Girardia pierremartini 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 : 114-120

Girardia pierremartini Souza & Leal-Zanchet sp. nov.

Etymology. The species name is dedicated to the Grupo Pierre Martin de Espeleologia as acknowledgement of numerous discoveries concerning caves in Brazil and especially in the Presidente Olegário region.

Material examined. Holotype: MZUSP PL.1645: “Lapa Fazenda São Bernardo” cave, Presidente Olegário, state of Minas Gerais (MG), Brazil, 30. September 2013, coll. M.E. Bichuette, sagittal sections on 9 slides.

Other specimens: Paratype MZU PL. 00210: “Lapa Fazenda São Bernardo” cave, Presidente Olegário, MG, Brazil, 30. September 2013, coll. M.E. Bichuette, sagittal sections on 11 slides, MZU PL. 00211: “Toca do Charco” cave, Presidente Olegário, MG, Brazil, 29. September 2013, coll. M.E. Bichuette, sagittal sections on 22 slides; MZU PL. 00212: “Toca do Charco” cave, Presidente Olegário, MG, Brazil, 29. September 2013, coll. M.E. Bichuette, sagittal sections on 13 slides.

Type-locality. “Lapa Fazenda São Bernardo” cave, Presidente Olegário, MG, Brazil.

Distribution. Known only from the Presidente Olegário karst area, MG, Brazil.

Diagnosis. This troglophilic Girardia species is characterized by ventral testes, a branched bulbar cavity with lateral funnel-shaped portions, obliquely oriented penis showing two distinct portions and a bursal canal smoothly inclining ventrally to communicate with the female canal.

Description. External morphology. Live specimens are brown and provided with two eyes. After fixation, the light-brown ground colour is covered by numerous dark-brown flecks that are homogeneously distributed over the dorsal surface and more sparsely distributed on the ventral surface. Head broadly triangular with moderately sized auricles after fixation ( Figs. 15–17 ); posterior tip pointed. Preserved specimens up to 8.5 mm long and 3 mm wide ( Table 2 ). Mouth located in the median third of the body and gonopore located in the posterior half of the body ( Table 2 , Figs. 15, 17 ).

Epidermis, cutaneous musculature and sensory organs. Epidermis constituted of a columnar epithelium ( Figs. 18–19 ), ciliated on the ventral body surface. The whole epidermis receives secretions of four types of glands: (1) xanthophil, rhabditogen secretion (rhammites), more densely distributed at the dorsal surface; (2) erythrophil, fine granular secretion; (3) xanthophil, fine granular secretion; and (4) cyanophil amorphous secretion. The erythrophil glands and a xanthophil, coarse granular secretion concentrate their openings medially at the anterior and posterior tips of the body. Numerous pigment cells are located under the dorsal and ventral epidermis.

Cutaneous musculature consisting of three layers, viz. a thin subepithelial circular layer, followed by an oblique layer with decussate fibers and a thicker layer of longitudinal muscle. Ventral musculature thicker than the dorsal musculature in the pre-pharyngeal region (20–25 µm thick and 6–15 µm thick, respectively).

The auricular sensory organs are lined with a densely ciliated, low cuboidal epithelium without rhammites, with scarce glands and with some insunk nuclei. The cutaneous musculature is very thin at the level of the sensory organs.

Digestive system. Pharynx cylindrical, pigmented; between about 1/5 and 1/4 of the body length. It is located approximately in the median third of the body. Mouth at the posterior end of the pharyngeal pouch. An esophagus, about 1/10 of the pharyngeal length, connects the pharynx with the intestine. Intestine with the usual tricladid form, with the anterior intestinal trunk extending onto the posterior half of the brain.

Male reproductive system ( Figs. 19–24 ). Abundant testicular follicles, 50–90 µm in diameter, arranged in various irregular rows on each side of the body. They are situated mainly ventrally ( Figs. 19–20 ), but may occupy the entire dorso-ventral space. Testes extend from about 0.8 mm to 1 mm from the anterior tip (equal to 13% or 14% of body length in sagittal sections), just behind the brain, to the posterior end of the body. Sperm ducts run posteriorly dorsally to the nerve cords and form spermiducal vesicles laterally to the pharynx. Laterally to the copulatory apparatus, the spermiducal vesicles ascend, in an almost straight vertical course, and penetrate the penis bulb. They diminish in diameter and bend to open dorsally into the large, branched and ample bulbar cavity ( Figs. 20–24 ). Each lateral branch of the bulbar cavity has the form of a funnel, narrowing to receive the opening of the sperm ducts. The long ejaculatory duct widens towards its opening at the tip of the blunt penis papilla. At the basis of the bulbar cavity, proximally to the transition to the ejaculatory duct, there is a diverticulum. The penis papilla (about 250–310 µm long and 310–400 µm wide at its basis), obliquely oriented in the male atrium, shows two parts, a basal portion constituted of a circular fold and a distal truncated portion ( Figs. 20, 23–24 ). The basal portion of the papilla contains the most distal part of the bulbar cavity and the distal portion is traversed by the ejaculatory duct.

