Dugesia insolita Stocchino & Sluys, 2024

Dugesia insolita Stocchino & Sluys , sp. nov.

urn:lsid:zoobank.org:act:9199C00D-BA4A-4DD4-BED2-16A3A1884BBE

Material examined

Holotype: RMNH.VER. 21536.1 (field code: GS32-Dmad5-6) one set of sagittal sections on 18 slides, Central High Plateau , Western Slope , small unnamed water course, tributary of Matsiatra River , Upper Mangoky basin, 4 km upstream of Ambohimahasoa (21°08 ʹ S 47°13 ʹ E), between Route National 7 to Fianarantsoa and Route national 25 to Ranomafana National Park , Madagascar, September 2011, coll. R. Manconi. GoogleMaps

Other specimens: Nine individuals were preserved in absolute ethanol, two of which (Dins1 and Dins2) were used for DNA extraction and sequencing in the study of Solà et al. (2022). GenBank accession numbers of the obtained sequences are given in Table 2 View Table 2 .

Etymology

The specific epithet, derived from the Latin adjective insolitus, unusual, uncommon, alludes to the remarkable situation that in this species the diaphragm has taken over the function of the penis papilla.

Diagnosis

A Dugesi a species characterized by the following features: high triangular head with a distinctly pointed auricle on either side; mouth opening at the posterior end of the pharyngeal pocket; vasa deferentia separately opening into anterior portion of the bulbar lumen; functional penis papilla a stubby, cone-shaped structure exhibiting an outbulging that provides it a shape in the form of a head of a spear; erythrophil penis glands discharging through the lining epithelium of the functional penis papilla; male atrium with three annular folds, the posterior-most one receiving longitudinal muscles that extend from the penis bulb; hyperplasic ovaries extending posterior to the level of the root of the pharynx; oviducts separately opening into the most proximal section of the bursal canal; poorly developed ectal reinforcement layer extending from about the knee-shaped bend in the bursal canal to about halfway between the bend and the copulatory bursa.

Geographical distribution

Known only from the type locality.

Habitat

Planarians were found under pebbles and boulders in shallow water of a rivulet in the relict Rainforests of the Atsinanana.

Reproduction and behaviour

At collection, all 20 specimens of D. insolita were asexual. After four months of rearing under standard laboratory conditions, a single asexual specimen underwent a sexualization process, thus producing an ex-fissiparous individual characterized by hyperplasic ovaries and presence of a copulatory apparatus. Asexual individuals exhibited post-pharyngeal fissioning.

Just after collection and during rearing, both in the field and in the laboratory, a particular kind of movement was observed. Besides the usual gliding type of locomotion, these planarians showed a swimming-like movement with up-and-down undulations of the body when they were not adhering to the substrate and moved, for example, from the water surface to the bottom of the container. Moreover, during their usual gliding the body margins showed an undulating movement.

Furthermore, on some occasions we observed a peculiar autotomy-like process when the worms were picked up softly with a paintbrush. Immediately after the planarians were touched, they quickly detached their tail, thus dividing at a post-pharyngeal level. The wound margins of the two resulting fragments were characterized by very irregular outlines, resembling those resulting from a forceful laceration. On one occasion, it happened that after this first division, touching of the anterior portion triggered another quick autotomy-like process, thus resulting in the detachment of another post-pharyngeal fragment. After these autotomy-like processes, each of the resulting fragments regenerated the missing body parts.

Description

Live animals with a high triangular head, provided with distinctly pointed auricles and two prominent eyes set in pigment-free patches ( Fig. 9 View Figure 9 ). Body size of live, fissiparous specimens ranged from 8 to 11 mm in length and 1.5–2 mm in width, while the single sexualized specimen measured 17 × 5 mm. Dorsal surface with numerous dark-brown specks and a dark mid-dorsal stripe that runs from behind the pharynx to the tip of the tail ( Fig. 9 View Figure 9 ). In some specimens this dorsal stripe runs from the anterior tip of the head to the very tip of the tail. Ventral surface pale and provided with dark brown specks, less numerous than on the dorsal surface.

Pharynx located at about halfway the length of the body, measuring about one-sixth of the body length. No extra layer of longitudinal muscles in the outer pharynx musculature. The mouth opening is located at the posterior end of the pharyngeal pocket.

