Girardia desiderensis Souza & Leal-Zanchet

Souza, Stella, Morais, Ana Laura, Bichuette, Maria Elina & Leal-Zanchet, Ana, 2016, Two new species of freshwater flatworms (Platyhelminthes: Tricladida: Continenticola) from South American caves, Zootaxa 4092 (1), pp. 107-121 : 108-114

Girardia desiderensis Souza & Leal-Zanchet sp. nov.

Etymology. The species name refers to its type-locality, the region of São Desidério, with one of the largest hydrogeological systems in South America.

Material examined. Holotype: MZUSP PL.1644: “Gruta da Baixa Fria” cave, São Desidério, state of Bahia (BA), Brazil, 4. November 2008, coll. M.E. Bichuette, sagittal sections on 19 slides.

Paratypes: “Gruta da Baixa Fria” cave, São Desidério, BA, Brazil, 4. November 2008, coll. M.E. Bichuette. MZU PL. 00208: sagittal sections on 19 slides; MZU PL. 00209: sagittal sections on 9 slides.

Type-locality. “Gruta da Baixa Fria” cave, São Desidério, BA, Brazil.

Distribution. Known only from the type-locality, São Desidério, BA, Brazil.

Diagnosis. Troglobitic Girardia species characterized by ventral testes, an ovoid, branched bulbar cavity; small, blunt penis papilla; broad bursal canal of the angled type.

Description. External morphology. Live specimens are eyeless and whitish. After fixation, pale yellow dorsal surface with scattered, fine pigmentation ( Fig. 6 ). Ventral surface whitish ( Fig. 7 ). Triangular head with moderately sized auricles; posterior tip rounded ( Figs. 6–7 ). Body up to 6.5 mm long and 3 mm wide ( Table 1 ). Mouth and gonopore located in the posterior half of the body ( Table 1 ; Fig. 7 ).

Epidermis, cutaneous musculature and sensory organs. Epidermis constituted of a columnar epithelium ( Figs. 8–9 ), ciliated on the ventral body surface. The whole epidermis receives xanthophil, rhabditogen secretion (rhammites), as well as sparse secretions of three types of glands: (1) xanthophil, coarse granular secretion; (2) erythrophil, fine granular secretion; (3) cyanophil amorphous secretion. Rhammites are more densely distributed at the dorsal surface ( Fig. 8 ). The xanthophil and erythrophil glands concentrate their openings medially at the anterior and posterior tips of the body.

Cutaneous musculature of three layers, viz. a thin subepithelial circular layer, followed by an oblique layer with decussate fibers and a thicker layer of longitudinal muscle. The cutaneous musculature is weakly developed; the ventral (13–16 µm thick) is slightly higher than the dorsal musculature (5–10 µm thick) in the pre-pharyngeal region.

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

Digestive system. Pharynx cylindrical, non-pigmented; about 1/4 of the body length. It is located in the median third of the body. Mouth at the posterior end of the pharyngeal pouch ( Fig. 10 ). An esophagus, between 1/8 to 1/9 of the pharyngeal length, connects the pharynx with the intestine ( Fig. 10 ). Intestine with the usual tricladid form, with the anterior intestinal trunk extending onto the brain.

Male reproductive system ( Figs. 9–14 ). Numerous testicular follicles, about 70–100 µm in diameter, arranged in various irregular ventral rows on either side of the body ( Figs. 9, 12–13 ). Testes extend from about 1 mm from the anterior tip (equal to 20–25% of body length in sagittal sections), just behind the ovaries ( Fig. 9 ), to the posterior end of the body. Sperm ducts dorsal to the nerve cords. They ascend laterally to the copulatory apparatus, in an almost straight vertical course, and separately penetrate the penis bulb. Close to their opening, the sperm ducts diminish in diameter, form a short loop, and turn anteriad. Each duct opens into lateral expansions of the bulbar cavity. This cavity is large, with an ovoid aspect in the sagittal plane, and shows two lateral ovoid expansions ( Figs. 12–14 ). A narrow and short ejaculatory duct traverses the blunt penis papilla (about 140–190 µm long and 110–125 µm wide at its basis), which may form a circular fold at the basis of the penis papilla (holotype and paratype MZU PL. 00208). The penis papilla is obliquely oriented in the male atrium ( Figs. 11, 13–14 ).

