Increasing diversity of land planarians (Platyhelminthes: Geoplanidae) in the Interior Atlantic Forest with the description of two new species and new records from Argentina Author Negrete, Lisandro Author Brusa, Francisco text Zootaxa 2017 2017-12-04 4362 1 99 127 journal article 31251 10.11646/zootaxa.4362.1.5 99c9eb85-21bc-470b-beda-e3f5fd023404 1175-5326 1076306 F72F750D-563E-4960-BAB3-CBEE139A288C Obama ladislavii ( Graff, 1899 ) ( Fig. 12 ) Material examined. MLP–He 7017, Salto Encantado Provincial Park, 25 February 2015 ; pre-pharyngeal region: transverse sections on 6 slides; pharynx: sagittal sections on 20 slides; copulatory apparatus: sagittal sections on 22 slides. MLP–He 7413, CIAR (27°26’40’’S, 54°56’24’’W), 11 November 2015 ; preserved in ethanol. MLP–He 7414, CIAR (27°26’40’’S, 54°56’24’’W), 13 October 2016 ; preserved in ethanol. Localities. Salto Encantado Provincial Park ( 27°03’31’’S , 54°49’44’’W ), and Centro de Investigación Antonia Ramos ( CIAR ) ( 27°26’40’’S , 54°56’24’’W ), Misiones province , Argentina ( Fig. 1 ). Description. External morphology. Body lanceolate, with the anterior body region gradually narrowing to the anterior tip, and posterior region ending abruptly ( Fig. 12A, B ). Dorsal surface yellow green, with black spots splattering the dorsum in two irregular rows at the level of the testes ( Fig. 12A ). Ventral surface golden yellow with margins yellow green ( Fig. 12B ). Once fixed, specimens become immediately beige. Eyes are uniserial and marginal around the anterior tip and up to 4mm from the tip. Then, they continue along the body margins in two or three irregular rows between 4 and 12mm from the anterior tip, and posteriorly become dorsal, reaching the posterior end. The maximum extension of the eyes on the dorsum is between 14 and 28mm from the anterior tip (28–57% relative to body length). When crawling, maximum length varied from 45 to 60mm . After fixation, the length of the specimen studied was 49mm , and maximum width was 6.5mm . The position of the mouth and gonopore relative to body length was 63% and 77% respectively. Internal morphology. Dorsal epidermis (50µm high) with three types of glandular secretion: abundant rhabdites and fine granular erythrophil secretion, and scarce fine granular cyanophil secretion. Ventral epidermis (40µm high), ciliated on the creeping sole (~100% of body width), with numerous small rhabdites occupying the apex of cells, and receiving fine granular erythrophil secretion and abundant cyanophil granules. Glandular margin absent. Cutaneous musculature, arranged with the three typical layers of Geoplaninae , a bit thicker ventrally (55µm thick) than dorsally (40µm thick). The thickness of the cutaneous musculature relative to body height at the prepharyngeal region is 5%. Parenchymal musculature organized in three layers, with loose fibres: a dorsal decussate layer with oblique fibres, a supra-intestinal transverse layer and a sub-intestinal transverse layer. The thickness of the parenchymal musculature relative to body height at the pre-pharyngeal region is ~3%. Pharynx cylindrical ( 3.2mm in length, 6% of body length), with dorsal insertion slightly displaced backwards (450µm). Mouth located at the level of the pharyngeal apex, in the middle third of the pharyngeal pouch ( 5.5mm long). Abundant erythrophil and xanthophil fine granules and less abundant cyanophil fine granules traverse the pharyngeal stroma and pierce the pharyngeal tip. Pharynx lined by ciliated cuboidal epithelium, followed by a subepithelial longitudinal muscle layer (5µm thick) and a subjacent circular layer (20µm thick). Pharyngeal lumen lined by ciliated columnar epithelium, with a subjacent muscle coat composed of circular fibres with longitudinal interspersed fibres (75–100µm thick). A short oesophagus is present (380µm long). Dorsal testes, located below the supra-intestinal parenchymal muscle layer, forming two irregular rows on each side of the body. At pre-pharyngeal level, sperm ducts located among sub-intestinal muscle fibres and dorso-medial to the ovovitelline ducts. Behind the pharynx, sperm ducts are dilated and full of spermatozoa, forming spermiducal vesicles ( Fig. 12C–E ). Their distal portions ascend and curve to the sagittal plane and open into also ascending paired portions of the prostatic vesicle ( Fig. 12C–E ). Paired tubular portions, obliquely oriented, join in an unpaired portion which runs to the dorsum, contouring the common muscle coat ( Fig. 12C, D ). The prostatic vesicle penetrates the antero-dorsal face of the penis bulb and, after a short intrabulbar tract, continues as ejaculatory duct inside the penis papilla ( Fig. 12C, D ). The ejaculatory duct traverses the penis papilla, running proximally sinuous and distally almost straight, opening near the tip of the penis, a bit ventrally displaced ( Fig. 12C, D ). The penis papilla projects obliquely from the dorsal wall of the male atrium, with its dorsal insertion posteriorly displaced ( Fig. 12C, D ). The male atrium ( 1.6mm long), without folded walls, is almost totally occupied by the penis papilla ( 1.4mm long) and broadly communicated with the female atrium ( Fig. 12C ). Distal portions of sperm ducts lined by a ciliated squamous epithelium, with a thin longitudinal muscle layer (2.5µm thick). Prostatic vesicle lined with a ciliated, columnar epithelium, pierced by fine erythrophil granules highly abundant in the unpaired portion ( Fig. 12F ). Musculature of prostatic vesicle composed of circular and some longitudinal fibres (15–25µm thick). The ejaculatory duct is lined by a ciliated columnar epithelium, filled with erythrophil fine granules, followed by a thin layer of circular fibres (2.5–5µm thick). The penis papilla is lined with non-ciliated columnar epithelium, followed by a circular muscle layer with some interspersed longitudinal fibres (10–15µm thick). Stroma of penis papilla with