The outer pharynx epithelium is underlain by a thin, subepidermal layer of circular muscle, followed by an equally thin layer of longitudinal muscle; this outer pharyngeal musculature is devoid of an extra, inner layer of longitudinal muscles. The lining epithelium of the pharyngeal lumen is underlain by a thick, subepithelial layer of circular muscles (~50 μm thick), followed by a much thinner layer of longitudinal muscles ( Fig. 10A View Figure 10 ).

The well-developed testes are located dorsally and extend from about the anterior level of the hyperplasic ovaries far into the tail region of the body.

The vasa deferentia are expanded to form spermiducal vesicles—filled with sperm—which, in addition, may show considerable outbulgings. Upon penetrating the muscular penis bulb, the vasa deferentia gradually decrease in diameter and after having become very narrow, the ducts separately open into the anterior portion of the bulbar lumen or seminal vesicle. The wall of the latter is highly pleated, with the folds projecting into its lumen. One of the larger folds forms an elongated papilla that projects posteriad into the lumen of a structure that functions as penis papilla (see below); through this fold runs a minute duct by means of which the seminal vesicle communicates with the lumen of the functional penis papilla. The latter is a stubby, cone-shaped structure that has a more or less horizontal orientation and projects into the atrial lumen ( Fig. 11 View Figure 11 ). At about one-third of the distance between its root and the tip, the papilla exhibits a fold or outbulging, thus providing the papilla—in sagittal view—with a hastate shape or the form of a head of a spear.

Erythrophil penis glands discharge through the lining epithelium of the functional penis papilla, which is surrounded by the funnel-shaped portion of a sclerotic spermatophore ( Figs 11 View Figure 11 , 12 View Figure 12 ). It is here surmised that the secretion of these penial glands contributed to the formation of the spermatophore, in the same way that normally in Dugesia species the spermatophore emanates from the penial diaphragm (see below). Therefore, it is here also surmised that the presumed penis papilla of D. insolita is actually homologous to the diaphragm (see below). Therefore, in the above and below we refer to the papilla as the functional penis papilla instead of the true penis papilla.

The large penis bulb is strongly muscular and consists of intermingled circular and longitudinal muscle fibres. From the outer surface of the penis bulb longitudinal fibres run posteriad and attach to the posterior atrial fold ( Fig. 11 View Figure 11 ).

The major part of the male atrium is lined with a tall epithelium, which, however, becomes very thin on its most anterior section near the base of the functional penis papilla. The atrium is surrounded by a subepithelial layer of circular muscles that is well-developed on its anterior section but rather thin on more posterior portions. The male atrium is provided with three annular folds or constrictions. One of the folds envelopes the root of the functional penis papilla, a second fold is located just anterior to the tip of the papilla, while the third, posterior fold is positioned at the posterior end of the male atrium. This third fold may indeed be considered as the separation between the male and the female atrium, since the openings of shell glands penetrate the dorsal epithelium of that part of the atrium that lies immediately posterior to this third fold. Basically, the openings of erythrophilic shell glands surround the opening of the bursal canal through the roof of the female atrium ( Fig. 11 View Figure 11 ).

The highly hyperplasic ovaries extend from ~800 μm behind the eyes and the brain to a little posterior (~700 μm) to the level of the root of the pharynx, occupying almost the entire dorsoventral space of the body ( Fig. 10B View Figure 10 ). The oocytes clearly exhibit a degenerative condition, as evidenced by vacuolization of their nuclei and cytoplasm.

The infranucleated oviducts run immediately dorsal to the ventral nerve cords; at the level of the gonopore the ducts turn medially to open—separately—into the most proximal section of the bursal canal, close to the opening of the latter through the roof of the female atrium. From the point of this opening, the bursal canal at first runs vertically towards the dorsal body surface and then exhibits a knee-shaped, anteriorly-directed bend, after which it follows its course more or less parallel to the dorsal and ventral body surfaces. The bursal canal communicates with a more or less ellipsoidal copulatory bursa that is located at a short distance behind the pharyngeal pocket and the mouth opening.

The bursal canal is lined with a tall, infranucleated epithelium. The long horizontal portion of the bursal canal is surrounded by a single, subepithelial layer of longitudinal muscles, followed by a slightly thicker layer of circular muscles. The short, vertical portion of the bursal canal is surrounded by a thin, subepithelial layer of circular muscles, followed by a layer of obliquely oriented fibres. An extra outer layer of longitudinal muscles forms a poorly developed ectal reinforcement layer that runs from about the knee-shaped bend in the bursal canal to about halfway between the bend and the copulatory bursa.