Sperm ducts lined with a ciliated, cuboidal epithelium, becoming flattened in the spermiducal vesicles; they are coated with a circular muscle layer (1–2 µm thick). The penis bulb, occupied by the large and branched bulbar cavity ( Figs. 20–24 ), consists of a loose connective tissue containing abundant gland necks of penial glands and interwoven muscle fibers. Bulbar cavity lined with a non-ciliated, columnar epithelium. The muscle fibres of the penis bulb may surround this cavity. Numerous penial glands with coarse granules containing a mixed secretion open into the bulbar cavity ( Figs. 20–23 ). This secretion has a cyanophil external part and an erythrophil internal core and it is usually seen in the bulbar cavity. In addition, erythrophil penial glands with coarse granules, densely disposed in the cytoplasm, open into the bulbar cavity. Both types of penial glands have extrabulbar cell bodies. Ejaculatory duct lined with cuboidal to columnar epithelium ( Fig. 20 ) which may contain sparse cilia. The muscularis of the ejaculatory duct is thin (about 2 µm thick), sometimes inconspicuous. Numerous erythrophil glands with fine granules and scarce glands with amorphous, slightly cyanophil secretion have their openings into the ejaculatory duct. Penis papilla covered with a non-ciliated, cuboidal to columnar epithelium with some insunk nuclei. Muscularis of penis papilla composed of a thick subepithelial layer of circular fibres (5–10 µm thick), followed by a layer of longitudinal fibres. The longitudinal layer is thicker in the distal portion of the penis papilla (10–12 µm thick) than in its basal portion (2–5 µm thick). Two types of penial glands, one with fine granular, erythrophil secretion and the other with amorphous, cyanophil secretion, open through the epithelium of the penis papilla. Both glands have intrapenial cell bodies. Male atrium lined with a non-ciliated, cuboidal to columnar epithelium with some insunk nuclei. The male atrial muscularis is constituted of a thin subepithelial layer of circular fibres (1–3 µm thick), followed by a thicker layer of longitudinal fibres (2–6 µm thick). Glands with slightly cyanophil amorphous secretion and erythrophil glands with fine granular secretion open sparsely into the male atrium. Cell bodies of these glands are located subepithelially or around fibers of the atrial musculature.

Female reproductive system ( Figs. 19–24 ). Vitellaria inconspicuous in the holotype and most other examined specimens. Ovaries ovoid ( Fig. 19 ), 120–170 µm in diameter. They are situated medially to the ventral nerve cords, with a small part dorsally to them, at about the same transversal level as the anteriormost testes and in close proximity to the brain, about 0.7 mm to 1 mm behind the anterior tip (equal to 13% or 14% of body length in sagittal sections). Ovovitelline ducts arising from the dorsal surface of the ovaries, but laterally or medially displaced, and running backwards dorsally to the nerve cords, following a sinuous path. Behind the gonoduct, the ovovitelline ducts turn towards the dorsal surface of the body and separately open into the most distal part of the bursal canal, in close proximity to each other. Copulatory bursa large and ovoid in most examined specimens, occupying the space between the penis bulb and the pharyngeal pouch ( Figs. 20–24 ). Bursal canal long, with its proximal half parallel to the dorsal surface of the body and its distal half smoothly inclining ventrally to opening into the large, vertically disposed female canal. Gonoduct almost straight ( Figs. 21 , 24 ).

Ovovitelline ducts lined with ciliated, cuboidal and nucleated epithelium with some insunk nuclei; it is covered mainly by circular muscle fibres (about 2 µm thick). Copulatory bursa lined with non-ciliated, columnar epithelium composed of cells with erythrophil secretion and cells with heavily stained, cyanophil secretion, receiving xanthophil, amorphous secretion as well as erythrophil, coarse granular secretion from glands with subepithelially located bodies. The bursa is covered by a thin muscle coat constituted of interwoven longitudinal and circular muscle fibres (3–8 µm thick). Bursal canal lined with a ciliated, columnar epithelium, the cells of which show irregular height ( Fig. 21 ). There are some insunk nuclei and cell bodies of xanthophil glands with amorphous secretion, as well as erythrophil glands with coarse granular secretion around the bursal canal. The latter concentrate their openings into the most distal part of the canal, around the openings of the ovovitelline ducts, and constitute the shell glands ( Figs. 21, 23–24 ). The muscularis of the bursal canal is constituted of interwoven circular and longitudinal muscle fibres (4–8 µm thick). In the holotype, there is sperm in the bursal canal and a few in the copulatory bursa. Female canal lined with a tall columnar epithelium and some insunk nuclei, the cells of which show irregular height and apocrine secretion. The muscularis of the female canal is constituted of interwoven circular and longitudinal fibres (8–12 µm thick). Abundant glands of two types open into the female canal: cyanophil glands with amorphous secretion and erythrophil glands with coarse granular secretion. Gonoduct lined with tall columnar epithelium, with some insunk nuclei, becoming ciliated close to the ventral surface of the body. The gonoduct is coated with a subepithelial layer of circular muscle (about 4–6 µm thick), followed by a layer of longitudinal muscle (about 5–10 µm thick). Abundant cement glands of two types discharge into the gonoduct, one xanthophil and the other erythrophil ( Figs. 21, 23 ). Both glands show coarse granular secretion. In addition, cyanophil glands with amorphous secretion and erythrophil glands with fine granular secretion also open into the gonoduct. These glands have long cell necks and cell bodies scattered in the mesenchyme, some of them adjacent to the muscle layers of the gonoduct ( Fig. 23 ).

