Girardia multidiverticulata, de Souza, Stella Teles, Morais, Ana Laura Nunes, Cordeiro, Livia Medeiros & Leal-Zanchet, Ana Maria, 2015

de Souza, Stella Teles, Morais, Ana Laura Nunes, Cordeiro, Livia Medeiros & Leal-Zanchet, Ana Maria, 2015, The first troglobitic species of freshwater flatworm of the suborder Continenticola (Platyhelminthes) from South America, ZooKeys 470, pp. 1-16 : 3-8

Taxon classification Animalia Tricladida Dugesiidae

Girardia multidiverticulata sp. n.

Material examined.

Holotype. MZUSP PL.1573: "Buraco do Bicho" cave, Bodoquena Plateau, Mato Grosso do Sul (MS), Brazil, July 2011, coll. L. M. Cordeiro & R. Borghezan, sagittal sections on 18 slides.

Paratypes. "Buraco do Bicho" cave, Bodoquena Plateau, MS, Brazil, July 2011, coll. L. M. Cordeiro & R. Borghezan. MZU PL.00184: sagittal sections on 61 slides; MZU PL.00185: sagittal sections on 8 slides; MZU PL.00186: transverse sections on 16 slides.

Etymology.

The species name refers to the multiple diverticula of the bulbar cavity.

Diagnosis.

Blind and unpigmented Girardia species characterized by a branched bulbar cavity with multiple irregular diverticula.

Description.

Live specimens unpigmented and eyeless (Figs 4-6). Head highly triangular with long and pointed auricles, which become moderately sized and almost rounded after fixation (Fig. 6); posterior tip rounded (Figs 4-6). Preserved specimens up to 20 mm long and 3 mm wide (Table 1). Mouth and gonopore located at the posterior half of the body (Table 1, Fig. 6).

Epidermis (Figs 7-10). Columnar epithelium, ciliated on the ventral body surface (Figs 7, 10). The whole epidermis receives secretions of three types of glands: (1) xanthophil, rhabidtogen secretion (rhammites); (2) erythrophil, fine granular secretion; (3) cyanophil amorphous secretion (Figs 7-10). Rhammites are more densely distributed at the dorsal surface (Fig. 7). The erythrophil glands and a fourth type of gland, with xanthophil, granular secretion, concentrate their openings medially at the anterior and posterior tips of the body (Figs 9-10) as well as at the body margins. Cyanophil glands become numerous towards the anterior tip (Fig. 9).

Cutaneous musculature (Figs 7-8). Three layers, viz. a thin subepithelial circular layer, followed by an oblique layer with decussate fibers and a thicker layer of longitudinal muscle. Dorsal and ventral cutaneous musculatures show similar height in the pre-pharyngeal region (10-13 µm thick in the holotype).

Sensory organs. The auricular sensory organs are lined with densely ciliated, low cuboidal epithelium, with insunk nuclei. Few secretory cells open through this epithelium. The cutaneous musculature is very thin at the level of the sensory organs.

Digestive system (Figs 5-6, 11-13). Pharynx cylindrical, non-pigmented; between about 1/4th and 1/6th of the body length. It is located in the posterior half or in the median third of the body (Figs 5-6). Mouth at the posterior end of the pharyngeal pouch (Fig. 11). Pharynx lined by cuboidal ciliated epithelium with insunk nuclei; pharyngeal lumen lined by non-ciliated, columnar epithelium with some insunk nuclei. Pharyngeal glands of the usual three types (xanthophil, cyanophil and erythrophil glands). Outer musculature of the pharynx constituted of a thin subepithelial layer of longitudinal muscle, followed by a thin layer of circular muscle, each about 4 µm thick in the holotype. Inner pharyngeal musculature composed of a thick subepithelial layer of circular muscle (30-60 µm thick in the holotype), followed by a layer of longitudinal muscle (15-20 µm thick in the holotype) (Figs 11-13). An esophagus, about 1/6 of the pharyngeal length, connects the pharynx with the intestine (Fig. 13). The esophagus is lined by a flat to cuboidal epithelium with insunk nuclei; it is coated with a thin muscularis containing circular fibers near the intestine, gradually becoming thicker towards the pharynx and similar to the inner pharyngeal musculature. Intestine with the usual tricladid form (Fig. 5), with the anterior intestinal trunk extending onto the posterior part of the brain.

Male reproductive system (Figs 10, 14-19, 24-26).

Numerous testicular follicles, 100-200 µm in diameter in the holotype, arranged in one irregular row on each side of the body. They are situated mainly ventrally (Fig. 15), but may occupy the whole body height; some are situated dorsally. Testes extend from about 2 mm from the anterior tip in the holotype (equal to 16% of body length), just behind the brain, to the posterior end of the body (Fig. 10). Sperm ducts form spermiducal vesicles laterally to the pharynx, diminishing in diameter close to their opening into the bulbar cavity (Fig. 16). Laterally to the copulatory apparatus, they ascend, forming a loop, and turn anteriad. Sperm ducts separately penetrate the penis bulb, and open laterally into the large, branched bulbar cavity which contains vari ous irregular diverticula (Figs 14, 16, 18, 24-26). The short ejaculatory duct narrows towards its opening at the tip of the penis papilla. The latter is a stubby, symmetrical cone, obliquely oriented in the male atrium (Figs 14, 18-19, 25-26).

