Searching for shelter in a ferruginous cave? A new species of Pasipha from a plateau in the Brazilian savanna (Platyhelminthes, Tricladida)

Abstract In a fauna survey in the eastern margin of Serra do Espinhaço Plateau, in an area belonging to the Brazilian savanna (Cerrado phytophysiognomy), a land flatworm was sampled in a ferruginous cave. Anatomical and histological analyses indicated that it belongs to a new species of the genus Pasipha, which is herein described. The new species shows an almost homogenous dark brown dorsal pigmentation, eyes spreading over the dorsal surface, a collar-shaped pharynx, and a prostatic vesicle with two portions separated by a canal. It differs from similar species mainly by anatomical and histological details of the ejaculatory duct, as well as male and female atria. The flatworm shows no troglomorphic traits and was collected once in the entrance zone of the cave. Hence, despite representing the first land flatworm species described from a Neotropical cave, we consider that its occurrence in the cave is probably occasional, using it as a shelter.

In a recent fauna survey in the eastern margin of Serra do Espinhaço Plateau, belonging to the Brazilian savanna (Cerrado phytophysiognomy), southeastern Brazil, a flatworm with elongate body and parallel margins was collected in a ferruginous cave. This specimen was assigned to the genus Pasipha and is herein described as a new species.

Materials and methods
A single specimen was collected during the day by direct sampling in the entrance zone of a ferruginous cave (CSS-0004) in Conceição do Mato Dentro (18°55'02.2"S, 43°25'42.4"W), at an altitude of 931 m a.s.l., in the state of Minas Gerais, southeastern Brazil (Fig. 1). The land flatworm was fixed in 70% ethyl alcohol during field work. The preserved specimen was analysed regarding colour pattern, body shape, and dimensions and then photographed under a stereomicroscope. Methods described by Rossi et al. (2015) were used for histological processing of the material and analysis of external and internal characters. The material was sectioned at intervals of 6 µm and stained with Goldner's Masson or Haematoxylin and Eosin. anterior region at the level of the ovaries: sagittal sections on 7 slides; pre-pharyngeal region in two fragments: transverse sections on 4 slides and sagittal sections on 6 slides; pharynx: sagittal sections on 5 slides; copulatory apparatus: sagittal sections on 8 slides.
Type-locality. Conceição do Mato Dentro, state of Minas Gerais (MG), Brasil. Diagnosis. Pasipha ferrariaphila is characterised by almost homogeneous dorsal pigmentation pattern, eyes spreading over the dorsal surface, collar-shaped pharynx, prostatic vesicle with two portions separated by a canal, ejaculatory duct long and spacious, male and female atria separated by a constriction and female atrium spacious, long and with a strongly developed circular musculature in its proximal part, resembling a sphincter.
Description. Body elongate with parallel margins; anterior tip rounded and posterior tip pointed (Figs 2-3). After fixation, length of 22 mm, maximal width of 2 mm, and maximal height 0.7 mm. Mouth at 77% of body length and gonopore at 82% of body length.
Fixed specimen with dorsal surface covered by fine, almost homogeneous dark brown pigmentation over light brown ground colour, which is discernible under stereomicroscope on a thin, almost imperceptible median stripe (Fig. 3). Ventral surface pale yellow.
Four gland types discharge through dorsal epidermis and body margins of prepharyngeal region: abundant rhabditogen cells with xanthophil secretion (Figs 5-7), numerous erythrophil glands with coarse granular secretion of two types (with ovoid or rounded granules), and sparse cyanophil glands with amorphous secretion. Creeping sole receives three types of glands: cyanophil glands with amorphous secretion, rhabditogen cells with small rhabdites, as well as scarcer glands with rounded erythrophil granules. Glandular margin absent (Fig. 5). Glands discharging through anterior tip of body similar to those of pre-pharyngeal region.
Cutaneous musculature with usual three layers (circular, oblique, and longitudinal layers), longitudinal layer with small bundles (Figs 6-7). Mc:h 10%. Thickness of cutaneous musculature similar to that of epidermis. Ventral musculature (about 30 µm) two times thicker than dorsal musculature (about 15 µm) at sagittal plane in pre-pharyngeal region. Thickness of cutaneous musculature gradually diminishes towards anterior tip.