Variability. Specimen MZU PL. 0 0 211 seems to be the only fully mature specimen, showing well developed vitellaria. All examined specimens contain a large, massive penis papilla constituted of the basal and distal portions described above, but the specimen MZU PL. 0 0 211, which is very much contracted, shows an everted pharynx and an extended penis papilla. Due to this extension, the papilla is more elongated, with twice the length than when in the resting stage, and the distal part of the bulbar cavity, as well as the ejaculatory duct, are longer than in other specimens. Both portions of the penis papilla are still recognized in this specimen, with the distal portion projected through the gonopore. It also shows signs of recent copula, since the bursa contains parts of a spermatophore with sperm mixed with erythrophil and cyanophil secretions in its lumen.

Comparative discussion. The trogophilic Girardia pierremartini differs from the majority of its hypogean congeners by having a heavily pigmented body and a pair of eyes, superficially resembling G. guatemalensis , as well as the troglophilic specimens of G. p a r a m e n s i s recorded in north Brazil (Mitchell & Kawakatsu 1973b; Kawakatsu & Froehlich 1992). It also differs from the hypogean G. mckenziei which only presents slight, minute pigmentation and two minute eyes (Mitchell & Kawakatsu 1973a).

Regarding the reproductive system, comparing G. pierremartini to other troglobitic species with ventral testes and a forked bulbar cavity, it has only superficial similarities with G. desiderensis . However, they differ mainly by the form of the bulbar cavity and the form and histological constitution of the bursal canal. The bulbar cavity has a characteristic form in G. desiderensis , as already commented, showing an ovoid aspect at the sagittal plane and ovoid lateral portions that maintain their diameter to receive the openings of the spermioducts. In contrast, G. pierremartini has funnel-shaped forked portions, gradually diminishing their diameter so as to receive the openings of the spermioducts. Furthermore, G. desiderensis has an angled bursal canal, whereas in G. pierremartini it smoothly inclines ventrally to communicate with the female canal. In addition, the relative abundance of the epidermal glands, the pigmentation of the pharynx, the musculature of the bursal canal, and penial glands distinguish both species one from another.

Considering the epigean species, similarly to G. desiderensis , the reproductive system of G. p i e r rem a r t i n i has overall similarities with those of some specimens of G. f es t a e, by having ventral testes, a penis papilla containing a circular fold, and a forked and ample bulbar cavity (Kawakatsu & Mitchell, 1984, Sluys 1992, Sluys et al. 2005). However, in G. pierremartini , the spermioducts ascend in an almost straight vertical course laterally to the copulatory apparatus, whereas in G. f es t a e they have a characteristic recurving course (Kawakatsu & Mitchell 1984, Sluys 1992). In addition, in G. f e s t a e, the bulbar cavity gradually narrows to form the ejaculatory duct, but, in G. pierremartini , the transition of the bulbar cavity to the ejaculatory duct is sharp and there is a diverticulum at the basis of the bulbar cavity. The anatomy of the bursal canal can also be used to distinguish both species, since G. festae has an angled bursal canal (Kawakatsu & Mitchell, 1984, Sluys 1992, Sluys et al. 2005), whereas the bursal canal of G. pierremartini smoothly inclines ventrally towards the female canal.

In conclusion, the troglophilic G. pierremartini shows a combination of features of its copulatory apparatus, namely ventral testes, a large, branched and ample bulbar cavity with a lateral funnel-shaped portion, a large and truncate papilla showing two different portions, and a bursal canal smoothly inclining ventrally to communicate with the female canal, among others, which distinguish it from its congeners.

Notes on ecology and distribution. In the karst area of São Desidério, specimens of G. desiderensis were directly sampled from small pools inside Baixa Fria cave, ca. 200 m from the cave entrance in the aphotic zone ( Fig. 3 ). Its abundance was low in comparison to other freshwater flatworms. The water body has a maximum depth of 0.2 m and shows slow water current and a bottom formed by silt and clay.