Very abundant and conspicuous cement glands discharge their orange-staining secretion into the short gonoduct.

Karyology

Metaphasic plates revealed that specimens constantly showed a set of 18 chromosomes. Chromosomes from six metaphasic plates could be arranged, according to their length, into nine groups of two chromosomes with a diploid chromosome set of 2n = 18; n = 9. The analysis within each pair of chromosomes revealed uniformity in both length and centromeric position, except for chromosome 1, in which the first element is always longer than its homologue. The first two chromosomes were clearly identifiable. Instead, from the third to the ninth pair, the differences in length were so small that contiguous chromosomes could be overlapping (such as chromosomes 3 and 5, 6 and 7, 8 and 9). Chromosomal length decreased gradually, with low standard deviation values. In contrast, the centromeric indices showed great variation, resulting in high standard deviations, as, for example, for chromosome numbers 4 and 6 ( Fig. 13 View Figure 13 ; Table 3 View Table 3 ).

The karyometric data indicate that the diploid chromosome complement is characterized by metacentric heterobrachial chromosomes, with the exceptions of chromosome 3, which is submetacentric, and chromosomes 1 and 9 that are metacentric, bordering on metacentric isobrachial ( Table 3 View Table 3 ).

Comparative discussion

Dugesia insolita is included in a phylogenetic clade encompassing all long-eared Dugesia from Madagascar with full support ( Fig. 2 View Figure 2 ). However, none of the phylogenetic approaches lent good support for the relationship of D. insolita to the rest of the long-eared populations in this lineage. Thus, its precise position within this group remains unresolved.

The two specimens from the same population of D. insolita are clustered together with full support in both ML and BI phylogenetic trees, indicating that they most probably also belong to this species.

Sperm transfer by means of a sclerotic spermatophore is a common phenomenon among freshwater planarians ( Sluys 1989, Sluys and Riutort 2018). Such spermatophores may be simply sac-shaped or may possess a long, narrow, and hollow stalk, the latter situation being the case in species of Dugesia , including D. insolita .

Spermatophores are formed in the ejaculatory duct running through the penis papilla, the secretions of various penial glands contributing to the formation of the spermatophore. In species of Dugesia , the diaphragm in the proximal section of the ejaculatory duct makes a different contribution to the formation of the spermatophore as compared with the middle and distal parts of the duct. The secretions discharged through the tip of the lining epithelium of the conus of the diaphragm form an antiagglutination layer at the inner surface of the stalk of the spermatophore, while the actual wall of the spermatophore is formed by secretions discharged through a more basal, anterior portion of the diaphragm, as well as the most proximal part of the ejaculatory duct ( Vreys et al. 1999).

This construction method results in the situation that during the formation process the funnel-shaped basal portion of the stalk of the spermatophore envelopes the diaphragm (cf. Vreys et al. 1997: fig. 2; 1999: fig. 4), a situation that, over the years, we have regularly observed in histological preparations of species of the genus Dugesia . A similar situation is present in D. insolita ( Figs 11 View Figure 11 , 12 View Figure 12 ) in which the funnel-shaped base of the spermatophore covers a cone-shaped structure that resembles a short penis papilla, the covering epithelium of which is penetrated by the openings of erythrophil penial glands that contribute to the formation of the stalk of the spermatophore. We have never encountered the situation that in species of Dugesia the penis papilla contributed in this way to the formation of the spermatophore, as the latter is always formed within the ejaculatory duct. Therefore, it is here surmised (see above) that this cone-shaped, penis-like structure is actually homologous to the diaphragm in other species of Dugesia —as it is without doubt that D. insolita belongs to this genus (see molecular results)—albeit it may function as a penis papilla. It should be noted that the conspicuous fold projecting into the lumen of the functional penis papilla and through which runs a minute duct, cannot be seen as phylogenetically homologous to the diaphragm, nor as being functionally equivalent to it, since it does not receive the secretion of penial glands and does not have any connection with the spermatophore.

In this context it is noteworthy that the epithelium of the first annular fold in the atrium, located at the base of the functional penis papilla, is much lower than that of the rest of the epithelium. Perhaps this lower epithelium signals that this proximal, basal fold actually is the remnant of what once was a normal penis papilla?

