Pasipha quirogai, Negrete & Brusa, 2017

Negrete, Lisandro & Brusa, Francisco, 2017, Increasing diversity of land planarians (Platyhelminthes: Geoplanidae) in the Interior Atlantic Forest with the description of two new species and new records from Argentina, Zootaxa 4362 (1), pp. 99-127 : 101-105

Pasipha quirogai sp. nov.

( Figs. 2–3 )

Type material. Holotype. MLP–He 7018, Osununú Private Reserve, 28 February 2015; cephalic region: transversal sections on 17 slides; anterior region at the level of the ovaries: sagittal sections on 8 slides; prepharyngeal region: transverse sections on 7 slides; pharynx and copulatory apparatus: sagittal sections on 18 slides.

Diagnosis. Elongated body with parallel margins; ~ 40mm in length in maximum extension; dorsal surface brown beige with median band golden yellow; thin brown beige line along the median band and thin graphite black para-median stripes; ventral surface light grey; pharynx cylindrical; extrabulbar prostatic vesicle piriform, with folded walls, and tubular paired proximal portion; male atrium richly folded, with small folds in its most proximal part; female genital canal opening into the dorso-caudal wall of the female atrium.

Type locality. Osununú Private Reserve (27°16’46’’S, 55°34’43’’W), near San Ignacio , Misiones province, Argentina ( Fig. 1 ).

Etymology. The name of this species is in honour to Horacio Quiroga (1878–1937), an Uruguayan writer who lived in San Ignacio town, in the vicinity of the type locality of the new species. He wrote wonderful stories in which, with the rainforest as setting, he used the supernatural to show the struggle of men and animals to survive.

Description. External morphology. Body elongate, with parallel margins, and flattened in cross section ( Fig. 2A, B ). The anterior tip is blunt, and the posterior tip ends sharply pointed. The dorsal surface is brown, with a median band light brown to yellowish, which is approximately 1/4 of body width, although it is a bit wider at the level of the pharynx and copulatory apparatus ( Fig. 2A ). The median band has a narrow darker brown longitudinal line. Thin black para-median stripes extend along the whole body, except near the cephalic apex ( Fig. 2A ). The ventral surface is light grey. After fixation, the dorsal pigment was conserved almost as in live, although a bit paler ( Fig. 2A ). The holotype was ~ 40mm long when crawling and 35.5mm after fixation. Its maximum width was 2.3mm, and its maximum height was 1mm. The mouth and gonopore were located at 24.7mm (70% of body length) and 29.6mm (83% of body length) of the anterior tip, respectively. The eyes, with clear halos, extend along the whole body ( Fig. 2A ), and are uniserial around the anterior tip and in the first millimetre of the body. Then, they sparse bi- and triserially, and, at 5mm from the anterior tip, they spread on the dorsal surface, reaching 6–8 rows in their maximum extension, which is at 12–15mm from the anterior tip. Towards the posterior body region, the eyes remain dorsal but scarcer and at the level of the pharynx they are less numerous (3–4 rows on each side of the body). At the level of the copulatory apparatus, they are more isolated, reaching the posterior end.

Internal morphology. Sensory pits (25µm deep), as simple invaginations of ventral epidermis, contour the anterior tip and extend along the body margins in a single row until 4mm from the anterior end ( Fig. 2B ). Four different types of secretory cells discharge through the dorsal epidermis (25µm high) at the pre-pharyngeal region: rhabditogen cells with xanthophil rhammites, which expand along the whole dorsal epidermis, abundant cells with fine granular erythrophil secretion, and less numerous cells with fine granular cyanophil secretion and erythrophil amorphous secretion ( Fig. 2B–D ). Glandular margin with abundant fine granular erythrophil and cyanophil secretions, and erythrophil and cyanophil amorphous secretions in less quantity ( Fig. 2B, C ). Ventral epidermis (15µm high) ciliated on the creeping sole, which occupies 95% of the body width. The same types of secretory cells discharge through the ventral epidermis as in the dorsal surface, although there are small dermal rhabdites ( Fig. 2B, C ). Fine granular cyanophil and erythrophil amorphous secretions are more abundant than in the dorsal epidermis, and fine granular erythrophil secretion is scarcer. The dorsal and ventral epidermis of the cephalic region receives the same types of secretory cells as in the pre-pharyngeal region, except for the dorsal epidermis, which receives abundant fine granular xanthophil secretion instead of erythrophil secretion ( Fig. 3A ). Also, erythrophil amorphous secretion is highly abundant in the creeping sole (70% of body width) ( Fig. 2B ).