In the karst area of Presidente Olegário, specimens of G. pierremartini were always abundant and co-occurred with amphipod crustaceans in both sampling areas. The pools showed a silt and clay bottom, with slow moving waters and depths of ca. 0.2 m, located in the twilight and photic zones, at variable distances from the caves’ entrance ( Figs. 4−5 ).

General discussion. The two new species herein described enhance the knowledge on triclad diversity in South American subterranean environments. One of them ( G. desiderensis ) represents the first Continenticola described for an area of Cerrado-Caatinga transition. Girardia desiderensis is also the first aquatic troglobitic species described for São Desidério karst area and Bahia state. This species occurs in the aphotic zone of a single cave (Baixa Fria) located at the boundary of a huge hydrogeological system (João Rodrigues), in a peripheral water body formed by phreatic water. Thus, G. desiderensis is restricted not only to a cave environment, but also to specific microhabitats. Regions with high diversity of troglobites in Brazil have been recognized, based not only on species richness, but also on a high proportion of these species in relation to the total subterranean richness, including also troglophiles and trogloxenes, as observed by Gallão & Bichuette (2015) for caves from Chapada Diamantina in the state of Bahia. Such areas should be considered to have conservation priority, since they represent unique elements of subterranean evolution.

Our knowledge of the aquatic cave fauna of the Presidente Olegário karst area is still incipient. One species of amphipod ( Hyalella veredae Cardoso & Bueno, 2014 ) was described for the Vereda da Palha cave, close to the sampling localities of G. pierremartini (Cardoso et al. 2014) . Thus, this flatworm represents the second freshwater species described for Presidente Olegário caves. Due to their heavily pigmented body, the presence of eyes, and a possible connection between the surface streams and the subterranean pools where the planarians occur, we consider the species as a troglophile.

It is noteworthy that the regions of São Desidério and Presidente Olegário are not part of any legally protected conservation area. In São Desidério, the agriculture is the most threatening pressure imposed on its cave fauna. The surroundings have very large plantations of soybean and cotton, while contamination of phreatic waters is documented for at least the urban area of the municipality. In Presidente Olegário there are at least 200 caves documented up to now (GPME, pers. comm.) and the main threats are gas extraction and hydro-electrical projects, which strongly impact the water streams and phreatic oscillation. In the latter case, the drainage can be entirely modified. According to Brazilian law, caves should be protected if they constitute habitats for rare or endemic troglobites and possible endangered species ( BRASIL, 2008). We suggest that these two regions should be protected through the creation of Conservation Units (e.g., Natural Monuments or Parks), and that these two flatworm species should be taken up in the Brazilian Red List as vulnerable organisms.

Figures

FIGURE 24. Girardia pierremartini. Sagittal composite reconstruction of the copulatory apparatus of the holotype. Arrows indicate the basal part of the penis papilla; the arrowhead indicates a diverticulum of the ejaculatory duct.

FIGURES 15 – 17. Girardia pierremartini. Photographs of preserved specimens: (15) paratype MZU PL. 0 0 210 in dorsal view; (16 – 17) specimen MZU PL. 0 0 212 in dorsal (16) and ventral (17) view. The posterior tip of the specimens is bent.

FIGURES 18 – 23. Girardia pierremartini, in sagittal sections: (18) ventral surface of the body of the holotype; (19) ovary and anterior testes of the holotype; (20 – 23) copulatory apparatus of the holotype (20 – 22) and of the specimen MZU PL. 0 0 212 (23)

FIGURES 1 – 5. Sampling areas of Girardia desiderensis and Girardia pierremartini in the karst areas of São Desidério, Bahia state, and Presidente Olegário, Minas Gerais state, Brazil: (1) location of the studied areas; (2) outcrops of Baixa Fria cave, São Desidério karst area (Photograph: J. E. Gallão); (3) sampling site of G. desiderensis in the aphotic zone of Baixa Fria cave, São Desidério karst area (Photograph: J. E. Gallão); (4) sampling location of G. pierremartini in the photic zone of Lapa da Fazenda São Bernardo cave, Presidente Olegário karst area (Photograph: Ericson C. Igual); (5) sampling site of G. pierremartini in the twilight zone of Toca do Charco cave, Presidente Olegário karst area (Photograph: Tamires Zepon).

Tables

TABLE 2. Measurements, in mm, of preserved specimens of Girardia pierremartini. 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. 1645	Paratype MZU PL.00210	Specimen MZU PL. 0 0 211	Specimen MZU PL. 0 0 212
Length	6	6	8	8.5
Width	2	2.5	3	2.5
DM	3 (50%)	3 (50%)	4 (50%)	4.5 (53%)
DG	4 (67%)	4 (67%)	5 (62%)	5 (59%)