et al. 2002) (Table 1). The first species is known only from the type locality, Nosy Bé Island, north-west of Madagascar. Dugesia myopa is reported from two localities, viz. Andranoboka Cave, northwest of Mahajanga, and from Andringitra, in the southeast of the island. In contrast, Dugesia milloti is recorded from widely scattered Madagascan localities, viz. Montagne d’Ambre, Ivohibe, Morafenobé, and occurs also on the island of Anjouan in the Comoros Archipelago (De Beauchamp 1952, De Vries 1988).

A recent field survey performed by one of us (R. Manconi) on the presence and distribution of Madagascan aquatic invertebrates in some unexplored lentic or lotic freshwaters of the High Plateau and the oriental slope (31 sites) yielded several new records of planarians, suggesting that species richness of this taxon in the island is underestimated.

In this paper we describe a new, and in some respects aberrant, species of Dugesia that was identified on the basis of morphological and karyological data. This contribution represents a first step of a more comprehensive faunistic and taxonomic study of planarian populations on Madagascar. An integrative taxonomic analysis, including molecular and morphological data, is in progress in order to analyze in more detail the problematic position of Madagascan freshwater triclads in an historical biogeographic scenario involving the splitting of Gondwana (cf. Sluys et al. 1998).

The collected specimens were transferred to the laboratory and were reared in glass bowls under semi-dark conditions at 18 +/- 2 °C; the worms were fed with fresh beef liver.

For morphological study specimens were fixed for 24 hours in Bouin’s fluid, dehydrated in an ascending ethanol series, cleared in clove oil, and embedded in synthetic paraffin. Serial sections were made at intervals of 6–8 µm and were stained with Mallory-Cason. Reconstructions of the copulatory apparatus were obtained by using a camera lucida attached to a compound microscope.

For karyological analyses metaphasic plates were obtained by the squashing method and also air drying (splashing), following Vacca et al. (1993). The squashing method was performed on single caudal regenerative blastemas of 10 specimens in order to verify the uniformity of the chromosome complement of the strain. The air drying method was performed on 15 intact specimens, thus yielding good metaphasic plates for karyometrical analysis. The chromosome complement was characterized on the basis of 6 metaphasic plates. Karyometric values were calculated after first arranging the chromosomes according to their gradually decreasing lengths. Relative length was calculated as chromosome length × 100/total length of the haploid genome. Centromeric index was calculated as length of short arm × 100/total length of the chromosome. Chromosomal nomenclature follows Levan et al. (1964).

The histological material is deposited at Naturalis Biodiversity Center, Leiden, The Netherlands (ZMA collection code), and in the Giacinta A. Stocchino collection (CGAS), University of Sassari.

Dugesia bifida differs from its congeners in its external morphology, in particular the head shape, the peculiar course of the oviducts at the level of the copulatory apparatus, including the presence of a common posterior extension, and in the asymmetrical openings of the vasa deferentia at about halfway along the seminal vesicle. Further, it is characterized also by a ventral course of the ejaculatory duct with a terminal opening, a large seminal vesicle, and unstalked cocoons.

Almost all known species of Dugesia are characterized by a distinctly triangular head with pointed auricles, whereas in Dugesia bifida the latter are much more smoothly rounded. The only other species of Dugesia with a peculiar external morphology is Dugesia milloti, which is characterized by a head with a high triangular shape and prominent, pointed auricles. The latter species is known only from Madagascar and from the island of Anjouan in the Comoros Archipelago (De Beauchamp 1952, De Vries 1988). It is remarkable that these two Dugesia species with an aberrant external morphology both occur on Madagascar.