Other notable features of D. insolita are (i) shell glands opening through the roof of the female atrium, (ii) the knee-shaped bend in the bursal canal, (iii) muscle fibres extending from the penis bulb to the posterior wall of the male atrium, (iv) the presence of hyperplasic ovaries, and (v) a highly triangular head with long and pointed auricles.

In species of Dugesia , shell glands usually open into the bursal canal, just ventral to the oviducal openings. However, frequently the openings of the shell glands are located very close to the point where the bursal canal communicates with the atrium, while occasionally part of the shell glands actually discharges into the female atrium, e.g. in D. hepta Pala et al., 1981 , D. superioris Stocchino & Sluys, 2013 , D. effusa Sluys, 2013 , and D. naiadis (see: Stocchino et al. 2005, 2013, Sluys et al. 2013). Dugesia insolita represents the most extreme condition, in which almost none of the shell glands open into the bursal canal and the majority of the glands open through the roof of the female atrium.

From its opening into the atrium, the bursal canal in species of Dugesia generally curves smoothly towards the anterior end of the body to open into the copulatory bursa. However, in some species the bursal canal bends much more abruptly in the anterior direction, as is the case in D. capensis Sluys, 2007 , D. bifida Stocchino & Sluys, 2014 (see: Sluys 2007, Stocchino et al. 2014), and also in D. insolita .

The situation in which longitudinal muscle fibres run posteriorly from the penis bulb and eventually attach to the wall of the atrium is an uncommon condition among species of Dugesia . This feature is more or less present in D. debeauchampi de Vries, 1988 and D. liguriensis de Vries, 1988 but a situation very similar to that in D. insolita is expressed in D. milloti de Beauchamp, 1952 (see: de Vries 1988).

Presence of hyperplasic ovaries is characteristic of normally asexually reproducing specimens that have sexualized, i.e. have developed reproductive organs, either in the field or under laboratory conditions. In a similar way, after 4 months of rearing, a single specimen in our laboratory culture of animals of the population from the small tributary of the Matsiatra River developed reproductive organs (specimen RMNH.VER.21536.1, described above), including aberrant hyperplasic ovaries. In our experience, hyperplasic ovaries and, sometimes, poorly developed testes are the only abnormalities in such sexualized individuals, so that their copulatory apparatus can be interpreted as representing the normal condition of the species.

A high triangular head with long, pointed auricles has been described as a uniquely characteristic feature of D. milloti ( de Vries 1988) , but now it is known also from D. insolita .

Despite the fact that D. insolita shares with D. milloti its characteristic head shape, the atrial folds, and the layer of longitudinal muscles that runs between the wall of the atrium and the penis bulb, the two species are distinctly different. Dugesia insolita is the only species of Dugesia in which the diaphragm has evolved into a functional penis papilla. Nevertheless, this diaphragm has retained its primary function, formation of the spermatophore (see above). In D. milloti , the ancestral condition is still present in which the spermatophore is formed within the ejaculatory duct, with the funnel-shaped base of the spermatophore enveloping the diaphragm (see: de Vries 1988: fig. 14A).

Karyology

As for karyology, D. insolita shows an eudiploid chromosome complement of 18 chromosomes with basic number n = 9. Among Dugesia species, this haploid number is shared by only six other species: D. sicula and D. maghrebiana Stocchino et al., 2009 , from the Mediterranean Region; D. arabica from Yemen; D. aethiopica , D. afromontana Stocchino & Sluys, 2012 , and D. bifida Stocchino & Sluys, 2014 from the Afrotropical Region (cf. Stocchino et al. 2002, 2004, 2009, 2012, 2014, Harrath et al. 2013) ( Table 4 View Table 4 ).

Dugesia insolita shares with D. bifida the condition in which fissiparous individuals exhibit a diploid chromosome complement, while in all of the other above-mentioned five species the fissiparous populations are always triploid and/or mixoploid, while species with sexual populations are diploid (viz., D. sicula and D. arabica ). However, D. insolita differs from D. bifida in that its diploid karyotype is characterized by metacentric chromosomes, excepting chromosome 3, which is submetacentric, while all chromosomes of D. bifida are metacentric. Dugesia insolita and D. bifida represent the easternmost records of species with a basic chromosomal number of n = 9.