Sperm ducts lined with a ciliated, cuboidal epithelium, becoming flattened in the spermiducal vesicles; they are coated with a circular muscle layer (3 µm thick in the holotype). The large penis bulb consists of a loose connective tissue containing abundant gland necks of penis glands and interwoven muscle fibers (Figs 14, 16). Bulbar cavity lined with a non-ciliated, cuboidal to flat epithelium, underlain with a weak and inconspicuous muscle layer. Numerous penis glands with extrabulbar cell bodies and mixed secretion open into the bulbar cavity (Figs 14, 16-17). This secretion has a cyanophil external part and an erythrophil internal core. In addition, few erythrophil penis glands with extrabulbar cell bodies open into the bulbar cavity. Ejaculatory duct lined with non-ciliated, columnar epithelium, and surrounded by a thin muscularis (about 3 µm thick in the holotype) composed of a subepithelial layer of circular muscle and a layer of longitudinal muscle. Erythrophil glands have abundant openings into the distal, narrow portion of this duct (Fig. 19). Penis papilla covered with a non-ciliated, columnar epithelium that becomes flat towards the tip of the papilla. Muscularis of penis papilla (5-9 µm thick in the holotype) composed of a thick subepithelial layer of circular fibres and a thin subjacent layer of longitudinal fibres (Fig. 19). Few penis glands with amorphous, cyanophil secretion and with fine granular, erythrophil secretion open through the epithelium of the penis papilla. Cyanophil glands with extrabulbar cell bodies; erythrophil glands with intrapapillar cell bodies. Male atrium lined with a non-ciliated, cuboidal to columnar epithelium, the cells of which have an irregular height and cyanophil cytoplasm (Fig. 19). The male atrial muscularis (4-5 µm thick in the holotype) is constituted of a thick subepithelial layer of circular fibres, followed by a thin layer of longitudinal fibres. Glands with cyanophil amorphous secretion and erythrophil glands with fine granular secretion open into the male atrium. Cyanophil glands with extrabulbar cell bodies, and erythrophil glands with subepithelial cell bodies.

Female reproductive system (Figs 8, 14, 17, 20-23).

Vitellaria well developed (Fig. 8), located between intestinal branches. Ovaries ovoid (Fig. 20), 150-200 µm in diameter in the holotype. They are situated dorsally to the ventral nerve cords, at about the same transversal level as the anteriormost testes and in close proximity to the brain (about 0.9 mm behind it in the holotype). Ovovitelline ducts arising from the lateral surface of the ovaries and running backwards dorsally to the nerve cords. At about the level of the gonoduct, the ovovitelline ducts turn medially, and separately open into the most distal, postero-ventral part of the bursal canal, in close proximity to each other. Copulatory bursa large and ovoid (Figs 14, 17). Bursal canal long, curving towards the ventral surface of the body and opening into the short female atrium (Figs 14, 17). Gonoduct almost straight (Fig. 14, 23).

Ovovitelline ducts lined with ciliated, cuboidal epithelium with insunk nuclei and covered mainly by circular muscle fibres (2-3 µm thick in the holotype). Copulatory bursa lined with non-ciliated, columnar epithelium composed of cells with erythrophil secretion and cells with heavily stained, cyanophil secretion; it is covered by a thin muscle coat constituted by interwoven longitudinal and circular muscle fibres (5-8 µm thick in the holotype). The bursa of the holotype contains sperm and cyanophil secretion in its lumen (Figs 17, 21); some spermatozoids are absorbed by its epithelial cells. Bursal canal lined with a ciliated, cuboidal to columnar epithelium with cyanophil cytoplasm (Fig. 21). The muscularis of the bursal canal (3-4 µm thick in the holotype) is constituted of interwoven circular and longitudinal muscle fibres (Fig. 21). There are some insunk nuclei and cell bodies of xanthophil glands around the copulatory bursa and bursal canal. Female atrium lined with a ciliated, tall columnar epithelium, the cells of which show irregular height. The muscularis of the female atrium (6 µm thick in the holotype) is constituted of a subepithelial layer of circular fibres, followed by a layer of longitudinal fibres (Fig. 22). Numerous glands with fine granular, erythrophil secretion (shell glands) and few cyanophil glands open into the female atrium. Gonoduct lined by ciliated, tall columnar epithelium, and coated with a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle (about 9 µm thick in the holotype) (Fig. 23). Abundant cement glands with coarse granular, xanthophil secretion (Fig. 23) and numerous glands with heavily stained, cyanophil amorphous secretion discharge into the gonoduct. Both cell types have long cell necks and their cell bodies are located in the mesenchyme. Few glands with fine, erythrophil secretion and subepithelial cell bodies also open into the gonoduct (Fig. 23).

Geographical distribution.

Known only from the type-locality ("Buraco do Bicho" cave), Bodoquena Plateau, Mato Grosso do Sul, Brazil.

Variability.

In paratype MZU PL.00186 with contracted body, the penis papilla protrudes into the gonoduct and the bulbar cavity formes two main proximal chambers and one large distal one (Fig. 24). The distal portion of the bursal canal and the female atrium of this paratype were elongated and protruded towards the ventral surface of the body (Fig. 24). Paratype MZU PL.00184 has a more elongate, conical and truncated penis papilla occupying almost the whole cavity of the male atrium (Fig. 25). Despite the fact that this specimen is mature, it has a small copulatory bursa with narrow cavity, probably due to a different physiological state in relation to the other specimens. In this paratype, stained with Hematoxyline/Eosine, the penis glands showed an amorphous, chromophobous secretion, shell glands were stained deep pink (Figs 25-26) and cement glands showed chromophobous, coarse granular secretion.