Sperm ducts lined with a ciliated, cuboidal epithelium, becoming flattened in the spermiducal vesicles; they are coated mainly with circular muscles (about 2 µm thick). The penis bulb consists of a loose connective tissue with necks of cyanophil penial glands and scarce interwoven muscle fibers ( Figs. 11–13 ). Bulbar cavity lined with a non-ciliated, cuboidal to columnar epithelium, which is underlain by a weak layer of interwoven circular and longitudinal muscles (about 2 µm thick). Abundant cyanophil penial glands, with coarse granular secretion and extrabulbar cell bodies, open into the bulbar cavity. This secretion is usually visible in the bulbar cavity as an amorphous mass ( Figs. 11–13 ). Ejaculatory duct lined with non-ciliated, flat to cuboidal epithelium; it is surrounded by a thin muscularis composed of interwoven circular and longitudinal muscles (about 1–2 µm thick). Cyanophil glands with coarse granular secretion and extrabulbar cell bodies and scarce xanthophil/ erythrophil glands with subepithelial cell bodies open into this duct. The penis papilla is covered with a nonciliated, cuboidal to columnar epithelium containing some insunk nuclei. Muscularis of the penis papilla composed of a subepithelial layer of circular fibres, followed by a layer of longitudinal fibres (about 6–7 µm thick), becoming thinner and consisting of interwoven circular and longitudinal muscles towards the tip of the papilla. Penial glands with amorphous, slightly cyanophil secretion and xanthophil glands with granular secretion open through the epithelium of the penis papilla. Cyanophil glands with extrabulbar cell bodies; xanthophil/erythrophil glands with subepithelial cell bodies. Male atrium lined with a non-ciliated, columnar epithelium with some insunk nuclei ( Figs. 11–13 ). The male atrial muscularis (3–4 µm thick) is constituted of a subepithelial layer of circular fibres, followed by a thin layer of longitudinal fibres. Glands with cyanophil amorphous secretion and subepithelial cell bodies open into the male atrium ( Fig. 13 ).

Female reproductive system ( Figs. 9, 11–14 ).Vitellaria well developed, located dorsally and between intestinal branches. Ovaries ovoid ( Fig. 9 ), about 90 µm in diameter. They are situated dorsally to the ventral nerve cords, in close proximity to the brain (between 0.1–0.7 mm behind it) and about 0.9–1.2 mm behind the anterior tip (equal to 18% to 23% of body length in sagittal sections). Ovovitelline ducts arising from the lateral surface of the ovaries, sometimes displaced to the dorsal surface, and running backwards dorsally to the sperm ducts and nerve cords. Just behind the gonoduct, the ovovitelline ducts turn dorsally to open separately into the most distal part of the bursal canal, in close proximity to each other. Copulatory bursa ovoid, located in close proximity to the pharyngeal pocket ( Figs. 11–12 ). Bursal canal short and broad, mainly horizontal disposed, showing a sharp bend just before communicating with the female canal ( Figs. 12 , 14 ). Gonoduct almost straight ( Figs. 12–14 ), slightly inclining forward to communicate with the atria.

Ovovitelline ducts lined with ciliated, cuboidal nucleated epithelium with some insunk nuclei. They are covered mainly by circular muscle fibres (about 2 µm thick). Copulatory bursa lined with non-ciliated, columnar epithelium with the usual morphology. The bursa of the holotype contains some spermatozoids absorbed by its epithelial cells ( Figs. 11–12 ). Bursal canal lined with a ciliated, columnar nucleated epithelium with irregular height ( Figs. 11–12 ) and some insunk nuclei, receiving cyanophil amorphous secretion. Glands with fine granular, strong xanthophil secretion (shell glands) open into the bursal canal distally to the ovovitelline duct openings ( Fig. 12 ). The muscularis of the bursal canal (about 5–10 µm thick) is constituted of interwoven circular and longitudinal fibres. Female canal lined with a tall columnar epithelium ( Fig. 12 ), the cells of which show irregular height and some insunk nuclei. The muscularis of the female canal (about 10 µm thick) is constituted of interwoven circular and longitudinal fibres. Few cyanophil glands with subepithelial cell bodies open into the female canal. Gonoduct lined by tall columnar epithelium with irregular height, ciliated in its ectal portion, and coated with a subepithelial layer of circular muscle, followed by a layer of longitudinal muscle (about 8–12 µm thick). Abundant cement glands with coarse granular, xanthophil secretion of two types (one coarser than the other, the coarser one with cromophobous secretion in the holotype) and glands with heavily stained, cyanophil amorphous secretion discharge into the gonoduct. These glands have long cell necks and their cell bodies are scattered in the mesenchyme, some of them adjacent to the muscle layers of the gonoduct ( Figs. 12–13 ).

Variability. All analysed specimens show well developed vitellaria, indicating that they are mature specimens. The occurrence of a circular fold at the basis of the penis papilla of the holotype ( Figs. 11 , 14 ) and paratype MZU PL. 0 0 208 may be due to contraction of the specimens during fixation, since this characteristic was not observed in paratype MZU PL. 0 0 209 ( Fig. 13 ).