Pharynx collar-shaped, nearly 8% of body length, occupies almost all length of pharyngeal pouch. Pharyngeal dorsal insertion posteriorly shifted next to end of pharyngeal pouch. Mouth in median third of pharyngeal pouch (Fig. 8). Oesophagus absent.
Testes in two irregular rows on either side of body, located close to dorsal cutaneous musculature (Fig. 7). Testes begin at the same transversal level as ovaries, about 3.5 mm from anterior tip (16% of body length), and extend to near root of pharynx. Sperm ducts dorsal to ovovitelline ducts, laterally displaced, forming spermiducal vesicles laterally to pharynx. Behind pharynx, spermiducal vesicles well developed and sinuous, extending laterally to penis bulb. These vesicles recurve, ascend, and, subsequently, open through lateral walls of proximal portion of prostatic vesicle (Fig. 9). Large prostatic vesicle extrabulbar and not forked, close to pharyngeal pouch. This vesicle shows two portions united by a narrow canal: proximal portion oval-elongate, with a spacious lumen; distal portion globose with a narrower lumen (Figs 9-11). Ejaculatory duct sinuous, with irregular contour and ample lumen, arising from posterior region of prostatic vesicle and thereafter ascending to open into proximal portion of male atrium. Male atrium long with folded walls (Figs 9-10). Proximal region of male atrium, about anterior 1/4 of male atrium length, with narrower lumen (Figs 9-10; 12). Distal region of male atrium communicates with female atrium through a constriction (Figs 9-10). Epithelial lining of prostatic vesicle ciliated and columnar, receiving coarse granular erythrophil or probably mixed secretion (erythrophil core and a chromophobic peripheral part), more abundant in its proximal portion. Distal portion of prostatic vesicle receives numerous amorphous, slightly cyanophil secretions. Muscularis of prostatic vesicle thick (20-35 µm thick), constituted of interwoven longitudinal, circular and some oblique fibres (Fig. 11). Canal uniting both portions of prostatic vesicle receives few coarse granular erythrophil secretions. Ejaculatory duct lined with ciliated, columnar epithelium, receiving openings from finely granular, cyanophil glands. Muscle coat of ejaculatory duct relatively thick (about 20 µm), mainly constituted of circular fibres. Male atrium lined with non-ciliated and erythrophil epithelium in distal region, ciliated and cyanophil in proximal region. Glands of distal region of two types: with coarse granular, erythrophil secretion and with amorphous, cyanophil secretion, whereas proximal region receives a third type with finely granular, erythrophil secretion. Muscularis of male atrium thick (50-60 µm), mainly comprised of circular fibres followed by some longitudinal fibres, diminishing in thickness and number of fibres in proximal region (20 µm).
Vitelline follicles, situated between intestinal branches, well-developed (Figs 5-7). Ovaries oval-elongate, measuring about 0.3 mm in length; they are situated dorsally to ventral nerve plate, about 3.5 mm from anterior tip (16% of body length). Ovovitelline ducts emerge dorsally from median third of ovaries and run posteriorly, close to median plane, immediately above ventral nerve plate. Distal sections of ovovitelline ducts run medially lateral to female atrium, with a slight asymmetry, the left ovovitel-  line duct contouring the atrial coat ventrally dislocated. They unite posteriorly to female atrium to form a C-shaped, ascending common glandular ovovitelline duct (Fig.  9). Female canal, horizontal, penetrates female muscle coat and opens into posteriormost part of female atrium. Female atrium ovoid with folded walls and narrow lumen (Figs 9-10; 13). Length of female atrium about 4/5 of male atrial length.
Female canal and atrium lined with columnar epithelium, sparsely ciliated in female canal. Glands of female atrium of two types: numerous glands with cyanophil, amorphous secretion and few glands with coarse granular erythrophil secretions. Female canal receives scant glands with coarse granular erythrophil secretion. Musculature of female atrium well developed, especially in proximal half (120 µm thick), composed mainly of circular fibres intermingled with some oblique fibres (Fig. 13).