The peculiar condition of the oviducts in Dugesia bifida lies in the fact that the ducts open into a common posterior extension, which anteriorly divides into two branches before opening into the vaginal section of the bursal canal. Such a condition was never reported before for the genus Dugesia. Usually, in this genus the oviducts run in caudal direction and at the level of the copulatory apparatus open symmetrically or asymmetrically into the vaginal part of the bursal canal. Exceptions to this rule are Dugesia myopa and Dugesia congolensis De Beauchamp, 1951 from the Afrotropical Region, Dugesia mertoni (Steinmann, 1914) from the Australasian region, Dugesia deharvengi Kawakatsu & Mitchell, 1989, and Dugesia andamanensis (Kaburaki, 1925) from the Oriental Region, in which the oviducts fuse to form a short common oviduct before opening into the bursal canal. Two other species, viz. Dugesia lindbergi De Beauchamp, 1959 from the Palaearctic and Oriental regions and Dugesia uenorum Kawakatsu & Mitchell, 1995 from the Australasian Region are characterized by a polymorphism concerning symmetrical openings and a common oviducal condition (cf. Sluys et al. 1998).

That in Dugesia bifida the common posterior extension belongs to the oviducts is demonstrated by the fact that the histological architecture of this common duct is the same as the rest of the oviducts. The continuation of the oviducts caudally to the copulatory apparatus may be functionally related to the presence of vitellaria in the tail region.

This common posterior extension of the oviducts in Dugesia bifida reminds one of the caudally branched oviducts reported for some other genera of the Dugesiidae viz. Spathula Nurse, 1950 from New Zealand and Australia, Reynoldsonia Ball, 1974, and Eviella Ball, 1977 from Australia, and three species of the genus Romankenkius Ball, 1974 from Tasmania (cf. De Vries and Sluys 1991, Sluys 2001). According to Sluys' (2001) phylogenetic analysis, presence of caudally branched oviducts could be considered as a synapomorphy for a group comprising the genera Spathula, Eviella, and Reynoldsonia, albeit that there are also three cases of parallelism, viz. Romankenkius sinuosus Sluys & Kawakatsu, 2001, Romankenkius libidinosus Sluys & Rohde, 1991, and Romankenkius pedderensis Ball, 1974 [note that this interpretation differs somewhat from that discussed by Sluys (2001, p. 70)].

Caudally branched oviducts occur also in other species belonging to different freshwater families, such as the dendrocoelid Macrocotyla glandulosa Hyman, 1956 and the cavernicolan Rhodax evelinae, Marcus, 1946 (cf. Sluys 2001).

Among African and Madagascan species of Dugesia absence of ectal reinforcement in Dugesia bifida is shared only with Dugesia aethiopica Stocchino et al., 2002 and Dugesia afromontana Stocchino & Sluys, 2012 (Stocchino et al. 2002, 2012).

From Madagascar only three species of Dugesia have been reported up to this moment, viz. Dugesia debeauchampi, Dugesia milloti, and Dugesia myopa (De Beauchamp 1952, De Vries 1988) (Table 1). In contrast to Dugesia bifida, a central course of the ejaculatory duct is displayed by these three species, while Dugesia myopa also has a short common oviduct and reduced eyes. Dugesia bifida shares with Dugesia milloti and Dugesia myopa a large diaphragm, whereas Dugesia debeauchampi has a small diaphragm.

As for life history, the life cycle of Dugesia bifida under laboratory conditions is comparable to that of other African species, such as Dugesia aethiopica and Dugesia afromontana, in which post–pharyngeal transverse fissioning occur continuously, while sexual reproduction followed by fertile cocoon deposition is less frequent, involving only a small percentage of individuals. In contrast to Dugesia aethiopica, ex-fissiparous specimens of Dugesia bifida do not retain the fissioning ability during their sexual state (cf. Stocchino and Manconi 2013).

In those species of Dugesia in which ex-fissiparous specimens develop from fissiparous strains, these sexualized individuals are characterized by the presence of hyperplasic ovaries and underdeveloped testes, in which germ cells show degenerative processes (cf. Stocchino et al. 2012). In contrast, in Dugesia bifida the testes are well developed in all specimens examined, without anomalies of the germ cells. Moreover, the hyperplasic ovaries in Dugesia bifida are not visible through the dorsal body wall of living animals, in contrast with other species, such as Dugesia sicula Lepori, 1948, Dugesia maghrebiana Stocchino et al., 2009, Dugesia aethiopica, Dugesia afromontana, and Dugesia arabica Harrath & Sluys, 2013 (Stocchino et al. 2012, Stocchino and Manconi 2013, Harrath et al. 2013). This may be due to the fact that in Dugesia bifida the hyperplasic ovaries are more weakly developed and more expanded in horizontal direction than in vertical direction.