Comparative discussion. Considering the lack of eyes and whitish body, in live specimens, the troglobitic Girardia desiderensis differs from the majority of its congeners, which are heavily pigmented, epigean organisms. After fixation, it resembles the hypogean Girardia mckenziei (Mitchell & Kawakatsu, 1973) , from Mexico, regarding the slight pigmentation, but this species has two minute eyes (Mitchell & Kawakatsu 1973a). Eyes and pigmentation are also absent in other troglobitic congeners, such as G. typhlomexicana (Mitchell & Kawakatsu, 1973) and G. barbarae (Mitchell & Kawakatsu, 1973) , both from Mexico, and G. multidiverticulata Souza et al., 2015 , from the Brazilian Cerrado ecosystem (Mitchell & Kawakatsu 1973b; Souza et al. 2015). However, the preserved specimens of these three species have unpigmented body, differing from the pale-yellow body, with scattered pigmentation, of preserved specimens of G. desiderensis . Regarding body length, mature specimens of G. desiderensis , with a maximal length of 6.5 mm, are smaller than those of G. multidiverticulata , with 12 mm and 20 mm lengths after fixation (Souza et al. 2015). Girardia desiderensis also differs from the hypogean Girardia guatemalensis (Mitchell & Kawakatsu, 1973) , from Mexico, as well as from the troglophilous specimens of G. paramensis (Fuhrmann, 1914) , reported for the state of Pará, in northern Brazil, which show heavily pigmented body and a pair of eyes (Mitchell & Kawakatsu 1973b; Kawakatsu & Mitchell 1981; Kawakatsu & Froehlich 1992).

Regarding the reproductive system, G. desiderensis can be differentiated from other troglobitic species, such as G. barbarae , G. mckenziei and G. typhlomexicana , which have dorsal and/or dorsoventral testes and do not show the distinctly angled bursal canal (Mitchell & Kawakatsu 1973a, b) as in G. desiderensis . Similar to Girardia desiderensis , the troglobitic G. guatemalensis and G. multidiverticula show ventral testes. However, both species differ from G. desiderensis by the form of the bulbar cavity and the position of the opening of the spermioducts. In G. guatemalensis , the single bulbar cavity receives the opening of the spermioducts in its very distal part, whereas in G. multidiverticulata the large bulbar cavity, with multiple diverticula, receives the opening of each sperm duct in one of its lateral diverticula.

In relation to the epigean species, the reproductive system of G. desiderensis has a similar gross morphology with that of some specimens of G. f e s t a e (Borelli, 1898), by having ventral testes, a forked bulbar cavity and an angled bursal canal (Kawakatsu & Mitchell 1984, Sluys 1992, Sluys et al. 2005). Girardia festae is a polymorphic species, originally described from Bahía Blanca, Argentina, which was later reported for other localities in South America (Kawakatsu & Mitchell 1984, Sluys 1992, Sluys et al. 2005), showing great variation in the anatomy of the bulbar cavity, penis papilla, ejaculatory duct and bursal canal. According to Sluys et al. (2005), the shape of the penis papilla is a well distinguishable feature of G. f e s t a e, whereas Kawakatsu & Mitchell (1984) considered that its papilla is highly variable. Girardia desiderensis shows a small and blunt penis papilla, which only sometimes contracts and forms a circular fold or annular zone, superficially resembling or not resembling the penis papilla of G. f es t a e, depending on the interpretation. However, the bulbar cavity of G. desiderensis shows a characteristic feature, which is its ovoid aspect at the sagittal plane and also ovoid lateral portions maintaining diameter to receive the openings of the spermioducts. In contrast, the single or forked, elongated or ovoid bulbar cavity of G. f es t a e gradually diminishes in diameter in order to receive the openings of the spermioducts (Kawakatsu & Mitchell 1984, Sluys 1992, Sluys et al. 2005).

Other species with large and forked bulbar cavity, such as G. anderlani (Kawakatsu & Hauser, 1983) and G. capacivasa Sluys, 2005 , have dorsal or dorsoventral testes, besides other differential characters (Kawakatsu et al. 1983, Sluys et al. 2005).

In conclusion, the troglobitic G. desiderensis shows a unique combination of characters of its external morphology, such as absence of eyes and body pigmentation, and of its copulatory apparatus, such as ventral testes, a small and blunt penis papilla, a broad bursal canal of the angled type, and, especially, an ovoid, branched bulbar cavity, which distinguish the new species from its congeners.

Figures

FIGURE 14. Girardia desiderensis. Sagittal composite reconstruction of the copulatory apparatus of the holotype. The arrow indicates the contracted penis papilla.

FIGURES 6 – 7. Girardia desiderensis. Photograph of a preserved specimen (paratype MZU PL. 00209) in dorsal (6) and ventral (7) view.

FIGURES 8 – 13. Girardia desiderensis, in sagittal sections: (8) dorsal surface of the body of paratype MZU PL. 00209; (9) ovary and anterior testes of paratype MZU PL. 00209; (10) pharynx of the holotype; (11 – 13) copulatory apparatus of the holotype (11 – 12) and paratype MZU PL. 0 0 209 (13).

Tables

TABLE 1. Measurements, in mm, of preserved specimens of Girardia desiderensis. DG: distance of gonopore from anterior end; DM: distance of mouth from anterior end. The numbers given in parentheses represent the position relative to body length.

	Holotype MZUSP PL. 1644	Paratype MZU PL.00208	Paratype MZU PL.00209
Length	6.5	6	5.5
Width	3	3	2
DM	5 (77%)	4 (67%)	4 (73%)
DG	5.5 (85%)	5 (83%)	4.5 (82%)