Cutaneous musculature with a subepidermal circular layer (2.5µm thick), an intermediate diagonal layer (10µm thick), and an internal longitudinal layer (50µm thick dorsally, 60µm thick ventrally). The thickness of the cutaneous musculature represents 13% of the body height (CMI). Parenchymal musculature thinner than the cutaneous one (9% of body height), constituted by a dorsal decussate layer (50µm thick) followed by a transverse supra-intestinal (15µm thick) and a sub-intestinal (25µm thick) muscle layer. In addition, dorso-ventral fibres accommodate among intestinal branches ( Fig. 2B, D, E ).

Pharynx cylindrical, 2mm in length (6% of body length), occupying most of the pharyngeal pouch (2.3mm in length) ( Fig. 2F ). Pharynx lined by ciliated cuboidal epithelium, followed by a longitudinal subepithelial muscle layer (5µm thick) and a subjacent circular layer (10µm thick). Pharyngeal lumen lined by ciliated columnar epithelium, followed by a circular subepithelial muscle layer (30µm thick) and a subjacent longitudinal layer (10µm thick). Two types of secretory cells, the cell bodies of which are located in the surrounding parenchyma anterior to the pharynx, discharge through the pharyngeal epithelium: abundant cells with fine granular erythrophil secretion and less abundant fine granular cyanophil secretion ( Fig. 2F ). Short oesophagus (350µm in length) lined by ciliated columnar epithelium, followed by a subepithelial circular layer (25µm thick) and a subjacent longitudinal layer (10µm thick). The oesophagus:pharynx ratio is 17%.

Dorsal mature testes ovoid in shape, arranged in three irregular rows on each side of the body and located immediately below the supra-intestinal parenchymal muscle layer ( Fig. 2B, D ). Testes extending from behind the ovaries to the proximity of the pharyngeal root (24% and 65% of body length from the anterior end, respectively), occupying up to 35% of the body height at the pre-pharyngeal region. Sperm ducts medial and slightly dorsal to the ovovitelline ducts, located among fibres of the sub-intestinal parenchymal muscle layer ( Fig. 2B, E ). At the level of the pharynx, sperm ducts are dilated, with their lumen full of spermatozoa, constituting spermiducal vesicles. Sperm ducts extend backwards, laterally to the prostatic vesicle, until the common muscle coat, and then bend antero-dorsally, and to the sagittal plane, and open into the forked parts of the prostatic vesicle ( Fig. 3A, E ). Extrabulbar prostatic vesicle, almost piriform in shape, located very close (~300µm) to the pharyngeal pouch ( Figs. 2F , 3E ). The lumen of the prostatic vesicle is highly irregular due to its folded walls, especially the distal portion, which has a narrow lumen ( Fig. 3A–D, F ). The proximal portion of the prostatic vesicle has tubular forked parts, and it is also distinguished from the distal unpaired portion by the type of glandular secretions that each receives (see below). The ejaculatory duct (~700µm long) runs sinuously within the common muscle coat and opens into the bottom of the male atrium ( Fig. 3D–F ). The male atrium (2.5mm long), three times larger than the female atrium, is highly folded with its lumen very narrow ( Fig. 3E, F ). The most proximal region is distinguished from the rest by having numerous small folds ( Fig. 3D–F ).

Sperm ducts lined by ciliated cuboidal epithelium, without underlying musculature at pre-pharyngeal level, while spermiducal vesicles exhibit squamous epithelium with a thin subepithelial longitudinal muscle layer (5µm thick). The lining epithelium of the prostatic vesicle is columnar and ciliated, followed by musculature composed of circular fibres intermingled with some longitudinal ones (25–35µm thick). The epithelium of the proximal portion, which involves the forked tubular parts, receives abundant fine granular erythrophil secretion, whereas the distal portion is traversed by abundant coarse granular xanthophil secretion ( Fig. 3A–D, F ). The cell bodies of these glands are located in the surrounding parenchyma anteriorly to the prostatic vesicle and laterally to the distal part of the pharyngeal pouch. The ejaculatory duct is lined by ciliated columnar epithelium, with subjacent muscle coat composed of circular and oblique fibres (10–15µm thick). The most proximal part of the male atrium, which exhibits small folds, is lined by columnar and ciliated epithelium. The rest of the atrial epithelium is columnar and non-ciliated. The muscularis of the male atrium is composed mainly of circular fibres with some longitudinal and oblique fibres (20–40µm thick). The whole atrial epithelium only receives abundant fine granular erythrophil secretion. Common muscle coat with longitudinal fibres (10–15µm thick).