The cocoons of Dugesia bifida are unstalked but firmly attached by cement to the substratum. The production of unstalked cocoons is an uncommon condition in the genus Dugesia, for which they are generally reported as being provided with a pedicel and a terminal plate (Gourbault 1972, Ball and Reynoldson 1981). Among dugesiids, stalked cocoons are known also from Schmidtea Ball, 1974 (cf. Gourbault 1972), Cura Strand, 1942 (Ball 1974, Grant et al. 2006), and Girardia dorotocephala (Woodworth, 1897) (cf. Gourbault 1972). In Spathula ochyra Ball & Tran, 1979 the cocoons are enclosed in a jelly-like dome, attached to the substratum (Grant et al. 2006). Unstalked cocoons that can be fastened to the substratum very lightly or attached firmly to it by cement, are reported for the dugesiid genus Neppia Ball, 1974, the planariid genera Phagocata Leidy, 1847 and Seidlia Zabusov, 1911, and the dendrocoelid genus Dendrocoelopsis Kenk, 1930. Specifically, unstalked cocoons have been reported for the planariid species Planaria torva (Müller, 1774), Polycelis nigra (Müller, 1774), Polycelis tenuis Ijima, 1884, Polycelis felina (Dalyell, 1814), and Crenobia alpina (Dana, 1776), and for the dendrocoelids Bdellocephala punctata (Pallas, 1774), Dendrocoelum lacteum (Müller, 1774), Dendrocoelum album (Steinmann, 1910), Dendrocoelum romanodanubialis (Codreanu, 1949), and Dendrocoelum vesiculosus Stocchino & Sluys, 2013, (Gourbault 1972, Ball 1974, Ball and Reynoldson 1981, Stocchino et al. 2013, Kawakatsu pers. comm., Stocchino pers. obs.).

As for karyology, Dugesia bifida shows a diploid chromosome complement of 18 chromosomes with basic number n = 9. Among Dugesia species this basic number is shared by only six other species: Dugesia sicula, Dugesia maghrebiana and Dugesia biblica Benazzi & Banchetti, 1972, from the Mediterranean region; Dugesia arabica from Yemen; Dugesia aethiopica and Dugesia afromontana from the Afrotropical region (cf. Stocchino et al. 2004, Stocchino et al. 2012, Harrath et al. 2013).

Dugesia bifida represents the easternmost record of a species with a basic chromosomal number n = 9. However, Dugesia bifida differs from all of these other species in that it is the only species in which fissiparous specimens exhibit a diploid chromosome complement. Dugesia sicula and Dugesia biblica have sexual and fissiparous populations with diploid and triploid chromosome complements, respectively (Pala et al. 1995, Stocchino et al. 2012). Dugesia afromontana is known only from two fissiparous populations with a triploid chromosome complement (Stocchino et al. 2012). Dugesia maghrebiana is represented by a fissiparous population, characterized by a condition of diffuse mosaicism or mixoploidy, with each individual having triploid and tetraploid cells (Stocchino et al. 2009). The only fissiparous population of Dugesia aethiopica shares with Dugesia maghrebiana the condition of mixoploidy, but chromosome complements of the former species are diploid and triploid. Dugesia arabica shows a combination of the above-mentioned conditions, in that it has diploid populations that reproduce sexually, triploid fissiparous populations, and mixoploid (diploid and triploid) populations reproducing sexually as well as by fission (Harrath et al. 2013).

With respect to this group of six species discussed above, it turns out that fissiparous populations are always triploid or mixoploid, while sexual populations are diploid.

The presence of well developed testes with very abundant sperm and weakly hyperplasic ovaries in ex-fissiparous specimens of Dugesia bifida may be related to its diploid condition, in that it allows a more regular meiosis. A similar condition was reported for Dugesia colapha Dahm, 1967 from Ghana, in which the fissiparous populations that produced ex-fissiparous individuals under laboratory conditions were characterized by regular diploid chromosome complements (2n = 16; n = 8). However, in this case, ex-fissiparous individuals were always sterile, despite their regular gametogenesis (Dahm 1967).