abundant fine granular erythrophil secretion and scarce cyanophil granules ( Fig. 12D ). Dorsal wall of the male atrium lined with columnar epithelium (up to 60µm high), and ventral wall lined with cuboidal epithelium ( Fig. 12D ), both pierced by erythrophil granules. Muscularis of male atrium with circular fibres with some interspersed longitudinal fibres (5–10µm thick). Abundant cyanophil fine granules extend from the dorsal to the antero-ventral side of the male atrium, spreading from the left wall of the atrium ( Fig. 12C, D, G ). Cyanophil granules also spread onto the dorsal part of the penis papilla ( Fig. 12D ). Vitelline follicles mature but scarce, located in the surrounding parenchyma among intestine branches. Ovovitelline ducts located immediately below the sub-intestinal parenchymal muscle layer. At the level of the gonopore, the ovovitelline ducts ascend to above the female atrium joining each other ( Fig. 12C, H ). The common glandular ovovitelline duct (200µm long) runs backwards and ventrally flexed ( Fig. 12C, D ) to communicate with the female canal (180µm long), which runs downwards and anteriorly ( Fig. 12C, D ). The female canal opens into the female atrium (600µm long), which is funnel-shaped in sagittal section ( Fig. 12C ). The ovovitelline ducts are lined with cuboidal ciliated epithelium with a subjacent thin circular muscle layer (2.5µm thick). Distal ascending portions of ovovitelline ducts receive erythrophil secretion from shell glands ( Fig. 12C, H ). Common glandular ovovitelline duct lined with ciliated columnar epithelium, pierced by shell glands ( Fig. 12C, D, H ), followed by a subjacent muscle layer consisting of circular and longitudinal fibres (10–15µm thick). Epithelial lining of the female canal, columnar and non-ciliated, with abundant fine granular erythrophil secretion, wrapped by longitudinal and circular muscle fibres (10µm thick). Female atrium lined with non-ciliated columnar epithelium of stratified appearance (60–75µm high) ( Fig. 12D ), which receives erythrophil granules and scarce xanthophil amorphous secretion. Muscularis with the same arrangement and thickness of the female genital canal. Gonopore canal lined with ciliated columnar epithelium, apically erythrophil, with abundant cyanophil granules. Remarks and comparative discussion. Obama ladislavii ( Graff, 1899 ) is one of the land planarian species most easily distinguished from others, due to the striking bright green pigmentation of its dorsum. The external aspect of specimens from Argentina ( Misiones ) matches well with that of Brazilians specimens, which have been reported in many localities of the southern region of this country ( Graff 1899 ; Froehlich 1959 ; Álvarez-Presas et al. 2015 ). Our specimens also exhibit black spots, which spread on the dorsum at the level of the testes, as observed in specimens from Santa Catarina and Rio Grande do Sul ( Froehlich 1959 ; Álvarez-Presas et al. 2015 ). According with the original description, maximum extension in living specimens ranges from 46 to 100mm ( Graff 1899 ). The specimens studied by us (~ 50mm in maximum extension, on average) are similar to those studied by Froehlich (1959) from Rio Grande do Sul (the type locality, among others) and Santa Catarina . Álvarez-Presas et al. (2015) , who re-described O. ladislavii based on material from new localities and specimens previously studied by Graff (1899) and material collected by Froehlich (1959) , found specimens with ~ 70–80mm in maximum extension. The relative position of the mouth and gonopore in relation to body length (63% and 77% respectively, in the specimen sectioned) is similar to that of the specimens analysed by Froehlich (1959) (average: 61% and 76%) and Álvarez- Presas et al. (2015) (average: 64% and 83%). Regarding the internal anatomy, the thickness of the cutaneous musculature relative to the body height at the pre-pharyngeal region of the Argentinean specimen is concordant with that of specimens studied by Álvarez-Presas et al. (2015) . However, these authors observed a glandular margin at the pre-pharyngeal region, not observed in the sectioned specimen from Argentina , although this could be due to variations in the fixation and staining protocols. The cylindrical pharynx presents insertions almost at the same level in sagittal view, with its apical portion located at the level of the mouth, as illustrated by Froehlich (1959) and Álvarez-Presas et al. (2015) . Similarly to specimens from the type locality (Taquara, Rio Grande do Sul ), the pharynx is pierced by erythrophil, xanthophil and cyanophil secretions, with the mouth in the middle of the pharyngeal pouch ( Álvarez-Presas et al. 2015 ). The body region at both the level of the ovaries and the level of the anteriormost testes could not be compared because the specimen could not be sectioned. In general, the aspect of the copulatory apparatus agrees with descriptions provided by Froehlich (1959) and Álvarez-Presas et al. (2015) but, as pointed out by the latter authors, we also found differences with the schematic reconstruction illustrated by Graff (1899) , namely: intrabulbar prostatic vesicle without paired portions, and ovovitelline ducts ascending behind the female atrium. Froehlich (1959) pointed out the presence of a dorsal fold of the male atrium separating it partially from the female atrium, absent in the specimen from Argentina and those studied by Álvarez-Presas et al. (2015) , in which both atria are broadly communicated. It is also noteworthy that both atria, particularly the female one, exhibit tall columnar epithelium (~100–200µm high) ( Froehlich 1959 ; Álvarez-Presas et al. 2015 ), although in the Argentinean specimen its maximum height (75µm) was lower than that in the Brazilian ones, similarly to the male atrium. This is probably because the specimen studied was not fully mature, evidenced by the scarcity of vitelline follicles.