Ovaries (500µm long and 200µm diameter) ovoid in shape, located between the sub-intestinal parenchymal muscle layer and the nervous plate ( Fig. 3G ), at 8mm from the anterior end (22% of body length). Ovovitelline ducts arising in the mid-dorsal third of the ovaries ( Fig. 3G ). Backwards, they are situated below the sub-intestinal parenchymal muscle layer ( Fig. 2B, E ). Behind the gonopore, the ovovitelline ducts run ventro-laterally to the female atrium, contouring the common muscle coat, and posteriorly ascend slightly and join in a common glandular ovovitelline duct (~300µm long). Common duct almost vertical and distally curved to open into female atrium through a short female canal (150µm long). Female canal opening into the dorso-caudal wall of the female atrium ( Fig. 3E, H ). The female atrium (800µm long) is an oblique cavity poorly folded and almost tubular in shape, partially separated from the male atrium by an oblique fold which hangs from the roof between both atria at the level of the gonopore ( Fig. 3E, F, H ). Vitelline follicles well developed, located among intestinal branches ( Fig. 2B, E ).

Ovovitelline ducts lined by ciliated cuboidal epithelium, followed by a thin circular muscle layer (5µm thick). Lining epithelium of common glandular ovovitelline duct columnar and ciliated, receiving abundant erythrophil amorphous secretion from shell glands ( Fig. 3F, H ); common duct coated by a muscle layer composed of circular fibres intermingled with some oblique fibres (20–25µm thick). Female canal lined by a ciliated columnar epithelium followed by a muscle layer (20µm thick) with the same arrangement as in the common ovovitelline duct. The female canal receives abundant fine granular erythrophil secretion and erythrophil amorphous secretion in less quantity ( Fig. 3H ). Lining epithelium of female atrium columnar and non-ciliated, filled with abundant fine granular erythrophil secretion and scarce erythrophil amorphous secretion. Cell bodies of these glands located in the surrounding parenchyma. Muscularis of the female atrium much thicker than that of the male atrium (~200µm thick), being composed of circular fibres with some interspersed longitudinal fibres, although thinner near the gonopore (25–50µm thick). Common muscle coat not well organized as in the male atrium, consisting of loose longitudinal fibres (5–10µm thick).

Parasitism. Nematode larvae were found in the anterior body region (inside the nervous plate and parenchyma very close to the ovaries) and inside the pharynx.

Comparative discussion. The new species here described exhibits the combination of diagnostic characters of the genus Pasipha Ogren & Kawakatsu, 1990 , namely: slender body with parallel margins; extrabulbar prostatic vesicle histologically differentiated according to the secretions it receives and commonly with a bifurcated proximal portion; ejaculatory duct opening directly into a long and richly folded male atrium; ovovitelline ducts ventral to the female atrium and joining each other behind it; female genital canal ventrally flexed ( Ogren & Kawakatsu 1990; Carbayo et al. 2013). Besides, species of Pasipha tend to exhibit a thick cutaneous musculature in relation to body height, varying approximately between 10% and 20% (values of CMI). The internal anatomy of the different species of Pasipha , mainly concerning the copulatory apparatus, is extremely similar, making difficult any comparison among them. So, they are usually distinguished by differences in the external appearance and histological details of the internal anatomy.