Ecology and distribution. Pasipha ferrariaphila is known only from its type locality. It was sampled in an area situated in the eastern margin of Serra do Espinhaço Plateau, in southeastern Brazil. The area is covered by Brazilian savanna on rocky outcrops, also known as rupestrian complexes (Rapini et al. 2008, Oliveira et al. 2018, which occur associated with quartzite, sandstone, and itabirite above 900 m of altitude along the Serra do Espinhaço (Giulietti et al. 2000). The sampling site is the entrance zone of a ferruginous cave, representing 80% of the cave area. The sampling place is a low cavity (1.6 m high) with an area of 37 m 2 and sandy soil covered by crushed ferruginous rocks. It is located in an area planned for mining activities, which is constituted by itabirite profoundly affected by such activities. Caves within iron formations are small and narrow, being formed by chemical, physical, and biological processes (Auler 2015). Ten samplings were conducted in the area between December 2010 and September 2014, but a single specimen of P. ferrariaphila was collected. Since this specimen shows no troglomorphic features and was collected only once in the entrance zone of the cave, we consider that its occurrence in the cave is probably occasional, using it as a temporary shelter.
Remarks. The holotype, directly fixed in 70% ethanol during field work, showed a coiled body with some artefacts (Fig. 2), such as loss of the epidermis in some body parts, numerous lacunae in the mesenchyme and ovaries. In spite of that, the anatomy and most histological aspects were relatively well preserved. Nevertheless, the specimen may have some distortion in its anatomical features. The flatworm was parasitised by helminths (Figs 6; 8; 11).
Comparative discussion. The new species herein described shows characteristics that match the diagnosis of the genus Pasipha, such as a body shape with parallel margins and prostatic vesicle with two portions receiving different secretions (Ogren andKawakatsu 1990, Carbayo et al. 2013). Pasipha ferrariaphila also shows a folded male atrium and the female canal presenting a post-flex condition with ventral approach, i.e., the ovovitelline ducts join behind the female atrium and the female canal arises from posterior region of the female canal.
With respect to colour pattern, by having an almost homogeneous dorsal pattern with a thin, nearly imperceptible median stripe, P. ferrariaphila differs from the five species mentioned above. Pasipha penhana and P. hauseri show a quite distinct median stripe, P. plana and P. rosea a light median stripe and P. velutina a marbled appearance (Schirch 1929, Riester 1938, E.M. Froehlich 1955, Froehlich 1959. Concerning internal anatomy, four of these species, namely P. plana, P. penhana, P. velutina, and P. rosea, present a not-forked prostatic vesicle with two distinct regions separated by a constriction or canal (Riester 1938, E.M. Froehlich 1955) similar to P. ferrariaphila. In contrast, P. hauseri stands apart in this group by having a forked proximal portion in its prostatic vesicle (Froehlich 1959). The new species shows a large and richly folded female atrium and, as usual in Geoplaninae, testes distributed pre-pharyngeally, differing from P. plana, which has a small, almost unfolded female atrium and testes almost reaching the level of the copulatory organs (E.M. Froehlich 1955). By having a horizontal female canal and an almost C-shaped common glandular ovovitelline duct, P. ferrariaphila differs from P. rosea, which shows a female canal with a C-shaped appearance and an almost horizontal common glandular ovovitelline duct (E.M. Froehlich 1955). In addition, the sperm ducts open anteriorly displaced into the proximal region of the prostatic vesicle of P. rosea, whereas in P. ferrariaphila, the openings of the sperm ducts occur into the posterior part of the proximal region of the prostatic vesicle. Froehlich (1955) describes the occurrence of a distinct circular musculature in the distal portion of the male atrium, similar to a sphincter, in both P. plana and P. rosea, which does not occur in P. ferrariaphila. In contrast, a strong circular musculature, resembling a sphincter, occurs in the proximal part of the female atrium of P. ferrariaphila.
Pasipha ferrariaphila shows a prostatic vesicle presenting pear-shaped proximal and distal regions of similar lengths with the distal part located above the proximal, differing from P. penhana and P. velutina (Riester 1938). In P. penhana, the prostatic vesicle is tubular-shaped with an elongate distal region (= drüsiger Teil des Ductus ejaculatorius according to Riester 1938) and a shorter proximal region (= Ductus seminalis according to Riester 1938). In P. velutina, both distal and proximal regions are tubular, giving an inverted U-shape to the prostatic vesicle (Riester 1938). In addition, besides the occurrence of a highly developed circular musculature in the proximal part of the female atrium, P. ferrariaphila differs from all species in this group by showing a longer and more spacious ejaculatory duct, as well as male and female atria separated by a constriction.
Etymology. The specific name is a composite of the Latin noun ferraria (iron mine) and a suffix from the Greek adjective phílos (affinity), referring to its sampling site.