In comparison with the other species of the genus, Pasipha quirogai sp. nov. externally resembles Pasipha mbya Negrete & Brusa, 2016 , species described for Argentina ( Negrete & Brusa 2016a). Both species exhibit a median yellowish band with black para-median stripes on the dorsum. However, in P. mbya , whose ground colour is dark grey, the median band is mottled with black spots, while in P. quirogai , whose ground colour is brown, there is a thin brownish line along the median band. Regarding the copulatory apparatus, they are also similar but P. quirogai sp. nov. can be differentiated from P. mbya in some details. The prostatic vesicle of both species is globose with folded walls, with the proximal portion bifurcated and receiving fine granular erythrophil secretion. Nevertheless, in P. mbya , the distal part of the prostatic vesicle is pierced by coarse erythrophil granules, whereas, in P. quirogai sp. nov., it receives coarse xanthophil granules. We also observed differences in the secretions that pierce the epithelium of the male atrium. In P. quirogai sp. nov., it receives only fine granular erythrophil secretion, whereas, in P. mbya , it is also pierced by amorphous erythrophil secretion and cyanophil granules ( Negrete & Brusa 2016a). Regarding the female system, P. quirogai sp. nov. is distinguished from P. mbya because the female atrium of P. quirogai sp. nov. is lined by a columnar epithelium, whereas that of P. mbya is tall columnar of pseudostratified aspect ( Negrete & Brusa 2016a).

Regarding the colour pattern, Pasipha quirogai sp. nov. also shares some similarity with the Brazilian species Pasipha penhana ( Riester, 1938) , Pasipha tapetilla ( Marcus, 1951) , Pasipha backesi Leal-Zanchet et al., 2012 , and Pasipha mesoxantha Amaral & Leal-Zanchet, 2016 , all of which exhibit a yellowish median band along the dorsum. Pasipha penhana and P. tapetilla differ from the new species because they exhibit lateral black stripes on a greyish background ( Riester 1938; Marcus 1951), absent in P. quirogai sp. nov. The dorsal colour pattern of P. backesi , unlike that of P. quirogai sp. nov., also shows lateral stripes of dark brown pigment ( Leal-Zanchet et al. 2012). Pasipha mesoxantha lacks para-median stripes, whereas, in P. quirogai sp. nov., they are present.

Regarding the copulatory apparatus, the new species can be differentiated from the above-mentioned Brazilian species due to some anatomical and histological details. In P. penhana and P. tapetilla , the prostatic vesicle is tubular and unforked, while in P. quirogai sp. nov., it is globose with the proximal portion forked, as P. backesi and P. mesoxantha . The epithelial lining of the male atrium of P. backesi and P. mesoxantha receives different secretions along its length, whereas that of P. quirogai sp. nov. receives only erythrophil granules. In addition, the female atrium of P. backesi and P. mesoxantha is oval-elongate with highly folded walls ( Amaral & Leal-Zanchet 2016; Leal-Zanchet et al. 2012), contrasting with the poorly folded atrium of P. quirogai sp. nov., which is almost tubular in shape. Also, the position of the common glandular ovovitelline duct differs between these species because in P. backesi and P. mesoxantha it is slightly ascendant and runs posteriorly, while in P. quirogai sp. nov. it is almost vertical and distally anteriorly flexed before opening into the female genital canal.

Figures

FIGURE 1. Map with the known distribution of Geoplana quagga, Obama ladislavii, and Paraba multicolor throughout Brazil (original extension of the Atlantic Forest in dark grey). In detail, the Argentine portion of the Atlantic Forest (Misiones province) with the new records of these species and localities of the new species here described. (CIAR) Centro de Investigaciones Antonia Ramos, (MO) Moconá Provincial Park, (OS) Osununú Private Reserve, (SA) San Antonio, (SE) Salto Encantado Provincial Park.

FIGURE 2. Pasipha quirogai sp. nov. (A) Photograph of preserved specimen (holotype), in dorsal view (arrowheads indicate the position of the mouth andgonopore, respectively).(B) Half of a transverse section at the cephalic region(onthe left) and at the pre-pharyngeal region (on the right). (C–E) Transverse sections of the pre-pharyngeal regionwith details:glandular margin (C), dorsal to intestine (D), ventral to intestine (E). (F) Sagittal section of the pharynx. Scale bars: A: 5mm, B: 200µm, C: 50µm, D, E: 100µm, F: 500µm.

FIGURE 3. Pasipha quirogai sp. nov. (A–D) Sagittal sections at the level of the prostatic vesicle (small folds of the male atrium indicated by arrows). (E) Schematic reconstruction of the copulatory apparatus in sagittal view. (F–H) Sagittal sections of thereproductive system:copulatory apparatus(F), ovary (G), detail of the femaleatrium (H).Scale bars:A–D, G, H: 200µm, E, F: 500µm.